Daisuke Yoshihara

PPAR-γ agonist ameliorates kidney and liver disease in an orthologous rat model of human autosomal recessive polycystic kidney disease

In autosomal recessive polycystic kidney disease (ARPKD), progressive enlargement of fluid-filled cysts is due to aberrant proliferation of tubule epithelial cells and transepithelial fluid secretion leading to extensive nephron loss and interstitial fibrosis. Congenital hepatic fibrosis associated with biliary cysts/dilatations is the most common extrarenal manifestation in ARPKD and can lead to massive liver enlargement. Peroxisome proliferator-activated receptor γ (PPAR-γ), a member of the ligand-dependent nuclear receptor superfamily, is expressed in a variety of tissues, including the kidneys and liver, and plays important roles in cell proliferation, fibrosis, and inflammation. In the current study, we determined that pioglitazone (PIO), a PPAR-γ agonist, decreases polycystic kidney and liver disease progression in the polycystic kidney rat, an orthologous model of human ARPKD. Daily treatment with 10 mg/kg PIO for 16 wk decreased kidney weight (% of body weight), renal cystic area, serum urea nitrogen, and the number of Ki67-, pERK1/2-, and pS6-positive cells in the kidney. There was also a decrease in liver weight (% of body weight), liver cystic area, fibrotic index, and the number of Ki67-, pERK1/2-, pERK5-, and TGF-β-positive cells in the liver. Taken together, these data suggest that PIO inhibits the progression of polycystic kidney and liver disease in a model of human ARPKD by inhibiting cell proliferation and fibrosis. These findings suggest that PPAR-γ agonists may have therapeutic value in the treatment of the renal and hepatic manifestations of ARPKD.

Metabolomic profiling of the autosomal dominant polycystic kidney disease rat model

BackgroundAutosomal dominant polycystic kidney disease (ADPKD) is an inherited systemic disease characterized by renal cyst expansion, resulting in renal failure. With the progression of renal damage, the accumulation of uremic compounds is recently reported to subsequently cause further renal damage and hypertension. Finding uremic toxins and sensitive markers for detecting the early stage of ADPKD is necessary to clarify its pathophysiological process and to prevent its progression. The aim of this study was to analyze the profile of uremic retention solutes of ADPKD by capillary electrophoresis–mass spectrometry (CE-MS) using the Han:SPRD rat model.MethodsTwo hundred and ninety-seven cations and 190 anions were comprehensively analyzed by CE-MS in Han:SPRD rats and control rats.ResultsWe found 21 cations and 19 anions that accumulated significantly in the heterozygous (Cy/+) ADPKD rat model compared with control rats. Among the compounds, increases in 5-methyl-2′-deoxycytidine, glucosamine, ectoine, allantoate, α-hydroxybenzoate, phenaceturate and 3-phenylpropionate and decreases in 2-deoxycytidine, decanoate and 10-hydroxydecanoate were newly identified in the ADPKD Cy/+ rats.ConclusionWe identified uremic retention solutes in ADPKD Cy/+ rats. Compounds related to ADPKD could be useful markers for detecting the early stage of ADPKD.

Polycystic kidney disease in Han:SPRD Cy rats is associated with elevated expression and mislocalization of SamCystin

Polycystic kidney disease (PKD) in Han:SPRD Cy rats is caused by a missense mutation in Anks6 (also called Pkdr1), leading to an R823W substitution in SamCystin, a protein that contains ankyrin repeats and a sterile alpha motif (SAM). The cellular function of SamCystin and the role of the Cy (R823W) mutation in cyst formation are unknown. In normal SPRD rats, SamCystin was found to be expressed in proximal tubules and glomeruli; protein expression was highest at 7 days of age and declined by ∼50-60% at 45-84 days of age. In Cy/+ and Cy/Cy kidneys, expression of SamCystin was lower than in +/+ kidneys at 3 and 7 days but became elevated at 21 days. Immunohistochemical analysis revealed that SamCystin was distributed on the brush border of proximal tubules in normal rat kidneys. In Cy/+ kidneys, there were robust SamCystin staining in cyst-lining epithelial cells and loss of apical localization, and increased number of PCNA-positive cells in cyst-lining epithelia. Verapamil, an L-type Ca(2+) channel blocker, accelerated PKD progression in this model and caused a further increase in the expression and abnormal distribution of SamCystin. We conclude that aberrant expression and mislocalization of R823W SamCystin lead to increased cell proliferation and renal cyst formation.

Telmisartan Ameliorates Fibrocystic Liver Disease in an Orthologous Rat Model of Human Autosomal Recessive Polycystic Kidney Disease

Human autosomal recessive polycystic kidney disease (ARPKD) produces kidneys which are massively enlarged due to multiple cysts, hypertension, and congenital hepatic fibrosis characterized by dilated bile ducts and portal hypertension. The PCK rat is an orthologous model of human ARPKD with numerous fluid-filled cysts caused by stimulated cellular proliferation in the renal tubules and hepatic bile duct epithelia, with interstitial fibrosis developed in the liver. We previously reported that a peroxisome proliferator activated receptor (PPAR)-γ full agonist ameliorated kidney and liver disease in PCK rats. Telmisartan is an angiotensin receptor blocker (ARB) used widely as an antihypertensive drug and shows partial PPAR-γ agonist activity. It also has nephroprotective activity in diabetes and renal injury and prevents the effects of drug-induced hepatotoxicity and hepatic fibrosis. In the present study, we determined whether telmisartan ameliorates progression of polycystic kidney and fibrocystic liver disease in PCK rats. Five male and 5 female PCK and normal control (+/+) rats were orally administered 3 mg/kg telmisartan or vehicle every day from 4 to 20 weeks of age. Treatment with telmisartan decreased blood pressure in both PCK and +/+ rats. Blood levels of aspartate amino transferase, alanine amino transferase and urea nitrogen were unaffected by telmisartan treatment. There was no effect on kidney disease progression, but liver weight relative to body weight, liver cystic area, hepatic fibrosis index, expression levels of Ki67 and TGF-β, and the number of Ki67- and TGF-β-positive interstitial cells in the liver were significantly decreased in telmisartan-treated PCK rats. Therefore, telmisartan ameliorates congenital hepatic fibrosis in ARPKD, possibly through the inhibition of signaling cascades responsible for cellular proliferation and interstitial fibrosis in PCK rats. The present results support the potential therapeutic use of ARBs for the treatment of fibrocystic liver disease in ARPKD patients.

Immortalized hepatocytes using human artificial chromosome.

The shortage of organ donors has impeded the development of human hepatocyte transplantation. Immortalized hepatocytes could provide an unlimited supply of transplantable cells. To determine whether immortalized hepatocytes could provide global metabolic support in end-stage liver disease, rat hepatocyte clones were developed by transduction with the gene encoding the Simian virus 40 T antigen (SVT) using the human artificial minichromosome (HAC). The SVLT sequence was excised by FRT recombination. Following HAC infusion, the transduced hepatocytes express SVT, blasticidine resistance (BS), and the PGK promoter TK gene. Forty-six cell clones were obtained and at least partially characterized, as previously described, for albumin, α-1-antitrypsin, glucose-6-phosphatase (G6Pase), dipeptidylpeptidase 4 (Dpp4), γ-glutamyltransferase 1 (Ggt), SVT, and β-actin expression using RT-PCR. Clones were also assessed for albumin secretion into the culture medium using ELISA. All of the cell line secreted approximately 10 mg/dl of albumin, which is equivalent to the amount secreted by primary hepatocytes. In further experiments, this cell line will be used for transplantable cells or artificial organ using HAC. These results represent an important step toward the development of immortalized hepatocytes.

Mitochondrial Abnormality Facilitates Cyst Formation in Autosomal Dominant Polycystic Kidney Disease

ABSTRACT Autosomal dominant polycystic kidney disease (ADPKD) constitutes the most inherited kidney disease. Mutations in the PKD1 and PKD2 genes, encoding the polycystin 1 and polycystin 2 Ca2+ ion channels, respectively, result in tubular epithelial cell-derived renal cysts. Recent clinical studies demonstrate oxidative stress to be present early in ADPKD. Mitochondria comprise the primary reactive oxygen species source and also their main effector target; however, the pathophysiological role of mitochondria in ADPKD remains uncharacterized. To clarify this function, we examined the mitochondria of cyst-lining cells in ADPKD model mice (Ksp-Cre PKD1flox/flox) and rats (Han:SPRD Cy/+), demonstrating obvious tubular cell morphological abnormalities. Notably, the mitochondrial DNA copy number and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) expression were decreased in ADPKD model animal kidneys, with PGC-1α expression inversely correlated with oxidative stress levels. Consistent with these findings, human ADPKD cyst-derived cells with heterozygous and homozygous PKD1 mutation exhibited morphological and functional abnormalities, including increased mitochondrial superoxide. Furthermore, PGC-1α expression was suppressed by decreased intracellular Ca2+ levels via calcineurin, p38 mitogen-activated protein kinase (MAPK), and nitric oxide synthase deactivation. Moreover, the mitochondrion-specific antioxidant MitoQuinone (MitoQ) reduced intracellular superoxide and inhibited cyst epithelial cell proliferation through extracellular signal-related kinase/MAPK inactivation. Collectively, these results indicate that mitochondrial abnormalities facilitate cyst formation in ADPKD.

Intrasplenic hepatocyte transplantation prolonged the survival in Nagase analbuminemic rats with liver failure induced by common bile duct ligation.

It has already been established that hepatocyte transplantation (HTx) in animal models, such as both chemically and surgically induced acute liver failure, liver-based metabolic disease, and cirrhosis, resulted in significant improvement of liver function and survival. However, the efficacy of hepatocyte transplantation in secondary cholestatic liver disease is not well known. In this study, we transplanted hepatocytes into the spleen of Nagase analbuminemic rats (NARs) with common bile duct ligation (CBDL) to evaluate the function of transplanted hepatocytes by both of serum albumin levels and total bilirubin levels. CBDL was carried out on NARs to induce liver failure. Lewis rat hepatocytes were transplanted in NARs 7 days after CBDL. Animals, in groups of four, underwent the following interventions: group 1—intrasplenic transplantation of 30 × 106 primary Lewis rat hepatocytes in NARs with CBDL (n = 4), group 2—intrasplenic injection of 0.5 ml DMEM in NARs with CBDL (n = 4); group 3—CBDL only (n = 4); group 4—intrasplenic transplantation of 30 × 106 primary Lewis rat hepatocytes in NARs (n = 4). Both bilirubin levels and albumin levels in NARs with CBDL were significantly improved post-HTx. Animals receiving hepatocyte transplantation survived longer than animals in nontransplant control groups. This study indicates that hepatocytes can be transplanted to temporarily provide life-supporting liver-specific metabolic function and prolong the survival in recipient rats with liver failure induced by CBDL.

Cyclooxygenase product inhibition with acetylsalicylic acid slows disease progression in the Han:SPRD-Cy rat model of polycystic kidney disease.

Renal cyclooxygenase (COX) derived eicosanoids are elevated and lipoxygenase (LOX) products are reduced in the Han:SPRD-Cy rat model of polycystic kidney disease (PKD). Selective COX2 inhibition reduces kidney disease progression, but COX1 levels also are elevated in this model. Since the effect of reducing the products of both COX isoforms and the role of LOX products is not known, weanling normal and diseased Han:SPRD-cy littermates were given either low dose acetylsalicylic acid (ASA), nordihydroguaiaretic (NDGA) or no treatment for eight weeks. Renal eicosanoids were altered in the diseased compared to normal cortex, with COX products being higher and LOX products being lower. ASA reduced COX products, cyst growth and kidney water content, while NDGA reduced LOX products without altering disease progression or kidney function. Hence, a human equivalent ASA dose equal to less than one regular strength aspirin per day slowed disease progression, while further reduction of LOX products did not worsen disease progression.

Global Gene Expression Profiling in PPAR-γ Agonist-Treated Kidneys in an Orthologous Rat Model of Human Autosomal Recessive Polycystic Kidney Disease.

Kidneys are enlarged by aberrant proliferation of tubule epithelial cells leading to the formation of numerous cysts, nephron loss, and interstitial fibrosis in polycystic kidney disease (PKD). Pioglitazone (PIO), a PPAR-γ agonist, decreased cell proliferation, interstitial fibrosis, and inflammation, and ameliorated PKD progression in PCK rats (Am. J. Physiol.-Renal, 2011). To explore genetic mechanisms involved, changes in global gene expression were analyzed. By Gene Set Enrichment Analysis of 30655 genes, 13 of the top 20 downregulated gene ontology biological process gene sets and six of the top 20 curated gene set canonical pathways identified to be downregulated by PIOtreatment were related to cell cycle and proliferation, including EGF, PDGF and JNK pathways. Their relevant pathways were identified using the Kyoto Encyclopedia of Gene and Genomes database. Stearoyl-coenzyme A desaturase 1 is a key enzyme in fatty acid metabolism found in the top 5 genes downregulated by PIO treatment. Immunohistochemical analysis revealed that the gene product of this enzyme was highly expressed in PCK kidneys and decreased by PIO. These data show that PIO alters the expression of genes involved in cell cycle progression, cell proliferation, and fatty acid metabolism.

Structure and function of the pancreas in the polycystic kidney rat.

Objectives Mutation in the Pkhd1 gene that encodes a ciliary protein, fibrocystin, causes multiple cysts in the kidneys and liver in the polycystic kidney (PCK) rat, a model for human autosomal recessive PCK disease. To clarify the role of primary cilia in the pancreatic duct, we examined the structure and function of the exocrine pancreas of PCK rats. Methods Pancreatic juice and bile were collected from anesthetized rats. Pancreatic ductal structure was analyzed by microdissection and immunohistchemistry. Results Histologically pancreatic acini were apparently normal, and no cysts were detected in the pancreas. Larger pancreatic ducts were irregularly dilated with enhanced expression of AQP1 in epithelial cells. The pancreatic duct of PCK rats exhibited significantly (P < 0.05) higher distensibility than that of wild-type (WT) rat at a physiological luminal pressure (3 cm H2O). Pancreatic fluid secretion stimulated with a physiological dose of secretin (0.03 nmol/kg per hour) in PCK rats was significantly smaller than that in WT, but the differences were not significant at higher doses. The amylase responses to carbamylcholine were not different between PCK and WT rats. Conclusions These findings suggest that fibrocystin/primary cilia–dependent mechanisms may play a role in the regulation of pancreatic ductal structure and fluid secretion.