U.S. patent application number 09/381031 was filed with the patent office on 2001-11-29 for hgf for treating acute renal failure.
Invention is credited to KUDO, IKUE, NAGANO, TOMOKAZU.
Application Number | 20010047079 09/381031 |
Document ID | / |
Family ID | 13768753 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010047079 |
Kind Code |
A1 |
KUDO, IKUE ; et al. |
November 29, 2001 |
HGF FOR TREATING ACUTE RENAL FAILURE
Abstract
The present invention provides a pharmaceutical composition for
treating acute renal failure caused by rhabdomyolysis comprising an
effective amount of HGF. The present invention provides a method of
treating acute renal failure caused by rhabdomyolysis comprising
administering an effective amount of HGF to a patient in need
thereof.
Inventors: |
KUDO, IKUE; (OSAKA-SHI,
JP) ; NAGANO, TOMOKAZU; (OSAKA-SHI, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
13768753 |
Appl. No.: |
09/381031 |
Filed: |
February 24, 2000 |
PCT Filed: |
March 12, 1998 |
PCT NO: |
PCT/JP98/01081 |
Current U.S.
Class: |
530/350 |
Current CPC
Class: |
A61P 43/00 20180101;
A61K 38/1833 20130101; A61P 13/00 20180101; A61P 13/02 20180101;
A61P 13/12 20180101 |
Class at
Publication: |
530/350 ;
514/12 |
International
Class: |
C07K 014/00; A61K
038/00; C07K 001/00; C07K 017/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 15, 1997 |
JP |
9/82235 |
Claims
1. A pharmaceutical composition for treating acute renal failure
caused by rhabdomyolysis comprising a therapeutically effective
amount of HGF and a pharmaceutically acceptable carrier.
2. A pharmaceutical composition for treating myoglobinuria
comprising a therapeutically effective amount of HGF and a
pharmaceutically acceptable carrier.
3. A pharmaceutical composition for treating acute renal failure
caused by rhabdomyolysis caused by a myolytic substance released by
external injury, compression damage, crash syndrome, burn,
infection, drug poisoning, muscle metabolic disease, overuse of
muscle, hypophosphatemia, or snake venom comprising a
therapeutically effective amount of HGF.
4. A method of treating acute renal failure caused by
rhabdomyolysis comprising administering a therapeutically effective
amount of HGF to a patient in need thereof.
5. A method of treating myoglobinuria comprising administering a
therapeutically effective amount of HGF to a patient in need
thereof.
6. A method of treating acute renal failure caused by
rhabdomyolysis caused by a myolytic substance released by external
injury, compression damage, crash syndrome, burn, infection, drug
poisoning, muscle metabolic disease, overuse of muscle,
hypophosphatemia, or snake venom comprising administering a
therapeutically effective amount of HGF to a patient in need
thereof.
7. A packaged product, comprising: a container; HGF contained
within said container in an amount therapeutically effective for
treating acute renal disease caused by rhabdomyolysis or
myoglobinuria; and instructions associated with said container
which indicate that said HGF can be used for treating acute renal
disease caused by rhabdomyolysis or myoglobinuria.
Description
BACKGROUND OF THE INVENTION
[0001] Acute renal failure is defined as having symptoms of
azotemia, electrolyte imbalance, uremia and the like caused by
acute renal dysfunction. Acute renal failure is classified into
prerenal acute renal failure, renal acute renal failure and
postrenal acute renal failure caused by renal dysfunction. Renal
acute renal failure is classified into (1) vasculitis, glomerular
lesion, (2) acute interstitial nephritis, (3) tubule obstruction
and (4) acute renal failure in a narrow sense. Acute renal failure
in a narrow sense is caused by acute tubular necrosis. The acute
renal failure in a narrow sense results from (1) ischemia, (2)
nephrotoxic substance, or (3) myolytic substance (e.g. myoglobin)
and so on.
[0002] Ischemic acute renal failure is caused by bleeding from
surgery, shock, external injury, burn and the like. Experimental
animal model for ischemic acute renal failure is exemplified by
renal artery ligation. In the rat model, BUN (blood urea nitrogen)
and serum creatinine are increased, HGF (Hepatocyte Growth Factor)
mRNA expression is enhanced 6 to 12 hours after ischemia, and then
HGF bioactivity in rat kidney and plasma is activated (American
Journal of Physiology, 1993; 265; 61-69).
[0003] Acute renal failure is also caused by a nephrotoxic
substance such as anti-biotic agent, antitumor agent, contrast
medium. An experimental animal model of acute renal failure caused
by a nephrotoxic substance is made by administration of a compound
such as mercurous chloride, cisplatin, and contrast medium to rats.
Mercurous chloride administered rats show an increase of BUN and
creatinine, enhancement of HGF mRNA expression and activity of HGF
(Nephron 1996; 73; 735), as reported on ischemia model. It is
suggested that HGF be involved in restoring a patient from renal
failure.
[0004] It is reported that renal dysfunction in experimental animal
models caused by ischemia or a nephrotoxic substance such as
mercurous chloride and cisplatin is recoverable by HGF
administration (American Journal of Physiology, 1994; 266; 129-134,
Proc. Natl. Acad. Sci. 1994; 91; 4537-4361). In both models, the
increase of BUN and creatinine is recovered rapidly by HGF
administration. It is considered that HGF acts as a renotropic
factor to enhance proliferation of damaged tubular cells.
[0005] Myolytic substances (such as myoglobin, phosphate,
potassium, and uric acid) are released by external injury,
compression damage (e.g. crash syndrome), burn, infection, drug
poisoning (e.g. alcohol, barbitur derivatives), muscle metabolic
disease, overuse of muscle, hypophosphatemia, snake venom and the
like and show toxicity to tubular cells. As a result, necrosis of
tubular cells occurs, and necrotic cells and protein (hyaline)
casts bring tubular obstruction. It is also reported that myoglobin
reduces renal blood flow. The above mentioned mechanism is
considered to cause acute renal failure caused by rhabdomyolysis
(Common Disease Series 17: Zinfuzen (renal failure), 57-61,
Nankoudo, (1991)).
[0006] Clinical symptoms of rhabdomyolysis are: (1) increase in
muscle derived enzyme such as creatine phosphate kinase, lactate
dehydrogenase, aldolase, glutamic-oxaloacetic transaminase; (2)
enhanced uptake of .sup.99mTC-pyrophosphate into injured muscle;
(3) presence of myoglobin; (4) BUN/serum creatinine ratio of 10-20
or under; (5) hyperuricemia; and (6) hyperphosphatemia,
hypocalcemia and hyperpotassemia.
[0007] The methods of treatment for rhabdomyolisis include fluid
replacement therapy, administering diuretics, fascia release
therapy, plasmapheresis, antiplatelet therapy, but these methods of
treatment are not adequate for a severe or poor prognosis. There is
no adequate method of treating or preventing acute renal failure
caused by rhabdomyolysis or pharmaceutical composition for treating
or preventing acute renal failure caused by rhabdomyolysis.
[0008] There is also no adequate method of treating or preventing
hemolytic uremic syndrome (HUS) or a pharmaceutical composition for
treating or preventing HUS. Clinical characterization of HUS is
thrombocytopenia, microangiopathic hemolytic anemia, and symptoms
of acute renal failure. The cause of HUS are infection (e.g.
O-157), hereditary, administration of drug (e.g. cyclosporin),
pregnancy, organ graft, SLE, hypertension, different group blood
transfusion and the like. Hemolysis is caused by the above
conditions, and then hemoglobin and the like are released, and HUS
occurs as a result.
[0009] A glycerol-induced animal model is well known for use in
modeling acute renal failure. When glycerol is injected into the
muscle of animals, hyperosmolarity of glycerol causes hemolysis in
muscle cells and intramuscular vessel in the animal model.
Myoglobin, hemoglobin, potassium and other rhabdomyolytic
substances are released into the blood and damage renal cells.
Dehydration in the animal model further enhances the toxicity of
the rhabdomyolytic substances. As a result severe decrease of renal
blood flow and ischemia are caused, and severe glomerular
filtration causes oliguresia and anuria. The part of muscle where
glycerol is administered becomes a place to store bodily fluid, and
systemic circulation, blood flow rate, and cardiac output are
reduced (JINSIKKAN MODEL (a model for renal disease), JIN TO
TOUSEKI (kidney and dialysis) Vol. 31 (1991) 395-399). If the model
animal is deprived of water before the administration of glycerol,
the effect of myolytic substances is enhanced and rhabdomyolytic
model is induced.
BRIEF DESCRIPTION OF THE INVENTION
[0010] The present invention provides a pharmaceutical composition
for treating acute renal failure caused by rhabdomyolysis
comprising a therapeutically effective amount of HGF.
[0011] The present invention provides a method of treating acute
renal failure caused by rhabdomyolysis comprising administering a
therapeutically effective amount of HGF to a patient, particularly
a human patient, in need thereof.
[0012] The present invention further provides a pharmaceutical
composition for treating myoglobinuria comprising a therapeutically
effective amount of HGF.
[0013] The present invention further provides a method of treating
acute renal failure caused by rhabdomyolysis caused by a myolytic
substance released by external injury, compression damage (e.g.
crash syndrome), burn, infection, drug poisoning (e.g. alcohol,
barbitur derivative), muscle metabolic disease, overuse of muscle,
hypophosphatemia, snake venom and the like comprising administering
a therapeutically effective amount of HGF to a patient in need
thereof.
[0014] The present invention further provides a pharmaceutical
composition for treating acute renal failure caused by
rhabdomyolysis caused by a myolytic substance released by external
injury, compression damage (e.g. crash syndrome), burn, infection,
drug poisoning (e.g. alcohol, barbitur derivative), muscle
metabolic disease, overuse of muscle, hypophosphatemia, snake venom
and the like comprising a therapeutically effective amount of
HGF.
[0015] The present invention further provides a pharmaceutical
composition for treating hemolytic uremic syndrome comprising a
therapeutically effective amount of HGF and a pharmaceutically
acceptable carrier.
[0016] The present invention further provides a method of treating
hemolytic uremic syndrome comprising administering a
therapeutically effective amount of HGF to a patient in need
thereof.
[0017] The present invention further provides a pharmaceutical
composition for treating hemolytic uremic syndrome caused by
infection (O-157), heredity, administration of drug (cyclosporin),
pregnancy, organ graft, SLE, hypertension, different group blood
transfusion and the like comprising a therapeutically effective
amount of HGF and a pharmaceutically acceptable carrier.
[0018] The present invention further provides a method of treating
hemolytic uremic syndrome caused by infection (e.g. O-157),
heredity, administration of drug (e.g. cyclosporin), pregnancy,
organ graft, SLE, hypertension, different group blood transfusion
and the like comprising administering a therapeutically effective
amount of HGF to a patient in need thereof.
BRIEF DESCRIPTION OF THE DRAWING
[0019] FIG. 1 is a graph showing the survival rate of the control
group and HGF administered group.
[0020] FIG. 2 is a graph showing the biochemical values of serum,
urine, and renal function one, three and six days after the
administration of glycerol.
DETAILED DESCRIPTION OF THE INVENTION
[0021] Hepatocyte growth factor (HGF), which can be used in the
present invention, has been already sold or is obtained by the
methods described below.
[0022] The methods of preparing HGF are well known to a person of
ordinary skill in the art. For example, HGF may be prepared by a
process comprising the steps of extracting from an organ such as a
liver, spleen, lung, bone marrow, brain, kidney, placenta, blood
cells such as platelets, white blood cells, plasma, serum and the
like of a mammal such as a rat, bovine, horse, sheep and the like,
and purifying the extract (FEBS Letters, 224, 312, 1987, Proc.
Natl. Acad. Sci. USA, 86, 5844, 1989).
[0023] HGF may also be prepared by a process which comprises the
steps of culturing primary cells or a cell line which produce(s)
HGF, obtaining extract from the cultured product (supernatant
fluid, cultured cells, etc), and purifying HGF from the
extract.
[0024] HGF may be prepared by genetic engineering method comprising
the steps of inserting a gene encoding HGF into an appropriate
vector, transfecting a host cell with the vector containing the HGF
gene, and purifying HGF from the supernatant of the cultured
transfected cells (for example Nature, 342, 440, 1989; Japanese
patent application KOKAI 5-111383; Japanese patent application
KOKAI 3-255096; Biochem. Biophys. Res. Commun., 163, 967,
1989).
[0025] The host cell is not limited, and various host cells
conventionally used in genetic engineering methods can be used,
which are, for example, Escherichia coli, Bacillus subtilis, yeast,
mold, fungi, plant and animal cells and the like.
[0026] A more specific process of preparing HGF from a living
tissue comprises the steps of administering carbon tetrachloride to
a rat intraperitoneally to induce rat hepatitis, removing a liver
from said rat and homogenizing, and purifying the HGF by a
conventional method of protein purification such as gel column
chromatography (such as S-Sepharose, heparinsepharose and the
like), HPLC and the like.
[0027] HGF may be prepared by a genetic engineering process
comprising the steps of transforming an animal cell (such as
Chinese Hamster Ovary (CHO) cells, mouse C127 cells, monkey COS
cells, Sf (Spodoptera frugiperda) cells and the like) with a gene
encoding an amino acid sequence of HGF, and purifying HGF from the
supernatant fluid of said cells.
[0028] HGF includes human HGF and mammalian HGF, preferable HGF is
a human HGF, and more preferable HGF is a human recombinant HGF
(Japanese patent application KOKAI H5-111383 (1993)).
[0029] HGF prepared by the above processes includes any HGF that
has substantially the same as the full-length HGF, such as a
partial deletion derivative of HGF, an amino acid substitution
derivative of HGF, an amino acid sequence insertion derivative of
HGF, a derivative of HGF caused by binding one or more amino acids
to N-terminus or C-terminus of the HGF amino acid sequence, or a
sugar chain deleted or substituted HGF.
[0030] HGF may be formulated in various ways such as in liquid
preparations, solid preparations, capsule preparations, depot
preparations and the like. HGF may be formulated for parenteral
administration for injection without any carrier or with an
appropriate conventional carrier and for oral administration with
an appropriate conventional carrier. The formulation for parenteral
administration for injection may be prepared by conventional
methods known to a person of ordinary skill in the art, such as a
method comprising the steps of dissolving HGF in an appropriate
solvent such as sterilized water, buffered solution, isotonic
sodium chloride solution and the like, sterilizing by filtration
and filling said solution in a sterilized bottle. An amount of HGF
in the parenteral formulation is from about 0.0002 to about 0.2
(W/W %), and preferred amount is from about 0.001 to about 0.1 (W/W
%). The formulation may be prepared by a conventional formulation
technique. An amount of HGF may be varied depending on the
formulation, the disease to be treated, the symptoms of patient and
the like.
[0031] HGF may be formulated in rectal compositions such as
suppositories or retention enemas, e.g. containing conventional
suppository bases such as cocoa butter, water soluble bases
(glycerinated gelatin, macrogols, etc.) or other glycerides.
[0032] HGF may be administered in a form for inhalation. For
administration by inhalation HGF in conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or
nebulizers, with the use of suitable propellants such as carbon
dioxide or other suitable gasses.
[0033] HGF may be administered using a conventional drug delivery
system well known to a person skilled in the art. Examples of the
preparations for a drug delivery system are microspheres
(nanoparticle, microparticle, microcupsule, bead, liposome,
multiple emulsion, etc.) and the like.
[0034] Preferably, a stabilizer may be added to the formulation.
Examples of a stabilizer include albumin, globulin, gelatin,
mannitol, glucose, dextran, ethylene glycol and the like. The
formulation of the present invention may include a necessary
additive such as an excipient, a solubilizer, an antioxidizing
agent, a pain-alleviating agent, an isotonic agent and the like.
The liquid formulation may be stored in a frozen condition, or
after removal of water by a process such as freeze-drying. The
freeze-dried preparations are dissolved in pure water for injection
and the like before use.
[0035] Effective dosages and schedules for administering HGF may be
determined empirically, and the determination is within the skill
of a person of ordinary skill in the art. The administration route
of the preparation may vary depending on the form of the
preparation. For example, the parenteral preparation may be
administered intravenously, intraarterially, subcutaneously or
intramuscularly. The amount of administration may vary depending on
the symptom, age, weight, etc. of the patient. A dose can be
selected from the range of from 0.1 .mu.g to 10 mg/kg of body
weight. The preferred range is from 1 .mu.g to 400 .mu.g/kg. The
preparation of HGF may be administered once or several (2 or 3)
times per day.
[0036] HGF may also be formulated as depot preparations. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or intramuscular
injection. Thus, for example, HGF may be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil), ion exchange resins, or as sparingly soluble
derivatives, for example as a sparingly soluble salt.
[0037] Effective dosages and schedule for administering the depot
preparation may be determined empirically, and the determination is
within the skill of a person of ordinary skill in the art. The
administration route of the depot preparation may vary depending on
the form of the preparation.
[0038] The preferred mode of administration of the depot
preparations is once for at least one week, preferably once for at
least one month.
[0039] It is an object of this invention to provide a packaged
product, kit or article of manufacture for treating acute renal
failure caused by rhabdomyolysis or myoglobinuria, which contains
HGF. The packaged product may comprise a container which contains a
therapeutically effective amount of HGF for treating acute renal
disease caused by rhabdomyolysis or myoglobinuria, and instructions
associated with the container which indicates that HGF can be used
for treating acute renal disease caused by rhabdomyolysis or
myoglobinuria. For example, the HGF can be placed into a sterile
vial, which can then be placed into another container such as a
box. Instructions for administering the drug in accordance with the
present invention can be placed on a label on the vial and/or on
the box and/or can be inserted in the box as a package insert.
EXAMPLES
[0040] The following examples are for illustrative purposes only
and are not to be construed as limiting the invention.
Example 1
[0041] Materials
[0042] Animals
[0043] Male wistar rats(seven weeks old) were purchased from NIHON
SLC (Shizuoka, Japan), and were raised preliminarily for three
days. Male wistar rats (7.5 weeks old, weight: about 200 g) were
used in the following examples.
[0044] Method of Acute Renal Failure Model
[0045] A 50% glycerol solution in saline was prepared and was
intramuscularly administered to rats (10 ml/kg) that were deprived
from water for 15 hours before the administration under anesthesia
caused by diethylether. 8 hours after the administration the rat
were able to drink water freely.
[0046] Agent Administered
[0047] Recombinant human hepatocyte growth factor (hHGF) was
diluted to 125 .mu.g/ml with an HGF solution. A dosage was 2 ml/kg
(250 .mu.g/ml) was administered via caudate vein.
[0048] HGF solution was 10 mM sodium citrate aqueous solution which
includes 0.3M NaCl and 0.01% Tween 80.
[0049] Administration
[0050] Rats were divided into two groups, one was the control group
and the other was hHGF administered group. hHGF solution was
administered to one group 1 hour before the glycerol administration
and hHGF solution was also administered to the group 1, 3, 5, 8,
24, and 36 hours after the glycerol administration. 1 ml of saline
was administered to the control group 1 and 3 hours after the
glycerol administration. 1 ml of water was also administered to the
control group 5 and 8 hours after the glycerol administration.
[0051] Observation of Symptoms
[0052] The survival rate of the rats was observed for 14 days after
the administration of the glycerol.
[0053] Results
[0054] Rats were induced with severe acute renal failure by the
glycerol administration and the value of serum BUN and serum
creatinine two days after much were increased (the value of serum
creatinine: control group=5.1.+-.1.2 mg/dl, HGF administration
group=4.2.+-.0.8 mg/dl).
[0055] FIG. 1 shows the survival rate of rats. In the control group
death of rats was observed from 3 days after the glycerol
administration, and 9 of 13 rats were dead within 9 days.
Immediately after the death, the rat underwent postmortal
examination, and the appearance of renal was observed to be quite
different. Necrosis of the uriniferous tubule in renal cortex was
observed. In the HGF administration group death was not observed
during the experiment. The survival rate of 14 days after the
glycerol administration was analyzed by .chi.-square test, and the
survival rate of the HGF administration group was significantly
higher (P<0.01) than the control group.
Example 2
[0056] Material
[0057] Experimental Animals
[0058] Male wistar rats (seven weeks old) were purchased from NIHON
SLC (Shizuoka, Japan), and were raised preliminarily for three
days. Male wistar rats (7.5 weeks old, weight: about 200 g) were
used in the following examples.
[0059] Method of Acute Renal Failure Model
[0060] A 50% glycerol solution in saline was prepared and was
intramuscularly administered to the rats (10 ml/kg) that were
deprived from water for 15 hours before the administration under
anesthesia by diethylether. 8 hours after the administration the
rats were able to drink water freely.
[0061] Agent Administered
[0062] Recombinant human hepatocyte growth factor (hHGF) was
diluted to 125 .mu.g/ml with saline. A dosage was 2 ml/kg (250
.mu.g/ml) was administered via caudate vein.
[0063] Administration
[0064] Rats were divided into two groups, one was the control group
(n=10) and the other was hHGF administered group (n=10). hHGF
solution was administered to one group 1 hour before the glycerol
administration and 1, 3, 5, 8, 24, and 36 hours after the glycerol
administration. 1 ml of saline was administered to the control
group 1 and 3 hours after the glycerol administration and 1 ml of
water was administered to the control group 5 and 8 hours after the
glycerol administration.
[0065] Measurement of Biochemical Value of Serum and Urine
[0066] Serum was obtained from arteria caudalis under anesthesia by
diethylether 1, 3 and 6 days after the glycerol administration. The
amount of serum urea nitrogen (BUN) and serum creatinine (Cre) were
measured by ultramicro multipurpose biochemical automatic analyzer,
CHEM1. 12-hour urine was collected 1, 3 and 6 days after the
glycerol administration using a metabolic cage. The amount of the
urine was measured. The amount of creatinine in the urine was
measured by ultramicro multipurpose biochemical automatic analyzer,
CHEM1. From the analysis of the data above clearance of endogenous
creatinine was derived from the equation described below. 1
Clearance of endogenous creatinine = Urine creatinine value ( mg /
dl ) .times. 12 - hour urine collection ( ml ) Serum creatinine
value ( mg / dl ) .times. urine collection time ( 720 min )
[0067] Observation of Symptoms
[0068] The survival of the rats was observed for fifteen days after
the administration of glycerol.
[0069] Statistical Processing
[0070] The survival rate of 15 days after glycerol administration
was analyzed by .chi.-square test.
[0071] Serum biochemical values obtained 1 and 3 days after the
glycerol administration was analyzed by t-test or Welch-test.
[0072] Results
[0073] Survival Rate
[0074] In the control group, the survival rate of rats was observed
from 3 days after the glycerol administration, and 7 of 10 rats
died within 9 days. In the HGF administration group, 2 of 10 rats
died during the experiment. The survival rate of the HGF
administration group was significantly higher (p<0.05) than the
control group.
[0075] The Biochemical Value of Serum and Urine and Function of
Kidney
[0076] FIG. 2 shows the biochemical value obtained from serum,
urine and function of the kidney.
[0077] In the control group, beginning one day after the glycerol
administration, significant increase in values of serum BUN and
creatinine was observed, and the values got worse for 6 days.
Endogenous clearance of creatinine was kept at 0.1 or below and
symptoms of severe renal failure were observed.
[0078] On the contrary, in the HGF administered group, a decrease
of serum BUN was observed for three days after the glycerol
administration, which was accompanied by significant suppression
(p<0.05) of acute renal failure. At the same time endogenous
clearance of creatinine was also significantly improved
(p<0.05).
Example 3
[0079] Materials
[0080] Animals
[0081] Male wistar rats(seven weeks old) were purchased from NIHON
SLC (Shizuoka, Japan), and were raised preliminarily for three
days. Male wistar rats (7.5 weeks old, weight: about 200 g) were
used in the following examples.
[0082] Acute Renal Failure Model
[0083] A 50% glycerol solution in saline was prepared and was
intramuscularly administered to the rats (10 ml/kg) that were
deprived from water for 15 hours before the glycerol administration
under anesthesia by diethylether. The rats were able to drink water
freely after 8 hours from the glycerol administration.
[0084] Agent Administered
[0085] Recombinant human hepatocyte growth factor (hHGF) was
diluted to 125 .mu.g/ml with an HGF solution. A dosage of 2 ml/kg
(250 .mu.g/ml) was administered via caudate vein.
[0086] Administration
[0087] Rats were divided into two groups, one was the control group
(n=8) and the other was hHGF administered group (n=8). hHGF
solution was administered to one group 1 hour before the glycerol
administration and 1, 3, 5, 8, 24, and 36 hours after the glycerol
administration. 1 ml of saline was administered to the control
group 1 and 3 hours after the glycerol administration and 1 ml of
water was administered to the control group 5 and 8 hours after the
glycerol administration.
[0088] Analysis of Renal Tissue
[0089] Rats were killed 3 days after the glycerol administration,
and the kidney was extracted, fixed by formalin phosphate buffer
and embedded in paraffin. Renal microtome was made from
paraffin-embedded tissue and was PAS stained. A light manifest
image of renal cortex of microtome of each rat was taken, and the
degree of renal cortex tubular cell necrosis was analyzed. Analysis
was made by scoring as weak, moderate and severe. The difference
between the control group and HGF administered group was determined
by .chi.-test.
[0090] Result
[0091] Tissue Damage of Renal Tubular Cells
[0092] The scores of renal tissue damage are shown in Table 1. In
the HGF administered group, damage was suppressed significantly in
contrast to the control group.
1 TABLE 1 Severe Moderate Weak Control group 6 1 1 HGF
administrated group 0 4 4
* * * * *