PREVENTION OR TREATMENT OF CHRONIC ORGAN INJURY

Abstract

The present disclosure is directed to novel therapeutic approaches for the prevention, treatment and/or delaying progression of chronic injury, progressive loss of functional parenchymal cells, or fibrosis of an organ. Specifically disclosed are agents for use in increasing homodimer-formation of ARNT in an organ in the prevention, treatment and/or delaying progression of chronic injury, progressive loss of functional parenchymal cells, or fibrosis of said organ, as further defined in the claims. In embodiments, said agent may be (i) an inhibitor of protein phosphatase 2A (PP2A) activity, (ii) an inhibitor of the transcriptional repressor complex FKBP12/YY1, or (iii) an expression construct, which is capable of over-expressing ARNT in said organ, as well as combinations of (i), (ii), and/or (iii).

Claims

1-36. (canceled)

37. A method for increasing homodimer-formation of ARNT in an organ of a subject to be treated in the prevention, treatment and/or delaying progression of chronic injury, progressive loss of functional parenchymal cells, or fibrosis of said organ, comprising the step of administering said agent to said subject.

38. The method of claim 37, wherein the increased homodimer-formation of ARNT is to increase the expression of ALK3.

39. The method of claim 37, wherein the increased homodimer-formation of ARNT is by an increased expression of ARNT.

40. The method of claim 37, wherein the increased homo-dimer formation of ARNT is to protect said organ against chronic injury, progressive loss of functional parenchymal cells, or fibrosis.

41. The method of claim 37, wherein said chronic injury, progressive loss of functional parenchymal cells, or fibrosis is caused by a chronic progressive disease, or by a long-term exposure to a substance which is toxic for said organ, or by a long-term ischemia.

42. The method of claim 37, wherein the organ is selected from the group consisting of kidney, heart, intestine, spleen, lung, liver, brain, spinal cord, skin, and pancreas.

43. The method of claim 37 wherein said method is: (i) for the prevention, treatment and/or for delaying progression of a condition selected from the group consisting of chronic kidney disease; or (ii) for the prevention of and/or for delaying progression to end-stage renal disease; or (iii) for the prevention, treatment, and/or for delaying progression of pulmonary fibrosis; or (iv) for the prevention, treatment, and/or for delaying progression of a fibrosis selected from the group consisting of cystic fibrosis, idiopathic pulmonary fibrosis, progressive massive fibrosis, liver cirrhosis, artrial fibrosis of the heart, endomyocardial fibrosis, glial scar of the brain, keloid of the skin, and Crohn's disease of the intestine; or (v) for the prevention, treatment, and/or for delaying progression of chronic cardiac injury; or (vi) for protecting from impairment of organ function or histopathological patterns of chronified injury, applied before, during, or after injury; or (vii) for the treatment or prevention of diabetes mellitus.

44. The method of claim 37, wherein said agent is an inhibitor of protein phosphatase 2A (PP2A) activity.

45. The method of claim 44, wherein said inhibitor is: (a) a siRNA, or (b) a vivo morpholino, or (c) a small molecule selected from the group consisting of (i) an oxabicycloheptane or oxabicycloheptene such as LB-100, LB-102 and LB-107, (ii) okadaic acid, (iii) fostriecin, and (iv) calyculin A.

46. The method of claim 37, wherein said agent is an inhibitor of the transcriptional repressor complex FKBP12/YY1.

47. The method of claim 46, wherein said agent is an inhibitor of FKBP12 or an inhibitor of YY1.

48. The method of claim 46, wherein said agent is an inhibitor of FKBP12 selected from the group consisting of a small molecule, a siRNA, and a vivo morpholino.

49. The method of claim 48, wherein said inhibitor of FKBP12 is: (a) a small molecule selected from the group consisting of (i) a pipecolic acid derivative such as GPI 1046, GPI 1044, GPI 1102, GPI 1116, or GPI 1206, (ii) rapamycin, FK 506 and derivatives thereof; or (b) a vivo morpholino comprising or consisting of the sequence shown in SEQ ID NO: 9.

50. The method of claim 46, wherein said agent is an inhibitor of YY1 selected from the group consisting of a vivo morpholino and a siRNA.

51. The method of claim 50, wherein said inhibitor of YY1 is a vivo morpholino comprising or consisting of the sequence shown in SEQ ID NO: 10.

52. The method of claim 37, wherein said agent is an expression construct, which is capable of over-expressing ARNT in said organ.

53. The method of claim 52, wherein said expression of ARNT is under the control of a constitutive promoter, an inducible promoter, or a promoter which selectively expresses ARNT in said organ.

54. The method of claim 37, wherein the agent is a combination of at least two of (i) an inhibitor of protein phosphatase 2A (PP2A) activity, (ii) an inhibitor of the transcriptional repressor complex FKBP12/YY1, and (iii) an expression construct, which is capable of over-expressing ARNT in said organ.

55. The method of claim 37, wherein the agent is a combination of (i) an inhibitor of protein phosphatase 2A (PP2A) activity, and (ii) an inhibitor of the transcriptional repressor complex FKBP12/YY1.

56. The method of claim 55, wherein the agent is a combination of (i) GPI 1046; (ii) FK-506 or a derivative thereof, and (iii) LB100.

Description

DESCRIPTION OF THE FIGURES

[0047] FIG. 1: Mice were challenged with UUO and treated with either vehicle buffer, FK506 (0.02, 0.075, 0.2, 5.0 mg/kg orally per day, respectively) or CsA (10 mg/kg orally per day) starting one day prior of surgery.

[0048] FIG. 2: (A) Tubular damage at day 10 after ureteral obstruction was semi-quantitatively scored using PAS-stained kidney sections (0=healthy, 1=mild, 2=moderate, 3=severe, n=6/group, data are presented as meanss.d., *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (B) The graph summarizes average means of relative tubulointerstitial fibrosis 3, 7 and 10 days after ureteral obstruction (n=6/group, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C) In mice receiving FK506, areas positive for Collagen-1 was assessed (n=6/group, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (D) The graph summarizes average means of areas positive for aSMA 3, 7 and 10 days after ureteral obstruction (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (E,F) Intrarenal expression levels of Collagen-1a1 and Acta2 (aSMA) were analyzed by qRTPCR in total kidney lysates (n=3-4/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0049] FIG. 3: Based on genome-wide transcriptional expression datasets for bioactive small molecules (accession number GSE5258), pathway analysis of differentially expressed genes induced in response to FK506 is shown (data are presented as process analyses of log.sub.10 p).

[0050] FIG. 4: (A,B) Mice were challenged with UUO and treated with either vehicle buffer, indicated concentrations of FK506 or CsA starting one day prior of surgery. Analyzed by qRT-PCR 10 days after ureteral obstruction, the bar graphs reflect relative mRNA expression levels of type I BMP receptors Alk3 and Alk6 (n=3/group, data are presented as meanss.d., ** p<0.1,** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C-E) Analyzed by immunoblotting of total kidney to lysates and immunostaining, type I BMP receptor Alk3 and pSmad1/5/8 was assessed, reflecting activation of BMP signaling (n=6/group, data are presented as meanss.d., * p<0.05, ** p<0.01, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0051] FIG. 5: Mice were challenged with UUO and treated with either vehicle buffer or low-dose FK506 (0.2 mg/kg orally per day) when specifically canonical pSmad1/5/8-dependent ALK3 signaling transduction was pharmacologically blocked with small molecule LDN-193189 (LDN, 3 mg/kg intraperitoneally per day).

[0052] FIG. 6: Alk3 and pSmad1/5/8 were analyzed by immunostaining (n=6/group, data are presented as meanss.d., * p<0.05, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0053] FIG. 7: (A,B) The panels show data for PAS-stained fibrotic kidney sections and sections immunolabelled with primary antibodies against Collagen-1 (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C,D) To elucidate the specific contribution of identified ALK3-dependent canonical BMP signaling responses to FK506-mediated protection, mice were challenged with UUO and treated with either vehicle buffer or low-dose FK506 (0.2 mg/kg orally per day, previously established as most effective) when specifically canonical pSmad1/5/8-dependent ALK3 signaling transduction was pharmacologically blocked with small molecule LDN-193189 (LDN, 3 mg/kg intraperitoneally per day). The graph summarizes average means of relative tubulointerstitial fibrosis and areas positive for aSMA in PAS stainings and sections immunolabelled with primary antibodies against aSMA 10 days after ureteral obstruction (n=6/group, data are presented as meanss.d., ** p<01, * p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0054] FIG. 8: (A-C) TECs were exposed to either DMSO alone (vehicle), DMSO containing indicated concentrations of FK506 (0.02, 0.2, 2, 20, 200 nM, respectively) or equimolar Cyclosporine A (CsA, 10 nM), mRNA expression levels of type I BMP receptors Alk3, Alk5 and Alk6 were analyzed by qRT-PCR (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0055] FIG. 9: (A) TECs were exposed to DMSO alone (vehicle) or DMSO containing indicated concentrations of FK506, representative photomicrographs of phosphorylated Smad1/5/8 complex (pSmad1/5/8) immunostainings overlayed with differential interference contrast (DIC, scale bars 25 m) are analyzed (n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (B) TECs were seeded at a density of 60,000 per well and proliferative activity was determined at indicated time points (n=3 independent to experiments, data are presented as meanss.d., *** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0056] FIG. 10: (A-D) Tubular epithelial cells (MCT) were transfected with either scrambled RNA (scrRNA), siRNA targeting Fkbp12 (Fkbp12kd), Fkbp25 (Fkbp25kd), Fkbp38 (Fkbp38kd, Fkbp52 (Fkbp52kd). Analyzed by qRT-PCR, the bar graphs summarize relative mRNA expression levels (n=3 independent experiments, data are presented as meanss.d., ** p<0.01, *** p<0.001, **** p<0.0001, values of p were calculated using Student's t test).

[0057] FIG. 11: As analyzed by qRT-PCR, Alk3 mRNA expression levels in TECs were analyzed after siRNA-mediated knockdown of Fkbp12 (Fkbp2ka), Fkbp25 (Fkbp25kd), Fkbp38 (Fkbp38kd) or Fkbp56 (Fkbp56kd, n=3 independent experiments, data are presented as meanss.d., **** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0058] FIG. 12: (A) As analyzed by co-immunoprecipitation after Fkbp12 pull-down (IP: Fkbp12), direct interaction between Fkbp12 and Alk3 was assessed. (B) As analyzed by co-immunoprecipitation after Fkbp12 pull-down (IP: Fkbp12), direct interaction between Fkbp12 and Alk3 was assessed.

[0059] FIG. 13: As analyzed by co-immunoprecipitation after Yy1 pull-down (IP: Yy1), direct interaction between Yy1 and Fkbp12 was assessed.

[0060] FIG. 14: Analyzed by qRT-PCR, the bar graphs summarize relative Yy1 mRNA expression levels (n=3 independent experiments, data are presented as meanss.d., * p<0.05, values of p were calculated using Student's t test).

[0061] FIG. 15: (A) Alk3 mRNA expression levels were assessed by qRTPCR after knockdown of either Yy1 (Yy1kd) or Fkbp12 (Fkbp12kd) and exposure to FK506 (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, # no significance, values of p were calculated using Student's t test). (B) Representative photomicrographs of pSmad1/5/8 immunostainings overlayed with differential interference contrast (DIC, scale bars 25 m) are analyzed (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, # no significance, values of p were calculated using Student's t test).

[0062] FIG. 16: Mice conditionally depleted for YY1 in TECs (yGTcre+;Yy1/f/1 and corresponding littermate controls (yGTcre;Yy1fl/fl) were challenged with UUO and treated with either vehicle buffer or FK506 (0.2 mg/kg orally per day) starting one day prior of surgery.

[0063] FIG. 17: (A-E) Representative photomicrographs of immunostainings for Alk3, pSmad1/5/8, PAS-stained fibrotic kidney sections, MTS and Collagen-1 in mice challenged with UUO are analyzed (n=3/group, data are presented as meanss.d., * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (F) Alk3 mRNA expression levels were analyzed by qRT-PCR (n=3/group, data are presented as meanss.d., p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0064] FIG. 18: (A) TECs were exposed to the protein translation blocker cycloheximide (CHX) one hour prior FK506 incubation, Alk3 mRNA expression was assessed by qRT-PCR (n=3 independent experiments, data are presented as meanss.d., **** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (B) BMP signaling responses reflected by nuclear pSmad1/5/8 accumulation was analyzed by immunostaining overlayed with differential interference contrast (DIC, scale bars 25 m, n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0065] FIG. 19: FK506-mediated transcriptional network alterations were analyzed by transcription factor array analysis and subsequent prediction of putative binding motifs within the ALK3 proximal promoter, log base 2 fold changes (log.sub.2 FC) are shown.

[0066] FIG. 20: Identified transcriptional factors induced by FK506 were validated by qRT-PCR upon FK506 exposure (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, **** p<0.0001, values of p were calculated using Student's t test in comparison to DMSO-treated control cells).

[0067] FIG. 21: Alk3 mRNA levels were analyzed by qRT-PCR after siRNA-mediated knockdown (n=3 independent experiments, data are presented as meanss.d., ** p<0.01, *** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0068] FIG. 22: (A-F) TECs were transfected with either scrambled RNA (scrRNA), siRNA targeting Ar (Arkd), Arnt (Arntkd), Cebpb (Cebpbkd, Creb1 (Creb1kd), Gata3 (Gata3kd) or Max (Maxkd). Analyzed by qRT-PCR, the bar graphs summarize relative mRNA expression levels in response to FK506 (200 pM, n=3 independent experiments, data are presented as meanss.d., * p<0.05, **** p<0.0001, # no significance, values of p were calculated using Student's t test). (G) Analyzed by qRT-PCR, Arnt over-expression (Arntoe) was assessed (n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, values of p were calculated using Student's t test).

[0069] FIG. 23: As analyzed by qRT-PCR, Arnt mRNA expression levels in TECs after siRNA-mediated knockdown of Fkbp12 (Fkbp12kd), Fkbp25 (Fkbp25kd), Fkbp38 (Fkbp38kd), Fkbp52 (Fkbp52kd) or Yy1 (Yy1kd) are shown (n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0070] FIG. 24: Binding of Yy1 to the Arnt proximal promoter was analyzed by Chromatin immunoprecipitation (ChIP) and subsequent target PCR after Yy1 pull-down (IP: Yy1, n=3 technical replicates, data are presented as meanss.d., ** p<0.01, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0071] FIG. 25: (A) As assessed by qRT-PCR, Alk3 mRNA expression levels after knockdown of either Fkbp12 (Fkbp12kd) or Yy1 (Yy1kd) are shown (n=3 independent experiments, data are presented as meanss.d., ** p<0.01, # no significance, values of p were calculated using Student's t test). (B) Arnt protein levels were analyzed by immunostaining overlayed is with differential interference contrast (DIC, scale bars 25 m, n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using Student's t test).

[0072] FIG. 26: Representative kidney sections of yGTcre+;Yy1fl/fl and yGTcre;Yy1fl/fl mice immunolabelled with primary antibodies against Arnt are analyzed (n=3/group, data are presented as meanss.d., ** p<0.01, *** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0073] FIG. 27: (A,B) Arnt protein levels were analyzed by immunoblotting and immunostaining (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C) Arnt mRNA from total kidney lysates was analyzed by qRT-PCR. (n=3-6/group, data are presented as meanss.d., *** p<0.001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0074] FIG. 28: (A) Alk3 mRNA was assessed by qRT-PCR after depletion of Arnt (Arntkd, n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (B) Efficacy of BMP signaling activation was analyzed by immunostaining overlayed with differential interference contrast (DIC, scale bars 25 m, n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0075] FIG. 29: (A,B) Alk3 and Alk6 mRNA expression levels analyzed by qRT-PCR are shown (n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using Student's t test). (C) BMP signaling responses in cultured TECs were assessed by pSmad1/5/8 immunostaining overlayed with differential interference contrast (DIC, scale bars 25 m, n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, values of p were calculated using Student's t test).

[0076] FIG. 30: (A,B) Impact of FK506 on hypoxic signaling and drug metabolism was analyzed by respective array analysis.

[0077] FIG. 31: (A,B) Efficacy of FK506 or Arnt over-expression to induce Alk3 mRNA expression levels in cultured TECs depleted for HIF1 (Hif1akd) or AHR (Ahrkd) was assessed by qRT-PCR (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0078] FIG. 32: (A-D) TECs were transfected with either scrambled RNA (scrRNA), siRNA targeting Hif1 (Hif1akd) or Ahr (Ahrkd). Analyzed by qRT-PCR, the bar graphs summarize relative mRNA expression levels in response to either FK506 (200 pM) or transgenic Arnt over-expression (Arntoe, n=3 independent experiments, data are presented as meanss.d., # no significance, values of p were calculated using Student's t test).

[0079] FIG. 33: (A-C) Analyzed by co-immunoprecipitation (CoIP) using antibodies to Arnt (IP: is Arnt), Arnt/Hif1 and Arnt/Ahr interactions were assessed in cultured TECs in response to FK506 (200 pM).

[0080] FIG. 34: (A,B) Analyzed by CoIP, homodimer formation was assessed in cultured TECs after EGFP-tagged (Arnt-EGFPoe) and myc-tagged (Arnt-mycoe) ARNT overexpression and pulldown of Arnt-EGFP (IP: EGFP) or Arnt-myc (IP: myc).

[0081] FIG. 35: Dimer formation of Arnt/Arnt, Arnt/Hif1, Arnt/Hif2 and Arnt/Ahr was assessed by native gel electrophoresis.

[0082] FIG. 36: Binding of Arnt to the Alk3 proximal promoter containing palindromic E-box binding motifs (5-CACGTG core sequence) was analyzed by ChIP and subsequent target PCR after Arnt pull-down (IP: Arnt, n=3 technical replicates, data are presented as meanss.d., ** p<0.01, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0083] FIG. 37: Analyzed by reporter assays, Alk3 proximal promoter activity was assessed in presence (Alk3 wt) or absence (Alk3mut, CACGTG (SEQ ID NO: 1) to TATATA (SEQ ID NO:2)) of the palindromic E-box motif (n=5 independent experiments, data are presented as meanss.d., * p<0.05, # no significance, values of p were calculated using Student's t test).

[0084] FIG. 38: Dimer formation of Arnt/Arnt, Arnt/Hif1, Arnt/Hif2 and Arnt/Ahr in total kidney lysates was assessed by native gel electrophoresis after Arnt pulldown.

[0085] FIG. 39: Mice were treated daily with either intraperitoneal administration of control in vivo-morpholinos (control-VMO), in vivo-morpholinos targeting translational start site of Fkbp12 (Fkbp12-VMO), Yy1 (Yy1-VMO) or Arnt (Arnt-VMO) starting two days prior of surgery. One day prior of UUO surgery, mice were orally treated with either vehicle buffer or FK506 (0.2 mg/kg orally per day).

[0086] FIG. 40: (A,B) As determined by immunostaining, intrarenal Fkbp12 and Yy1 were assessed (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C) Analyzed by immunoblotting, intrarenal Arnt in mice challenged with UUO is shown. (D) Mice were treated daily with either intraperitoneal administration of control in vivo-morpholinos (control-VMO) or in vivo-morpholinos targeting translational start site of Fkbp12 (Fkbp12-VMO) starting two days prior of surgery, intrarenal Fkbp12 was analyzed by immunoblotting of total kidney lysates.

[0087] FIG. 41: (A-D) Representative photomicrographs of kidney sections labelled for Arnt (scale bars 25 m), PAS (scale bars 50 m), MTS (scale bars 50 m) and Collagen-1 (scale bars 25 m) are shown (n=6/group, data are presented as meanss.d., *** p<0.001, **** p<00001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (E) Expression levels of Collagen-1a1 was assessed by qRT-PCR (n=3-4/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (F-H) Representative photomicrographs of kidney sections immunolabelled for Alk3, pSmad1/5/8 or aSMA are analyzed (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (I) Expression levels of Acta2 (encoding aSMA) was assessed by qRT-PCR (n=3-4/group, data are presented as meanss.d., *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0088] FIG. 42: (A,B) TECs were exposed to DMSO alone (vehicle), DMSO containing FK506 (200 pM) or GPI-1046 (10 M), relative Arnt and Alk3 mRNA expression levels were analyzed by qRT-PCR (n=3 independent experiments, data are presented as meanss.d., * p<0.05, **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C,D) Representative photomicrographs of Arnt and phosphorylated Smad1/5/8 complex (pSmad1/5/8) immunostainings overlayed with differential interference contrast (DIC) are analyzed (n=3 independent experiments, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0089] FIG. 43: Mice were challenged with UUO and treated with either vehicle buffer or GPI-1046 (10 mg/kg subcutaneously per day) starting one day prior of surgery.

[0090] FIG. 44: (A,B) Analyzed by qRT-PCR 10 days after ureteral obstruction, intrarenal Arnt and Alk3 mRNA expression levels are shown (n=4/group, data are presented as meanss.d., *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C,D) Intrarenal Alk3 (scale bars 25 m) and pSmad1/5/8 (scale bars 25 m) were analyzed by immunostaining (n=6/group, data are presented as meanss.d., * p<0.05, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (E,F) The panels show representative photomicrographs of PAS-stained fibrotic kidney sections (scale bars 50 m) and sections immunolabelled with primary antibodies against Collagen-1 (scale bars 25 m, n=6/group, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (G,H) Mice were challenged with UUO and treated with either vehicle buffer or GPI-1046 (10 mg/kg subcutaneously per day, respectively) starting one day prior of surgery, representative kidney sections stained for MTS or immunolabelled with aSMA are analyzed (n=6/group, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0091] FIG. 45: (A) Mice were challenged with UUO and treated with either vehicle buffer, FK506 (0.2 mg/kg orally per day) or GPI-1046 (30 mg/kg orally per day) starting one day prior of UUO surgery, representative kidney sections immunolabeled with CD45 are analyzed (n=6/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (B) Intrarenal NFATc1 and NFATc2 were assessed by immunoblotting.

[0092] FIG. 46: Mice were challenged with UUO and administered either vehicle buffer or GPI-1046 (30 mg/kg orally per day) starting three days after surgery.

[0093] FIG. 47: (A,B) Analyzed by qRT-PCR, intrarenal Arnt and Alk3 mRNA expression levels were assessed (n=4/group, data are presented as meanss.d., * p<0.05, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C,D) Representative photomicrographs of immunostainings for Alk3 and pSmad1/5/8 are analyzed (n=6/group, data are presented as meanss.d., **p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0094] FIG. 48: (A,B) Representative photomicrographs of immunostainings for PAS-stained fibrotic kidney sections and Collagen-1 are analyzed (n=6/group, data are presented as meanss.d., **p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (C,D) Mice were challenged with UUO and treated with either vehicle buffer, FK506 (0.2 mg/kg orally per day) or GPI-1046 (30 mg/kg orally per day) starting three days after UUO surgery, representative kidney sections stained for MTS or immunolabelled with aSMA are analyzed (n=6/group, data are presented as meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0095] FIG. 49: (A) Analyzed by qRT-PCR, Arnt mRNA expression levels are shown in response to FK506 (0.2 mg/kg orally per day) or GPI-1046 (30 mg/kg orally per day, n=3/group, data are presented as meanss.d., * p<0.05, ** p<0.01, **** p<0.0001, # no significance, values of p were calculated using oneway ANOVA with Bonferroni post-hoc analysis in comparison to vehicle-treated control mice).

[0096] FIG. 50: (A-D) Abundance of Fkbp12 and Yy1 was analyzed by immunostaining (scale bars 25 m) in AT II-induced cardiomyopathy and CCI4-mediated liver injury (n=5-7/group, data are presented as meanss.d., * p<0.05, ** p<0.01, *** p<0.001, # no significance, values of p were calculated using Student's t test).

[0097] FIG. 51: (A,B) Mice were challenged with AT II delivered by osmotic minipumps or intraperitoneal injections of CCI4, vehicle buffer or GPI-1046 (10 mg/kg subcutaneously per day) were administered starting one day prior.

[0098] FIG. 52: (A-F) Representative photomicrographs of immunostainings for Alk3, pSmad1/5/8, PAS-stained fibrotic kidney sections, MTS, Collagen-1 and aSMA in mice challenged with AT II are analyzed (n=6/group, data are presented as meanss.d., *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (G-L) Representative photomicrographs of immunostainings for Alk3, pSmad1/5/8, MTS-stained fibrotic kidney sections, Sirius red, Collagen-1 and aSMA in mice challenged with CCI4 are analyzed (n=5-7/group, data are presented as meanss.d., ** p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (M,N) Mice were treated with GPI-1046 (10 mg/kg subcutaneously per day) and challenged with AT II, Arnt and Alk3 mRNA expression levels in total hearts (n=4/group, data are presented as meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis). (0-R) Systolic, diastolic, mean arterial pressure (MAP) and heart rate beats per minute (BPM) of mice treated with GPI-1046 (10 mg/kg subcutaneously per day) and challenged with AT II are shown (n=6/group, data are presented as aligned dot plots with meanss.d., **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis comparing indicated pairs of columns). (S,T) Measurements of aspartate and alanine aminotransferases in mice challenged with CCI4 are shown (n=5-6/group, data are presented as aligned dot plots with meanss.d., **** p<0.0001, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0099] FIG. 53: (A) Human TEC cultures were exposed to DMSO alone (vehicle) or DMSO containing indicated concentrations of FK506, ALK3 mRNA expression was analyzed by qRT-PCR (n=3 independent experiments). (B) Human TEC cultures were exposed to DMSO alone (vehicle) or DMSO containing FK506 (200 pM), representative photomicrographs of Arnt immunostainings overlayed with differential interference contrast (DIC) are analyzed (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, values of p were calculated using Student's t test). (C) Analyzed by qRT-PCR, ALK3 mRNA expression levels are shown (n=3 independent experiments, data are presented as meanss.d., *** p<0.001, values of p were calculated using Student's t test). (D) Analyzed by SDS-PAGE and subsequent immunoblotting, ALK3 was assessed in response to FK506. (E) Representative photomicrographs of phosphorylated Smad1/5/8 complex (pSmad1/5/8) immunostainings overlayed with differential interference contrast (DIC) are analyzed (n=3 independent experiments, data are presented as meanss.d., ** p<0.01, values of p were calculated using Student's t test).

[0100] FIG. 54: (A-E) In a small cohort of kidney transplant recipients with comparable histological patterns and immunosuppressive regimens including either CsA or FK506, representative photomicrographs of kidney sections stained for PAS, MTS, immunolabelled for Collagen-1 or aSMA are analyzed (measurements were done in 10 visual fields, data are presented as meanss.d., # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0101] FIG. 55: (A-I) In a small cohort of kidney transplant recipients with comparable histological patterns and immunosuppressive regimens including either CsA or FK506, kidney sections immunolabelled with primary antibodies against ARNT, ALK3, pSmad1/5/8, FKBP12 and YY1 are shown, mRNA expression levels were assessed by qRT-PCR (measurements were done in 10 visual fields for immunostainings and in technical triplicates for qRT-PCR, data are presented as meanss.d., ** p<0.01, *** p<0.001, **** p<0.0001, # no significance, values of p were calculated using one-way ANOVA with Bonferroni post-hoc analysis).

[0102] FIG. 56: (A) In injured TECs, FKBP12/YY1 complexes repress ARNT (B) FK506 or GPI-1046 disrupt FKBP12/YY1 interaction, in turn associated with release from transcriptional repression of ARNT (C) ARNT subsequently mediates transcriptional ALK3 induction independent of HIF1 or AHR. (D) Transcriptional ALK3 induction is associated with activation of canonical BMP signaling responses reflected by nuclear translocation of phosphorylated Smad1/5/8.

[0103] FIGS. 57 & 58: While PP1 inhibition did not affect ARNT/HIF1 homodimer formation, blocking PP2A was associated with enhanced ARNT/HIF1 homodimer formation.

[0104] FIG. 59: LB-100 enhances protection by FK506/GPI-1046.

EXAMPLES

[0105] Materials and Methods

[0106] Human Kidney Specimens.

[0107] The use of parts of human specimens for research purposes as approved by the Ethics Committee of the University Medicine Gttingen, clinical data are presented in Table 6.

[0108] Animals.

[0109] All studies and inclusion/exclusion of animals were performed according to the German animal care and ethics legislation and had been carried out with the approval of the local government authorities (LAVES) and the University Medicine Gttingen. Experimental protocols are detailed below. B6;12954-Yy1tm2Yshi/J (referred as Yy1fl/fl) mice were obtained from Jackson Laboratory (Bar Harbor, USA), Tg(Ggt1-cre)M3Egn/J (referred as yGTcre+) mice were previously described and genetic backgrounds were identical when comparing experimental groups (53, 152).

[0110] Unilateral Ureteral Obstruction (UUO).

[0111] Eight to twelve weeks-old C57/6, yGTcre;Yy1fl/fl and yGTcre+;Yy1fl/fl mice were anesthetized with isoflurane inhalation (2-3%), analgesia was performed by subcutaneous injection of 0.1 mg/kg body weight per day Buprenorphine. The ureter was separated from the surrounding tissues and two ligatures were placed about 5 mm apart in upper two-thirds of the left ureter to obtain reliable obstruction. Mice were sacrificed 3, 7 or 10 days after ureteral obstruction for further analyses, as described before (53, 153-155).

[0112] Angiotensin II (AT II)-Induced Cardiac Hypertrophy and Fibrosis.

[0113] Eight to twelve weeks old C57BL/6 mice were anesthetized with isoflurane inhalation (2-3%), analgesia was performed by subcutaneous injection of 0.1 mg/kg body weight Buprenorphine per day. Osmotic minipumps (Alzet, Cupertino, USA) were loaded with AT II to continuously deliver 1.44 g/kg body weight per day and implanted subcutaneously (123). Mice were sacrificed 14 days after implantation for further analyses.

[0114] Tetrachlormethan (CCI14)-Induced Liver Fibrosis.

[0115] Eight to twelve weeks-old C57BL/6 mice were intraperitoneally injected with 0.25 (first injection), 0.5 (second injection) and 1 mL/kg body weight CCI4 (25% v/v dissolved in sterile oil) twice a week (63). Mice were sacrificed after 42 days for further analyses.

[0116] Blood Pressure Measurements.

[0117] Measurements of blood pressure were performed using a tail cuff system, systolic, diastolic, mean arterial pressure (MAP) and heart rate were recorded (156).

[0118] FK506/CsA Preparation and Treatment.

[0119] FK506 and Cyclosporine A (CsA) were purchased as powders with a purity of >98% (Abcam Biochemicals, Cambridge, UK). FK-506 and CsA stock solutions (0.2 mg/mL) were prepared by dissolving the compound in saline (0.9% NaCl) containing 1.25% PEG40 Castor Oil (spectrum chemicals &laboratory products, USA) and 2% ethanol. On the basis of an average drinking volume of 3 mL and a body weight of 20 g per mouse, FK506 and CsA stock solutions were diluted in glucose-water (5%) and orally applied. One day before surgery, mice were treated orally with either vehicle buffer glucose (5%), with 0.02, 0.075, 0.2, 5.0 mg/kg body weight per day FK506, or 10 mg/kg body weight per day CsA, respectively. Solutions were changed once a day and mice were sacrificed at indicated time points.

[0120] FK506 blood Concentration Measurements.

[0121] FK506 concentration in whole blood samples of mice was measured using colorimetric FK506 Elisa Kit (Abnova, Taipei, Japan) according to the manufacturer's protocol. Briefly, 25 L of whole blood samples and standard solutions containing 0, 2, 10 and 50 ng/mL FK506 were analyzed by OD measurements at 450 nanometer (nm) wavelength.

[0122] Ldn-193189 Treatment.

[0123] Mice were injected intraperitoneally with 3 mg/kg body weight per day LDN-193189 (LDN, Sigma, St. Louis, USA) in DMSO twice daily starting one day prior of surgery, control mice received equivalent volume of vehicle DMSO.

[0124] In Vivo-Morpholino (VMO) Treatment.

[0125] Mice were injected intraperitoneally with 12.5 mg/kg body weight in vivo-morpholinos (Gene Tools, Philomath, USA) in saline at a final volume of 100 L every other day starting two days prior of surgery (114), sequence alignments were performed using NCBI Nucleotide Blast and are listed in Tables 3-5 (157). A control in vivo-morpholino that targets a human -globin intron mutation was used as standard control (116). In vivo-morpholino sequences are listed in Table S.

[0126] Gpi-1046 Treatment.

[0127] Mice were injected subcutaneously with 10 mg/kg body weight per day GPI-1046 (Santa Cruz Biotechnology, Dallas, USA) in DMSO once daily starting one day prior of surgery, control mice received equivalent volume of vehicle DMSO. For oral administration, GPI-1046 stock solution was diluted in glucose-water (5%) and orally applied on the basis of an average drinking volume of 3 mL and a body weight of 20 g per mouse. Three days after challenging with UUO, mice were treated orally with either vehicle buffer glucose (5%) or 30 mg/kg body weight per day GPI-1046, respectively.

[0128] LB-100 Treatment.

[0129] Mice were injected intraperitoneally with 2 mg/kg body weight LB-100 (LDN, Sigma, St. Louis, USA) every alternate day starting at the day of surgery, control mice received equivalent volume of vehicle buffer.

[0130] Histology.

[0131] Paraffin-embedded specimens were sectioned at 3 m, periodic acid-Schiff (PAS), Masson's Trichrome Stain (MTS) and Sirius Red/Fast green was performed at the University Medicine Gttingen. For morphometric analysis of interstitial fibrosis, fibrotic areas were assessed by using cellSens (Olympus, Tokyo, Japan) software. Ten visual fields were selected randomly for each MTS stained section at 200 magnification and the relative interstitial fibrotic area was evaluated by using a 10 mm2 graticule. Tubular damage was analysed after PAS stain and graded according to a semi-quantitative score of 0 to 3 (0: normal, 1: mild, 2: moderate, 3: severe) at 400 magnification in a total number of 100 tubules per section (158). To evaluate collagen deposition, sections were stained with Sirius red in a saturated aqueous solution of picric acid containing 0.1% Direct Red 80 (Sigma, St. Louis, USA), ten visual fields were selected randomly for each section at 400 magnification and evaluated by using a 10 mm2 graticule.

[0132] Cardiomyocyte Diameter.

[0133] Cardiomyocyte diameters were determined from 100 random fibers in PAS-stained heart sections using cellSens (Olympus, Tokyo, Japan) software.

[0134] Immunohistochemistry.

[0135] Paraffin-embedded specimens were deparaffinized in xylene and rehydrated in ethanol containing distilled water. Tissue sections were stained using polyclonal antibodies against ALK3 (sc-20736, Santa Cruz Biotechnology, Dallas, USA) and ARNT (3718S, Cell Signaling, Danvers, USA), peroxidase labeling was performed using Vectastain Universal Elite ABC Kit (Vector Laboratories, Burlingame, USA) according to the manufacturer's protocol. AEC Substrate-Chromogen (Dako, Glostrup, Denmark) was applied for peroxidase visualization according to the manufacturer's protocol. Nuclear counterstain was performed by using Mayer's Hematoxylin Solution (Sigma, St. Louis, USA).

[0136] Immunofluorescence.

[0137] For immunofluorescent staining, primary antibodies against ARNT (5537S, Cell Signaling, Danvers, USA), Hif1 (H6535, Sigma, St. Louis, USA), Hif2 (ab20654, Abcam Biochemicals, Cambridge, UK), phosphorylated Smad1/5/8 (pSmad1/5/8, sc-12353, Santa Cruz Biotechnology, Dallas, USA), CD45 (550539, BD Biosciences, Franklin Lakes, USA), Collagen-1 (ab34710, Abcam Biochemicals, Cambridge, UK), -smooth muscle actin (aSMA, A5228, Sigma, St. Louis, USA), FKBP12 (ab2918, Abcam Biochemicals, Cambridge, UK) and YY1 (ab12132, Abcam Biochemicals, Cambridge, UK) were used, secondary antibodies were labeled with Alexa Fluor 488 or 568 (Life Technologies, Carlsbad, USA). Renal basement membranes were stained with antibodies against Collagen-4 (1340-30, SouthernBiotech, Birmingham, USA), cardiac cell membranes with antibodies against WGA (W11261, Life Technologies, Carlsbad, USA). Nuclear staining was performed using 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, USA). Relative areas positive for Collagen-1, aSMA, tubular nuclei positive for ARNT and pSmad1/5/8 were quantified in 10 high power fields per section at 400 magnification.

[0138] Cell Culture.

[0139] HK-2 (ATTC, Manassas, USA) are immortalized proximal tubule epithelial cells derived from normal adult human kidney. The murine proximal tubular epithelial cell line MCT was generated from the renal cortex of SJL mice (75). All cells were routinely tested negative for the presence of mycoplasma contamination. None of the cell lines used in this manuscript is listed in the ICLAC and NCBI Biosample database of misidentified cell lines. Cells were cultured in Dulbecco's modified Eagle's (DMEM, Gibco, Carlsbad, USA) medium supplemented with 100 g/mL penicillin, 100 g/mL streptomycin and 10% heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, USA) at 37 C. in 5% CO2. For RNA extractions, cells were seeded in 6 well culture plates at 105 cells per well in antibiotic free standard growth medium. After 24 hours, cells were stimulated with FK506 (Abcam Biochemicals, Cambridge, UK) or Cyclosporin A (CsA, Sigma, St. Louis, USA) dissolved in DMSO at indicated concencentrations. Cells were harvested for further analysis 6 hours after incubation. To examine de novo protein syntheses, cells were again plated in 6 well culture plates as initially described and then pre-treated with the translation blocker Cycloheximide (CHX, 10 g/mL, Sigma, St. Louis, USA). After one hour of incubation, 200 pM FK506 was applied for additional 6 hours.

[0140] In Vitro Transfection.

[0141] One night before transfection, MCT cells were seeded in 6 well culture plates at a concentration of 1.5-210.sup.5 per well in antibiotic-free DMEM (Gibco, Carlsbad, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Sigma, St. Louis, USA). For knockdown experiments, 60 pmol siRNA (Santa Cruz Biotechnology, Dallas, USA) or scrambled siRNA (scrRNA, Santa Cruz Biotechnology, Santa Cruz, USA) was transfected, for over-expression experiments, 2 g plasmid DNA was transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA). After 4 hours of incubation, transfection medium was replaced by antibiotic-free medium and cells recovered overnight. For stimulation experiments, cells were stimulated the day after with 200 M FK506 (Abcam Biochemicals, Cambridge, UK) dissolved in DMSO and harvested after 6 hours of incubation for RNA and protein analysis.

[0142] Alk3 Promoter Constructs.

[0143] Site-directed mutagenesis of the palindromic E-box motif CACGTG (SEQ ID NO: 1) to TATATA (SEQ ID NO: 2) within the proximal ALK3 promoter was performed using a QuikChange XL Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, USA). The original ALK3 promoter construct (Gene Universal Inc., Newark, USA) was fragmented into two parts (335 and 794 bp) using PCR amplification and restriction digestion. The 335 bp fragment containing the E-box binding site was amplified using primers with KpnI (primer sequence: 5-GGGGGTACCGAGGTAGTGACAGTTCTT-3; SEQ ID to NO: 3) and BsmI cutting sites (primer sequence: 5-CCCTGCATTCATTACTCA-3; SEQ ID NO: 4). The purified PCR products were cloned into pGEM-T-Easy (Promega, Madison, USA) vector for site-directed mutagenesis (primer sequence: 5-GTGATCCGGAGAACGCTATATACTCCACGTTCCTCCCG-3 (SEQ ID NO: 5) and 5-GTAGCGAAAGCCTGG CG-3; SEQ ID NO: 6). The reaction was carried out according to is the manufacturer's recommendation containing 10 ng of DNA template, 5 L of 10 reaction buffer, 125 ng of primers, 1 L of dNTP mix, 3 l of QuikSolution and 1 L of PfuTurbo DNA polymerase in a final volume of 50 L. The thermal cycling condition was initiated with a denaturating step at 95 C. for 3 minutes followed by 25 cycles containing 95 C. for 50 seconds, 60 C. for 50 seconds, 68 C. for 4 minutes and a final extension at 68 C. for 7 minutes. 1 L Dpn1 (2.5 U/L) was added and incubated for 2 hours at 37 C. to remove the original template from the reaction. The PCR reaction was transformed into XL10-Gold Ultracompetent Cells (Agilent Technologies, Santa Clara, USA). The resulting 335 bp mutated fragment was digested with KpnI/BsmI restriction enzymes and subcloned to a pGL3-basic vector (Promega, Madison, USA) together with the BsmI/HindIII digested 794 bp fragment. All plasmids were carefully sequenced to confirm that mutations were placed at the proper position.

[0144] Promoter Analysis.

[0145] For promoter analysis, cells were co-transfected at 75% confluence with 4 g plasmid DNA, 4 g of promoter construct DNA and 0.1 g renilla luciferase internal control vector pGL4.73 (Promega, Madison, USA) in 6-well plates using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer's instructions. Growth medium was not replaced during incubation. After 48 hours, transfected cells were washed in PBS. Firefly and renilla luciferase activity of 20 L cell extract was determined using the Dual-Luciferase Reporter Assay System (Promega, Madison, USA) according to the manufacturer's instructions. Signals were normalized to renilla luciferase for each sample.

[0146] RNA Isolation.

[0147] Total RNA was extracted from cells using TRIzol Reagent (Life technologies, Carlsbad, USA), tissue was shredded using TissueLyser LT (Qiagen, Hilden, Germany). Subsequent RNA purification procedure was performed by PureLink RNA Mini Kit (Ambion, Carlsbad, USA) according to the manufacturer's protocol.

[0148] Quantitative Real-Time PCR Quantification (qRT-PCR).

[0149] For SYBR-based real-time PCR, cDNA synthesis was performed by using DNase I digestion (Invitrogen, Carlsbad, USA) and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, USA) according to the manufacturer's protocol. 1 L of reverse-transcribed cDNA was added to the reaction mixture containing the primer pair (200 nmol/L each) and diluted 2 Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, USA) in a final volume of 20 L for each PCR reaction. The real-time PCR reactions were performed in a 96-well reaction plate using the StepOne Plus Real-Time System (Applied Biosystems, Carlsbad, USA) and were done in triplicates. An initiation step at 95 C. for 20 seconds was followed by 40 cycles at 95 C. for 3 to seconds and 60 C. for 30 seconds, with one cycle of dissociation at 95 C. for 15 seconds, 60 C. for 60 seconds, and 95 C. for 15 seconds. The intercalation of SYBR Green dye and its fluorescent signal is directly proportional to the amount of amplified DNA and was transformed into the cycle threshold (Ct). For normalization, the Ct values of the housekeeping genes Gapdh and Actb were subtracted from the Ct values of the gene of interest to generate the dCt values. The relative expression levels were calculated using the equation 2.sup.ddCt. Oligonucleotide sequences are listed in Table 9.

[0150] RT2 Profile PCR Array.

[0151] To compare qPCR-validated cDNA samples after FK506 treatment and DMSO control, gene expression profiling was performed using commercially available plates (PAHS-075Z/PAMM-002Z/PAMM032Z, SABiosciences, Qiagen, Hilden, Germany). HK-2 cells were plated in 6 well culture plates as previously described and stimulated with 150 ng/mL FK506, MCT cells with 200 pM FK506. After 6 hours, cells were dissolved and RNA was isolated, digested and reverse transcripted. The 25 ng cDNA equivalent of total RNA was added to the reaction mixture containing diluted 2 RT.sup.2 SYBR Green ROX qPCR Mastermix (Qiagen, Hilden, Germany) in a final volume of 25 L for each well of the RT2 Profiler PCR. PCR reactions were performed under recommended thermal cycling conditions (10 min at 95 C., 15 s at 95 C., 1 min at 60 C. for 40 cycles). To verify PCR specificity, dissociation curve analysis was generated. Relative levels of mRNA expression were normalized in all the samples with expression levels of included housekeeping genes, and data analysis was done using an web-based analysis software provided by SABiosciences. Transcription factor binding sites within the ALK3 proximal promoter was performed 5000 basepairs relative to transcriptional start site using TRANSFAC database (98).

[0152] Western Blot Analyses.

[0153] Tissue and cells were homogenized in NP40 lysis buffer (Life technologies, Carlsbad, USA) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland). After sonication, protein samples were resolved by a 4-12% Bis-Tris polyacrylamide gel electrophoresis system (Novex, Carlsbad, USA) and transferred onto a nitrocellulose membrane (GE Healthcare, Freiburg, Deutschland), followed by a blocking step with 5% dry milk or 5% bovine serum albumin (BSA) in TBS-T (TBS pH 7.2, 0.1% Tween-20) to prevent unspecific bindings. After incubation with respective primary antibodies against ALK3 (ABD51, Merck Millipore, Billerica, USA) and ALK6 (ABD50, Merck Millipore, Billerica, USA), pSmad1/5/8 (13820, Cell Signaling, Danvers, USA), Arnt (3718, Cell Signaling, Danvers, USA), Fkbp12 (ab2918, Abcam Biochemicals, Cambridge, UK), NFATc1 (sc-7294, Santa Cruz Biotechnology, Dallas, USA), NFATc2 (sc-7295, Santa Cruz Biotechnology, Dallas, USA), -actin (A5316, Sigma, St. Louis, USA) and Gapdh (5G4, HyTest, Turku, Finland), secondary HRP-conjugated antibodies were used (Dako, Glostrup, Denmark). Luminescence was detected by using chemiluminescent substrate (Cell Signaling, Danvers, USA) on a ChemiDoc XRS system (Bio-Rad, Hercules, USA). Native protein samples were prepared with Native PAGE Sample Prep kit (Novex, Carlsbad, USA) according to the manufacturer's instruction. Non-denaturating native gel electrophoresis was performed with Native PAGE 3-12% Bis-Tris Protein gel (Novex, Carlsbad, USA).

[0154] Co-Immunoprecipitation (CoIP).

[0155] CoIP was performed with Protein G Immunoprecipitation Kit (Roche, Basel, Switzerland). Protein A/G PLUS Agarose beads (Santa Cruz Biotechnology, Santa Cruz, USA) were used for the lysate pre-cleaning and pull-down. For each CoIP, 210.sup.7 MCT cells have been used following the manufacturer's instructions. Yy1 (ab12132, Abcam Biochemicals, Cambridge, UK), Fkbp12 (ab2918, Abcam Biochemicals, Cambridge, UK), Arnt (3718, Cell Signaling, Danvers, USA), GFP (MA5-15256, Thermo Fisher Scientific, Waltham, USA) and myc-tag antibodies (2276, Cell Signaling, Danvers, USA) were used for immunoprecipitation, detection of co-immunoprecipitated Fkbp12 (ab12132, Abcam Biochemicals, Cambridge, UK), Hif1 (H6535, Sigma, St. Louis, USA), Ahr (MA1-514, Thermo Fisher Scientific, Waltham, USA), GFP (MA5-15256, Thermo Fisher Scientific, Waltham, USA) and myc-tag (2276, Cell Signaling, Danvers, USA) was performed by immunoblotting.

[0156] Chromatin Immunoprecipitation (ChIP).

[0157] DNA and protein interaction was performed with the OneDay ChIP Kit (Diagenode, Seraing, Belgium) according to the manufacturer's instructions. 210.sup.7 MCT cells have been used for each ChIP reaction. Cell lysates were sonicated using an ultrasonic processor S-4000 (Misonix, Farmingdale, USA). Immunoprecipitation was performed with a ChIP grade antibody against Yy1 (ab12132, Abcam Biochemicals, Cambridge, UK) and Arnt (3718, Cell Signaling, Danvers, USA). Enriched DNA was analyzed by qRT-PCR with EpiTect ChIP qPCR primers for genomic Arnt (Qiagen, Hilden, Germany), oligonucleotide sequences for genomic Alk3 are listed in Table 10.

[0158] Analyses of Publicly Available Array Datasets.

[0159] Datasets provided publicly were analyzed according to general recommendations (159). For gene ontology analysis, 5% of most significant up-regulated genes in response to FK506 were extracted from Nephroseq database (nephroseq.org) based on genome-wide transcriptional expression datasets for bioactive small molecules (accession number GSE5258) (44, 45), and process analysis was performed using Gene Ontology enRIchment anaLysis and visuaLizAtion tool (GORILLA) using a value of p<0.001 threshold (46, 47). Protein-protein interactions were extracted from Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) using highest confidence (score 0.900) (91), prediction of transcription factors regulating 5% of most significant up-regulated genes in response to FK506 extracted from Nephroseq database based on genome-wide transcriptional expression datasets for bioactive small molecules (accession number GSE5258) was performed using Predicting ASsociated Transcription factors from Annotated Affinities (PASTAA) within 200 basepairs upstream transcriptional start site, maximum affinity level and values of p<0.05 (44, 45, 92). Human transcriptome array data are shown as log.sub.2 median centered intensities extracted from Nephroseq database (accession numbers GSE69438, GSE66494, GSE35487, GSE30566, GSE21785, GSE1563, GSE3526) (127-132, 141), and log.sub.2 expression values extracted from GEO2R (accession numbers GSE48944 and GSE14964) (133, 139).

[0160] Statistical Analysis.

[0161] The numbers of individual mice and independent experiments are indicated in the corresponding figure legends. For single group comparison, Student's t test was used to determine statistical significance. One-way ANOVA with Bonferroni post-hoc analysis was used for multiple comparisons of samples to determine statistical significance. Linear regression was performed comparing indicated pairs of parameters, r.sup.2 and values of p are indicated in the corresponding graphs. Statistical significance was defined as values of p<0.05, indicated as * p<0.05, ** p<0.01, *** p<0.001 or **** p<0.0001. Prism 5 software (GraphPad, La Jolla, USA) was used for statistical analysis.

Example 1Identification of the FKBP12/YY1/ARNT Signaling Axis, which Controls Endogenous BMP Signaling Responses Via Transcriptional Regulation of Canonical BMP Receptor ALK3

[0162] Low-Dose FK506 Protects the Kidney from Chronic Organ Injury Dependent on Enhanced ALK3 Signaling

[0163] Based on previous reports that FK506 preconditioning regimens effectively protect the kidney from acute experimental injuries (19-22), the inventors first aimed to validate organ protection upon low-dose FK506 administration in a murine model of chronic renal injury. For this purpose, the inventors challenged C57BL/6 mice with the non-immunological, mechanical model of unilateral ureter obstruction (UUO), resulting in injury of the tubular epithelium and severe interstitial fibrosis within 10 days after ureteral obstruction. Based on previous regimens (19-31), the inventors administered low-dose FK506 (0.02, 0.075 and 0.2 mg/kg orally per day) to mice starting one day prior to challenge with UUO (FIG. 1). As controls, the inventors also included standard-immunosuppressive dose FK506 (5.0 mg/kg orally per day) and Cyclosporine A (CsA, 10 mg/kg orally per day) (40, 41), an alternative CNI immunosuppressant that acts via dimerizing with cycophilin A (independent of FKBPs) (42, 43).

[0164] Histopathological analysis demonstrated that FK506 reduced both, chronic tubular injury and interstitial fibrosis with an optimum dose of 0.075 and 0.2 mg/kg FK506 per day (FIG. 2A-F). In contrast, administration of CsA failed to attenuate tubular injury or interstitial fibrosis (FIG. 2A-F), suggesting that observed anti-fibrotic and pro-regenerative efficacy of low-dose FK506 was mediated by mechanisms independent of calcineurin phosphatase inhibition. Administration of low-dose FK506 (0.02, 0.075 and 0.2 mg/kg orally per day) resulted in picomolar, sub-immunosuppressive FK506 blood concentrations of 24573, 45271 and 53536 g/mL, respectively.

[0165] To gain insights into underlying mechanisms, the inventors next performed gene set enrichment analysis of transcriptional expression datasets for bioactive small molecules (accession number GSE5258) with evidence that FK506 induces expression of genes involved in BMP signaling responses (GO terms GO:0071772 and GO:0071773, FIG. 3 and Table 1) (44-47), including the proto-typical type I BMP receptor ALK3 (synonym type I BMP receptor serine/threonine kinase, BMPR1A). These findings were in line with previous reports demonstrating that lowdose FK506 is a strong inducer of endogenous BMP signaling responses (48-51), mediated by enhanced transcription and increased signaling of type I BMP receptors (29, 52). Multiple independent studies demonstrated that induction of BMP signaling through administration of type I BMP receptor ALK3 ligands protected against fibrosis and loss of functional parenchymal cells in various organs including kidney (53-57), but also heart (58-61), liver (62, 63), lung (64, 65), brain (66, 67), and intestine (68). Furthermore, it was demonstrated that protective activity of BMPs is specifically mediated by canonical BMP receptor ALK3, and that suppressed ALK3 expression limits regenerative capacity of injured tubular epithelium (53).

[0166] Based on identification of FK506-induced ALK3 expression in transcriptional profiling datasets and known protective activity of ALK3-mediated BMP signaling, the inventors next explored if observed reno-protective effect of low-dose FK506 was due to enhanced ALK3 transcription and subsequently enhanced BMP signaling responses. Canonical BMP signaling in general is characterized by nuclear translocation of phosphorylated signal transducer proteins pSmad1, pSmad5 and/or pSmad8 (69-71). Activity of protective canonical BMP signaling in kidney, heart, lung and liver depends on presence of the type I BMP receptor ALK3, inducing BMP signaling upon ligand binding and dimerization with type II BMP receptor BMPR2, subsequently mediating anti-fibrotic and pro-regenerative BMP signaling responses (29, 53-56, 58-68). To explore a possible causal link between low-dose FK506, ALK3-dependent BMP signaling and reno-protection, the inventors analyzed ALK3 receptor expression and downstream Smad1/5/8 phosphorylation (pSmad1/5/8) in kidneys of mice which had received low-dose FK506. Analysis of UUO-challenged murine kidneys revealed that FK506 specifically induced expression of ALK3, but not of related type I BMP receptor ALK6 (FIG. 4A-C). FK506-induced ALK3 expression was associated with nuclear pSmad1/5/8 accumulation (correlating with protective efficacy of low-dose FK506, FIG. 4C-E), whereas non-protective CsA failed to induce ALK3 expression and pSmad1/5/8 (correlating with failure of CsA to protect injured kidneys, FIG. 4A-E). To further substantiate the observed link between low-dose FK506, ALK3 and reno-protection, the inventors treated UUO-challenged mice with dorsomorphin derivate small molecule LDN-193189 (LDN, 3 mg/kg intraperitoneally per day), a specific BMP type I receptor kinase inhibitor and blocker of ALK3-dependent signal transduction (FIG. 5) (48, 72-74). LDN did not impact efficacy of FK506 to induce ALK3 expression, but effectively inhibited downstream pSmad1/5/8 signaling responses (FIG. 6). Inhibition of canonical BMP signaling responses completely blocked FK506-mediated reno-protection (FIG. 7A-D), confirming that FK506 elicits protection from chronic organ failure by induction of canonical BMP signaling responses.

[0167] In summary, the inventors' studies demonstrated that low-dose FK506 protected UUO-challenged kidneys from chronic injury, and that such reno-protection was due to increased ALK3 expression and subsequently enhanced BMP signaling responses towards endogenously present ALK3 ligands. The inventors' data did not provide explanation how FK506 could specifically induce ALK3 transcription to this point.

[0168] YY1 Inks Immunophilin FKBP12 and ALK3 transcription in Response to FK506

[0169] The inventors next aimed to gain insights into the molecular mechanisms underlying observed increased ALK3 transcription upon low-dose FK506 administration. Because within the kidney, tubular epithelial cells (TECs) have been established as primary targets of ALK3-mediated BMP signaling (53), and because the inventors had observed robust protection of the tubular epithelium upon FK506 treatment in the inventors' initial studies, the inventors decided to focus here on TECs. The inventors exposed murine TECs to different concentrations of FK506 ranging from standard nanomolar dosages used for immunosuppression in transplant patients (2-200 nM as compared to immunosuppressive doses ranging from 6.2 to 18.7 nM) (75, 76), down to picomolar concentrations (0.02-0.2 nM) reflecting FK506 regimens which the inventors had used in the inventors' murine studies. FK506 induced optimal ALK3 transcription at concentrations from 0.2 nM to 2 nM, whereas higher concentrations had no further enhancing effects (FIG. 8A). In contrast, expression levels of the related type I BMP receptor ALK6 or its counterpart ALK5 (synonym TGFBR1, mediating pro-fibrotic TGF signaling) (77-79), were not increased (FIG. 8B,C). Increased ALK3 transcription in TECs was associated with accumulation of pSmad1/5/8 and proliferative activity (FIG. 9A,B), both indicating functional activation of canonical BMP signaling (80-83). FK506 elicits biological function by complexing with distinct FK506-binding proteins (FKBPs) characterized by prolyl isomerase activity and acting as folding chaperones for proteins containing proline residues (84, 85). FKBP-FK506 complexes are best known for inhibiting calcineurin phosphatase activity, but numerous additional proteins are targeted by distinct FK506-FKBP complexes, most notably including FKBP family members FKBP12, FKBP25, FKBP38 and FKBP56 (84, 85). Because the inventor's studies had revealed that observed protective activity of low-dose FK506 was independent of calcineurin phosphatase inhibition, the inventors next aimed to identify involved FKBP family members and subsequently targeted proteins. To identify FKBP family members involved in mediating FK506-induced ALK3 transcription, the inventors next depleted aforementioned FKBPs in cultured TECs by siRNA-mediated knockdown experiments (FIG. 1A-D). Whereas knockdown of FKBP25, FKBP38 and FKBP56 had no significant effects on ALK3 transcription, depletion of FKBP12 (Fkbp12kd) induced ALK3 mRNA expression without further addition of FK506 to culture media (FIG. 11). Previous reports established that FKBP12 is capable of inhibiting ALK3-dependent receptor signaling by direct interaction (29). Such interaction was not detectable in cultured TECs (FIG. 12A,B), suggesting that the immunophilin FKBP12 suppresses ALK3-dependent receptor signaling by repressing ALK3 transcription to regulate canonical BMP signaling responses. However, FKBPs including FKBP12 do not possess DNA binding properties themselves, but are known to interact with a subset of transcriptional regulators to impact gene expression (86-90), and FK506 is known to elicit biological functions by interaction with such FKBP/transcriptional regulator complexes (87). Based on these prerequisites, the inventors hypothesized that FKBP12 alters ALK3 transcription by interacting with a to-be-determined transcriptional regulator, and that FK506-induced ALK3 expression and canonical BMP signaling responses via interaction with this FKBP12/transcriptional regulator complex. Among FKBP12 interacting proteins (91), the inventors identified Yin Yang 1 (YY1) predicted to regulate transcription of differentially expressed genes induced in response to FK506 (accession number GSE5258, and Table 2) (44, 45, 92). YY1 is a GLI-Kruppel family member known to either repress or activate a diverse number of gene promoters (93-95). Repressive function of YY1 on transcription of select genes is dependent on its interaction with adaptor proteins such as FKBPs, which increases its DNA binding properties (86-90). The inventors hypothesized that disruption of repressive FKBP12/YY1 complexes by FK506 is involved in enhanced ALK3 transcription and activation of canonical BMP signaling responses. Co-immunoprecipitation using antibodies to YY1 (IP: Yy1) confirmed direct interaction of YY1 and FKBP12 in cultured TECs, while such FKBP12/YY1 interaction was not detectable when FK506 was added to culture media (FIG. 13). While in untreated TECs, FKBP12 and YY1 were equally present and detection of FKBP12/YY1 complexes correlated with low ALK3 expression levels, depletion of YY1 (Yy1kd) by siRNA-mediated knockdown (FIG. 14) was equally effective in induction of ALK3 transcription and subsequent BMP signaling activation as compared to FKBP12-depleted cells (Fkbp12kd, FIG. 15A,Band Figure A). In addition, supplementation of FK506 to culture media did not additionally induce ALK3 transcription and BMP signaling responses when YY1 had been depleted (FIG. 15A,B), indicating that FK506-mediated ALK3 induction is primarily dependent on YY1.

[0170] To further substantiate that tubular YY1 is equally involved in transcriptional ALK3 repression in injured kidneys, the inventors next generated mice conditionally depleted for YY in TECs (referred as yGTcre+;Yy1fl/fl). In mice challenged with UUO (FIG. 16), conditional YY depletion in TECs resulted in robust induction of BMP signaling responses, protection from tubular injury and renal fibrogenesis based on enhanced ALK3 transcription (FIG. 17A-F). When YY1 was depleted in TECs, administration of FK506 had no additive effects (FIG. 17A-F), indicating that FK506-mediated anti-fibrotic and pro-regenerative efficacy is dependent on presence and modulation of YY1 signaling in TECs. In summary, the inventors' studies demonstrated that presence of FKBP12, YY1 and their respective complex formation correlated with low ALK3 expression. The inventors' studies further revealed that depletion of FKBP12/YY1 complexes by knockdown of either FKBP12 or YY1 induced ALK3 transcription, suggesting that observed transcriptional induction of ALK3 upon exposure to FK506 was mediated by disruption of the FKBP12/YY1 repressor complex.

[0171] ARNT/HIF1 Causally Links Disruption of FKBP12/YY1 Complexes to Increased ALK3 Transcription

[0172] In control experiments in which Cycloheximide (CHX, 10 g/mL) was added to cell culture media to block protein translation, FK506 failed to induce ALK3 transcription and also activation of canonical BMP signaling responses (nuclear pSmad1/5/8, FIG. 18A,B). These observations suggested that de novo translation of yet unknown mediator(s) is required to induce ALK3 transcription after release from FKBP12/YY1 transcriptional is repressor complexes. Evidence that ALK3 transcription is not directly regulated by modulation of FKBP12/YY1 complexes is additionally supported by absence of putative YY1 binding motifs within the ALK3 proximal promoter region (96, 97). To identify factors linking disruption of FKBP12/YY1 complexes and ALK3 transcription, the inventors next used an unbiased, array-based approach to analyze expression levels of distinct transcriptional factors in TEC cultures in response to FK506. Based on computational prediction of putative transcriptional factor binding sites (98), the inventors identified 6 candidate transcriptional factors with a binding motif within the ALK3 proximal promoter at least 2-fold induced upon FK506 exposure: AR, ARNT, CEPB, CREB1, GATA3 and MAX (FIG. 19A,B). While qRT-PCR confirmed increased mRNA expression levels of all identified targets upon exposure to FK506 (FIG. 20), only siRNA-mediated depletion of ARNT (Arntkd) prevented transcriptional ALK3 induction in response to FK506, whereas depletion of AR, CEBPB, CREB1, GATA3 or MAX had no significant impact on ALK3 expression (FIG. 21 and FIG. 22A-F). Furthermore, the inventors observed robust ARNT induction upon depletion of either FKBP12 (Fkbp12kd) or YY1 (Yy1kd) (FIG. 23 and FIG. 10A,E), suggesting ARNT as possible link between FKBP12/YY1 complex disruption and enhanced ALK3 transcription. These observations were further supported by transcriptional expression datasets with robust induction of ARNT and enrichment of ARNT-regulated genes induced in response to FK506 (accession number GSE5258, Table 2) (44, 45, 92). To further explore the role of ARNT transcription as causal link between FKBP12/YY1 complex disruption and increased ALK3 expression, the inventors performed ARNT ChIP PCR using antibodies to YY1 for immunoprecipitation (IP: Yy1). ChIP demonstrated binding of YY1 to its putative motif within the ARNT proximal promoter in cultured TECs that had been maintained in control media without FK506 (with low ALK3 expression levels, FIG. 24). Addition of FK506 to culture media (associated with enhanced ALK3 transcription) reduced YY1 binding to the ARNT proximal promoter (FIG. 24), associated with transcriptional ARNT induction (FIG. 25A,B). Observed induction of ARNT upon depletion of either FKBP12 or YY1 was not further enhanced by FK506, supplementation of culture media with FK506 did not additionally induce ARNT mRNA and protein levels in experiments in which ARNT expression had been induced by FKBP12 (Fkbp12ko) or YY1 depletion (Yy1ko, FIG. 25A,B), supporting that FK506-mediated ARNT induction is dependent on modulation of a repressive FKBP12/YY1 signaling axis. These findings in cultured TECs were further confirmed by significantly increased ARNT expression levels in yGTcre+;Yy1fl/fl conditional knockout mice (FIG. 26, correlating with enhanced ALK3 transcription and renoprotection). Furthermore, renal ARNT expression was significantly increased in mice treated with low-dose FK506, but not in mice that had been given CsA (FIG. 27A-C, again correlating with enhanced ALK3 transcription and reno-protection).

[0173] To further substantiate the causal link between ARNT and ALK3 induction, the inventors next analyzed efficacy of FK506 to enhance ALK3 transcription when ARNT induction in response to FK506 was blocked in cultured TECs (Arntkd, FIG. 22B). When ARNT induction was depleted, FK506 failed to induce ALK3 transcription (FIG. 28A) or nuclear pSmad1/5/8 accumulation (FIG. 28B). In addition, transgenic ARNT over-expression in cultured TECs (Arntoe, FIG. 22G) alone was sufficient to specifically induce type I BMP receptor ALK3 mRNA expression and nuclear pSmad1/5/8 accumulation (FIG. 29A-C), confirming that ARNT effectively mediates transcriptional ALK3 induction and downstream BMP signaling responses.

[0174] ARNT (synonym HIF1) is a member of the PAS domain family and heterodimerizes with other PAS family members to form heterodimeric transcription factors, classically HIF-1 in hypoxia responses the dioxin receptor AHR in xenobiotic signaling (99-112). Furthermore, ARNT forms homodimers with itself and elicits transcriptional activation by binding to the Ebox core sequence CACGTG and the inventors next aimed to further dissect mechanisms underlying FK506-induced, ARNT-dependent transcriptional ALK3 activation. The inventors first performed hypoxia- and drug metabolism mRNA expression arrays on TECs exposed to FK506, revealing that FK506 exposure did not markedly induce pathways involved in hypoxic signaling or drug metabolism (including xenobiotic signaling), while ARNT induction was confirmed (FIG. 30A,B). These observations were further confirmed by preserved effectiveness of FK506 or transgenic ARNT over-expression to induce ALK3 transcription when either HIF1 (Hif1akd) or AHR (Ahrkd) were depleted (FIG. 31A,Band FIG. 32A-D). In addition, co-immunoprecipitation using antibodies to ARNT (IP: Arnt) did not provide evidence for ARNT/HIF1 (hypoxic signaling) or ARNT/AHR (xenobiotic signaling) interaction in cultured TECs in response to FK506 (FIG. 33A-C). As these studies suggested that FK506-induced ALK3 transcription is independent of heterodimeric ARNT activation of hypoxia or xenobiotic signaling, the inventors next aimed to further investigate contribution of ARNT homodimer signaling. To elucidate capacity of ARNT homodimer formation in cultured TECs, the inventors next generated EGFP-tagged (Arnt-EGFPoe) and myc-tagged (Arnt-mycoe) ARNT overexpression plasmids and confirmed formation of ARNT homodimers by coimmunoprecipitation with presence of Arnt-myc after pulldown of Arnt-EGFP (IP: EGFP) and Arnt-EGFP after Arnt-myc pulldown (IP: myc, FIG. 34A,B). Analysis of endogenous ARNT by native gel electrophoresis revealed presence of ARNT dimers within TECs cultivated under standard conditions (FIG. 35). In addition, formation of ARNT dimers was further enhanced upon exposure to FK506 (FIG. 35). The inventors did not detect HIF1, HIF2 or AHR as constituent of ARNT dimers, further suggesting that transcriptional responses to FK506 in TECs are mediated by homodimerized ARNT (FIG. 35) (102-112). To further substantiate involvement of ARNT homodimers in transcriptional ALK3 regulation, the inventors performed ALK3 ChIP-PCR to analyze ARNT binding to the ARNT homodimer CACGTG target sequence within the proximal ALK3 promoter. Upon exposure to FK506 (associated with high ARNT expression and enhanced ALK3 transcription), the inventors detected significantly increased ARNT binding to its respective motif specific for ARNT homodimers (FIG. 36) (111, 112). Transcriptional ALK3 induction was further confirmed by reporter assays, as ARNT overexpression effectively induced ALK3 proximal promoter activity (Alk3 wt FIG. 37) (113). In contrast, ARNT failed to induce ALK3 transcription when the palindromic E-box motif was disrupted (CACGTG to TATATA, Alk3mut, FIG. 37). In summary, the inventors' studies demonstrate that FK506-induced ALK3 transcription is mediated by ARNT homodimers that bind to its established palindromic E-box motif within the ALK3 promoter, independent of hypoxic of xenobiotic signaling responses requiring heterodimer formation. To verify if observed effects of FK506-induced ALK3 transcription correlating with ARNT homodimer formation in cultured TECs translate to mice, the inventors also analyzed kidneys of mice which had been challenged with UUO and which had been treated with FK506. Analysis of total kidney lysates by immunoprecipitation, native gel analysis and subsequent immunoblotting revealed increased formation of ARNT dimers without interaction with HIF1, HIF2 or AHR (FIG. 38), confirming a critical role of ARNT homodimerization.

Example 2Selective Modulation of FKBP12/YY1 Signaling Constituents Effectively Modulates Protective ARNT/HIF1 within Chronically Injured Kidneys

[0175] To elucidate therapeutical implication of the inventors' findings, the inventors next selectively modulated constituents of identified FKBP12/YY1/ARNT signaling axis in mice challenged with UUO by administration of in vivo morpholinos (VMO) targeting translational start sites (FIG. 39 and Table 3) (114, 115). As compared to control-VMO targeting a human R-globin intron mutation (116), administration of VMO targeting FKBP12 (Fkbp12-VMO), YY1 (Yy1-VMO) or ARNT (Arnt-VMO) effectively reduced intrarenal protein levels of their respective transcript (FIG. 40A-D). Administration of Fkbp12-VMO, Yy1-VMO or FK506 equally induced intrarenal ARNT, ALK3 and nuclear pSmad1/5/8 immunostaining, associated with attenuated tubular injury and tubulointerstitial fibrosis (FIG. 41A-I). Administration of FK506 had no additive effects on ALK3 expression, nuclear accumulation of pSmad1/5/8, tubular injury or interstitial fibrosis in cohorts of mice that had also received Fkbp12-VMO or Yy1-VMO (FIG. 41A-I). In addition, administration of Fkbp12-VMO or Yy1-VMO did not further enhance the effect of FK506 on ALK3 expression or reno-protection (FIG. 41A-I), mirroring the effect of genetic YY1 depletion which the inventors had observed in yGTcre+;Yy1fl/fl conditional knockout mice. In contrast, FK506 failed to induce intrarenal ALK3-dependent BMP signaling responses when intrarenal ARNT induction was depleted (FIG. 41A-I), correlating with failure of FK506 to protect kidneys from tubular injury and progressive fibrotic disease (FIG. 41A-I).

[0176] In summary, these studies demonstrate that FKBP12/YY1/ARNT signaling constituents can be effectively targeted with in vivo morpholino approaches to selectively modulate intrarenal ARNT/HIF1 and protective BMP signaling within chronically injured kidneys.

Example 3Selective Targeting of FKBP12 by Non-Immunosuppressive FKBP12 Inhibitor GPI-1046 Effectively Modulates FKBP12/YY1/ARNT Signaling and Protects from Chronic Renal Failure

[0177] The inventors' studies suggested that observed effect of picomolar FK506 was mediated specifically via its interaction with FKBP12. However, FK506 by no means is specific to FKBP12, suggesting that a more specific drug would be an attractive opportunity (84, 85). Because the inventors' data suggested that picomolar FK506 elicited reno-protective properties independent of calcineurin inhibition, the inventors next explored efficacy of a specific small molecule FKBP12 inhibitor, 3-(3-pyridyl)-1-propyl-(2S)-1-(3,3-dimethyl-1,2-dioxopentyl)-2-pyrrolidinedine carboxylate (GPI-1046), an FK506 derivate without immunosuppressive properties (117), to modulate intrarenal FKBP12/YY1/ARNT/ALK3 signaling and to pre-empt chronic renal injury (118-122). Exposure of cultured TECs to GPI-1046 (10 M) was equally effective in transcriptional induction of ARNT and ALK3 mRNA expression levels (FIG. 42A,B), associated with activation of canonical BMP signaling responses (FIG. 42C,D). Based on the inventors' previous regimen, the inventors next administered GPI-1046 (10 mg/kg subcutaneously per day) one day prior to UUO challenge (FIG. 43) (118-122). As compared to FK506, non-immunosuppressive GPI-1046 equally induced intrarenal ARNT and ALK3 transcription, BMP signaling responses and protected from tubular injury and renal fibrogenesis (FIG. 44A-H), further supporting that specifically inhibition of immunophilin FKBP12 is involved in modulating intrarenal ARNT/ALK3 signaling and protection from injury independent of calcineurin modulation or immunosuppressive properties and in line with previous reports (29, 117). This is further confirmed by uniform immune cell infiltration in cohorts of mice administered either picomolar FK506 or nonimmunosuppressive GPI-1046 (FIG. 45A), not affecting intrarenal NFAT signaling (FIG. 45B). Upon establishing that the known protective effectiveness of picomolar FK506 is mediated by induction of an FKBP12/YY1/ARNT/ALK3 signaling axis which subsequently induced canonical BMP signaling, and upon establishing that this protective mechanism can be specifically induced by the FKBP12 inhibitor GPI 1046, the inventors next aimed to test if administration of GPI-1046 was also effective to attenuate chronic kidney disease progression when administration is initiated after manifestation of kidney injury. For this reason, the inventors next administered FK506 or GPI-1046 three days after challenging with UUO (FIG. 46). Because previous studies established that GPI-1046 also provides oral bioavailability (118, 119), the inventors administered FK506 or GPI-1046 orally for better comparison (0.2 mg/kg or 30 mg/kg orally per day, respectively). Both, FK506 and small molecule FKBP12 inhibitor GPI-1046 were equally effective in inducing intrarenal ARNT and ALK3 transcription (FIG. 47A,B), associated with enhanced canonical BMP signaling responses (FIG. 47C,D). Activation of intrarenal BMP signaling responses attenuated tubular injury and renal fibrogenesis (48A-D).

[0178] In summary, the present studies revealed that interventional induction of an FKBP12/YY1/ARNT/ALK3 signaling axis after manifestation of kidney lesions is still beneficial. The studies further demonstrate that an FKBP12/YY1/ARNT/ALK3 signaling axis can be either induced by picomolar FK506, but also by oral administration of the specific FKBP12 inhibitor GPI-1046.

Example 4Pharmacological Modulation of an FKBP12/YY1/ARNT/ALK3 Signaling Axis Protects from Chronic Injury in the Heart and Liver

[0179] Because effectiveness of FK506 had been documented in various parenchymal organs (18-35), the inventors finally aimed to explore whether identified FKBP12/Y1/ARNT/ALK3 signaling axis could be equally targeted in organs other than the kidney. For this reason the inventors next analyzed ARNT mRNA expression levels in the kidney, heart, brain, spinal cord, skin, bladder, liver, lung, pancreas and intestine harvested from mice administered FK506 or GPI-1046 (0.2 mg/kg or 30 mg/kg orally per day, respectively). In response to FK506 and GPI-1046, transcriptional ARNT induction was present in the kidney (confirming previous results), but also in the heart, brain, spinal cord, skin, liver, lung and intestine (FIG. 49). Based on robust ARNT induction observed in the heart and liver in response to FK506 and GPI-1046, previous reports that chronic heart failure and liver fibrosis is equally associated with loss of ALK3-dependent BMP signaling responses (61-63), and based on previous studies which reported that FK506 protects from cardiac and hepatic injury and fibrosis (23-26), the inventors next hypothesized that small molecule FKBP12 inhibitor GPI-1046 could equally induce ALK3-dependent BMP signaling responses and protect fibrogenesis in models of chronic cardiac and hepatic injuries. Therefore, the inventors next analyzed presence of an FKBP12/YY1/ARNT/ALK3 signaling axis in rodent models of cardiac fibrosis after continuous minipump delivery of angiotensin II (AT II) and carbon tetrachloride (CCI4)-induced liver failure (63, 123). FKBP12 and YY1 (FIG. 50A-D) were detectable in chronically injured hearts and livers, suggesting that repressive FKBP12/YY1 complexes were equally present. To elucidate if identified FKBP12/YY1/ARNT/ALK3 signaling axis could be equally targeted to protect from chronic heart and liver failure and fibrosis, the inventors next administered GPI-1046 (10 mg/kg subcutaneously per day) one day prior to administration of AT II (FIG. 51A) or CCL4 (FIG. 51B). GPI-1046 was equally effective in induction of activation of ALK3-dependent canonical BMP signaling responses and protection from fibrogenesis in both organ systems (FIG. 52A-T), in line with a substantial body of literature demonstrating that induction of canonical BMP signaling responses mediates anti-fibrotic and pro-regenerative capacity in cardiac and hepatic pathologies (58-63).

[0180] In summary, small molecule FKBP12 inhibitor GPI-1046 equally induced intrarenal ARNT/ALK3 and downstream canonical BMP signaling responses, associated with protection from chronic organ failure. Furthermore, an FKBP12/YY1/ARNT/ALK3 signaling axis is also present in the heart and liver, and can be therapeutically targeted with small molecule FKBP12 inhibitor GPI-1046. Because FK506 impacts additional pathways (124-126), direct targeting of FKBP12 with small molecule inhibitors including GPI-1046 may have beneficial effects with regard of organ protection.

Example 5Evidence for Presence of FKBP12 and YY1 in Human Pathologies and its Modulation with Subsequent ARNT/ALK3 Induction by FK506 in Kidney Allografts

[0181] With regard of translational implications of the inventors' findings, the inventors next aimed to gain insights into presence of FKBP12/YY1 among different human pathologies: diabetic nephropathy (ID #S3060), hypertensive nephrosclerosis (ID #S3170), FSGS (ID #S3584), autoimmune hepatitis (ID #P11150/17), liver fibrosis (ID #P9446/17), liver cirrhosis (ID #P14203/17), and lung fibrosis (ID #7183/17). FKBP12 and YY1 were equally detectable in various human diseases including injured kidneys (diabetic nephropathy, hypertensive nephrosclerosis, focal-segmental glomerulosclerosis/FSGS), hearts (myocardial infarction, aortic valve stenosis, diabetic cardiomyopathy), livers (autoimmune hepatitis, liver fibrosis, liver cirrhosis) and lungs (lung fibrosis) as prerequisite of identified organ protection mediated by FK506 or GPI-1046. To elucidate whether modulation of an FKBP12/YY1/ARNT/ALK3 signaling axis was not only limited to mice but similarly effective in humans, the inventors next exposed human proximal tubular epithelial cells (HK2 cells) to FK506 and analyzed efficacy to induce ARNT and ALK3-dependent BMP signaling responses. Exposure of human TEC cultures to previous established picomolar FK506 equally induced ARNT and ALK3 transcription (FIG. 53A-C), associated with nuclear accumulation of pSmad1/5/8 (FIG. 53D,E), indicating that FK506 also induces ARNT and ALK3-dependent canonical BMP signaling responses in human TECs. Because FK506 has been in clinical use for decades to prevent rejection of kidney transplants, raising the question of induction of the FKBP12/YY1/ARNT/ALK3 signaling axis within allografts of patients on FK506 containing immunosuppressive regimen. Because FK506 is used at high immunosuppressive doses (with well established adverse toxicity likely masking beneficial effects) and not at low doses at which the inventors observed optimal reno-protection in rodents, the inventors focused in the inventors' analysis on the induction of ARNT expression and subsequent ALK3-mediated BMP signaling responses. For this reason, the inventors analyzed kidney allograft biopsies matched for comparable kidney function, chronic tubular injury and extent of interstitial fibrosis from patients that had either received immunosuppressive regimens based on FK506 (acting via FKBP12) or CsA (acting via cyclophilin, FIG. 54A-E and Table 6). When biopsies were normalized for fibrosis and tubular injury, immunolabelling of ARNT, ALK3 and pSmad1/5/8 were significantly increased in kidney biopsies of patients on an FK506-based immunosuppressive regimen as compared to CsA (FIG. 55A-C). Transcriptional induction of intrarenal ARNT and ALK3 was confirmed by analysis of mRNA expression levels (FIG. 55D,E). Immunolabelling further confirmed presence of FKBP12 and YY1 in all allografts is (FIG. 55 F-I), confirming presence of FKBP12/YY1 within chronically injured kidneys and in line with the inventors' observation in cultured TECs and mice.

[0182] In summary, retrospective analysis of renal allografts revealed presence of all constituents involved in identified FKBP12/YY1/ARNT signaling pathway, even though potential beneficial effects are likely masked by CNI toxicity at the immunosuppressive doses that were used. The inventors' analysis also provided evidence for enhanced ALK3 transcription by FK506, and that such increased ALK3 expression correlates with increased ARNT levels.

DISCUSSION

[0183] Progression of CKD is still an unmet biomedical challenge, because once chronic lesions have manifested, no effective therapies are available as of yet for clinical use. Prompted by various studies across multiple organs which demonstrated that FK506 effectively protects various organ systems (18-35), the inventors here aimed to gain insights into the molecular mechanisms underlying successful protection, and to explore whether such pathways could be utilized to inhibit progression of already established chronic kidney injury. The inventors provide evidence that FK506 protects from injury by transcriptional induction of type I BMP receptor ALK3, in line with previous studies demonstrating that low-dose FK506 is capable to induce transcriptional activation and signaling of type I BMP receptors (29, 48-52). The Inventors identified a novel protective mechanism that is controlled by the transcription factor ARNT/HIF1, which effectively inhibits progression of chronic kidney injury by transcriptional ALK3 induction, the principal mediator of anti-fibrotic and pro-regenerative BMP signaling responses. The inventors further report that ARNT expression itself is controlled by the FKBP12/YY1 transcriptional repressor complex (FIG. 56A), and that disruption of such FKBP12/YY1 complexes by picomolar FK506 at sub-immunosuppressive doses, small molecule FKBP12 inhibitor GPI-1046, or by direct targeting of FKBP12/YY1 using in vivo-morpholinos increases ARNT (FIG. 56B). Subsequent activation of ALK3-dependent canonical BMP signaling responses by ARNT homodimer formation (independent of HIF1 or AHR) attenuates chronic organ failure in models of chronic kidney, cardiac and liver injuries (FIG. 56C,D).

[0184] To the inventors' knowledge, this is the first report linking FKBP12/YY1 repressor complexes to ARNT transcription and subsequent canonical BMP signaling activation, suggesting FKBP12/YY1 interaction and ARNT as novel therapeutic targets. Furthermore, this is the first report linking FK506 to disruption of FKBP12/YY1 repressor complexes, providing novel mechanistic insights into the protective activity of FK506. This newly identified FKBP12/YY1/ARNT/ALK3 signaling axis is supported by mining of public expression profiling databases in various organs: in context of the kidney, an inverse correlation between intrarenal ALK3 expression and FKBP12/YY1 is confirmed by several array datasets performed in different renal pathologies (accession numbers GSE69438 and GSE66494) (127, 128), and independently confirmed in cohorts of IgA nephropathy (accession number GSE35487) (129), diabetic kidney disease (accession numbers GSE30566 and GSE21785) (130, 131), allograft nephropathy (accession number GSE1563) (132), and microdissected renal tubules from diseased kidneys (accession number GSE48944) (133). A link between FK506 treatment, disruption of FKBP12/YY1 repressor complexes, ARNT and ALK3 is further confirmed by observations that immunosuppressive regimens including FK506 were associated with enhanced intrarenal ARNT and ALK3 transcription when compared to alternative CsA (accession number GSE1563) (132). Hence, identification of the FKBP12/YY1/ARNT/ALK3 signaling axis causally connects two lines of research to protect parenchymal tissues: Several independent studies highlighted efficacy of low-dose FK506 administration to protect against acute experimental injuries including the kidney (19-22), heart (23-25) and liver (26-28), albeit underlying mechanisms were poorly understood. On the other hand, several pre-clinical studies established beneficial effects of ALK3-mediated BMP signaling in various organs including the kidney (53-57), heart (58-61) and liver (62, 63), and the small molecule ALK3 agonist THR-184 has recently completed successful clinical testing to circumvent acute kidney injury and progressive kidney disease (ClinicalTrials.gov identifier NCT01830920) (53).

[0185] At the mechanistic level, the inventors' observation that interaction with FKBP12 turns YY1 into a transcriptional repressor of ALK3, whereas disruption of such interaction voided such repressive activity, is supported by recent findings of FKBP12 enrichment in injured kidneys (134-136), that FKBP12 and YY1 have been described as robust repressors of canonical BMP signaling responses (137, 138), and that interaction of YY1 with specific adaptor proteins (including FKBP12) determines efficacy of transcriptional regulation in a gene context-dependent manner (86-90, 93-95). Transcriptome array datasets performed in HeLa cells confirmed transcriptional ARNT induction when YY1 was depleted (accession number GSE14964) (139). This is supported by publicly available ChIP sequencing array datasets for YY1 target loci (YY1TargetDB) revealing direct binding of YY1 repressor to the ARNT proximal promoter (140). Observed negative correlation between ARNT and FKBP12/YY1 is not limited to the kidney, array datasets from various human tissues identified strongest correlation between ARNT and YY1 compared to other transcriptional factors with known binding sites within the ARNT proximal promoter and confirmed inverse correlation in addition to FKBP12 (accession number GSE3526) (141). When clustered for distinct organ systems, inverse correlation between ARNT and FKBP12/YY1 was primarily detectable in renal, cardiovascular and digestive tissues (confirming the inventors' findings), but also in central nervous systems (accession number GSE3526, Table 7) (141). In this context, previous reports implicate that activation of an YY1 signaling axis is detectable in renal, cardiac, hepatic and pulmonary pathologies, and YY1 depletion protects from chronic organ failure (142-146). The inventors' studies do not preclude the possibility of YY1 effects independent of FKBP12 since YY1 has been shown to mediate fibrosis in the lung, in part through binding to Collagen and SMA promoters (142). Nevertheless, modulation of newly identified FKBP12/YY1/ARNT signaling axis and associated induction of ALK3-dependent canonical BMP signaling responses may be a promising target in chronic failure of multiple organ systems. In this regard, low-dose FK506 has already entered clinical testing (ClinicalTrials.gov identifier NCT01647945) and shown promise in pulmonary arterial hypertension patients to induce protective BMP signaling responses (147). This in line with a substantial body of literature demonstrating that induction of BMP signaling mediates antifibrotic and pro-regenerative capacity in various organs including kidney (53-57), heart (58-61), liver (62, 63), lung (64, 65), brain (66, 67), and intestine (68).

[0186] To the inventors' knowledge, this is the first report of a causal contribution of dynamic ARNT expression to protection of the kidney or of any other parenchymal organ. In line with reports that ARNT enables proliferation and survival (148, 149), an array-based approach has recently linked loss of ARNT in TECs to accelerated susceptibility towards kidney injury (150). These observations were independently observed in cardiac pathologies (151), further supporting a protective and pro-regenerative role of ARNT. The inventors' data suggests that ARNT homodimerizes to elicit its function to induce ALK3 transcription. ARNT is a member of the PAS domain family predominantly known to heterodimerize with other PAS family members to for heterodimeric transcription factors, classically with an a subunit of HIF or the dioxin receptor AHR to mediate hypoxia or xenobiotic responses by targeting genomic E-box motifs (99-110). In contrast to ARNT heterodimers, the biological role of ARNT homodimers is less known. Current models suggest that E-box motifs contain two half-sites, with each partner's basic region determining half-site specificity and binding properties, and recent studies established that the palindromic E-box motif CACGTG is the critical binding site specific for ARNT homodimers (in contrast to asymmetric E-box motifs in hypoxic/xenobiotic response elements) (111, 112). As ARNT in context of hypoxia or xenobiotic signaling is usually not rate-limiting as it is present in relative excess and physiological changes in protein levels are not significantly affected by hypoxia or xenobiotic responses, the inventors' studies suggest that the fate decision of homodimerization over heterodimerization is in part regulated by endogenous ARNT levels and critical for regulation of FKBP12/YY1/ARNT transcriptional responses. As ARNT homodimerization potentially provides another therapeutic target, additional research is warranted to explore the underlying mechanisms. Observed beneficial efficacy of FK506 was counter-intuitive at first sight, because decades of use as immunosuppressant upon kidney transplantation had revealed its calcineurin inhibitor nephrotoxicity, thereby limiting its clinical use (38, 39). However, the inventors' studies established that FK506-induced organ protection is independent of calcineurin inhibition, and that FKBP12/YY1/ARNT/ALK3 signaling is achieved at picomolar doses which are far below the nanomolar immunosuppressive regimens. Hence, the inventors provide mechanistic evidence for why protective activity of FK506 in interventional therapeutic regimens has remained elusive thus far. Efficacy of the specific FKBP12 inhibitor GPI-1046 to protect and attenuate disease progression in kidney, heart and is liver by induction of FKBP12/YY1/ARNT/ALK3 signaling validated observed efficacy of FK506, and also provides a more specific tool from a translational perspective.

[0187] In summary, this newly identified FKBP12/YY1/ARNT/ALK3 signaling axis and its modulation by FK506 or small molecule FKBP12 inhibitor GPI-1046 is supported by various transcriptome array datasets across numerous organs. Finally, the inventors' data demonstrate that low-dose FK506, which has been found to effectively ameliorate acute multiple organ failure (18), independently confirmed in various organs including kidney (19-22), heart (23-25), liver (26-28), lung (18, 29), brain (30, 31), spinal cord (32, 33), skin (34), and intestine (35), is also effective in protection from chronic organ failure. The inventors are aware that FK506 impacts additional pathways (124-126), and thus it is attractive to speculate that direct targeting of FKBP12/YY1 with in vivo-morpholino approaches or small molecule inhibitors including GPI-1046 may have beneficial effects in organ protection.

Example 6Selective PP2A Inhibition Increases Endogenous ARNT Associated with Enhanced ARNT/HIF1 Homodimer Formation

[0188] Based on previous reports that combined inhibition of PP1/PP2A with Okadaic acid is capable to induce ARNT/HIF1 homodimer formation and transactivation activity by directly blocking ARNT dephosphorylation (163), the inventors first confirmed enhanced ARNT/HIF1 homodimer formation upon PP1/PP2A inhibition (FIG. 57A). To gain insights into involvement of distinct protein phosphatases, the inventors next specifically blocked PP1 with Tautomycin or PP2A with LB-100 and analyzed efficacy of ARNT/HIF1 homodimer formation (164, 165). While PP1 inhibition did not affect ARNT/HIF1 homodimer formation (FIG. 57B), blocking PP2A was associated with enhanced ARNT/HIF1 homodimer formation (FIG. 57C), indicating PP2A as therapeutical target. These results were further confirmed by mass spectrometry with enhancement of ARNT homodimer formation in the presence of PP2A inhibitor LB-100 (FIG. 57D). See also FIG. 58.

[0189] Because LB-100 has been recently developed for in vivo usage to overcome the toxicity of PP2A inhibitors, the inventors next analyzed efficacy of previous established preconditioning regimens with either low-dose FK506 (0.2 mg/kg s.c.) or GPI-1046 (10 mg/kg s.c.) in combination with LB-100 (2 mg/kg). The inventors did not observe any injury in parenchymal organs including kidney, heart, liver, lung, spleen, intestine, pancreas or brain, in line with previous reports (166). Rather, LB-100 enhances protection by FK506/GPI-1046 (FIG. 59). These observations are in line with previous studies reporting protection from myocardial infarction and fibrosis after administration of PP2A inhibitor Fostriecin (167).

[0190] In summary, the inventors report that selective PP2A inhibition by LB-100 effectively increases endogenous ARNT by protection from degradation, associated with enhanced ARNT/HIF1 homodimer formation. Enforced ARNT/HIF1 homodimer formation is associated with enhanced protection from chronic organ failure in kidney, heart and liver. In summary, the inventors report a novel approach to induce ARNT/HIF1R homodimer formation, ultimately associated with attenuation of chronic organ failure. Identifying key molecules involved in enforced ARNT homodimer formation show promise in attenuating chronic organ failure of parenchymal organs including kidney, heart and liver.

Example 7Low-Dose FK506 and GPI-1046 Protect the Kidney in a Diabetic Model of Chronic Kidney Injury

[0191] With regard of translational implications of our findings, we next aimed to gain insights into presence of FKBP12/YY1 among different human pathologies. FKBP12 and YY1 were equally detectable in various human diseases including injured diabetic nephropathy, hypertensive nephropathy, focal-segmental glomerulosclerosis, autoimmune hepatitis, liver fibrosis, liver cirrhosis and lung fibrosis (histoimmunological data due to bad printing-reproducibility not shown herein) as prerequisite of identified organ protection mediated by ARNT homodimer formation, e.g. as mediated by FK506 or GPI-1046. Based on these observations, we next hypothesized that FK506 and small molecule FKBP12 inhibitor GPI-1046 could equally protect functional parenchyma in an experimental models of diabetic nephropathy (168). Diabetes was induced in 8 to 12 weeks old C57BL/6N mice by a single intraperitoneal injection of streptozotocin (STZ) at 200 mg/kg in 10 mmol/L citrate buffer (pH 4.5), citrate buffer alone was injected as control (168). FK506 (0.2 mg/kg body weight) and GPI-146 (10 mg/kg body weight) in DMSO once daily were applied by subcutaneous injections starting one day prior of STZ administration. At day 3 after STZ injection, diabetes was confirmed by urine dipstick. Mice were sacrificed 8 weeks after STZ injection for further analyses. While streptozotocin (STZ) administration to mice was associated with chronic tubular injury, picomolar FK506 (0.2 mg/kg subcutaneously per day) and GPI-1046 (10 mg/kg subcutaneously per day) were equally effective in protection of functional parenchyma from chronic injury (immunohistology data not shown).

TABLE-US-00001 TABLE 1 Pathway analysis in response to FK506 (accession number GSE5258). GO term description enrichment p value GO:2000404 regulation of T cell migration 61.20 4.81e.sup.4 GO:2000406 positive regulation of T cell 61.20 4.81e.sup.4 migration GO:2000403 positive regulation of 61.20 4.81e.sup.4 lymphocyte migration GO:1900025 negative regulation of substrate 43.71 9.73e.sup.4 adhesion-dependent cell spreading GO:0000060 protein import into nucleus, 14.23 5.27e.sup.4 translocation GO:0071772 response to BMP 12.24 8.99e.sup.4 GO:0071773 cellular response to BMP 12.24 8.99e.sup.4 stimulus GO:0035023 regulation of Rho protein 10.41 3.83e.sup.4 signal transduction GO:0043547 positive regulation of GTPase 8.63 9.17e.sup.6 activity GO:0046578 regulation of Ras protein 8.33 3.14e.sup.4 signal transduction GO:0051056 regulation of small GTPase 7.85 1.09e.sup.4 mediated signaling transduction GO:0043087 regulation of GTPase activity 7.77 2.50e.sup.5 GO:0044236 multicellular organismal metabolic 4.54 2.76e.sup.4 process GO:0044763 single-organism cellular process 1.02 7.32e.sup.4

TABLE-US-00002 TABLE 2 Motif enrichment analysis of differentially expressed genes induced in response to FK506 (accession number GSE5258). transcription factor association score p value E2F-1/E2F-2 6.747 1.30e.sup.5 C-ETS-1 6.450 2.70e.sup.5 MAZ 5.380 2.43e.sup.4 MAX 5.380 2.43e.sup.4 E2F-1 4.705 1.01e.sup.3 TFII-I 4.593 1.34e.sup.3 SPZ1 4.557 1.45e.sup.3 AP-2 4.460 1.75e.sup.3 PAX-5 4.344 2.22e.sup.3 MAZR 4.254 2.71e.sup.3 USF1 4.201 2.94e.sup.3 EGR-1/EGR-2 4.139 3.20e.sup.3 PEA3 3.950 4.92e.sup.3 ATF6 3.887 5.72e.sup.3 ARNT 3.823 6.58e.sup.3 SP1 3.785 6.93e.sup.3 SP1/SP3 3.778 7.02e.sup.3 SP1/SP2 3.777 7.04e.sup.3 YY1 3.776 7.06e.sup.3 STRA13 3.762 7.31e.sup.3 AHR/ARNT 3.725 7.89e.sup.3 PAX-9A 3.719 7.95e.sup.3 RELA 3.638 9.37e.sup.3 ATF-1/ATF- 23.524 1.15e.sup.2 MAX 3.519 1.17e.sup.2 C-MAF 3.509 1.17e.sup.2 ATF3 3.465 1.29e.sup.2 TEL-2/TEL-2 3.457 1.32e.sup.2 n-MYC 3.435 1.38e.sup.2 DP-1/E2F-1 3.422 1.44e.sup.2 CHCH 3.368 1.53e.sup.2 MOVO- 3.363 1.56e.sup.2 NERF-1 3.304 1.76e.sup.2 USF1 3.272 1.93e.sup.2 NF-B1/NF-B2 3.191 2.28e.sup.2 SREBP-1 3.178 2.36e.sup.2 ELF-1 3.146 2.50e.sup.2 CREB/CREB- 3.101 2.61e.sup.2 BRCA1 3.085 2.67e.sup.2 NRF-1 3.076 2.70e.sup.2 ZF5 3.070 2.75e.sup.2 HIF-1 2.996 3.31e.sup.2 USF1 2.989 3.36e.sup.2 CREB 2.989 3.36e.sup.2 GABP-/GABP-1 2.978 3.43e.sup.2 AHR 2.954 3.56e.sup.2 AP-2 2.853 4.25e.sup.2 ATF4 2.819 4.54e.sup.2 EGR-4 2.811 4.58e.sup.2 ELK-1 2.780 4.89e.sup.2 AP-2 2.769 4.96e.sup.2

TABLE-US-00003 TABLE 3 Sequence alignment of Fkbp12-VMO. maximal total query description score score cover E value identity accession Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NR_126058.1 (Fkbp1a), transcript variant 3, non-coding RNA Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NM_001302080.1 (Fkbp1a), transcript variant 6 mRNA Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NM_001302079.1 (Fkb1a), transcript variant 5, mRNA Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NM_001302078.1 (Fkbp1a), transcript variant 4, mRNA Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NM_001302077.1 (Fkbp1a), transcript variant 2, mRNA Mus musculus FK506 binding protein 1a 50.1 50.1 100% 3.00E06 100% NM_008019.3 (Fkb1a), transcript variant 1, mRNA PREDICTED: Mus musculus pregnancy 32.2 32.2 64% 0.65 100% XM_006540007.3 specific glycoprotein 19 (Psg19), transcript variant X1, mRNA PREDICTED: Mus musculus pregnancy- 32.2 32.2 64% 0.65 100% XM_017322233.1 specific glycoprotein 22 (Psg22), transcript variant X1, mRNA Mus musculus pregnancy-specific 32.2 32.2 64% 0.65 100% NM_001004152.2 glycoprotein 22 (Psg22), mRNA Mus musculus pregnancy specific 32.2 32.2 64% 0.65 100% NM_011964.2 glycoprotein 19 (Psg19), mRNA Mus musculus aminolevulinic acid 32.2 32.2 64% 0.65 100% NM_001102446.1 synthase 2, erythroid (Alas2), transcript variant 2, mRNA Mus musculus aminolevulinic acid 32.2 32.2 64% 0.65 100% NM_009653.3 synthase 2, erythroid (Alas2), transcript variant 1, mRNA PREDICTED: Mus musculus NHS-like 2 30.2 30.2 76% 2.6 95% XM_006527721.3 (Nhsl2), transcript variant X4, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509985.3 (Dnm2), transcript variant X18, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_017313129.1 (Dnm2), transcript variant X15, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509982.2 (Dnm2), transcript variant X14, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509981.2 (Dnm2), transcript variant X13, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_017313128.1 (Dnm2), transcript variant X12 mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_017313127.1 (Dnm2), transcript variant X11, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_017313126.1 (Dnm2), transcript variant X10, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509980.2 (Dnm2), transcript variant X9, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509978.2 (Dnm2), transcript variant X7, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_017313125.1 (Dnm2), transcript variant X5, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509976.2 (Dnm2), transcript variant X4, mRNA PREDICTED: Mus musculus discs, large 30.2 30.2 76% 2.6 95% XM_006503105.3 (Drosophila) homolog-associated protein 3 (Dlgap3), transcript variant X3, mRNA PREDICTED: Mus musculus discs, large 30.2 30.2 76% 2.6 95% XM_006503104.3 (Drosophila) homolog-associated protein 3 (Dlgap3), transcript variant X2, mRNA PREDICTED: Mus musculus discs, large 30.2 30.2 76% 2.6 95% XM_011240537.2 (Drosophila) homolog-associated protein 3 (Dlgap3), transcript variant X1, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XR_374058.3 (Dnm1), transcript variant X18, misc_RNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XR_001780678.1 (Dnm1), transcript variant X16, misc_RNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XR_001780675.1 (Dnm1), transcript variant X15, misc_RNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_017315328.1 (Dnm1), transcript variant X10, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497659.3 (Dnm1), transcript variant X9, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497658.3 (Dnm1), transcript variant X8, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497654.3 (Dnm1), transcript variant X5, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516407.3 motif containing 9 (Trim9), transcript variant X12, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XR_381560.3 motif containing 9 (Trim9), transcript variant X11, misc_RNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516406.3 motif containing 9 (Trim9), transcript variant X10, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516405.3 motif containing 9 (Trim9), transcript variant X9, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516404.3 motif containing 9 (Trim9), transcript variant X8, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516403.3 motif containing 9 (Trim9), transcript variant X7, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516402.3 motif containing 9 (Trim9), transcript variant X6, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_017315265.1 motif containing 9 (Trim9), transcript variant X5, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_017315264.1 motif containing 9 (Trim9), transcript variant X4, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516401.3 motif containing 9 (Trim9), transcript variant X3, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_017315263.1 motif containing 9 (Trim9), transcript variant X2, mRNA PREDICTED: Mus musculus tripartite 30.2 30.2 60% 2.6 100% XM_006516400.3 motif-containing 9 (Trim9), transcript variant X1, mRNA PREDICTED: Mus musculus armadillo 30.2 52.5 96% 2.6 91% XM_006529960.3 repeat containing 9 (Armc9), transcript variant X11, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XR_374057.2 (Dnm1), transcript variant X17, misc_RNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XR_374055.2 (Dnm1), transcript variant X14, misc_RNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497662.2 (Dnm1), transcript variant X13, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497661.2 (Dnm1), transcript variant X12, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497660.2 (Dnm1), transcript variant X11, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497653.2 (Dnm1), transcript variant X7, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497655.2 (Dnm1), transcript variant X6, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497652.2 (Dnm1), transcript variant X4, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497651.2 (Dnm1), transcript variant X3, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497650.2 (Dnm1), transcript variant X2, mRNA PREDICTED: Mus musculus dynamin 1 30.2 30.2 60% 2.6 100% XM_006497649.2 (Dnm1), transcript variant X1, mRNA Mus musculus discs, large (Drosophila) 30.2 30.2 76% 2.6 95% NM_001302081.1 homolog-associated protein 3 (Dlgap3), transcript variant 2, mRNA Mus musculus discs, large (Drosophila) 30.2 30.2 76% 2.6 95% NM_198618.5 homolog-associated protein 3 (Dlgap3), transcript variant 1, mRNA Mus musculus dynamin 1 (Dnm1), 30.2 30.2 60% 2.6 100% NR_125959.1 transcript variant 3, non-coding RNA Mus musculus dynamin 1 (Dnm1), 30.2 30.2 60% 2.6 100% NM_001301737.1 transcript variant 2, mRNA Mus musculus dynamin 1 (Dnm1), 30.2 30.2 60% 2.6 100% NM_010065.3 transcript variant 1, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509983.1 (Dnm2), transcript variant X16, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509979.1 (Dnm2), transcript variant X8, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509977.1 (Dnm2), transcript variant X6, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509975.1 (Dnm2), transcript variant X3, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509974.1 (Dnm2), transcript variant X2, mRNA PREDICTED: Mus musculus dynamin 2 30.2 30.2 60% 2.6 100% XM_006509973.1 (Dnm2), transcript variant X1, mRNA PREDICTED: Mus musculus fat mass and 30.2 30.2 60% 2.6 100% XM_006531036.1 obesity associated (Fto), transcript variant X1, mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_001286388.1 (Trim9), transcript variant 6, mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_001286387.1 (Trim9), transcript variant 5, mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_001286386.1 (Trim9), transcript variant 4, mRNA Mus musculus dynamin 2 (Dnm2), 30.2 30.2 60% 2.6 100% NM_001253894.1 transcript variant 4, mRNA Mus musculus dynamin 2 (Dnm2), 30.2 30.2 60% 2.6 100% NM_007871.2 transcript variant 3, mRNA Mus musculus dynamin 2 (Dnm2), 30.2 30.2 60% 2.6 100% NM_001039520.2 transcript variant 2, mRNA Mus musculus dynamin 2 (Dnm2), 30.2 30.2 60% 2.6 100% NM_001253893.1 transcript variant 1, mRNA Mus musculus fat mass and obesity 30.2 30.2 60% 2.6 100% NM_011936.2 associated (Fto), mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_001110203.1 (Trim9), transcript variant 3, mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_001110202.1 (Trim9), transcript variant 2, mRNA Mus musculus tripartite motif-containing 9 30.2 30.2 60% 2.6 100% NM_053167.3 (Trim9), transcript variant 1, mRNA Mus musculus WAP, FS, Ig, KU, and NTR- 30.2 30.2 60% 2.6 100% NM_001100454.1 containing protein 1 (Wfikkn1), mRNA PREDICTED: Mus musculus intestinal cell 28.2 28.2 56% 10 100% XM_006511305.3 kinase (Ick), transcript variant X2, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XR_001778478.1 hydroxysteroid dehydrogenase like 1 (Hsdl1), transcript variant X7, misc_RNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XM_006531397.3 hydroxysteroid dehydrogenase like 1 (Hsdl1), transcript variant X4, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XM_006531395.3 hydroxysteroid dehydrogenase like 1 (Hsdl1), transcript variant X3, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XM_006531394.3 hydroxysteroid dehydrogenase like 1 (Hsdl1), transcript variant X2, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XM_006531393.3 hydroxysteroid dehydrogenase like 1 (Hsdl1), transcript variant X1, mRNA PREDICTED: Mus musculus kinesin family 28.2 28.2 56% 10 100% XM_017312585.1 member C3 (Kifc3), transcript variant X7, mRNA PREDICTED: Mus musculus kinesin family 28.2 28.2 56% 10 100% XM_006530723.3 member C3 (Kifc3), transcript variant X6, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XR_001778116.1 uncharacterized LOC108167423 (LOC108167423), transcript variant X2, ncRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XR_001778115.1 uncharacterized LOC108167423 (LOC108167423), transcript variant X1, ncRNA PREDICTED: Mus musculus IQ motif 28.2 52.5 80% 10 100% XR_001785567.1 containing GTPase activating protein 1 (Iqgap1), transcript variant X3, misc_RNA PREDICTED: Mus musculus IQ motif 28.2 28.2 56% 10 100% XR_391347.3 containing GTPase activating protein 1 (Iqgap1), transcript variant X2, misc_RNA PREDICTED: Mus musculus IQ motif 28.2 52.5 80% 10 100% XM_006540950.3 containing GTPase activating protein 1 (Iqgap1), transcript variant X1, mRNA PREDICTED: Mus musculus chloride 28.2 28.2 56% 10 100% XM_006505477.3 channel, voltage-sensitive 1 (Clcn1), transcript variant X3, mRNA PREDICTED: Mus musculus adrenergic 28.2 28.2 56% 10 100% XR_880393.2 receptor kinase, beta 2 (Adrbk2), transcript variant X3, misc_RNA PREDICTED: Mus musculus adrenergic 28.2 28.2 56% 10 100% XR_389274.3 receptor kinase, beta 2 (Adrbk2), transcript variant X1, misc_RNA PREDICTED: Mus musculus predicted 28.2 50.5 76% 10 100% XM_006530360.3 gene 15800 (Gm15800), transcript variant X11, mRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XM_006530359.3 gene 15800 (Gm15800), transcript variant X10, mRNA

TABLE-US-00004 TABLE 4 Sequence alignment of Yy1-VMO. maximal total query description score score cover E value identity accession PREDICTED: Mus musculus YY1 50.1 50.1 100% 3.00E06 100% XM_006515820.3 transcription factor (Yy1), transcript variant X1, mRNA Mus musculus YY1 transcription factor 50.1 50.1 100% 3.00E06 100% NM_009537.3 (Yy1), mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495691.3 (Dst), transcript variant X30, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495690.3 (Dst), transcript variant X29, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314868.1 (Dst), transcript variant X28, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495688.3 (Dst), transcript variant X27, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314848.1 (Dst), transcript variant X26, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495685.2 (Dst), transcript variant X24, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495684.3 (Dst), transcript variant X23, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314811.1 (Dst), transcript variant X22, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495682.2 (Dst), transcript variant X21, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314794.1 (Dst), transcript variant X20, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495680.3 (Dst), transcript variant X19, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495679.3 (Dst), transcript variant X18, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495678.3 (Dst), transcript variant X17, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314750.1 (Dst), transcript variant X16, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314741.1 (Dst), transcript variant X15, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495674.3 (Dst), transcript variant X14, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314722.1 (Dst), transcript variant X13, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314716.1 (Dst), transcript variant X12, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314712.1 (Dst), transcript variant X11, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314706.1 (Dst), transcript variant X10, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495669.3 (Dst), transcript variant X9, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314689.1 (Dst), transcript variant X8, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314687.1 (Dst), transcript variant X7, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314676.1 (Dst), transcript variant X6, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314665.1 (Dst), transcript variant X5, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314651.1 (Dst), transcript variant X4, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_006495666.3 (Dst), transcript variant X3, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314635.1 (Dst), transcript variant X2, mRNA PREDICTED: Mus musculus dystonin 28.2 28.2 56% 10 100% XM_017314621.1 (Dst), transcript variant X1, mRNA Mus musculus active BCR-related gene 28.2 28.2 56% 10 100% NM_198895.2 (Abr), transcript variant 3, mRNA Mus musculus dystonin (Dst), transcript 28.2 28.2 56% 10 100% NM_133833.3 variant 3, mRNA Mus musculus dystonin (Dst), transcript 28.2 28.2 56% 10 100% NM_134448.3 variant 2, mRNA Mus musculus dystonin (Dst), transcript 28.2 28.2 56% 10 100% NM_001276764.1 variant 1, mRNA Mus musculus insulin-like 3 (Insl3), mRNA 28.2 28.2 56% 10 100% NM_013564.7 PREDICTED: Mus musculus SREBF 26.3 26.3 52% 40 100% XM_006512085.3 chaperone (Scap), transcript variant X3, mRNA PREDICTED: Mus musculus SREBF 26.3 26.3 52% 40 100% XM_006512084.3 chaperone (Scap), transcript variant X1, mRNA PREDICTED: Mus musculus synuclein, 26.3 26.3 52% 40 100% XM_006526192.3 alpha interacting protein (synphilin) (Sncaip). transcript variant X2, mRNA PREDICTED: Mus musculus HMG box 26.3 26.3 52% 40 100% XM_017317779.1 domain containing 3 (Hmgxb3), transcript variant X1, mRNA PREDICTED: Mus musculus exocyst 26.3 26.3 52% 40 100% XM_017315474.1 complex component 3 (Exoc3), transcript variant X3, mRNA PREDICTED: Mus musculus exocyst 26.3 26.3 52% 40 100% XM_006517191.2 complex component 3 (Exoc3), transcript variant X1, mRNA PREDICTED: Mus musculus SREBF 26.3 26.3 52% 40 100% XM_006512083.2 chaperone (Scap), transcript variant X2, mRNA Mus musculus SREBF chaperone (Scap), 26.3 26.3 52% 40 100% NM_001001144.3 transcript variant 1, mRNA Mus musculus SREBF chaperone (Scap), 26.3 26.3 52% 40 100% NM_001103162.2 transcript variant 2, mRNA Mus musculus forkhead box F1 (Foxf1), 26.3 26.3 52% 40 100% NM_010426.2 mRNA Mus musculus MARVEL (membrane- 26.3 26.3 52% 40 100% NM_028584.3 associating) domain containing 3 (Marveld3), transcript variant 1, mRNA Mus musculus MARVEL (membrane- 26.3 26.3 52% 40 100% NM_212447.2 associating) domain containing 3 (Marveld3), transcript variant 2, mRNA Mus musculus insulinoma-associated 1 26.3 26.3 52% 40 100% NM_016889.3 (Insm1), mRNA Mus musculus serine (or cysteine) 26.3 26.3 52% 40 100% NM_008871.2 peptidase inhibitor, clade E, member 1 (Serpine1), mRNA Mus musculus HMG box domain 26.3 26.3 52% 40 100% NM_134134.2 containing 3 (Hmgxb3), transcript variant 2, mRNA Mus musculus HMG box domain 26.3 26.3 52% 40 100% NM_178277.1 containing 3 (Hmgxb3), transcript variant 1, mRNA Mus musculus ATPase, H+ transporting, 26.3 26.3 52% 40 100% NM_007510.2 lysosomal V1 subunit E1 (Atp6v1e1), mRNA PREDICTED: Mus musculus poly (ADP- 26.3 26.3 52% 40 100% XR_001778888.1 ribose) polymerase family, member 3 (Parp3), transcript variant X4, misc_RNA PREDICTED: Mus musculus poly (ADP- 26.3 26.3 52% 40 100% XM_006511719.3 ribose) polymerase family, member 3 (Parp3), transcript variant X2, mRNA PREDICTED: Mus musculus cDNA 26.3 26.3 52% 40 100% XM_011241784.2 sequence BC017158 (BC017158), transcript variant X3, mRNA PREDICTED: Mus musculus DEAD (Asp- 26.3 26.3 52% 40 100% XR_878688.2 Glu-Ala-Asp) box polypeptide 54 (Ddx54), transcript variant X1, misc_RNA PREDICTED: Mus musculus actin filament 26.3 26.3 52% 40 100% XM_017318171.1 associated protein 1-like 2 (Afap1l2), transcript variant X5, mRNA PREDICTED: Mus musculus actin filament 26.3 26.3 52% 40 100% XM_017318170.1 associated protein 1-like 2 (Afap1l2), transcript variant X4, mRNA PREDICTED: Mus musculus actin filament 26.3 26.3 52% 40 100% XM_017318169.1 associated protein 1-like 2 (Afap1l2), transcript variant X2, mRNA PREDICTED: Mus musculus actin filament 26.3 26.3 52% 40 100% XM_017318168.1 associated protein 1-like 2 (Afap1l2), transcript variant X1, mRNA PREDICTED: Mus musculus predicted 26.3 26.3 52 40 100% XR_001779509.1 gene, 31348 (Gm31348), transcript variant X4, ncRNA PREDICTED: Mus musculus predicted 26.3 26.3 52% 40 100% XR_001779508.1 gene, 31348 (Gm31348), transcript variant X3, ncRNA PREDICTED: Mus musculus predicted 26.3 26.3 52% 40 100% XR_373640.3 gene, 31348 (Gm31348), transcript variant X1, ncRNA Mus musculus poly (ADP-ribose) 26.3 26.3 52% 40 100% NM_001311150.1 polymerase family, member 3 (Parp3), transcript variant 1, mRNA Mus musculus poly (ADP-ribose) 26.3 26.3 52% 40 100% NM_145619.3 polymerase family, member 3 (Parp3), transcript variant 2, mRNA PREDICTED: Mus musculus actin filament 26.3 26.3 52% 40 100% XM_006527024.2 associated protein 1-like 2 (Afap1l2), transcript variant X3, mRNA PREDICTED: Mus musculus poly (ADP- 26.3 26.3 52% 40 100% XM_006511720.1 ribose) polymerase family, member 3 (Parp3), transcript variant X3, mRNA PREDICTED: Mus musculus poly (ADP- 26.3 26.3 52% 40 100% XM_006511718.1 ribose) polymerase family, member 3 (Parp3), transcript variant X1, mRNA Mus musculus linterleukin-1 receptor- 26.3 26.3 68% 40 94% NM_001177973.1 associated kinase 1 (Irak1), transcript variant 1, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_001177975.1 associated kinase 1 (Irak1), transcript variant 3, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_001177976.1 associated kinase 1 (Irak1), transcript variant 2, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_001177974.1 associated kinase 1 (Irak1), transcript variant 5, mRNA Mus musculus actin filament associated 26.3 26.3 52% 40 100% NM_001177797.1 protein 1-like 2 (Afap1l2), transcript variant 2, mRNA Mus musculus actin filament associated 26.3 26.3 52% 40 100% NM_001177796.1 protein 1-like 2 (Afap1l2), transcript variant 1, mRNA Mus musculus actin filament associated 26.3 26.3 52% 40 100% NM_146102.2 protein 1-like 2 (Afap1l2), transcript variant 3, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_001168240.1 associated kinase 1 binding protein 1 (Irak1bp1), transcript variant 2, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_022986.4 associated kinase 1 binding protein 1 (Irak1bp1), transcript variant 1, mRNA Mus musculus interleukin-1 receptor- 26.3 26.3 68% 40 94% NM_008363.2 associated kinase 1 (Irak1), transcript variant 4, mRNA Mus musculus DEAD (Asp-Glu-Ala-Asp) 26.3 26.3 52% 40 100% NM_028041.2 box polypeptide 54 (Ddx54), mRNA Mus musculus tet methylcytosine 24.3 24.3 48% 159 100% NM_001347313.1 dioxygenase 3 (Tet3), transcript variant 1, mRNA PREDICTED: Mus musculus RIKEN 24.3 24.3 48% 159 100% XR_390177.3 cDNA C330020E22 gene (C330020E22Rik), transcript variant X13, ncRNA PREDICTED: Mus musculus RIKEN 24.3 24.3 48% 159 100% XR_882416.2 cDNA C330020E22 gene (C330020E22Rik), transcript variant X12, ncRNA PREDICTED: Mus musculus RIKEN 24.3 24.3 48% 159 100% XR_882415.2 cDNA C330020E22 gene (C330020E22Rik), transcript variant X11, ncRNA PREDICTED: Mus musculus RIKEN 24.3 24.3 48% 159 100% XR_882414.2 cDNA C330020E22 gene (C330020E22Rik), transcript variant X5, ncRNA PREDICTED: Mus musculus RIKEN 24.3 24.3 48% 159 100% XR_882413.2 cDNA C330020E22 gene (C330020E22Rik), transcript variant X10, ncRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_006511257.3 domain containing 33 (Ccdc33), transcript variant X8, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_017313448.1 domain containing 33 (Ccdc33), transcript variant X7, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_006511254.3 domain containing 33 (Ccdc33), transcript variant X6, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_017313447.1 domain containing 33 (Ccdc33), transcript variant X5, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_017313446.1 domain containing 33 (Ccdc33), transcript variant X4, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_017313445.1 domain containing 33 (Ccdc33), transcript variant X3, mRNA PREDICTED: Mus musculus coiled-coil 24.3 24.3 48% 159 100% XM_017313444.1 domain containing 33 (Ccdc33), transcript variant X2, mRNA PREDICTED: Mus musculus DIS3 like 24.3 24.3 48% 159 100% XM_006510982.3 exosome 3-5 exoribonuclease (Dis3l), transcript variant X3, mRNA PREDICTED: Mus musculus DIS3 like 24.3 24.3 48% 159 100% XM_006510983.3 exosome 3-5 exoribonuclease (Dis3l), transcript variant X2, mRNA PREDICTED: Mus musculus asparaginyl- 24.3 24.3 48% 159 100% XR_001785558.1 tRNA synthetase 2 (mitochondrial)(putative) (Nars2), transcript variant X16, misc_RNA PREDICTED: Mus musculus asparaginyl- 24.3 24.3 48% 159 100% XR_001785557.1 tRNA synthetase 2 (mitochondrial)(putative) (Nars2), transcript variant X15, misc_RNA PREDICTED: Mus musculus asparaginyl- 24.3 24.3 48% 159 100% XR_001785556.1 tRNA synthetase 2 (mitochondrial)(putative) (Nars2), transcript variant X14, misc_RNA PREDICTED: Mus musculus asparaginyl- 24.3 24.3 48% 159 100% XR_001785555.1 tRNA synthetase 2 (mitochondrial)(putative) (Nars2), transcript variant X13, misc_RNA Mus musculus FERM domain containing 24.3 24.3 48% 159 100% NM_001347086.1 4A (Frmd4a), transcript variant 4, mRNA

TABLE-US-00005 TABLE 5 Sequence alignment of Arnt-VMO. maximal total query description score score cover E value identity accession PREDICTED: Mus musculus aryl 50.1 50.1 100% 3.00E06 100% XR_001783644.1 hydrocarbon receptor nuclear translocator (Arnt), transcript variant X5, misc_RNA PREDICTED: Mus musculus aryl 50.1 50.1 100% 3.00E06 100% XR_001783643.1 hydrocarbon receptor nuclear translocator (Arnt), transcript variant X4, misc_RNA PREDICTED: Mus musculus aryl 50.1 50.1 100% 3.00E06 100% XM_006500931.3 hydrocarbon receptor nuclear translocator (Arnt), transcript variant X2, mRNA PREDICTED: Mus musculus aryl 50.1 50.1 100% 3.00E06 100% XM_006500930.3 hydrocarbon receptor nuclear translocator (Arnt), transcript variant X1, mRNA PREDICTED: Mus musculus aryl 50.1 50.1 100% 3.00E06 100% XM_006500933.2 hydrocarbon receptor nuclear translocator (Arnt), transcript variant X6, mRNA Mus musculus aryl hydrocarbon receptor 50.1 50.1 100% 3.00E06 100% NM_009709.4 nuclear translocator (Arnt), transcript variant 2, mRNA Mus musculus aryl hydrocarbon receptor 50.1 50.1 100% 3.00E06 100% NM_001037737.2 nuclear translocator (Arnt), transcript variant 1, mRNA PREDICTED: Mus musculus UDP- 30.2 30.2 60% 2.6 100% XM_011248200.2 GlcNAc:betaGal beta-1,3-N- acetylglucosaminyltransferase 4 (B3gnt4), transcript variant X3, mRNA PREDICTED: Mus musculus UDP- 30.2 30.2 60% 2.6 100% XM_011248199.2 GlcNAc:betaGal beta-1,3-N- acetylglucosaminyltransferase 4 (B3gnt4), transcript variant X1, mRNA PREDICTED: Mus musculus 28.2 28.2 56% 10 100% XM_006533962.2 uncharacterized LOC666331 (LOC666331), transcript variant X1, mRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_878497.1 gene, 35562 (Gm35562), transcript variant X11, ncRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_878495.1 gene, 35562 (Gm35562), transcript variant X10, ncRNA Mus musculus uncharacterized 28.2 28.2 56% 10 100% NM_001256318.1 LOC666331 (LOC666331), mRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_872158.2 gene, 29862 (Gm29862), transcript variant X5, ncRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_872157.2 gene, 29862 (Gm29862), transcript variant X4, ncRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_380917.3 gene, 29862 (Gm29862), transcript variant X3, ncRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_001779668.1 gene, 29862 (Gm29862), transcript variant X2, ncRNA PREDICTED: Mus musculus predicted 28.2 28.2 56% 10 100% XR_872156.2 gene, 29862 (Gm29862), transcript variant X1, ncRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_017321986.1 growth factor I receptor (Igflr), transcript variant X6, mRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_006540645.3 growth factor I receptor (Igf1r), transcript variant X5, mRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_006540644.3 growth factor I receptor (Igf1r), transcript variant X4, mRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_006540643.3 growth factor I receptor (Igf1r), transcript variant X3, mRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_006540642.3 growth factor I receptor (Igf1r), transcript variant X2, mRNA PREDICTED: Mus musculus insulin-like 26.3 26.3 52% 40 100% XM_006540641.3 growth factor I receptor (Igf1r), transcript variant X1, mRNA PREDICTED: Mus musculus 26.3 26.3 84% 40 100% XM_017320601.1 argininosuccinate lyase (Asp, transcript variant X2, mRNA PREDICTED: Mus musculus 26.3 26.3 52% 40 100% XR_390344.2 transmembrane protein 68 (Tmem68), transcript variant X4, misc_RNA PREDICTED: Mus musculus 26.3 26.3 52% 40 100% XM_006538270.3 transmembrane protein 68 (Tmem68), transcript variant X3, mRNA PREDICTED: Mus musculus 26.3 26.3 52% 40 100% XM_006538269.3 transmembrane protein 68 (Tmem68), transcript variant X2, mRNA PREDICTED: Mus musculus 26.3 26.3 52% 40 100% XR_001784202.1 transmembrane protein 68 (Tmem68), transcript variant X1, misc_RNA PREDICTED: Mus musculus neuregulin 2 26.3 26.3 52% 40 100% XM_006525461.3 (Nrg2), transcript variant X1, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_011245554.2 factor 20 (Tcf20), transcript variant X6, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_011245553.2 factor 20 (Tcf20), transcript variant X5, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_011245552.2 factor 20 (Tcf20), transcript variant X4, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_006520732.2 factor 20 (Tcf20), transcript variant X2, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_011245551.2 factor 20 (Tcf20), transcript variant X1, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515988.3 transcription factor 8 (Sp8), transcript variant X8, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515987.3 transcription factor 8 (Sp8), transcript variant X7, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515986.3 transcription factor 8 (Sp8), transcript variant X6 mRNA PREDICTED: Mus musculus trans-acting 26.3 11. 52% 40 100% XM_006515985.3 transcription factor 8 (Sp8), transcript variant X5, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515984.3 transcription factor 8 (Sp8), transcript variant X4, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_011244124.2 transcription factor 8 (Sp8), transcript variant X3, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515983.3 transcription factor 8 (Sp8), transcript variant X2, mRNA PREDICTED: Mus musculus trans-acting 26.3 26.3 52% 40 100% XM_006515982.3 transcription factor 8 (Sp8), transcript variant X1, mRNA PREDICTED: Mus musculus transcription 26.3 26.3 52% 40 100% XM_006520733.1 factor 20 (Tcf20), transcript variant X3, mRNA PREDICTED: Mus musculus sodium 26.3 26.3 52% 40 100% XM_006511997.1 channel, voltage-gated, type V, alpha (Scn5a), transcript variant X4, mRNA PREDICTED: Mus musculus sodium 26.3 26.3 52% 40 100% XM_006511996.1 channel, voltage-gated, type V, alpha (Scn5a), transcript variant X3, mRNA PREDICTED: Mus musculus sodium 26.3 26.3 52% 40 100% XM_006511995.1 channel, voltage-gated, type V, alpha (Scn5a), transcript variant X2, mRNA PREDICTED: Mus musculus sodium 26.3 26.3 52% 40 100% XM_006511993.1 channel, voltage-gated, type V, alpha (Scn5a), transcript variant X1, mRNA Mus musculus sodium channel, voltage- 26.3 26.3 52% 40 100% NM_001253860.1 gated, type V, alpha (Scn5a), transcript variant 2, mRNA Mus musculus sodium channel, voltage- 26.3 26.3 52% 40 100% NM_021544.4 gated, type V, alpha (Scn5a), transcript variant 1, mRNA Mus musculus trans-acting transcription 26.3 26.3 52% 40 100% NM_177082.4 factor 8 (Sp8), mRNA Mus musculus BCL2-associated 26.3 26.3 52% 40 100% NM_013863.5 athanogene 3 (Bag3), mRNA Mus musculus transcription factor 20 26.3 26.3 52% 40 100% NM_001114140.1 (Tcf20), transcript variant 1, mRNA Mus musculus transcription factor 20 26.3 26.3 52% 40 100% NM_013836.3 (Tcf20), transcript variant 2, mRNA Mus musculus transmembrane protein 68 26.3 26.3 52% 40 100% NM_028097.3 (Tmem68), mRNA Mus musculus insulin like growth factor I 26.3 26.3 52% 40 100% NM_010513.2 receptor (Igf1r), mRNA Mus musculus receptor tyrosine kinase- 26.3 26.3 52% 40 100% NM_013846.3 like orphan receptor 2 (Ror2), mRNA Mus musculus tRNA methyltransferase O 24.3 24.3 48% 159 100% NM_001347095.1 (Trmo), transcript variant 2, mRNA Mus musculus transforming growth factor, 24.3 24.3 48% 159 100% NM_009367.4 beta 2 (Tgfb2), transcript variant 1, mRNA Mus musculus transforming growth factor, 24.3 24.3 48% 159 100% NM_001329107.1 beta 2 (Tgfb2), transcript variant 2, mRNA PREDICTED: Mus musculus predicted 24.3 24.3 48% 159 100% XR_001778574.1 gene, 33746 (Gm33746), transcript variant X3, ncRNA PREDICTED: Mus musculus predicted 24.3 24.3 48% 159 100% XR_379069.3 gene, 33746 (Gm33746), transcript variant X2, ncRNA PREDICTED: Mus musculus predicted 24.3 24.3 48% 159 100% XR_001778573.1 gene, 33746 (Gm33746), transcript variant X1, ncRNA PREDICTED: Mus musculus cytosolic 24.3 24.3 48% 159 100% XM_011248490.2 thiouridylase subunit 2 (Ctu2), transcript variant X3, mRNA PREDICTED: Mus musculus cytosolic 24.3 24.3 48% 159 100% XM_011248489.2 thiouridylase subunit 2 (Ctu2), transcript variant X2, mRNA PREDICTED: Mus musculus cytosolic 24.3 24.3 48% 159 100% XR_878872.2 thiouridylase subunit 2 (Ctu2), transcript variant X1, misc_RNA PREDICTED: Mus musculus nuclear 24.3 24.3 48% 159 100% XM_006531193.3 factor of activated T cells 5 (Nfat5), transcript variant X3, mRNA PREDICTED: Mus musculus nuclear 24.3 24.3 48% 159 100% XM_006531192.3 factor of activated T cells 5 (Nfat5), transcript variant X2, mRNA PREDICTED: Mus musculus nuclear 24.3 24.3 48% 159 100% XM_006531191.3 factor of activated T cells 5 (Nfat5), transcript variant X1, mRNA PREDICTED: Mus musculus glycerol-3- 24.3 24.3 48% 159 100% XM_011242104.2 phosphate acyltransferase 4 (Gpat4), transcript variant X1, mRNA PREDICTED: Mus musculus predicted 24.3 24.3 48% 159 100% XR_869909.2 gene, 39079 (Gm39079), ncRNA PREDICTED: Mus musculus RIKEN cDNA 24.3 24.3 48% 159 100% XM_006508331.3 9430038|01 gene (9430038|01Rik), transcript variant X4, mRNA PREDICTED: Mus musculus RIKEN cDNA 24.3 24.3 48% 159 100% XM_006508329.3 9430038|01 gene (9430038|01Rik), transcript variant X3, mRNA PREDICTED: Mus musculus RIKEN cDNA 24.3 24.3 48% 159 100% XM_011241949.2 9430038|01 gene (9430038|01Rik), transcript variant X2, mRNA PREDICTED: Mus musculus RIKEN cDNA 24.3 24.3 48% 159 100% XM_011241948.2 9430038|01 gene (9430038|01Rik), transcript variant X1, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540405.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X15 mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250732.2 induced proliferation-associated 1 like 3 (Sipa1l3 transcript variant X14, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540404.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X13, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540402.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X11, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540401.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X10, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540400.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X9, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_006540399.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X8, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250731.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X7, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250730.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X6, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250729.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X5, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250728.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X4, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM _006540397.3 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X3, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_017312346.1 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X2, mRNA PREDICTED: Mus musculus signal- 24.3 24.3 48% 159 100% XM_011250727.2 induced proliferation-associated 1 like 3 (Sipa1l3), transcript variant X1, mRNA PREDICTED: Mus musculus acid 24.3 24.3 48% 159 100% XM_011250772.2 phosphatase, testicular (Acpt), transcript variant X5, mRNA PREDICTED: Mus musculus acid 24.3 24.3 48% 159 100% XM_017321902.1 phosphatase, testicular (Acpt), transcript variant X4, mRNA PREDICTED: Mus musculus acid 24.3 24.3 48% 159 100% XM_017321901.1 phosphatase, testicular (Acpt), transcript variant X3, mRNA PREDICTED: Mus musculus acid 24.3 24.3 48% 159 100% XM_006540531.3 phosphatase, testicular (Acpt), transcript variant X2, mRNA PREDICTED: Mus musculus acid 24.3 24.3 48% 159 100% XM_006540530.3 phosphatase, testicular (Acpt), transcript variant X1, mRNA PREDICTED: Mus musculus tRNA 24.3 24.3 48% 159 100% XM_017320423.1 methyltransferase O (Trmo), transcript variant X4, mRNA PREDICTED: Mus musculus tRNA 24.3 24.3 48% 159 100% XM_017320422.1 methyltransferase O (Trmo), transcript variant X1, mRNA PREDICTED: Mus musculus RIKEN cDNA 24.3 24.3 48% 159 100% XM_017320219.1 1700024P16 gene (1700024P16Rik), transcript variant X1, mRNA PREDICTED: Mus musculus glial cell line 24.3 24.3 48% 159 100% XR_001780826.1 derived neurotrophic factor family receptor alpha 4 (Gfra4), transcript variant X3, misc_RNA PREDICTED: Mus musculus neuropilin 24.3 24.3 48% 159 100% XM_011247081.2 (NRP) and tolloid (TLL)-like 1 (Neto1), transcript variant X12, mRNA Mus musculus N-acetylglucosamine-1- 24.3 24.3 48% 159 100% NM_001346737.1 phosphotransferase, gamma subunit (Gnptg), transcipt variant 2, mRNA

TABLE-US-00006 TABLE 6 Kidney transplant patient characteristics. % pre- Creatinine BUN eGFR ID# fibrosis medication disease [mg/dl] [mg/dl] [mL/min] S3752 25-30 CsA FSGS 3.3 56 25 S3564 20 FK506 nephro- 3.2 74 16 sclerosis S3896 15-20 FK506 ADPKD 3.0 97 23

TABLE-US-00007 TABLE 7 In publicly available datasets (accession number GSE3526), expression levels of ARNT inversely correlated with FKBP12 and YY1 expression levels is not limited to the kidney (renal cortex), but also evident in cardiovascular, digestive and central nervous systems (slope, r.sup.2 and values of p were calculated by linear regression). system comparison slope r.sup.2 p value renal cortex ARNT vs. FKBP12 2.6 0.4 0.9618 0.0193 ARNT vs. YY1 0.6 0.0 0.9922 0.0039 cardiovascular ARNT vs. FKBP12 1.8 0.8 0.2843 0.0606 ARNT vs. YY1 1.1 0.5 0.3012 0.0521 digestive ARNT vs. FKBP12 1.2 0.5 0.1439 0.0093 ARNT vs. YY1 0.7 0.3 0.1152 0.0210 central nervous ARNT vs. FKBP12 0.9 0.3 0.0590 0.0015 ARNT vs. YY1 1.1 0.2 0.2368 <0.0001 lymphatic ARNT vs. FKBP12 0.9 0.7 0.1129 0.2032 ARNT vs. YY1 0.7 0.8 0.0503 0.4037 reproductive ARNT vs. FKBP12 0.6 0.5 0.0345 0.2931 ARNT vs. YY1 0.7 0.4 0.1119 0.0531 endocrine ARNT vs. FKBP12 0.6 0.7 0.0290 0.4055 ARNT vs. YY1 2.0 0.6 0.2917 0.0044 peripheral nervous ARNT vs. FKBP12 0.4 0.7 0.0169 0.5447 ARNT vs. YY1 0.5 0.3 0.0911 0.1517 respiratory ARNT vs. FKBP12 1.7 1.4 0.1708 0.2689 ARNT vs. YY1 0.6 0.4 0.2667 0.1546

TABLE-US-00008 TABLE8 Invivo-morpholinosequences. SEQ VMO sequence IDNO supplier control- 5-CCTCTTACCTCAG 7 GeneTools, VMO TTACAATTTATA-3 Philomath,USA(1) Arnt-VMO 5-AAGAGCCACTCCG 8 GeneTools, CAGATTAGGCAC-3 Philomath,USA(1) Fkbp12- 5-AGATGGTCTCCAC 9 GeneTools, VMO CTGCACTCCCAT-3 Philomath,USA(1) Yy1-VMO 5-TGTAGAGGGTGTC 10 GeneTools, GCCCGAGGCCAT-3 Philomath,USA(1)

TABLE-US-00009 TABLE9 OligonucleotidesequencesforqRT-PCR. forwardprimersequence SEQ gene reverseprimersequence IDNO supplier Acta2 5-CTCTTCCAGCCATCTTTCATTG-3 11 PrimerDesign, (mouse) 5-GTTGTTAGCATAGAGATCCTTCCT-3 12 Southampton,UK Actb undisclosed PrimerDesign, (mouse) undisclosed Southampton,UK Ahr 5-GCCCTTCCCGCAAGATGTTAT-3 13 EurofinsMWG (mouse) 5-TCAGCAGGGGTGGACTTTAAT-3 14 Operon(2) Alk3 5-TGTCATTCTAGCCATGTTTTACC-3 15 PrimerDesign, (mouse) 5-ACCAAGGATCAGATGTGAGAC-3 16 Southampton,UK ALK3 5-GGACATTGCTTTGCCATCATAG-3 17 PrimerDesign, (human) 5-GGGCTTTTGGAGAATCTTTGC-3 18 Southampton,UK Alk5 5-TCTGCATTGCACTTATGCTGA-3 19 EurofinsMWG (mouse) 5-AAAGGGCGATCTAGTGATGGA-3 20 Operon(2) Alk6 5-GCGGCCTATGCCATTTACAC-3 21 EurofinsMWG (mouse) 5-AGTCTCGATGGGCGATTGC-3 22 Operon(3) Ar 5-AAGAGCCGCTGAAGGGAAA-3 23 PrimerDesign, (mouse) 5-GAGACGACAAGATGGGCAAAT-3 24 Southampton,UK Arnt 5-CCTTCAGTGCTATGTCTCTTCC-3 25 PrimerDesign, (mouse) 5-CAGTCTCAGGAGGAAAGTTGGA-3 26 Southampton,UK ARNT 5-AGAGAGACTTGCCAGGGAAAAT-3 27 PrimerDesign, (human) 5-AGTTCTGTGATGTAGGCTGTCA-3 28 Southampton,UK Cebpb 5-ACGGGACTGACGCAACAC-3 29 PrimerDesign, (mouse) 5-AACAAAAACAAAACCAAAAACATCAAC-3 30 Southampton,UK Col1a1 5-ATGGATTCCCGTTCGAGTACG-3 31 EurofinsMWG (mouse) 5-TCAGCTGGATAGCGACATCG-3 32 Operon(4,5) Creb1 5-TTGAGTAAGGCTGAGCATGATC-3 33 PrimerDesign, (mouse) 5-TCTTAACTTTAAACTGCGGAACAC-3 34 Southampton,UK Fkbp12 5-CTATGCCTATGGAGCCACCG-3 35 EurofinsMWG (mouse) 5-ATCCACGTGCAGAGCTAAGG-3 36 Operon(6) FKBP12 5-GTGGAAACCATCTCCCCAGG-3 37 EurofinsMWG (human) 5-CCATCTTCAAGCATCCCGGT-3 38 Operon(6) Fkbp25 5-TTCTGCAGGATCACGGTTCA-3 39 EurofinsMWG (mouse) 5-TGGTCCTTATTAGCAGTCTTGGC-3 40 Operon(6) Fkbp38 5-GCTGGGAGACTGCGATGTTA-3 41 EurofinsMWG (mouse) 5-GTATGGGCTCCTGCTGCC-3 42 Operon(6) Fkbp52 5-ACCGCGTACTTCAAGGAAGG-3 43 EurofinsMWG (mouse) 5-ACCGGAGAAGCTAGACTCGT-3 44 Operon(6) Gapdh undisclosed PrimerDesign, (mouse) undisclosed Southampton,UK GAPDH undisclosed PrimerDesign, (human) undisclosed Southampton,UK Gata3 5-GAAGACTTTATTGTACCTGGATAGC-3 45 PrimerDesign, (mouse) 5-TGGACATCAGACTTAGTGGTTTC-3 46 Southampton,UK Hif1a 5-TCACCAGACAGAGCAGGAAA-3 47 EurofinsMWG (mouse) 5-GCGAAGCTATTGTCTTTGGG-3 48 Operon(7) Max 5-GTGAGTGAGTGAGCGAGTGA-3 49 PrimerDesign, (mouse) 5-GGAGGGGTGGAGGGAAGG-3 50 Southampton,UK Yy1 5-GCCCTTTCAGTGCACATTCG-3 51 EurofinsMWG (mouse) 5-CTCCGGTATGGATTCGCACA-3 52 Operon(6) YY1 5-AACAGGCATCCCGAGTTCAG-3 53 EurofinsMWG (human) 5-GCGGTGGTACAGATGCTTCA-3 54 Operon(6)

TABLE-US-00010 TABLE10 OligonucleotidesequencesforChIP. forwardprimersequence SEQ gene reverseprimersequence IDNO supplier Arnt 5-GACTTCAGTTCAGCCGGCTC 55 Eurofins motif TC-3 MWG (mouse) 5-CTCTGGTTCTGGCCCGCCGG 56 Operon GAGG-3

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