Hisahide Takahashi

Background. The appearance of interstitial fibrosis in polycystic kidneys is emblematic of progressive disease. Matrix forming this scar tissue is derived from local renal cells in response to cystogenesis. We investigated the phenotype of collagen-producing cells in the cystic kidneys of DBA/2-pcy mice to better characterize the spectrum of interstitial cells associated with renal fibrogenesis. Methods. The extent of interstitial fibrosis and the number of fibroblasts in cystic kidneys were first quantitated over time using computer-assisted image analysis. Subsequently, antisera to four cell protein markers were studied by coexpression immunohistochemistry during progression of fibrosis using confocal microscopy. The antisera included fibroblast-specific protein 1 (FSP1) for fibroblast phenotype, alpha-smooth muscle actin (alpha-SMA) for contractile phenotype, vimentin (VIM) for mes enchymal phenotype, and heat shock protein 47 (HSP47) for interstitial collagen-producing phenotype. Results. Interstitial fibrosis in cystic kidneys gradually increased throughout the 30-week observation period of our study. With progression of cystogenesis, most of the tubules in pcy mice either dilated or disappeared with time. FSP1(+) fibroblasts were distributed sparsely throughout the renal interstitium of young pcy and wild-type mice. Their number increased in the widening fibrotic septa by 18 weeks of age and persisted through 30 weeks of the study interval. Some epithelia among remnant tubules trapped within fibrotic septa around adjacent cysts also acquired the phenotype of FSP1(+), HSP47(+) collagen-producing fibroblasts, suggesting a possible role for epithelial-mesenchymal transformation (EMT) in this process. Most FSP1(+) fibroblasts were alpha-SMA(-), but HSP47(+), suggesting they were producing collagen proteins for the extracellular matrix. alpha-SMA(+), FSP1(-), HSP47(+) or HSP47(-) cells were also observed, and the latter tended to distribute independently in a linear pattern, reminiscent of vasculature adjacent to forming cysts. VIM+ expression was not observed in alpha-SMA(+) cells. Conclusions. Many nonoverlapping as well as fewer overlapping populations of FSP1(+) and alpha-SMA(+) cells shared in the collagen expression associated with progressive fibrogenesis in pcy mice undergoing cystogenesis. Some FSP1(+) fibroblasts are likely derived from tubular epithelium undergoing EMT, while alpha SMA(+), VIM- cells probably represent vascular smooth muscle cells or pericytes surviving vessel attenuation during the chaos of fibrogenesis. Importantly, not all interstitial cells producing collagens are alpha SMA(+).

Epithelial proliferation, extracellular matrix remodeling, and interstitial inflammation are central elements in the pathogenesis of slowly progressive polycystic kidney disorders. Probucol, an antioxidant that lowers plasma cholesterol, has been shown to decrease smooth muscle cell proliferation and macrophage accumulation in blood vessels and to prevent restenosis after coronary angioplasty, We determined in 30-day-old male BDF1-pcy hybrid mice (derived from mating DBA/2FG-pcy and C57BL/6FG-pcy) the effect of probucol administered in the diet (1%) for 200 days on kidney weight relative to body weight (KW/BW), cyst expansion, renal interstitial fibrosis, and serum urea nitrogen (SUN) concentration. Animals were fed a moderately high-protein diet (HPD, 36%) to accentuate the development of renal cysts and to promote interstitial fibrosis. Probucol decreased serum cholesterol from 68 to 16 mg/dL but had no effect on food intake or body weight. Probucol decreased relative kidney size from 4.16% +/- 0.55% to 2.64% +/- 0.12% KW/BW (P < 0.01), SUN from 30.5 +/- 1.8 to 25.9 +/- 1.0 mg/dL (P < 0.05), cystic index from 2.45 +/- 0.11 to 1.36 +/- 0.10 (P < 0.01), and fibrosis index from 2.40 +/- 0.11 to 1.82 +/- 0.08 (P < 0.01). We conclude that probucol ameliorates the progressive deterioration in renal function and structure in pcy mice ingesting a relatively high level of dietary protein. (C) 2000 by the National Kidney Foundation, Inc.

A polycystic kidney rat model is being established from a Crj:CD (SD) rat strain. Unlike existing animal models of polycystic kidney disease, this mutant rat has a completely polycystic liver. Mating experiments revealed that the phenotype is controlled by an autosomal recessive gene. We propose that this gene be tentatively called the "rpc" gene.

A polycystic kidney rat model is being established from a Crj:CD (SD) rat strain. Unlike existing animal models of polycystic kidney disease, this mutant rat has a completely polycystic liver. Mating experiments revealed that the phenotype is controlled by an autosomal recessive gene. We propose that this gene be tentatively called the "rpc" gene.

The Han:SPRD strain is an SD-background strain known to be a model of polycystic kidney disease (PKD) expressed through an autosomal dominant gene (Cy). However, different genotypes of this strain cannot be identified in the neonatal period. First, to establish an accurate method of determining the genotypes (Cy/Cy, Cy/+, +/+) which cause different disease progressions, we used polymorphic markers on rat chromosome 5. PCR products of tissue DNA templated with D5Rat9 showed distinct patterns on electrophoresis indicating three genotypes. Second, to determine whether the same locus plays a major role in expressing PKD, we performed linkage analyses ina a [BN x (BN x Han:SPRD)F-1] backcross. Cy/Cy and Cy/+ also caused PKD in a BN background. In this backcross, we discovered that D5Rat11 is located closer to the Cy locus than DSMgh10, which is regarded as one of the closest loci. We conclude that D5Rat9 and D5Rat11 are useful markers for deter-mining the presence of the Cy allele, which is regarded as the gene responsible for PKD.

Background Recombinant human erythropoietin (rHuEPO) is primarily used for patients with anemia associated with endstage renal disease. We evaluated the efficacy of EPO gene therapy using adenovirus vector for chronic renal failure mice expressing severe renal anemia. Methods. Recombinant HuEPO gene transfer to mesothelial cells was performed in vitro and in vivo. Recombinant replication-deficient adenoviruses containing rHuEPO cDNA (AdCMVEPO), E. coli lacZ gene (AdCMVlacZ), or an nonexogenous gene (AdNull as control vector) driven by the cytomegalovirus promotor/enhancer were constructed. The oligosaccharides associated with the rHuEPO from AdCMVEPO-treated mesothelial cells were analyzed. For in vivo study, the DBA/2FG-pcy mouse, a model for human autosomal recessive polycystic kidney disease resulting in chronic renal failure with progressive anemia, was used. Results. The sialylated oligosaccharides associated with the rHuEPO produced in AdCMVEPO-treated mesothelial cells occupied 78 +/- 0.7% of the total oligosaccharide pool. A single intraperitoneal administration of AdCMVEPO induced rHuEPO synthesis in the peritoneal cells and a marked increase in erythrocyte production. The maximal increase in hematocrit (43 +/- 4%) was observed on day 28, and it remained elevated for 40 days. Conclusion. These results indicate that intraperitoneal administration of AdCMVEPO improves renal anemia in mice with chronic renal failure and that the mesothelial cell is an appropriate target cell for gene transfer.

多発性嚢胞腎症D2-pcyマウスに1.0%NH4Cl- 20%しょ糖液を8週齢から1週間摂取させると,飼料摂取量は増加したが,体重,摂水量,尿量は低下し,腎重量は対照レベルを下回る傾向を示した.腎の組織変化の程度ならびに尿素窒素の血中濃度は対照と同レベルであった.尿中アンモニア窒素は著しく増加し,尿pHは対照レベルより更に低下したが,滴定酸及び無機リンの尿中排泄に変化はみられずNH4Cl摂取の嚢胞形成に及ぼす有意な影響は認めなかった

The ultrastructure of the parathyroid gland (PTG) of the UM-X7.1 cardiomyopathic hamster (the UM-X7.1) was qualitatively and quantitatively compared with that of the normal hamster. In the UM-X7.1, the Golgi apparatus and rER were well developed compared with those of the control hamster. Morphometric analysis revealed that the mean values of the Golgi apparatus, rER and large vacuolar bodies were significantly greater in the UM-X7.1 than in the normal hamster and the mean value of lipid droplets was significantly less in the UM-X7.1 than in the normal hamster. Ultrastructurally, we consider that in the UM-X7.1, the synthesis and release of the parathyroid hormone may be activated by an excessive amount of circulating catecholamine, and the functional activity of the PTG may be higher than that in the normal hamster.

Oxidative stress is suggested as a significant causative factor for pathogenesis of neuronal degeneration on spinal cord of human ALS. We measured some enzymic activities implicating neuronal degeneration process, such as cytochrome c oxidase (CO), superoxide dismutase (SOD), and transglutaminase (TG) in spinal cord of an animal model of ALS, motor neuron degeneration (Mnd) mouse, a mutant that exhibits progressive degeneration of lower spinal neurons during developmental growth, and compared them with age-matched control C57BL/6 mice. CO activity in Mnd spinal cord decreased during early postnatal period, while SOD activity reduced in later stage. In Mnd tissue, TG activity in lumbar cord was increasing during early stage, but tended to decline in later period gradually. These biochemical alterations became evident prior to the appearance of clinical motor dysfunction which were observed in later stages of development in Mnd spinal cord.

We analyzed protein fractions extracted from the spinal cord of the motor neuron degeneration (Mnd) mouse, a mutant that exhibits progressive degeneration of lower spinal motor neurons, by one-and two-dimensional polyacrylamide gel electrophoresis (PAGE) after solubilization of the tissue with medium containing sodium dodecyl sulfate (SDS)-urea during growth of the animal, in comparison with those of age-matched controls (C57BL/6). Several protein spots were detected around a region of pi 5.6-6.0 and molecular mass of 35-50 kDa in Mnd spinal cord tissue on the two-dimensional PAGE separation profile with Coomassie brilliant blue staining, while only a few spots around the same region were found in the control spinal cord. These spots were all immunoreactive with an antibody against glial fibrillary acidic protein (GFAP), a cytoskeleton filamentous protein specific to astroglial cells. The protein spot with molecular mass of 50 kDa showed immunoreactivity with anti-GFAP antibody, had a blocked amino-terminus, and is assumed to be intact GFAP. Several protein spots with slightly smaller molecular masses of 35 to 48 kDa lacked the head domain of the GFAP molecule as a result of cleavage at the 29th and 56th residues from the amino terminus. In Mild spinal cord tissue, the densities of the immunoreactive GFAP bands with smaller molecular masses increased with development, and became dominant at the time of the appearance of behavioral paralytic gait around 6 to 7 months of age. These results suggest that the increased GFAPs devoid of head domains are related to the degenerative loss of motor neurons in the Mnd spinal cord. Histopathological and GFAP immunohistochemical examination of Mnd spinal cord preparation demonstrated progressive degenerative loss of motor neurons, and considerable increases in number of GFAP-stained astrocytes in the ventral horn at 7 to 9 months of age. These processes of degenerative loss of motor neurons and proliferation of reactive astrocytes with increased levels of fragmented GFAP in the Mnd spinal cord during development seem to be characteristic and preceded the deterioration of motor activities in this animal model of amyotrophic lateral sclerosis. (C) 1998 Elsevier Science B.V.

多発性嚢胞腎症マウスに細胞分裂抑制薬タキソールを週1回腹腔内注射して腎の機能,形態に及ぼす影響を検討した. 1)生後12日目の動物は初回投与150μgで半数が死亡したが,100μgでは体重の増加抑制,脱毛がみられたもののこの投与量に耐え,11週間の投与期間中,多尿及び尿pHの低下が抑制され,嚢胞の拡張遅延がみられた.投与終了後の血清尿素窒素,尿量及び尿中クレアチンに有意差は認められなかった. 2)生後21日目から150μgを9週間投与したが,DMSO投与群と殆ど相異がなく,タキソール投与群の検査では,血清尿素窒素の有意低下がみられたが,腎嚢胞の拡張進展に影響はみられなかった

1)細胞増殖抑制剤パクリタキセルの構造類似物質であるドセタキセルを,多発性嚢胞腎症(PKD)の進行が比較的緩やかであるpcyマウスに投与したが,PKDの進行を抑制することはできなかった. 2)パクリタキセルと抗ファイブロジェニック効果を有する抗炎症薬メチルプレドニゾロンをpcyマウスに併用投与したところ,高死亡率と低体重と共に,顕著なPKD進行の抑制が認められた. 3)嚢胞発生関連因子に基づいた投薬実験は,嚢胞発生機序の解析に寄与すると考える

The effects of feeding a soy protein isolate or genistein, an isoflavonoid present in soy protein, on cyst development were examined in the DBA/2FG-pcy (pcy) mouse, an accepted animal model of polycystic kidney disease, before the appearance of clinical symptoms, In study 1, 60-day-old male pcy mice were evenly divided into two groups and fed semipurified diets, based on casein or a soy protein isolate (15 g protein/100 g diet) for 90 days, In study 2, the animals were fed a casein-based diet (25 g casein/100 g diet) with or without genistein (0.05 g/100 g diet) for 60 days, In study 1, total kidney weight and kidney weight relative to body weight were significantly reduced (by 24% to 25%) in the animals fed the soy protein-based diet, relative to the casein-fed group, as was kidney water content (by 38%), In addition, mean cyst volume, as measured by morphometry, were lower (by 25%) in kidneys from the soy protein-fed group, No differences were found between these two groups with respect to final body weight, plasma creatinine, and protein content; however, plasma urea values were significantly lower in the soy protein-fed animals, Genistein supplementation of a casein-based diet in study 2 did not reduce the renal enlargement and cyst development associated with progression of polycystic kidney disease, These results suggest that soy protein is effective in retarding cyst development in the pcy mouse and that this beneficial effect may be unrelated to its genistein content. (C) 1998 by the National Kidney Foundation, Inc.

Evidence from in vitro studies indicates that increased proliferation of epithelial cells and secretion of fluid by these cells may be important factors in the progressive enlargement of renal cysts. The rate of cellular proliferation and fluid secretion by cyst epithelium in vitro can be strikingly accelerated by cyclic adenosine 3'5' monophosphate (cAMP) and agonists that lead to the production of this nucleotide. The extent to which renal cAMP content is increased in polycystic kidneys is unknown. In the current study, we determined the amount of this nucleotide in intact kidneys, cyst fluid, plasma, and urine in nonazotemic mice (DBA/2FG-pcy/pcy) with a slowly progressive form of inherited polycystic kidney disease (PKD). In 45 pcy/pcy mice studied 20, 45, or 70 days after birth, the total kidney cAMP content was 0.22 +/- 0.01, 0.46 +/- 0.02, and 0.90 +/- 0.05 pmol/mg tissue, respectively. By contrast, in 37 control DBA/2J mice the levels of cAMP at identical times remained relatively constant at 0.22 +/- 0.01, 0.21 +/- 0.01, and 0.29 +/- 0.01 pmol/mg tissue, respectively. In 70-day-old nonazotemic pcy/pcy mice with normal serum levels of parathyroid hormone, cAMP generated by the kidneys (nephrogenous cAMP) was 22.9 +/- 2.8 nmol/100 mL creatinine clearance, compared with 6.5 +/- 1.3 in normal animals of the same age (P < 0.001). The cyst fluids of 70-day-old pcy/pcy mice contained a lipid that increased transepithelial secretion of fluid by MDCK monolayers from a baseline of 0.012 +/- 0.002 to 0.136 +/- 0.008 mu L/cm(2)/hr (P < 0.05). This lipid also stimulated cellular proliferation by monolayers of cultured MDCK and LLC-PK1 cells 2.5- and 7.9-fold (P < .05), respectively, and stimulated cAMP accumulation by these cells 1.6- and 2.0-fold (P < .05), respectively. These studies illustrate that renal cAMP production and excretion increase in concert with the cystic enlargement of the kidneys in DBA/2FG-pcy/pcy mice and identify a lipid cAMP agonist in murine renal cystic kidney disease. (C) 1997 by the National Kidney Foundation, Inc.

The A/J inbred strain of mice spontaneously develops cleft lip and/or palate (CL/P), Ovarian transplantation and several test-matings with C57BL/6 strain mice (0% CL/P) were performed to investigate a relationship between mother and fetus in regard to this order. The fetuses obtained showed that regardless of whether the genetic background of the mother was A/J or F1, CL/P was produced only in cases where the fetuses were genetically pure A/J, CL/P frequency was 7.46% in A/J fetuses developed in A/J mothers; it was 5.88% in F1 mothers with transplanted A/J ovaries, Isolated cleft palate (CP) and cleft lip with cleft palate (CLP) were found in A/J mothers, However, the frequency of CP was lower in F1 mothers than in A/J mothers, These results suggest that the occurrence of CLP depends more on the genetic constitution of the fetus than on maternal environmental factors, and the occurrence of CP was influenced by the mother's condition.

The murine polycystic kidney disease gene, pcy, is an autosomal recessive trait located on chromosome 9. To determine the genetic locus of pcy, 222 intraspecific backcross mice were obtained by mating C57BL/6FG-pcy and Mus molossinus. Restriction fragment length polymorphism analysis of 70 of the 222 backcross progeny showed that pcy, dilute coat color (d), and cholecystokinin (Cck) were located in the order d-pcy-Cck from the centromere. Simple sequence repeat length polymorphism analysis of DNA of all 222 backcross mice was carried out using four markers which were located near the central regions of d and Cck. One and eight recombinations were detected between D9Mit24 and pcy and between D9Mit16 and pcy, respectively. However, no recombinant was observed among pcy, D9Mit14, and D9Mit148. These findings strongly suggest that D9Mit14 and D9Mit148 are located near the pcy gene and are good markers for chromosomal walking to this gene.

The DBA/2FG-pcy mouse has a form of slowly progressive kidney disease that appears similar in many respects to that seen in the autosomal dominant form of human polycystic kidney disease. This study was designed to assess how the expression of extracellular matrix component genes is regulated in a model of murine polycystic kidney disease and control DBA/2 mice at 8, 16, and 30 weeks of age. The mRNA levels encoding for collagen IV, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, fibronectin, and collagens I and III increased with the progression of cystic lesions in the kidney of DBA/2FG-pcy mice. At 30 weeks of age, mRNA levels for collagen IV, laminin B1 and B2, heparan sulfate proteoglycan, fibronectin, and collagens I and III were increased 8.1-fold, 7.0-fold, 7.0-fold, 9.8-fold, 7.0-fold, 5.5-fold, and 5.4-fold, respectively, compared to those of control DBA/2 mice. An immunofluorescence study revealed the irregular staining for collagen IV, laminin, heparan sulfate proteoglycan, and collagens I and III around the cysts. These data suggest that changes in the expression of basement membrane components and interstitial collagens are associated with the development of polycystic kidney disease.

Polycystic kidney disease in adult laboratory animals and humans is associated with enlarged kidneys and a progressive decline of renal function, resulting in death from uremia. Interstitial inflammation and fibrosis typically are observed in association with the development of renal insufficiency. To determine whether amelioration of interstitial inflammation and fibrosis may diminish cyst expansion/kidney enlargement and stabilize renal function, we administered methylprednisolone, an anti-inflammatory drug with antifibrogenic effects, to mice and rats with hereditary polycystic kidney disease. The experiment was repeated once for each species. Mice were studied both in America and in Japan. Weanling male and female mice (DBA/FG pcy/pcy [cystic] and +/+ [normal], n = 87 and 20, respectively) and rats (Han:SPRD Cy/+ and +/+, n = 70 and 33, respectively) were administered methylprednisolone (1 to 2 mg/kg/d) in the drinking water for 100 days (mice) or 42 days (rats). Control animals drank distilled water. In normal DBA +/+ mice, methylprednisolone had no effect on serum urea nitrogen (SUN) levels, kidney weight, or kidney/body weight. Untreated male and female mice developed cystic kidneys and azotemia to an equal extent. Methylprednisolone administered in America to mice with renal cystic disease decreased kidney weight, kidney/body weight, SUN levels, volume density of cysts, and severity of interstitial fibrosis. In Japan, methylprednisolone decreased kidney weight and SUN levels of animals with cystic disease, but the effect on kidney/body weight did not reach statistical significance. In contrast to mice, male rats developed more severe renal cystic changes and were more azotemic than female rats. Methylprednisolone administered to male rats with cystic disease decreased SUN levels, kidney weight, kidney/body weight, volume density of cysts, and severity of interstitial fibrosis. Methylprednisolone had no effect on kidney/body weight or SUN levels in female rats with renal cystic disease. In normal Han:SPRD (+/+) rats of both sexes, kidney and body weight were decreased by methylprednisolone, but kidney/body weight and SUN levels were unchanged. On the basis of this study, we conclude that methylprednisolone decreased the extent of renal enlargement, reduced renal interstitial fibrosis, and preserved kidney function in mice and rats with relatively severe forms of inherited polycystic kidney disease. (C) 1995 by the National Kidney Foundation, Inc.

The objective of these studies was to examine the effects of early dietary protein restriction on disease progression and survival in the DBA/2FG-pcy (pcy) mouse model of polycystic kidney disease. Male pcy mice of 70 days of age were fed either a normal protein (NP, 25% casein) or a low-protein (LP, 6% casein) diet for 105 days. At the end of the dietary treatment, kidney weight, kidney weight relative to body weight, and kidney water contents were almost 50% lower, and relative renal phospholipid and triglyceride contents were almost 50% higher, in mice fed the LP diet, indicating a marked reduction in the progression of cystic disease. Morphometric analyses also revealed a lower total and percent cyst area in kidneys derived from mice on the LP compared with the NP diet. There were no significant differences in final body weight, urine volume and osmolality, GFR, proteinuria, or plasma levels of protein and urea between these two groups. In a second study, it was found that all mice fed an NP diet from 70 days of age onward had died by 310 days of age, compared with a 42% survival rate in LP-fed mice at this age. Overall, the mean lifespan for pcy mice on the LP diet was 24% longer than that for those mice on the NP diet(310 +/- 20 versus 251 +/- 16 days; P < 0.01). These studies demonstrate that the introduction of a diet that has a reduced level of protein, yet one that provides adequate amounts of protein to prevent any signs of dietary deficiency, is effective in slowing down the progression of polycystic kidney disease in pcy mice. More important, in the long term, survival in pcy mice can be significantly improved by mice being fed the LP diet. Thus, early dietary protein reduction initiated in pcy mice before the manifestation of clinical symptoms of disease results in the attenuation of polycystic kidney disease progression.

Preneoplastic and neoplastic liver cell lesions, induced by EHEN (N-ethyl-N-hydroxyethylnitrosamine) in rats, were investigated to establish the numbers of simultaneously expressed altered enzyme phenotypes within the lesion cells. The lesions were divided into 5 classes on the basis of altered expression in one or more of the following 5 enzymes: glutathione S-transferase placental form, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, adenosine triphosphatase, and gamma-glutamyl transpeptidase. Class 1 lesions contained cells expressing one altered enzyme. Similarly, class 2, 3, 4 and 5 lesions had cells simultaneously expressing 2, 3, 4, and 5 enzyme alterations, respectively. Four histopathological categories of lesions, ACF (altered cell foci) (274 lesions), HN (hyperplastic nodules) (47 lesions), HCC (hepatocellular carcinomas) (99 lesions) and THC (transplanted hepatocellular carcinomas) (5 lesions) were studied. Proliferation potential was assessed in terms of 5-bromo-2'-deoxyuridine (BrdU) incorporation. The distribution profiles of classes 1 to 5 showed a clear reciprocal change from low class (I to 2 enzymes) predominance in ACF to high class (4 to 5 enzymes) predominance in HN. Increase of BrdU labeling indices was clearly correlated with progression from HN to HCC. Only a small population of class 5 ACF showed a high BrdU labeling index, indicating particular potential for further development. Thus, the stages of EHEN-induced neoplasia were found to be characterized by gradual increase in the number of altered enzyme phenotypes, with acquisition of proliferative potential being associated with further progression towards malignant conversion.