Identification of a new target molecule for a cascade therapy of polycystic kidney

Autosomal dominant polycystic kidney disease is a systemic disorder that primary affects the kidney which is characterized by the formation of fluid-filled cysts in both kidneys that leads to progressive renal failure. Mutated genes, polycystin-1 and polycystin-2, are identified, and evidence has emerged that polycystins are ion channels or regulators of ion channels. In spite of extensive characterization of polycystins, how polycystin channel signaling may be involved in cyst formation in ADPKD is still unclear. We found a mutant mouse which exhibits polycystic kidney and bone deformity in the course of making a transgenic mouse carrying the Drosophila sex-lethal gene. We identified a mutated gene Makorin1 by positional cloning. Makorin1 carries a typical RING-finger motif, suggesting that Makorin1 belongs to ubiquitinase E3 family. Makorin1 would open a new avenue to understand pathogenesis of polycystic kidney, and become a new therapeutic target of polycystic kidney.     

The SGP-2 gene is developmentally regulated in the mouse kidney and abnormally expressed in collecting duct cysts in polycystic kidney disease.

Sulfated glycoprotein-2 (SGP-2) is a secreted, dimeric, glycosylated protein synthesized by a number of different epithelial cell types. Although its function is not yet understood, SGP-2 has been hypothesized to be involved in such diverse processes as the promotion of cell-cell interactions, spermatogenesis, modulation of the complement system, and programmed cell death. We have now found that the SGP-2 gene is developmentally regulated in the mouse kidney. SGP-2 gene expression is first detected in the condensing nephrogenic mesenchyme and is subsequently down-regulated during the maturation of the glomerular epithelia, proximal tubules, and collecting ducts. SGP-2 continues to be expressed in the mature kidney in distal tubules and in the urothelial lining of the calyx and papilla. We have also examined the expression of the SGP-2 gene in polycystic kidneys of the C57BL/6J-cpk mouse, a model of autosomal recessive polycystic kidney disease in which there is development of epithelial-lined cysts arising primarily from the collecting duct system. Abnormally high levels of SGP-2 mRNA were found in the cyst wall epithelium of polycystic kidneys. The expression of the SGP-2 gene in normal development suggests that it plays a role in differentiating epithelial structures; and the abnormally high levels of SGP-2 gene expression in polycystic kidneys suggests that the cells lining cysts are not fully differentiated. It is possible, therefore, that polycystic kidney disease is caused by a defective developmental process in which there is a delay in terminal differentiation.     

Strain difference in expression of the adult-type polycystic kidney disease gene, pcy, in the mouse

DBA/2FG-pcy and C57BL/6FG-pcy congenic strains were established by transferring the polycystic kidney disease gene, pcy, to DBA/2 and C57BL/6 mice. We carried out pathological and hematological examinations of these strains at 4, 8, 16 and 30 weeks of age. In DBA/2FG-pcy mice more than 8 weeks of age, macroscopic renal cysts were observed on the surface of both kidneys. Their kidneys weight was significantly greater than in DBA/2 mice at all ages examined. Microscopic renal cysts were evenly distributed at 4, 8 and 16 weeks of age. At 30 weeks of age, the kidneys were filled with numerous polycysts. In C57BL/6FG-pcy mice, no macroscopic renal cysts were found until the animals were 30 weeks old, and the weight of their kidneys was greater than in B6 mice of the same age. From 8 weeks of age on, a limited number of microscopic renal cysts was observed, and many renal cysts were found adjacent to the enlarged Bowman's capsules. With age, the red blood cell count and hematocrit level decreased while the platelet count increased in both strains, with greater changes occurring in DBA/2FG-pcy mice than in C57 BL/6FG-pcy mice. These findings demonstrate that polycystic kidney disease exhibits strain differences in animals with a DBA/2 and C57BL/6 background. Our results suggest that phenotypic expression of the pcy gene in the mouse depends on genetic background, and that variations in the severity of human polycystic kidney disease may be explained, at least in part, by individual differences in genetic background.     

Characterization of a novel polycystic kidney rat model with accompanying polycystic liver.

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.     

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Phenotypical and structural characterization of the Arabidopsis mutant involved in shoot apical meristem

An Arabidopsis mutant induced by T-DNA insertion was studied with respect to its phenotype, micro-structure of shoot apical meristem (SAM) and histo-chemical localization of the GUS gene in comparison with the wild type. Phenotypical observation found that the mutant exhibited a dwarf phenotype with smaller organs (such as smaller leaves, shorter petioles), and slower development and flowering time compared to the wild type. Optical microscopic analysis of the mutant showed that it had a smaller and more flattened SAM, with reduced cell layers and a shortened distance between two leaf primordia compared with the wild type. In addi-tion, analysis of the histo-chemical localization of the GUS gene revealed that it was specifically expressed in the SAM and the vascular tissue of the mutant, which suggests that the gene trapped by T-DNA may function in the SAM, and T-DNA insertion could influence the functional activity of the related gene in the mutant, lead-ing to alterations in the SAM and a series of phenotypes in the mutant.     

Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli

Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome. The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine. Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test. The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient. These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients. The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes. One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient. These data allow to conclude that ilv7434 mutation is located within the ilvA gene.     

The WD repeat-containing protein IFTA-1 is required for retrograde intraflagellar transport

The assembly and maintenance of cilia require intraflagellar transport (IFT), a microtubule-dependent bidirectional motility of multisubunit protein complexes along ciliary axonemes. Defects in IFT and the functions of motile or sensory cilia are associated with numerous human ailments, including polycystic kidney disease and Bardet-Biedl syndrome. Here, we identify a novel Caenorhabditis elegans IFT gene, IFT-associated gene 1 (ifta-1), which encodes a WD repeat-containing protein with strong homology to a mammalian protein of unknown function. Both the C. elegans and human IFTA-1 proteins localize to the base of cilia, and in C. elegans, IFTA-1 can be observed to undergo IFT. IFTA-1 is required for the function and assembly of cilia, because a C. elegans ifta-1 mutant displays chemosensory abnormalities and shortened cilia with prominent ciliary accumulations of core IFT machinery components that are indicative of retrograde transport defects. Analyses of C. elegans IFTA-1 localization/motility along bbs mutant cilia, where anterograde IFT assemblies are destabilized, and in a che-11 IFT gene mutant, demonstrate that IFTA-1 is closely associated with the IFT particle A subcomplex, which is implicated in retrograde IFT. Together, our data indicate that IFTA-1 is a novel IFT protein that is required for retrograde transport along ciliary axonemes.     

Glucocorticoid teratogenesis in the developing nephron

Although the induction of polycystic kidney disease by neonatal glucocorticoid treatment has been extensively documented, there are no data on induction of polycystic kidney disease with fetal exposure to glucocorticoids. We injected groups of pregnant Swiss Webster albino mice subcutaneously with 250 mg/kg of hydrocortisone acetate on individual days from days 1 to 19 of gestation. A control group received an equal volume of saline. Histologic analysis of 1,522 kidneys from the offspring of these animals revealed no evidence of polycystic kidneys in the control group or in offspring of animals injected before day 11 of pregnancy. A bimodal distribution of cystic kidney disease was noted in the remaining animals, with highest prevalence after injection on day 12 (50.8%) and day 17 (34.3%). We conclude that 250 mg/kg of glucocorticoids may induce polycystic kidney disease in utero, but possibly only during critical periods of metanephric development.     

拟南芥茎顶端分生组织相关突变体的表型与结构分析

以拟南芥野生型(C24)和T-DNA插入诱发的突变体(155系)为材料,通过表型分析、组织切片、GUS基因表达的组织化学定位等研究方法对155系的形态结构和生长发育进行了较为细致的观察分析,结果发现:(1)T-DNA插入诱发的155系突变体植株矮化,叶片等器官体积减小,营养生长阶段延长,发育较C24缓慢;(2)同一时期155系的茎顶端分生组织面积较C24减小,顶端平坦,细胞层数减少,两侧叶原基基部之间的距离缩短,呈现出发育迟缓、从茎顶端分生组织向花分生组织转变延迟等特征;(3)GUS基因特异性地在155系茎顶端分生组织和维管组织中表达.结果表明,T-DNA诱捕基因可能在茎顶端分生组织中发挥作用,由于T-DNA的插入使该基因的功能受到了影响,进而影响了155系中茎顶端分生组织的发育模式,产生了155系的一系列表型改变.     

C-fos expression is hypersensitive to serum-stimulation in cultured cystic kidney cells from the C57BL/6J-cpk mouse.

Cystic kidneys of the C57BL/6J-cpk murine model of polycystic kidney disease show a marked overexpression of the proto-oncogenes c-fos, c-myc, and c-Ki-ras, consistent with an increased rate of cell proliferation and an altered state of differentiation. To determine if cystic cells have increased responsiveness to stimulation with mitogenic agents, quiescent primary cultures from normal and cystic cpk kidneys were treated with fetal bovine serum (FBS), 8-bromo-cAMP (cAMP), or epidermal growth factor (EGF). The level of c-fos induction following stimulation by FBS was found to be dramatically higher in cystic cells than in normal cells; whereas induction by cAMP or EGF was essentially the same in both cell types and much less than that seen in FBS-stimulated cells. To determine if this serum hypersensitivity reflects an increased proliferative state in vivo, c-fos induction was examined in cultures derived from normal kidneys stimulated to regenerate by folic acid-induced acute renal injury. As with cystic kidneys, the folic acid-injured kidneys showed increased c-fos responsiveness to FBS in cell culture. These experiments suggest that cystic and regenerating kidneys have an altered phenotypic state in vivo that is manifested in cell culture by serum hypersensitivity. However, whereas the folic acid-injured kidneys ultimately reestablish normal kidney function, cystic kidneys further progress to renal failure, suggesting that cystic epithelial cells are locked in this altered state of differentiation.     

Progesterone induced mesenchymal differentiation and rescued cystic dilation of renal tubules of Pkd1(-/-) mice

Autosomal dominant polycystic kidney disease (ADPKD), the most common hereditary disease affecting the kidneys, is caused in 85% of cases by mutations in the PKD1 gene. The protein encoded by this gene, polycystin-1, is a renal epithelial cell membrane mechanoreceptor, sensing morphogenetic cues in the extracellular environment, which regulate the tissue architecture and differentiation. However, how such mutations result in the formation of cysts is still unclear. We performed a precise characterization of mesenchymal differentiation using PAX2, WNT4 and WT1 as a marker, which revealed that impairment of the differentiation process preceded the development of cysts in Pkd1(-/-) mice. We performed an in vitro organ culture and found that progesterone and a derivative thereof facilitated mesenchymal differentiation, and partially prevented the formation of cysts in Pkd1(-/-) kidneys. An injection of progesterone or this derivative into the intraperitoneal space of pregnant females also improved the survival of Pkd1(-/-) embryos. Our findings suggest that compounds which enhance mesenchymal differentiation in the nephrogenesis might be useful for the therapeutic approach to prevent the formation of cysts in ADPKD patients.     

Prenatal diagnosis of Meckel syndrome

Summary The three main features of Meckel syndrome are encephalocele, polycystic kidneys, and polydactyly. Prenatal diagnosis of a fetus with Meckel syndrome was made in the 16th week of gestation by means of amniotic fluid alpha₁ fetoprotein estimation. The indication for amniocentesis was a previous child with an occipital meningocele and polycystic kidneys. Interpretation of the alpha₁-fetoprotein value (240 g/ml) was difficult due to fetal blood contamination. Prenatal diagnosis is indicated in any pregnancy following the birth of a child with only two major symptoms of Meckel syndrome.     

The transcriptional profile of the kidney in Tsc2 heterozygous mutant Long Evans (Eker) rats compared to wild-type

Hereditary renal cell carcinoma (RCC) in Eker rats results from an inherited insertional mutation in the Tsc2 tumor suppressor gene and provides a valuable experimental model to characterize the function of the Tsc2 gene product, tuberin in vivo. The Tsc2 mutation predisposes the Eker rat to develop renal tumors at an early age. The exact mechanism of Tsc2 mediated tumor suppression is not known, however, there is evidence that it is most likely mediated by changes in cell cycle regulation via the PI3K/Akt pathway. The present study was designed to identify if gene expression was different in Tsc2 heterozygous mutant rat kidney compared to wild-type and if any of those differences are associated with tumorigenesis. cDNA microarray analysis of the untreated Tsc2 (+/-) mutant Long Evans (Eker) rat was compared to the Tsc2 (+/+) wild-type Long Evans rat to search for patterns that might be indicative of the intrinsic role of Tsc2. Of 4395 genes queried, 3.2% were significantly altered in kidneys from heterozygous mutant rats, of which 110 (76%) were up-regulated and 34 (24%) were down-regulated relative to the wild-type. The genes with altered expression belonged to the functional categories of cell cycle regulation, cell proliferation, cell adhesion and endocytosis. Many of these genes appear to be directly or indirectly regulated by the PI3K/Akt pathway. In addition to the PI3K/Akt pathway, other signaling pathways were also differentially expressed in Tsc2 mutant Eker rat kidneys compared to wild-type rats. The gene expression profiles of the Tsc2 heterozygous mutant and wild-type animals highlights new pathways for investigation that may be associated with the tumorigenic activity of tuberin loss and correlate with the enhanced susceptibility of the Tsc2 mutant animal's tendency to develop renal cell carcinoma.