METHOD FOR PREVENTING AND TREATING DRUG-INDUCED RENAL INJURY
20190328848 ยท 2019-10-31
Assignee
Inventors
Cpc classification
International classification
Abstract
The present invention relates to a method for preventing and/or treating a drug-induced renal tissue injury and its related conditions in a subject, comprising administering an effective amount of plasminogen to the subject. The present invention further relates to a medicament, a pharmaceutical composition, an article of manufacture, and a kit comprising plasminogen which are useful for preventing and/or treating a drug-induced renal tissue injury and its related conditions in a subject.
Claims
1-66. (canceled)
67. A method for preventing and/or treating a drug-induced renal tissue injury and its related conditions in a subject, comprising administering an effective amount of plasminogen to the subject.
68. The method of claim 67, wherein the drug is a nephrotoxic drug.
69. The method of claim 68, wherein the drug is a renal excretory drug.
70. The method of claim 67, wherein the drug comprises a chemotherapeutic drug, an antihypertensive drug, a hypolipidemic drug, a hypoglycemic drug, a nonsteroid anti-inflammatory drug, an antibiotic drug, and an antiviral drug.
71. The method of claim 67, wherein the plasminogen promotes repair of an injured renal tissue.
72. The method of claim 67, wherein the plasminogen alleviates fibrosis of the injured renal tissue.
73. The method of claim 67, wherein the plasminogen alleviates apoptosis of the injured renal tissue.
74. The method of claim 67, wherein the drug-induced renal tissue injury in the subject is an acute renal tissue injury.
75. The method of claim 67, wherein the drug-induced renal tissue injury in a subject is a chronic renal tissue injury.
76. The method of claim 67, wherein the plasminogen promotes recovery of renal function.
77. A method for protecting a subject's kidney from drug-induced injury or alleviating the drug-induced injury to the kidney, comprising administering an effective amount of plasminogen to the subject before, simultaneously with, and/or after administration of the drug.
78. The method of claim 77, wherein the drug is cisplatin.
79. The method of claim 77, wherein the chronic kidney disease is a chronic renal injury induced by a chemotherapeutic drug, an antihypertensive drug, a hypolipidemic drug, a hypoglycemic drug, a nonsteroid anti-inflammatory drug, an antibiotic drug, and an antiviral drug.
80. The method of claim 77, wherein the plasminogen can promote clearance of urea nitrogen and/or creatinine by the kidney.
81. The method of claim 77, wherein the plasminogen can reduce fibrosis of the injured renal tissue.
82. The method of claim 67, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No. 2, and still has the plasminogen activity.
83. The method of claim 67, wherein the plasminogen is a protein that comprises a plasminogen active fragment and still has the plasminogen activity.
84. The method of claim 67, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen or their variants that retain the plasminogen activity.
85. The method of claim 67, wherein the plasminogen can be administered in combination with one or more drugs selected from an antihypertensive drug, a hypolipidemic drug, an antibiotic drug, an anticoagulant drug, a thrombolytic drug, a diuretic drug, an anti-tumor drug, a hypoglycemic drug, a non-steroidal anti-inflammatory drug, an immunomodulatory drug, an anti-infective drug, an antiviral drug, a hormone, and an active ingredient of a natural product.
86. The method of claim 67, wherein the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains the plasminogen activity.
87. The method of claim 67, wherein the plasminogen is administered to the subject at a dosage of 1-100 mg/kg, 1-50 mg/kg, or 1-10 mg/kg, daily, every other day, or weekly.
88. The method of claim 87, wherein the dosage of the plasminogen is preferably repeated at least once.
89. The method of claim 87, wherein the plasminogen is preferably administered at least daily.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
Example 1
Plasminogen Promotes Repair of a Renal Injury Cused by Cisplatin
[0232] Ten healthy 8-9-week-old male C57 mice were randomly divided into two groups, five mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. After the grouping was completed, a chemotherapy-induced injury model was established by single intraperitoneal injection of cisplatin at 10 mg/Kg body weight.sup.[33]. After the model was established, mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner. The day when the experiment began was Day 0, and the mice were weighed and grouped. The mice were injected with cisplatin intraperitoneally from day 1 for model establishment. Plasminogen or vehicle PBS was administered to the mice within 3 hours after completion of model establishment, and the administration period was 7 days. Mice were sacrificed on Day 8, and kidneys were fixed in 10% neutral formalin fixative for 24-48 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 pm. The sections were dewaxed and rehydrated and washed with water once. The sections were repaired with citric acid for 30 minutes, and gently rinsed with water after cooling at room temperature for 10 minutes. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Goat anti-mouse IgM (HRP) antibody (Abcam) was added to the sections dropwise, incubated for 1 hour at room temperature and washed with PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0233] IgM antibodies play an important role during the clearance of apoptotic and necrotic cells, and the local level of IgM antibodies at the injury site in tissues and organs are positively correlated with the degree of injury.sup.[29,30]. Therefore, detection of local level of IgM antibodies in tissues and organs can reflect the injury of the tissues and organs.
[0234] The results showed that the IgM-positive expression in the group administered with plasminogen (
Example 2
Plasminogen Alleviates Renal Fibrosis in Cisplatin Chemotherapeutic Injury Model Mice
[0235] Ten healthy 8-9-week-old male C57 mice were randomly divided into two groups, five mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. After the grouping was completed, a chemotherapy-induced injury model was established by single intraperitoneal injection of cisplatin at 10 mg/Kg body weight.sup.[33]. After the model was established, mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0 1 mL/mouse/day via tail vein injection, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via tail vein injection. The day when the experiment began was Day 0, and the mice were weighed and grouped. The mice were injected with cisplatin intraperitoneally from day 1 for model establishment. Plasminogen or vehicle PBS was administered to the mice within 3 hours after completion of model establishment, and the administration period was 7 days. The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Rabbit anti-mouse IV collagen antibody (Abcam) was added to the sections dropwise, incubated at 4 C. overnight, and washed with TBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with TBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washing with water three times, the sections were counterstained with hematoxylin for 30 seconds, returned to blue with running water for 5 minutes, and washed with TBS once. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0236] The results showed that the renal type IV collagen-positive expression in the control group administered with vehicle PBS (
Example 3
Plasminogen Protects the Kidney in a Chronic Renal Injury Model
[0237] Twenty 8- to 9-week-old PLG.sup./ mice and six PLG.sup./ mice were taken. PLG.sup.+/+ mice were randomly divided into two groups, 10 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. Mice in the group administered with plasminogen, the control group administered with vehicle PBS, and the PLG.sup./ group were fed with a 0.25% purine diet (Nantong TROPHIC) every day to establish the chronic renal injury model.sup.[26]. The day of model establishment was recorded as Day 1, and administration began at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner, both lasting for 10 consecutive days for model establishment. PLG.sup./ mice were not treated. The mice were sacrificed on Day 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections were 3 m thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were dehydrated with alcohol gradient, permeabilized with xylene, sealed with a neutral gum, and observed under an optical microscope at 200 (
[0238] The results showed that kidneys of PLG.sup./ mice (
Example 4
Plasminogen Repairs Renal Fibrosis in a Chronic Renal Injury Model
[0239] Twelve 8- to 9-week-old male PLG.sup.+/+ mice and six PLG.sup./ mice were taken. PLG.sup.+/+ mice were randomly divided into two groups, 6 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1, and administration began at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner, both lasting for 10 consecutive days for model establishment. PLG.sup./ mice were not treated. The mice were sacrificed on Day 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections was 3 m thick. The sections were dewaxed and rehydrated and washed with water once. After stained with 0.1% Sirius red for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200.
[0240] Sirius red staining allows for long-lasting staining of collagen. As a special staining method for pathological sections, Sirius red staining can show the collagen tissue specifically.
[0241] The results showed that the collagen deposition in the group administered with plasminogen (
Example 5
Plasminogen Promotes the Expression of Apoptosis Inhibitory Protein Bcl-2 in Kidneys of Mice Having a Chronic Renal Injury
[0242] Eighteen 8- to 9-week-old male PLG.sup.+/+ mice were randomly divided into three groups, 6 mice in each of the blank control group, the group administered with plasminogen, and the control group administered with vehicle PBS. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1, and administration began at the same time. The blank control group was fed with a normal maintenance diet. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner, both lasting for 10 consecutive days for model establishment. Mice in the blank control group received no treatment. The day of model establishment and administration was recorded as Day 1. The mice were sacrificed on Day 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Rabbit anti-mouse Bcl-2 antibody (Abcam) was added to the sections dropwise, incubated at 4 C. overnight, and washed with 0.01 M PBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with alcohol gradient, permeabilization with xylenehe, and sealing with a neutral gum, the sections were observed under an optical microscope at 200.
[0243] Bcl-2 is an apoptosis inhibitory protein, and its expression will be down-regulated under the action of an apoptosis stimulating factor[.sup.27, 28]. The Bcl-2 immunohistochemical results showed that the renal Bcl-2-positive staining of the group administered with plasminogen (
Example 6
Plasminogen Improves Local Injuries of Kidneys of Mice with a Chronic Renal Injury
[0244] Eighteen 8- to 9-week-old male PLG mice were randomly divided into three groups, 6 mice in each of the blank control group, the group administered with plasminogen, and the control group administered with vehicle PBS. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1, and administration began at the same time. The blank control group was fed with a normal maintenance diet. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner, both lasting for 10 consecutive days for model establishment. The blank control group received no treatment. The mice were sacrificed on Day 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Goat anti-mouse IgM (HRP) antibody (Abcam) was added to the sections dropwise, incubated for 1 hour at room temperature and washed with PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0245] IgM antibodies play an important role during the clearance of apoptotic and necrotic cells, and the local level of IgM antibodies in damaged tissues and organs is positively correlated with the degree of injury.sup.[29,30]. Therefore, detection of local level of IgM antibodies in tissues and organs can reflect the extent of injury of the tissues and organs.
[0246] The results showed that the renal IgM-positive staining of mice in the group administered with plasminogen (
Example 7
Plasminogen Reduces the Expression of Renal Fibrin in Mice with a Chronic Renal Injury
[0247] Twelve 8- to 9-week-old male PLG mice and six PLG.sup./ mice were taken. PLG mice were randomly divided into two groups, 6 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1, and administration began at the same time. Both model establishment and administration lasted for a period of 4 days. The blank control group was fed with a normal maintenance diet. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0 1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner. PLG.sup./ mice received no treatment. The mice were sacrificed on Day 5. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Rabbit anti-mouse fibrin antibody (Abcam) was added to the sections dropwise, incubated at 4 C. overnight, and washed with PBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0248] Fibrinogen is the precursor of fibrin, and in the presence of tissue injury, as a stress response to the body's injury, fibrinogen is hydrolyzed into fibrin and deposited at the injury site.sup.[31,32]. Therefore, the local fibrin level at the injury site can be used as a sign of the degree of injury.
[0249] The results showed that the renal fibrin-positive staining in the control group administered with vehicle PBS (
Example 8
Plasminogen Alleviates Renal Fibrosis in Diabetic Mice
[0250] Ten 24- to 25-week-old male db/db mice were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 31 consecutive days. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were sacrificed after administration of plasminogen for 31 days. The kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Rabbit polyclonal antibody (Abcam) against IV collagen was added to the sections dropwise, incubated at 4 C. overnight, and washed with TBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with TBS twice. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0251] Diabetic nephropathy is a chronic complication of diabetes mellitus, and glomerular sclerosis and renal interstitial fibrosis are typical pathological changes.sup.[34]. The experimental results of the present invention showed that the positive staining of IV collagen in the group administered with plasminogen (
Example 9
Plasminogen Alleviates Renal Fibrosis in Diabetic Mice
[0252] Ten 26-week-old male db/db mice were randomly divided into two groups, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 35 consecutive days. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were sacrificed on Day 36. The kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 m. The sections were dewaxed and rehydrated and then put into a potassium dichromate solution overnight. The sections were stained with iron hematocylin for 3 to 5 minutes, and flushed with running water. The sections were differentiated with 1% hydrochloric acid in alcohol, treated with ammonia water for 1 second, and rinsed with water. The sections were stained in ponceau acid fuchsin fluid for 8 minutes, and rinsed rapidly in water. The sections were treated with 1% phosphomolybdic acid aqueous solution for about 2 minutes, and counterstained with aniline blue solution for 6 minutes. The sections were rinsed with 1% glacial acetic acid for about 1 minute. The sections were sealed after dehydration with absolute ethanol, and permeabilization with xylene, and were observed under an optical microscope at 200.
[0253] Masson staining can reveal tissue fibrosis. The results showed that in the control group administered with vehicle PBS (
Example 10
Plasminogen Lowers Renal Fibrosis in Systemic Sclerosis Mice
[0254] Ten 12-week-old male C57 mice were weighed and then randomly divided into two groups, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. All mice were injected with bleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis.sup.[35], and plasminogen or PBS was administered on the same day and this day was recorded as Day 1. The administration lasted for 21 consecutive days. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were sacrificed on Day 22. The kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections was 3 m thick. The sections were dewaxed and rehydrated and washed with water once. After stained with 0.1% Sirius red in saturated picric acid for 30 min, the sections were flushed with running water for 2 min. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol, returned to blue with ammonia water, flushed with running water, dried and sealed with a neutral gum. The sections were observed under an optical microscope at 200.
[0255] The results showed that in the bleomycin-induced systemic sclerosis mouse model, the collagen fibrosis in the kidney in the control group administered with vehicle PBS (
Example 11
Plasminogen Promotes the Repair of Renal Function in Chronic Renal Injury Model Mice
[0256] Ten 8- to 9-week-old PLG.sup.+/+ mice and six PLG.sup./ mice were taken. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1. The model establishment lasted for a period of 10 days. On Day 11, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the concentration of urea nitrogen in the serum. The content of urea nitrogen was detected using a urea nitrogen detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C013-2) according to the method of the urea nitrogen detection kit.
[0257] The results showed that the concentration of urea nitrogen in sera in the PLG.sup.+/+ group was remarkably lower than that in the PLG.sup./ group, and the statistical difference was significant (
Example 12
Plasminogen Promotes the Repair of Renal Function in Chronic Renal Injury Model Mice
[0258] Twenty 8- to 9-week-old PLG.sup.+/+ mice and six PLG.sup./ mice were taken. PLG.sup.+/+ mice were randomly divided into two groups, 10 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. The modelling method for the chronic renal injury model was the same as described in Example 1. The day of model establishment was recorded as Day 1, and administration began at the same time, for an administration period of 4 days. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. PLG.sup./ mice received no treatment. On Day 5, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the concentration of creatinine in the serum. The serum creatinine concentration was detected using a creatinine detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C011-2) according to the method of the detection kit.
[0259] The results showed that the concentration of creatinine in sera of mice in each of the control group administered with vehicle PBS and the group administered with plasminogen was remarkably lower than that in the PLG.sup./ group, and the statistical difference was significant. In addition, the concentration of creatinine in sera in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS (
Example 13
Plasminogen Promotes the Repair of Renal Function in Acute Renal Injury Model Mice
[0260] Nine 7-week-old male C57 mice were randomly divided into two groups, 5 mice in the group administered with plasminogen, and 4 mice in the control group administered with vehicle PBS. All mice received a single intraperitoneal injection of a folate (sigma A7876) solution at 250 mg/kg body weight to induce an acute renal injury.sup.[36]. Folate was dissolved in 0.3 mol/L NaHCO.sub.3. The day of model establishment was recorded as Day 1, and plasminogen or vehicle PBS was administered at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 7 days. On Day 8, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the concentration of urea nitrogen in the serum. The content of urea nitrogen was detected using a urea nitrogen detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C013-2) according to the method of the urea nitrogen detection kit.
[0261] The results showed that the concentration of urea nitrogen in sera in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was nearly significant (P=0.06) (
Example 14
Plasminogen Lowers Fat Deposition in Kidney of 3% Cholesterol Hyperlipemia Model Mice
[0262] Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fat diet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia.sup.[37,38]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with the 3% cholesterol high-fat diet. Another five male C57 mice of the same week age were taken as the blank control group, and were fed with a normal maintenance diet during the experiment. 50 L of blood was taken from each mouse three days before administration, and the total cholesterol was detected. The model mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, i.e., the group administered with plasminogen, and the control group administered with vehicle PBS, 8 mice in each group. The first day of administration was recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 30 days. The mice were sacrificed on Day 31. The kidneys were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4 C. overnight, respectively, and embedded in OCT. The frozen sections were 8 m thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 400.
[0263] Oil red O staining can show lipid deposition and reflect the extent of lipid deposition.sup.[37]. The results showed that the fat deposition in kidney (indicated by arrow) of mice in the group administered with plasminogen (
Example 15
Plasminogen Ameliorates Renal Injuries in Folate-Induced Acute Renal Injury Model Mice
[0264] Fifteen 7-week-old male C57 mice were randomly divided into three groups, 3 mice in the blank control group, 7 mice in the group administered with plasminogen, and 5 mice in the control group administered with vehicle PBS. Mice in the group administered with plasminogen and the control group administered with vehicle PBS received a single intraperitoneal injection of a folate (sigma A7876) solution at 250 mg/kg body weight to induce the acute renal injury model.sup.[36]. Mice in the blank control group received a single intraperitoneal injection of NaHCO.sub.3 solution of corresponding volume. Folate was dissolved in 0.3 mol/L NaHCO.sub.3 solution. The day of model establishment was recorded as Day 1, and plasminogen or vehicle PBS was administered at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 7 days. The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The tissue sections were 3 m thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were dehydrated with alcohol gradient, permeabilized with xylene, sealed with a neutral gum, and observed under an optical microscope at 200.
[0265] The results showed that in the blank control group (
Example 16
Plasminogen Promotes the Expression of Bcl-2 in Kidneys of Folate-Induced Acute Renal Injury Model Mice
[0266] Twelve 7-week-old male C57 mice were randomly divided into two groups, 7 mice in the group administered with plasminogen, and 5 mice in the control group administered with vehicle PBS. All mice received a single intraperitoneal injection of a folate (sigma A7876) solution at 250 mg/kg body weight to induce an acute renal injury.sup.[36]. Folate was dissolved in 0.3 mol/L NaHCO.sub.3. The day of model establishment was recorded as Day 1, and plasminogen or vehicle PBS was administered at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 7 days. The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Rabbit anti-mouse Bcl-2 antibody (Abcam) was added to the sections dropwise, incubated at 4 C. overnight, and washed with 0.01 M PBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with 0.01 M PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with alcohol gradient, permeabilization with xylenehe, and sealing with a neutral gum, the sections were observed under an optical microscope at 200.
[0267] The Bcl-2 immunohistochemical results showed that the renal Bcl-2-positive staining in the group administered with plasminogen (
Example 17
Plasminogen Reduces Renal Injuries in Folate-Induced Acute Renal Injury Model Mice
[0268] Twelve 7-week-old male C57 mice were randomly divided into two groups, 7 mice in the group administered with plasminogen, and 5 mice in the control group administered with vehicle PBS. All mice received a single intraperitoneal injection of a folate (sigma A7876) solution at 250 mg/kg body weight to induce an acute renal injury.sup.[36]. Folate was dissolved in 0.3 mol/L NaHCO.sub.3 solution. The day of model establishment was recorded as Day 1, and plasminogen or vehicle PBS was administered at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 7 days. The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 m. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Goat anti-mouse IgM (HRP) antibody (Abcam) was added to the sections dropwise, incubated for 1 hour at room temperature and washed with PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200.
[0269] The results showed that the renal IgM-positive staining of mice in the group administered with plasminogen (
Example 18
Plasminogen Lowers Renal Fibrosis in 3% Cholesterol Hyperlipemia Model Mice
[0270] Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fat diet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia.sup.[37,38]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with the 3% cholesterol high-fat diet. Another five male C57 mice of the same week age were taken as the blank control group, and were fed with a normal maintenance diet during the experiment. 50 L of blood was taken from each mouse three days before administration, and the total cholesterol was detected. The model mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, i.e., the group administered with plasminogen, and the control group administered with vehicle PBS, 8 mice in each group. The first day of administration was recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were administered for 30 days. After the mice were administered on day 30, the mice were sacrificed on Day 31. The kidneys were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The sections was 3 m thick. The sections were dewaxed and rehydrated and washed with water once. After stained with 0.1% Sirius red in saturated picric acid for 30 min, the sections were flushed with running water for 2 min. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol, returned to blue with ammonia water, flushed with running water, dried and sealed with a neutral gum. The sections were observed under an optical microscope at 200.
[0271] The results showed that the collagen deposition in kidney (indicated by arrow) in the group administered with plasminogen (
Example 19
Plasminogen Reduces Renal Injuries in Ischemic Reperfusion-Induced Acute Renal Injury Model Mice
[0272] Nine 7- to 9-week-old male PLG.sup.+/+ mice were randomly divided into three groups, 3 mice in each of the sham operation group, the group administered with plasminogen, and the control group administered with vehicle PBS. All mice were anesthetized by intraperitoneal injection of pentobarbital sodium at 50 mg/kg body weight. Incisions were made in the abdomens of the mice in the group administered with plasminogen and the control group administered with vehicle PBS for the exposure of kidneys, bilateral arteries and veins were isolated and clamped by vascular clamps, then the kidney were moved back into the abdominal cavity, and the wound was closed for 45 min. After the time was up, the kidneys were exposed again, the vascular clamps were removed, the renal situations were observed, and the wounds were sutured after confirmation of reperfusion. In the sham operation group, an incisions was made in the abdomen for the exposure of the kidney only without ischemic treatment, and the wounds were sutured after the time was up.sup.[39]. After the operation was completed, each mouse received an intraperitoneal injection of 1 mL of normal saline at 37 C. The body temperature were kept at 36.5 C. to 38 C. during the operation. The day of model establishment was recorded as Day 1, and plasminogen or vehicle PBS was administered at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 7 days. Sham-operated mice received no injection treatment The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The tissue sections were 3 m thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were dehydrated with alcohol gradient, permeabilized with xylene, sealed with a neutral gum, and observed under an optical microscope at 200.
[0273] The results showed that in the sham operation group (
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