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PubMed1992

Characterization of an endothelial protein in the developing rabbit kidney.

A new protein (EnPo 1 antigen) abundant on endothelial cells and glomerular podocytes has been characterized by means of the mouse monoclonal antibody EnPo 1. Following electrophoretical separation of rabbit kidney homogenates EnPo 1 recognized a protein with a molecular weight of 110 kDa and an isoelectric point of 5.9 in Western blots. Using immunohistological techniques, the EnPo 1 antigen has been localized in high concentrations on glomerular podocytes of different developmental stages. Furthermore, the EnPo 1 antigen was expressed on endothelial cells of all adult rabbit organs tested so far. Detailed analysis of neonatal rabbit kidney revealed the abundance of EnPo 1 antigen on both differentiated vessels as well as on immature endothelial cells and endothelium of the microvasculature. Thus, for the first time a marker for in situ investigations of angiogenic processes within the mammalian kidney is available. Analysis of kidney cryosections by confocal laser scan microscopy revealed a direct connection between mature and differentiating vessels in the outer kidney cortex. Furthermore, two differentially organized cell populations discriminated by their EnPo 1 binding pattern were localized in the embryonic renal cortex. Morphologically, these cells were not distinguishable from other mesenchymal cells.

Animals↗
PubMed1979

Cellular aspects of rabbit Masugi nephritis. IV. Cell quantitation of proliferative glomerulonephritis.

Diffuse proliferative glomerulonephritis was induced in the rabbit with injection of goat nephrotoxic serum, and cell dynamics in the glomerulus were quantitatively investigated. The mean total cell number of a normal glomerulus in cross-section was 54.8, consisting of 24.1 endothelial cells, 16.0 podocytes, and 14.3 mesangial cells. In 5 selected nephritic rabbits, the mean total cell number of a glomerulus at the florid stage (7 to 12 experimental days) was 2.4 times as great as the control. It was revealed that the cells consisted of 27.9 endothelial cells, 14.9 podocytes, 21.1 mesangial cells, and 57.8 monocytic cells, indicating that about 76 per cent of the increased cells were of monocytic origin. Mesangial cells showed moderate increase in number (1.5 times) which persisted until 100 days, whereas endothelial cells increased only slightly. The monocytic cells promptly disappeared at 40 days correlating well with resolution of the proliferative change. Although the cellular compositions varied in each case, it was concluded that monocytes were essential in producing proliferative glomerular changes in the reversible type of Masugi nephritis.

Animals↗
PubMed2004

Key molecular events in puromycin aminonucleoside nephrosis rats.

Nephrin, podocin and alpha-actinin are all involved in proteinuria, but it is unclear which molecular event plays a crucial role during the development of proteinuria. Immunofluorescence staining and real-time quantitative polymerase chain reaction were used to study the glomerular expression of these molecules in puromycin aminonucleoside (PAN) nephrosis. Morphometric methods were applied to evaluate the podocyte foot process (FP) morphology. Two days after PAN injection, nephrin and podocin staining became discontinuous, podocin intensity decreased and FP swelled. Nephrin protein and mRNA decreased at day 5. Both podocin and nephrin intensity decreased dramatically when heavy proteinuria occurred, but nephrin mRNA was regained. When proteinuria disappeared, podocin recovered whereas nephrin did not (P = 0.02); alpha-actinin intensity increased (P = 0.009) and the distribution changed. The podocyte FP volume density correlated negatively with nephrin (r = -0.78, P = 0.0001) and podocin immunofluorescence intensity (r = -0.76, P = 0.0001). We conclude that, before the onset of proteinuria, the first response was the nephrin and podocin distribution change, podocin protein decrease and swollen FP; the podocin quantitative change was earlier than nephrin. Podocin and nephrin distribution and the protein level was associated with proteinuria more closely than their mRNA level. The delayed alpha-actinin induction might be a reparative response.

Actinin↗
PubMed2004

Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy.

BACKGROUND: Recent disclosure of podocyte proteins has unraveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we addressed the role of nephrin, CD2-associated protein (CD2AP), and podocin together with the integrity of the slit diaphragm in the pathogenesis of proteinuria of patients with diabetes and nephropathy. METHODS: Nephrin mRNA and protein expression were evaluated in parallel in adult diabetic patients by in situ hybridization and immunohistochemistry. For comparison, nondiabetic patients with minimal change nephrosis and normal control patients were evaluated. CD2AP and podocin expression by immunohistochemistry was also assessed. The filtration slit was analyzed by morphometry and transmission electron microscopy. RESULTS: Extracellular nephrin mRNA and protein were markedly reduced in diabetic patients. No changes were found in patients with minimal change versus controls. CD2AP and podocin were comparable in all subjects. Ultrastructural analysis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a frequency of the filtration slits comparable to control patients. CONCLUSION: Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.

Adaptor Proteins, Signal Transducing↗
PubMed2004

DDR1-deficient mice show localized subepithelial GBM thickening with focal loss of slit diaphragms and proteinuria.

BACKGROUND: Type IV collagen in basement membranes is a ligand for the receptor tyrosine kinase discoidin domain receptor 1 (DDR1). DDR1 is expressed in renal cells and regulates cell adhesion and proliferation ex vivo. The interaction between type IV collagen and cell surface receptors is believed important for normal renal function as well as significant in chronic renal diseases and we therefore analyzed mice with a targeted deletion of DDR1. METHODS: Homozygous DDR1 knockout mice were compared to heterozygous and wild-type animals. The quantitative and qualitative amount of proteinuria was measured by urine-microelectrophoresis. Structural changes of the kidneys were determined by immunohistochemistry, light microscopy, and electron microscopy. RESULTS: Compared to heterozygous littermates, adult DDR1 knockout mice showed a selective middle- to high-molecular proteinuria of up to 0.3 g/L and urinary acanthocytes. There was no evidence of uremia with no change in serum urea in the first 9 months of age. Little apparent change in renal morphology was detected using light microscopy. However, electron microscopy showed a localized, subepithelial, mushroom-like isodense thickening of the glomerular basement membrane (GBM). Within these areas, a focal loss of the podocytic slit diaphragms occurred. CONCLUSION: The loss of cell-matrix communication in DDR1-deficient podocytes appears to result in excess synthesis of basement membrane proteins leading to disturbed anchorage of foot processes and disruption of the slit diaphragm. Our data suggest that the interaction between type IV collagen and DDR1 plays an important role in maintaining the structural integrity of the GBM.

Animals↗
PubMed2004

Proteinuria in rats induced by serum from patients with collapsing glomerulopathy.

BACKGROUND: Primary collapsing glomerulopathy recurs postransplant, raising the possibility of circulating factors implicated in the pathogenesis of the disease. METHODS: To determine the presence of circulating factors in collapsing glomerulopathy patients, we tested serum from those patients in an in vivo assay. Eleven groups of rats received serum from collapsing glomerulopathy patients, idiopathic focal segmental glomerulosclerosis (FSGS) or healthy subjects in its native form, isolated IgG, or serum without IgG. The presence of proteinuria and creatinine clearance were determined. Histopathologic analysis included light, immunofluorescence, and electron microscopy. RESULTS: Collapsing glomerulopathy rats developed proteinuria while rats injected with serum from FSGS and healthy subjects did not. Rats injected with serum of collapsing glomerulopathy in its native form developed marked proteinuria (99.2 +/- 42 mg/24 hours at day 5, P= 0.0001, compared to the baseline), and decreased in creatinine clearance. Rats receiving isolated IgG or serum without IgG from collapsing glomerulopathy developed mild proteinuria (46.5 +/- 8.4 mg/24 hours and 30.9 +/- 11 mg/24 hours, respectively, at day 5 (P= 0.0001). Glomerular tuft retraction and podocyte damage were seen only in collapsing glomerulapthy rats. No abnormalities were found in rats injected with serum from FSGS or healthy subjects. CONCLUSION: Circulating factors in the serum of collapsing glomerulopathy patients produce podocyte damage, whereas such factors are not present in noncollapsing FSGS. IgG eluates from collapsing glomerulopathy produce proteinuria when injected into the rat. Such factors remain in the circulation when serum of patients is adsorbed into protein A, raising the possibility that there are more than one circulating factor present in patients with collapsing glomerulopathy.

Adolescent↗
PubMed2004

Glomerular expression of the ATP-sensitive P2X receptor in diabetic and hypertensive rat models.

BACKGROUND: The molecular identification and characterization of the adenosine triphosphate (ATP)-sensitive family of P2 receptors is comparatively new. There are two main subgroups, each with several subtypes and widespread tissue distribution, including the kidney. A unique member of the P2X subgroup of P2 receptors is the ATP-gated ion channel P2X(7), which on activation can cause cell blebbing, cytokine release, and cell death by necrosis or apoptosis. We report expression of this receptor in normal rat kidney and in two chronic models of glomerular injury: streptozotocin-induced (STZ) diabetes and ren-2 transgenic (TGR) hypertension. METHODS: At different time points in these models, we used a polyclonal antibody to the P2X(7) receptor and immunohistochemistry to determine its expression and distribution. We also used Western blotting and real-time polymerase chain reaction (PCR) to detect changes in P2X(7) receptor protein and mRNA expression, respectively. RESULTS: We found only low-level glomerular immuno-staining for the P2X(7) receptor in normal rat kidney, but intense P2X(7) receptor immunostaining of glomeruli in kidneys from diabetic animals at 6 and 9 weeks, and in hypertensive animals at 12 weeks. In diabetic animals, real-time PCR demonstrated a approximately tenfold increase in glomerular P2X(7) receptor mRNA relative to control, and Western blotting confirmed an increase in protein. Immunohistochemistry and immunoelectron microscopy showed staining of glomerular podocytes, which was both intracellular and at the plasma membrane. CONCLUSION: We conclude that the P2X(7) receptor is not expressed appreciably under normal conditions, but that following glomerular injury it is significantly up-regulated, mainly in podocytes, though also in endothelial and mesangial cells, of animals with STZ-induced diabetes mellitus or TGR hypertension. Although the exact function and regulation of this receptor remain unclear, its association with inflammatory cytokine release and cell death suggests that increased expression might be involved in the pathogenesis of glomerular cell injury or repair.

Adenosine Triphosphate↗
PubMed2005

Phosphate binder therapy for attainment of K/DOQI bone metabolism guidelines.

Hyperphosphatemia in patients with chronic kidney disease leads to secondary hyperparathyroidism and renal osteodystrophy, and it is independently associated with mortality risk. The exact mechanism by which hyperphosphatemia increases mortality risk is unknown, but it may relate to enhanced cardiovascular calcification. The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (K/DOQI) Clinical Practice Guidelines for Bone Metabolism and Disease in Chronic Kidney Disease recommends maintenance of serum phosphorus below 5.5 mg/dL, calcium-phosphorus (Ca x P) product less than 55 mg(2)/dL(2), intact parathyroid hormone (iPTH) 150 pg/mL to 300 pg/mL, and bicarbonate (HCO(3)) greater than 22 mEq/L. Although calcium-based phosphate binders (CBPB) are cost effective, there are long-term safety concerns pertaining to their postulated role in the progression of cardiovascular calcification. Sevelamer hydrochloride has been recommended as an alternative noncalcium phosphate binder. Results from the Calcium Acetate Renagel Evaluation (CARE) study indicate that calcium acetate is more effective than sevelamer hydrochloride in controlling serum phosphorous, Ca x P product, and HCO(3) in hemodialysis patients. In the Treat-to-Goal study, dialysis patients treated with sevelamer hydrochloride had slower progression of coronary and aortic calcification than patients treated with CBPB. The mechanism underlying the beneficial effect of sevelamer hydrochloride is unknown but may relate to decreased calcium loading, or to dramatic reductions in low-density lipoprotein (LDL) cholesterol in sevelamer hydrochloride-treated patients. At present, evidence incriminating CBPB in the progression of cardiovascular calcification in end-stage renal disease (ESRD) remains largely circumstantial. As calcium acetate is more efficacious and cost effective than sevelamer hydrochloride, it remains an accepted first-line phosphate binder. This review examines these issues and provides rational guidelines for the use of CBPB in patients on maintenance hemodialysis.

Bone and Bones↗
PubMed2005

p27(Kip1) Knockout mice are protected from diabetic nephropathy: evidence for p27(Kip1) haplotype insufficiency.

BACKGROUND: High glucose up-regulates the mesangial cell expression of p27(Kip1), an inhibitor of cyclin-dependent kinases/cyclin complexes. Previous in vitro studies using cultured mesangial cells from p27(Kip1-/-) mice demonstrated that these cells do not undergo high glucose-mediated cellular hypertrophy. Since glomerular hypertrophy is an early feature of diabetic nephropathy and may precede the development of glomerulosclerosis, interference with p27(Kip1) expression may attenuate diabetic nephropathy. However, it is unclear whether deletion of p27(Kip1) protects the kidneys of diabetic nephropathy in vivo. METHODS: Type 1 diabetes mellitus was induced in p27(Kip1+/+), p27(Kip1+/-), and p27(Kip1-/-) mice by injection of streptozotocin (STZ). Mice were studied for 6 weeks. Animals injected with citrate buffer only served as controls. At the end of the experiments, urine was collected, albuminuria was determined with an enzyme-linked immunosorbent assay (ELISA), and blood glucose concentrations were measured. Kidneys were perfusion-fixed for quantitative morphologic analysis with glutaraldehyde and for immunohistochemical studies with formaldehyde. Glomerular cell number and volume were analyzed. Glomerulosclerosis, tubulointerstitial, and vascular damage indices were semiquantitatively assessed according to standard methodology. Quantitative glomerular parameters (cell numbers and volumes of endothelial, mesangial, and epithelial cells) were measured on semithin sections. Expression of transforming growth factor-beta1 (TGF-beta1), laminin, and collagen type IV were determined by immunohistochemical staining. RESULTS: In contrast to animals only injected with citrate buffer, mice that received STZ developed hyperglycemia. There was no significant difference in the degree of hyperglycemia among p27(Kip1+/+), p27(Kip1+/-), and p27(Kip1-/-) mice. Diabetic p27(Kip1+/+), but not control p27(Kip1+/+) animals, developed albuminuria. Albuminuria was significantly reduced in diabetic p27(Kip1+/-) and more profoundly in p27(Kip1-/-) animals. Diabetic p27(Kip1+/+) mice revealed a significant increase in mean glomerular volume at 6 weeks. The volumes of mesangial and endothelial cells and podocytes all increased, whereas cell numbers were reduced, consistent with cell hypertrophy. Glomerular, endothelial, mesangial and podocyte hypertrophy were reduced in diabetic p27(Kip1+/-) and p27(Kip1-/-) animals. Diabetic p27(Kip1) (+/+) animals had significantly increased glomerulosclerosis, tubulointerstium, and vascular damage indices compared to nondiabetic p27(Kip1+/+) controls. Diabetic p27(Kip1-/-) mice exhibited significantly less structural damage than diabetic wild-type animals. Diabetic p27(Kip1+/-) animals revealed intermediate glomerulosclerosis, tubulointerstium, and vascular damage values. Immunohistological stainings demonstrated increases in TGF-beta1, collagen type IV, and laminin expression in kidneys of diabetic p27(Kip1+/+) animals compared to nondiabetic p27(Kip1+/+) controls. Staining intensity for type IV collagen and laminin, but not for TGF-beta1, was significantly lower in diabetic p27(Kip1-/-) mice. CONCLUSION: Deletion of p27(Kip1) attenuates the functional and morphologic features of diabetic nephropathy. Although deletion of p27(Kip1) abolished some parameters of diabetic glomerular hypertrophy, the significant reduction of TGF-beta1 expression in the tubulointerstitium indicates that other protective mechanisms could be operative. The p27(Kip1) gene is haplo-insufficient because diabetic p27(Kip1)+/- mice exhibited an intermediate degree of functional and structural renal injury. Our data shows that p27(Kip1) plays an important role in diabetic nephropathy.

Albuminuria↗
PubMed2005

Immunolocalization of fibroblast growth factor-1 (FGF-1), its receptor (FGFR-1), and fibroblast-specific protein-1 (FSP-1) in inflammatory renal disease.

BACKGROUND: The fibroblast growth factor (FGF) family has functions in development, cell proliferation, migration, and differentiation. While FGF-2 induces fibrosis, the role of FGF-1 in inflammation and fibrosis is less defined. We examined the expression of FGF-1 and FGF receptor (FGFR-1) to determine if renal diseases with varying etiologies of inflammation, including lupus nephritis (LN), acute interstitial nephritis (AIN) and acute rejection superimposed on chronic allograft nephropathy (CAN), showed varying patterns of expression. We also examined the expression of fibroblast-specific protein-1 (FSP-1), which has been linked to epithelial-mesenchymal transition (EMT) and fibrosis, to determine whether it was linked to potential profibrotic and inflammatory FGF-1 mechanisms. METHODS: Proliferative LN (PLN) (N= 12), nonproliferative lupus nephritis (NPLN) (N= 5), AIN (N= 6), CAN (N= 4), and normal kidneys (N= 3) were studied. FGF, FGFR-1, and FSP-1 were localized by immunohistochemistry, and intensity scored on a 0 to 3+ scale. Double staining with CD68 and separate immunohistochemical staining for CD4 and CD8 with serial sections analysis were done to identify if T lymphocytes or macrophages showed staining for FGF-1 and FGFR-1 or FSP-1. RESULTS: In normal kidneys, FGF-1 was expressed in mesangial cells (0.67 +/- 0.58), glomerular endothelial (0.67 +/- 0.58), visceral, and parietal epithelial cells (1.67 +/- 0.58). FGFR-1 showed a similar pattern of staining but also was expressed in tubular epithelium, and arterial endothelium and smooth muscle. Expression of FGF-1 was increased over normal in glomerular parenchymal cells only in CAN in podocytes (2.30 +/- 0.58 vs. 3.00 +/- 0.00) (P < 0.05) and parietal epithelial cells (1.67 +/- 0.58 vs. 2.25 +/- 0.50) (P < 0.05). Infiltrating glomerular and interstitial inflammatory cells in diseased glomeruli also expressed FGF-1 and FGFR-1. Tubular cells expressed slightly increased FGFR-1 in renal diseases vs. normal, whereas tubules remained negative for FGF-1 in diseased kidneys. FSP-1 expression was prominent in the interstitium in all kidneys with interstitial inflammation, and most prominent in CAN. Interstitial FSP-1+ cells were consistent with a myofibroblast-type morphology, and did not stain with CD-68. FSP-1 expression was closely associated with inflammatory cells expressing FGF-1 and FGFR-1. FSP-1 also showed positivity within crescents and occasional podocytes in PLN. CONCLUSION: The expression of FGF-1 and FGFR-1 in infiltrating lymphocytes and macrophages, and of FGFR-1 in tubules, is supportive, but does not prove causality, of the possibility that FGF-1 might have both autocrine and paracrine functions in renal inflammation. However, the initial stimulus for renal inflammation, whether immune complex, hypersensitivity or rejection, did not alter expression patterns of FGF-1 or its receptor. The colocalization of inflammatory infiltrates with interstitial fibrosis supports the possibility of a contribution of FGF-1 for chemotaxis and associated fibrosis, further supported by interstitial FSP-1 expression closely associated with these inflammatory cells expressing FGF-1 and FGFR-1.

Acute Disease↗
PubMed2005

Pathways to nephron loss starting from glomerular diseases-insights from animal models.

Studies of glomerular diseases in animal models show that progression toward nephron loss starts with extracapillary lesions, whereby podocytes play the central role. If injuries remain bound within the endocapillary compartment, they will undergo recovery or be repaired by scaring. Degenerative, inflammatory and dysregulative mechanisms leading to nephron loss are distinguished. In addition to several other unique features, the dysregulative mechanisms leading to collapsing glomerulopathy are particular in that glomeruli and tubules are affected in parallel. In contrast, in degenerative and inflammatory diseases, tubular injury is secondary to glomerular lesions. In both of the latter groups of diseases, the progression starts in the glomerulus with the loss of the separation between the tuft and Bowman's capsule by forming cell bridges (parietal cells and/or podocytes) between the glomerular and the parietal basement membranes. Cell bridges develop into tuft adhesions to Bowman's capsule, which initiate the formation of crescents, either by misdirected filtration (proteinaceous crescents) or by epithelial cell proliferation (cellular crescents). Crescents may spread over the entire circumference of the glomerulus and, via the glomerulotubular junction, may extend onto the tubule. Two mechanisms concerning the transfer of a glomerular injury onto the tubulointerstitium are discussed: (1) direct encroachment of extracapillary lesions and (2) protein leakage into tubular urine, resulting in injury to the tubule and the interstitium. There is evidence that direct encroachment is the crucial mechanism. Progression of chronic renal disease is underlain by a vicious cycle which passes on the damage from lost and/or damaged nephrons to so far healthy nephrons. Presently, two mechanisms are discussed: (1) the loss of nephrons leads to compensatory mechanisms in the remaining nephrons (glomerular hypertension, hyperfiltration, hypertrophy) which increase their vulnerability to any further challenge (overload hypothesis); and (2) a proteinuric glomerular disease leads, by some way or another, to tubulointerstitial inflammation and fibrosis, accounting for the further deterioration of renal function (fibrosis hypothesis). So far, no convincing evidence has been published that in primary glomerular diseases fibrosis is harmful to healthy nephrons. The potential of glomerular injuries to regenerate or to be repaired by scaring is limited. The only option for extracapillary injuries with tuft adhesion is repair by formation of a segmental adherent scar (i.e., segmental glomerulosclerosis).

Animals↗
PubMed2001

Proteinuria and perinatal lethality in mice lacking NEPH1, a novel protein with homology to NEPHRIN.

A high-throughput, retrovirus-mediated mutagenesis method based on gene trapping in embryonic stem cells was used to identify a novel mouse gene. The human ortholog encodes a transmembrane protein containing five extracellular immunoglobulin-like domains that is structurally related to human NEPHRIN, a protein associated with congenital nephrotic syndrome. Northern analysis revealed wide expression in humans and mice, with highest expression in kidney. Based on similarity to NEPHRIN and abundant expression in kidney, this protein was designated NEPH1 and embryonic stem cells containing the retroviral insertion in the Neph1 locus were used to generate mutant mice. Analysis of kidney RNA from Neph1(-/-) mice showed that the retroviral insertion disrupted expression of Neph1 transcripts. Neph1(-/-) pups were represented at the expected normal Mendelian ratios at 1 to 3 days of age but at only 10% of the expected frequency at 10 to 12 days after birth, suggesting an early postnatal lethality. The Neph1(-/-) animals that survived beyond the first week of life were sickly and small but without edema, and all died between 3 and 8 weeks of age. Proteinuria ranging from 300 to 2,000 mg/dl was present in all Neph1(-/-) mice. Electron microscopy demonstrated NEPH1 expression in glomerular podocytes and revealed effacement of podocyte foot processes in Neph1(-/-) mice. These findings suggest that NEPH1, like NEPHRIN, may play an important role in maintaining the structure of the filtration barrier that prevents proteins from freely entering the glomerular urinary space.

Amino Acid Sequence↗
PubMed1997

Expression of the Wilms' tumour gene WT1 in the developing human and in paediatric renal tumours: an immunohistochemical study.

AIMS: The Wilms' tumour gene (WT1) product is expressed during the development of the urogenital system. This study was undertaken to evaluate four anti-WT1 antibodies and use the most specific one to examine the expression of WT1 in formalin fixed, paraffin wax embedded tissues from human embryos, fetuses, and paediatric renal neoplasms. METHODS: The antibodies were assessed on paraffin sections of fetal kidney and by western blotting. Immunohistochemical techniques were optimised and performed on a range of embryonic, fetal, and infant tissues from 35 days post-conception to three months of age, and on a selection of paediatric renal neoplasms. RESULTS: The antibodies tested were found to vary in their specificity. Anomalous expression in smooth muscle was seen with one batch of a commercial polyclonal antibody. WT1 protein was detected in both the metanephros and the mesonephros, the spleen, the gonads, and in the peritoneal mesothelium in fetuses. WT1 was expressed in nuclei and was strongest in the podocytes of fetal kidney. The podocytes of infant glomeruli were also positive. There was focal positive staining in Wilms' tumours, nephrogenic rests, and in a cystic partially differentiated nephroblastoma. Staining of nuclei was seen in one of two rhabdoid tumours of the kidney. No positive staining was seen in other renal tumours. CONCLUSIONS: WT1 is detected readily in formalin fixed material. There were differences in specificity between batches of the polyclonal antibodies used. The distribution of the WT1 gene product in tissues and tumours reflected previous findings with in situ hybridisation studies of WT1 mRNA.

Antibodies↗
PubMed2002

PDZ domain-mediated interaction of rabbit podocalyxin and Na(+)/H(+) exchange regulatory factor-2.

The transmembrane sialoglycoprotein podocalyxin is thought to be essential in the fine interdigitating foot process structure of the podocyte. The intracellular COOH-terminal amino acids Asp-Thr-His-Leu (DTHL) of podocalyxin comprise a putative ligand for a type I PSD95-Dlg-zona occludens-1 (PDZ) domain. A 20-amino acid synthetic peptide containing this motif was used to screen a cDNA library, and clones of rabbit Na(+)/H(+) exchange regulatory factor-2 (NHERF-2) were obtained. In vitro analysis demonstrated that each PDZ domain of NHERF-2 could bind podocalyxin independently. NHERF-2 coprecipitated from glomerular extracts with podocalyxin, and podocalyxin and NHERF-2 colocalized in the glomerular capillary loops, indicating that podocalyxin and NHERF-2 may interact in vivo. Podocalyxin peptide missing the terminal leucine (-DTHL) failed to interact with NHERF-2 in vitro. Podocalyxin localized to the apical membrane of transfected Madin-Darby canine kidney (MDCK) cells. However, mutant podocalyxin (missing a functional DTHL COOH-terminal motif) showed cytoplasmic and apical membrane localization in transfected cells and was also less stable at the apical membrane, as assessed by confocal microscopy and biotinylation studies. Mutant podocalyxin did lower the transepithelial resistance of MDCK cell monolayers, albeit to a lesser extent than full-length podocalyxin. We conclude that podocalyxin can interact with both PDZ domains of NHERF-2 and that this interaction requires the intact COOH terminus of podocalyxin, which is also responsible for the efficient apical localization of podocalyxin in transfected MDCK cells. These results suggest that the interaction of podocalyxin with NHERF-2 may function to efficiently retain podocalyxin at the apical surface of the podocyte and provide a mechanism linking podocalyxin to the actin cytoskeleton.

Amino Acid Motifs↗
PubMed2005

Temporal expression profile and distribution pattern indicate a role of connective tissue growth factor (CTGF/CCN-2) in diabetic nephropathy in mice.

Connective tissue growth factor (CTGF) is overexpressed in diabetic nephropathy (DN) and has therefore been implicated in its pathogenesis. The objective of the present study was to determine the tissue distribution of increased CTGF expression and the relationship of plasma, urinary, and renal CTGF levels to the development and severity of DN. We studied the relationship between CTGF and renal pathology in streptozotocin (STZ)-induced diabetes in C57BL/6J mice. Diabetic and age-matched control mice were killed after 1, 2, 4, and 9 wk of diabetes. In addition, key parameters of diabetes and DN were analyzed in 10-mo-old diabetic ob/ob mice and their ob/+ littermates. STZ-induced diabetic mice showed a significantly increased urinary albumin excretion after 1 wk and increased mesangial matrix score after 2 wk. Increased renal fibronectin, fibronectin ED-A, and collagen IValpha1 expression, as well as elevated plasma creatinine levels, were observed after 9 wk. After 2 wk, CTGF mRNA was upregulated threefold in the renal cortex. By 9 wk, CTGF mRNA was also increased in the heart and liver. In contrast, transforming growth factor-beta1 mRNA content was significantly increased only in the kidney by 9 wk. Renal CTGF expression was mainly localized in podocytes and parietal glomerular epithelial cells, and less prominent in mesangial cells. In addition, plasma CTGF levels and urinary CTGF excretion were increased in diabetic mice. Moreover, albuminuria strongly correlated with urinary CTGF excretion (R = 0.83, P < 0.0001). Increased CTGF expression was also demonstrated in type 2 diabetic ob/ob mice, which points to a causal relationship between diabetes and CTGF and thus argues against a role of STZ in this process. The observed relationship of podocyte and urinary CTGF to markers of DN suggests a pathogenic role of CTGF in the development of DN.

Amyloid↗
PubMed1987

Structure and development of the glomerular capillary wall and basement membrane.

The renal glomerular epithelium, Bowman's capsule, and tubule originate from a condensate of mesenchymal cells induced to undergo epithelial differentiation by a branch of the uretic bud. These nephrogenic cells aggregate and begin synthesizing the basement membrane molecules collagen type IV, heparan sulfate proteoglycans, and laminin as shown by immunofluorescence microscopy. Soon, the primitive nephron is invaginated by mesenchymal cells that establish the glomerular endothelium. Electron microscopy, metabolic labeling, and immunocytochemical techniques show that the endothelium and epithelium of early stage glomeruli each synthesize a basement membrane that appears to fuse, giving rise to the glomerular basement membrane (GBM). As development progresses, however, bulk GBM biosynthesis by the endothelium greatly diminishes or ceases. In contrast, GBM assembly by the epithelial podocytes continues and segments of new GBM appear beneath developing foot processes. In vivo labeling experiments with anti-laminin antibodies have shown that this new GBM derived from podocytes is subsequently spliced into existing GBM as capillary loop diameters expand. Molecular mechanisms for basement membrane fusion or splicing are not presently known but may involve partial enzymatic digestion and specific binding interactions among GBM components. The developing glomerular capillary wall, which filters plasma from very early stages, becomes decreasingly permeable to perfused macromolecules such as ferritin or immunoglobulin as the glomerulus matures. Evidence from immunolabeling studies showing that some monoclonal IgGs bind to the GBM only at specific developmental stages also indicates that temporal biochemical changes take place during GBM assembly. Such changes could include molecular rearrangement during basement membrane fusion and splicing and/or enzymatic and compositional modifications during maturation of the filtration barrier.

Animals↗
PubMed1996

Cellular localization of low-molecular-weight kininogen and bradykinin B2 receptor mRNAs in human kidney.

Kininogen is the precursor of the kinin peptide, which binds to kinin receptors and mediates a broad spectrum of physiological effects. To understand the function of kinin in the kidney, we have identified the cellular localization of the human low-molecular-weight (LMW) kininogen and bradykinin B2 receptor mRNAs in the human kidney by in situ hybridization histochemistry. Kininogen mRNA was found in the juxtaglomerular cells, mesangial areas, epithelium of parietal and visceral (podocytes) layers of Bowman's capsule, proximal and distal tubules, thin and thick segments of Henle's loop, collecting ducts, and the endothelial cells of the blood vessels. B2 receptor mRNA was colocalized with kininogen mRNA in the kidney except the podocytes. The most intense signals were observed in the distal tubules and collecting ducts for both kininogen and B2 receptor mRNAs. No signals were observed in the interstitial cells and macula densa. Control sections did not stain with either the kininogen or B2 receptor sense riboprobe. A Northern blot showed that the expression of LMW kininogen is in the liver and the kidney. Reverse transcription-polymerase chain reaction Southern blot showed expression of B2 receptor mRNA in the endothelial cells, renal proximal tubular cells, and kidney. Our results show the sites of action of kinin in the human kidney and provide further insight into the physiological role of the kallikrein-kinin system on renal function.

Base Sequence↗
PubMed2001

Glomerular laminin isoform transitions: errors in metanephric culture are corrected by grafting.

Glomerular basement membrane (GBM) assembly and maturation are marked by the replacement of laminin-1 (containing alpha 1-, beta 1-, and gamma 1-chains) with laminin-11 (consisting of alpha 5-, beta 2-, and gamma 1-chains). Similarly, the alpha 1- and alpha 2-chains of type IV collagen are replaced by collagen alpha 3-, alpha 4-, and alpha 5(IV)-chains. The cellular origins of these molecules and mechanisms for isoform removal and substitution are unknown. To explore glomerular laminin isoform transitions in vitro, we assessed metanephric organ cultures. Standard culture conditions do not support endothelial cell differentiation, and glomerular structures that form in vitro are avascular. Nevertheless, extensive podocyte development occurs in these cultures, including the formation of foot processes and assembly of a GBM-like matrix. Here, we show that the podocyte-specific markers, glomerular epithelial protein 1 and nephrin, which are normally expressed in capillary loop stage glomeruli in vivo, are also expressed by glomerular figures that form in organ culture. However, the GBM-like segments that form in vitro do not undergo normal laminin isoform switching. Instead, both laminin alpha 1- and alpha 5-chains are present, as is the beta 1-chain, but not beta 2. When avascular organ-cultured kidneys are grafted into anterior eye chambers, however, kidney-derived angioblasts establish extensive vasculature by 6 days, and glomeruli are lined by endothelial cells. We evaluated embryonic day 12 (E12) vascular endothelial growth factor receptor (Flk1)-lacZ kidneys that had first been grown in organ culture for 6--7 days and then grafted into wild-type mice. Correct laminin isoform substitution occurred and correlated with the appearance of endothelial cells expressing Flk1. Our findings indicate that endothelial cells, and/or factors present in the circulation, mediate normal GBM laminin isoform transitions in vivo.

Amino Acid Sequence↗