Characterization and cell distribution of polycystin, the product of autosomal dominant polycystic kidney disease gene 1.

BACKGROUND: In a majority of cases, autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations within a putative open reading frame of the PKD1 gene. The encoded protein, polycystin, is predicted to span the plasma membrane several times and contains extracellular domains, suggestive of a role in cell adhesion. The cellular distribution and function of polycystin is not known. MATERIALS AND METHODS: We selected as immunogens two conserved 15 amino acid peptides: P1, located in a predicted extracellular region of polycystin, and P2, located in the C-terminal putative cytoplasmic tail. The anti-peptide antibodies from immunized rabbits were affinity purified on peptide-coupled resins and their specificity confirmed by their selective binding to recombinant polycystin fusion proteins. Western blotting and immunohistochemistry were used to characterize the size, tissue, and cell distribution of polycystin. RESULTS: A high-molecular mass protein (about 642 kD) was detected by Western blotting in rat brain tissue. A few additional bands, in the 100- to 400-kD range, probably representing tissue-specific variants and/or proteolytic fragments, were recognized in human and rat tissues. Polycystin was abundantly expressed in fetal kidney epithelia, where it displayed basolateral and apical membrane distribution in epithelial cells of the ureteric buds, collecting ducts, and glomeruli. In normal human adult kidney, polycystin was detected at moderate levels and in a cell surface-associated distribution in cortical collecting ducts and glomerular visceral epithelium. Expression of polycystin was significantly increased in cyst-lining epithelium in ADPKD kidneys, but was primarily intracellular. CONCLUSIONS: Polycystin appears to be a developmentally regulated and membrane-associated glycoprotein. Its intracellular localization in the cyst-lining epithelium of ADPKD kidneys suggests an abnormality in protein sorting in this disease.     

Sonic hedgehog regulates proliferation and differentiation of mesenchymal cells in the mouse metanephric kidney

Signaling by the ureteric bud epithelium is essential for survival, proliferation and differentiation of the metanephric mesenchyme during kidney development. Most studies that have addressed ureteric signaling have focused on the proximal, branching, ureteric epithelium. We demonstrate that sonic hedgehog is expressed in the ureteric epithelium of the distal, non-branching medullary collecting ducts and continues into the epithelium of the ureter -- the urinary outflow tract that connects the kidney with the bladder. Upregulation of patched 1, the sonic hedgehog receptor and a downstream target gene of the signaling pathway in the mesenchyme surrounding the distal collecting ducts and the ureter suggests that sonic hedgehog acts as a paracrine signal. In vivo and in vitro analyses demonstrate that sonic hedgehog promotes mesenchymal cell proliferation, regulates the timing of differentiation of smooth muscle progenitor cells, and sets the pattern of mesenchymal differentiation through its dose-dependent inhibition of smooth muscle formation. In addition, we also show that bone morphogenetic protein 4 is a downstream target gene of sonic hedgehog signaling in kidney stroma and ureteral mesenchyme, but does not mediate the effects of sonic hedgehog in the control of mesenchymal proliferation.     

MUC1 marks collecting tubules,renal vesicles,comma- and S-shaped bodies in human developing kidney

MUC1 is a transmembrane glycoprotein, apically expressed in most epithelial cells, used in the differential diagnosis of carcinomas and for discrimination of tumors of non-epithelial origin showing epithelioid features. Little attention has been paid so far though, on its possible significance in embryonic tissues. A preliminary study from our group revealed MUC1 expression in the cap mesenchymal cells during human nephrogenesis, suggesting a role for MUC1 in the process of mesenchymal-to-epithelial transition. This study aimed at investigating the expression pattern of MUC1 in various developing structures of human fetal kidney. Expression of MUC1 was examined in kidneys of 5 human fetuses. MUC1 immunoreactivity was detected in ureteric bud tips, in collecting tubules, in cap mesenchymal cells undergoing the initial phases of mesenchymal-to-epithelial transition, in renal vesicles, comma-bodies, and S-shaped bodies. Our previous preliminary report suggested a role for MUC1 in the initial phases of the process of mesenchymal-to-epithelial transition. The present data suggest that MUC1 expression characterizes multiple structures during human nephrogenesis, from the ureteric bud, to the initial phases of mesenchymal-to-epithelial transition and that MUC1 should be added to the genes activated during the process of mesenchymal-to-epithelial transition in the cap mesenchyme of human kidney.Key words: MUC1, immunohistochemistry, fetal kdney, nephrogenesis, renal vesicles, comma and S-shaped bodies, collecting tubules.     

Immunohistochemical localisation of TRA-1-60, TRA-1-81, GCTM-2 and podocalyxin in the developing baboon kidney

The baboon is an ideal animal model to study human kidney development. The aim of the current study was to use immunohistochemistry to localise the antigens TRA-1-60, TRA-1-81, GCTM-2 and podocalyxin in the developing baboon kidney where nephrogenesis was still on-going and in kidneys where nephrogenesis was complete. Fixed kidney sections from baboons delivered at 125, 140, 175 and 185 days gestation (term = 185 days) were immuno-labelled with antibodies directed against TRA-1-60, TRA-1-81, GCTM-2 and podocalyxin. In kidneys with on-going nephrogenesis (125 and 140 days gestation), TRA-1-60, TRA-1-81 and GCTM-2 were specifically localised to the apical plasma membrane of the epithelium of the ureteric ampullae and the collecting ducts, while podocalyxin immunostaining was not detected. In kidneys where nephrogenesis was complete (175 and 185 days gestation) localisation of these markers was again very specifically localised to the collecting ducts. In conclusion, although further experimentation is required to confirm the identity of the specific cell types marked by these antibodies, this study provides new insight into the distribution of commonly utilised stem cell antibodies in the developing baboon kidney.     

Phenolsulphotransferase: localization in kidney during human embryonic and fetal development

Summary The aim of our study was to localize phenolsulphotransferase (PST) in the developing mesonephric and metanephric kidneys of the human embryo and fetus using immunohistochemical methods with an antibody preparation recognizing members of the human phenolsulphotransferase enzyme family. In embryonic and early fetal development of the metanephric kidney, PST is located primarily in derivatives of the ureteric bud such as the ureter, pelvis, calyces and collecting ducts. This predominance declines by mid-fetal life: first, as nephrons evolve and develop they become increasingly PST-immunoreactive such that in mature metanephric kidney, the proximal tubules are highly PST-reactive, with other elements of the nephron also immunopositive (albeit at lower reactivities) and secondly, with the formation of an immunonegative transitional epithelium in ureter, pelvis and calyces, the reactivity retained in collecting ducts is only a small proportion of the total. The distribution of PST immunoreactivity is relatively uniform in proximal tubular cells throughout development, in contrast to collecting ducts, where, in fetal life, this reactivity is displaced to apices and bases by intracellular glycogen deposits. Mesonephric kidney tubules and the mesonephric duct are PST-immunoreactive and although mesonephric immunopositivity overlaps with that in the developing metanephric kidney the renal contribution to sulphation is absent or low at a time when the developing conceptus is most vulnerable to the potential toxic effects of teratogens.     

Insights into the Renal Pathogenesis in Schimke Immuno-Osseous Dysplasia: A Renal Histological Characterization and Expression Analysis

Schimke immuno-osseous dysplasia (SIOD) is a pleiotropic disorder caused by mutations in the SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a-like-1 (SMARCAL1) gene, with multiple clinical features, notably end-stage renal disease. Here we characterize the renal pathology in SIOD patients. Our analysis of SIOD patient renal biopsies demonstrates the tip and collapsing variants of focal segmental glomerulosclerosis (FSGS). Additionally, electron microscopy revealed numerous glomerular abnormalities most notably in the podocyte and Bowman’s capsule. To better understand the role of SMARCAL1 in the pathogenesis of FSGS, we defined SMARCAL1 expression in the developing and mature kidney. In the developing fetal kidney, SMARCAL1 is expressed in the ureteric epithelium, stroma, metanephric mesenchyme, and in all stages of the developing nephron, including the maturing glomerulus. In postnatal kidneys, SMARCAL1 expression is localized to epithelial tubules of the nephron, collecting ducts, and glomerulus (podocytes and endothelial cells). Interestingly, not all cells within the same lineage expressed SMARCAL1. In renal biopsies from SIOD patients, TUNEL analysis detected marked increases in DNA fragmentation. Our results highlight the cells that may contribute to the renal pathogenesis in SIOD. Further, we suggest that disruptions in genomic integrity during fetal kidney development contribute to the pathogenesis of FSGS in SIOD patients.     

Immunohistochemical localisation of glucose-6-phosphatase in developing human kidney

The objective of our study was to determine the cellular localisation of glucose-6-phosphatase in developing human kidney using monospecific antiserum and a standard immunohistochemical method (peroxidase-antiperoxidase, PAP) on formalin fixed and paraffin embedded tissue. In embryonic and early fetal development of the metanephric kidney, glucose-6-phosphatase is located primarily in derivatives of the ureteric bud such as the pelvis, calyxes and collecting ducts. In mid-fetal life as nephrons evolve and develop they become increasingly immunoreactive to glucose-6-phosphatase, such that in mature metanephric kidney the proximal tubules are highly reactive for glucose-6-phosphatase with other elements of the nephron also immunopositive albeit at lower reactivities. In addition the parietal layer of Bowman's capsule and some cells of the visceral layer are immunopositive. Only with the development of nephrons does the early predominance of glucose-6-phosphatase immunoreactivity to ureteric bud derivatives change: in mature kidney the reactivity in the collecting ducts is a small proportion of the total. In proximal tubular cells the distribution of glucose-6-phosphatase immunoreactivity is relatively uniform throughout development in contrast to collecting ducts where in fetal life this reactivity is displaced to the apices and basal areas by intracellular glycogen deposits. The mesonephric kidney has a similar pattern of glucose-6-phosphatase immunoreactivity to that of metanephric kidney. The availability of monospecific antiserum to glucose-6-phosphatase and immunohistochemical methods now allows an alternative approach to cellular localisation. Many of the difficulties in the fixation of tissue and assay of glucose-6-phosphatase activity inherent in conventional histochemical methods are avoided by such methods.     

Kir1.1 expression in embryonic kidney epithelia

K(+) channels may regulate cell cycling, cell volume, and cell proliferation. We have recently shown a role for an inwardly rectifying K(+) channel, Kir6.1/SUR2(B), in the regulation of cell proliferation during early kidney development. Here, we show that the protein of a further K(+) channel, Kir1.1 (ROMK), is also developmentally expressed in prenatal rat kidney epithelia. In the embryonic stage, Kir1.1 protein was localized to the plasma membrane of ureteric buds and collecting ducts, and of nephron stages up to the comma-shaped body. Experimental increase in cAMP upregulated Kir1.1b (ROMK2) mRNA abundance in ureteric buds. Kir1.1 protein was restricted to the distal nephron during later postnatal development and adulthood, as has been reported. In conclusion, we demonstrate redundancy of Kir channel expression in early embryonic kidney which could suggest that Kir1.1 acts in a similar way as Kir6.1/SUR2(B) to promote cell proliferation or other developmental functions.     

Regulation of ureteric bud outgrowth by Pax2-dependent activation of the glial derived neurotrophic factor gene.

The outgrowth of the ureteric bud from the posterior nephric duct epithelium and the subsequent invasion of the bud into the metanephric mesenchyme initiate the process of metanephric, or adult kidney, development. The receptor tyrosine kinase RET and glial cell-derived neurotrophic factor (GDNF) form a signaling complex that is essential for ureteric bud growth and branching morphogenesis of the ureteric bud epithelium. We demonstrate that Pax2 expression in the metanephric mesenchyme is independent of induction by the ureteric bud. Pax2 mutants are deficient in ureteric bud outgrowth and do not express GDNF in the uninduced metanephric mesenchyme. Furthermore, Pax2 mutant mesenchyme is unresponsive to induction by wild-type heterologous inducers. In normal embryos, GDNF is sufficient to induce ectopic ureter buds in the posterior nephric duct, a process inhibited by bone morphogenetic protein 4. However, GDNF replacement in organ culture is not sufficient to stimulate ureteric bud outgrowth from Pax2 mutant nephric ducts, indicating additional defects in the nephric duct epithelium of Pax2 mutants. Pax2 can activate expression of GDNF in cell lines derived from embryonic metanephroi. Furthermore, Pax2 protein can bind to upstream regulatory elements within the GDNF promoter region and can transactivate expression of reporter genes. Thus, activation of GDNF by Pax2 coordinates the position and outgrowth of the ureteric bud such that kidney development can begin.     

Expression of Sprouty genes 1, 2 and 4 during mouse organogenesis.

We demonstrate that Sprouty genes 1, 2 and 4 are expressed in several developing organs of the craniofacial area and trunk, including the brain, cochlea, nasal organs, teeth, salivary gland, lungs, digestive tract, kidneys and limb buds. In organs such as the semicircular canal, Rathke's pouch, nasal organs, the follicle of vibrissae and teeth, Sprouty1 and Sprouty2 are expressed in the epithelium and Sprouty4 in the mesenchyme or neuronal tissue, while in the lung Sprouties1, 2 and 4 are all expressed mainly in the epithelial tissue. In the kidney, Sprouty1 is prominent in the ureteric bud whereas Sprouty2 and 4 are expressed in both the ureteric bud and the kidney mesenchyme and glomeruli deriving from it. The expression profiles suggest roles for these Sprouties in the epithelial-mesenchymal interactions that govern organogenesis.     

Intercalated cells as a probable source for the development of renal oncocytoma

Renal oncocytoma is a distinct type of epithelial tumor said to arise from the collecting duct system. Here we show that in nine of ten oncocytomas the tumor cells expressed an analog of the erythrocyte anion exchanger band 3. In the normal kidney band 3 is confined to the basolateral surface of the majority of intercalated cells which comprise up to 50% of the cortical collecting duct epithelium. Carbonic anhydrase c is another protein abundant in intercalated cells, and this was also expressed in six of the ten oncocytomas investigated. Immunoreactivity specific for band 3 and carbonic anhydrase c was not detected in any of the 20 renal cell carcinomas examined. At favourable section planes direct transitions between normal collecting ducts and oncocytic tubules were observed. These findings suggest that oncocytomas may develop from intercalated cells of the collecting duct epithelium.     

Developmental mucin gene expression in the gastroduodenal tract and accessory digestive glands. II. Duodenum and liver, gallbladder, and pancreas.

Studies were undertaken to provide information regarding cell-specific expression of mucin genes and their relation to developmental and neoplastic patterns of epithelial cytodifferentiation. In situ hybridization was used to study mRNA expression of mucin genes in duodenum and accessory digestive glands (liver, gallbladder, pancreas) of 13 human embryos and fetuses (6. 5-27 weeks' gestation), comparing these with normal and neoplastic adult tissues. These investigations demonstrated that the pattern of mucin gene expression in fetal duodenum reiterated the patterns we observed during gastric and intestinal ontogenesis, with MUC2 and MUC3 expression in the surface epithelium and MUC6 expression associated with the development of Brünner's glands. In embryonic liver, MUC3 was already expressed at 6.5 weeks of gestation in hepatoblasts. As in adults, MUC1, MUC2, MUC3, MUC5AC, MUC5B, and MUC6 were expressed in fetal gallbladder, whereas MUC4 was not. In contrast, MUC4 was strongly expressed in gallbladder adenocarcinomas. MUC5B and MUC6 were expressed in fetal pancreas, from 12 weeks and 26 weeks of gestation, respectively. Surprisingly, MUC3 which is strongly expressed in adult pancreas, was not detected in developmental pancreas. Taken together, these data show complex spatio-temporal regulation of the mucin genes and suggest a possible regulatory role for mucin gene products in gastroduodenal epithelial cell differentiation.     

The role of GDNF in patterning the excretory system

Mesenchymal-epithelial interactions are an important source of information for pattern formation during organogenesis. In the developing excretory system, one of the secreted mesenchymal factors thought to play a critical role in patterning the growth and branching of the epithelial ureteric bud is GDNF. We have tested the requirement for GDNF as a paracrine chemoattractive factor by altering its site of expression during excretory system development. Normally, GDNF is secreted by the metanephric mesenchyme and acts via receptors on the Wolffian duct and ureteric bud epithelium. Misexpression of GDNF in the Wolffian duct and ureteric buds resulted in formation of multiple, ectopic buds, which branched independently of the metanephric mesenchyme. This confirmed the ability of GDNF to induce ureter outgrowth and epithelial branching in vivo. However, in mutant mice lacking endogenous GDNF, kidney development was rescued to a substantial degree by GDNF supplied only by the Wolffian duct and ureteric bud. These results indicate that mesenchymal GDNF is not required as a chemoattractive factor to pattern the growth of the ureteric bud within the developing kidney, and that any positional information provided by the mesenchymal expression of GDNF may provide for renal branching morphogenesis is redundant with other signals.