Nephrin Gene Research Articles

Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy.

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivariate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

Induction of nephrin gene expression by selective cooperation of the retinoic acid receptor and the vitamin D receptor

Nephrin is a key molecule involved in the structure and function of the slit diaphragm in the glomerulus. We previously reported that all-trans retinoic acid (ATRA) and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induced expression of nephrin in murine podocytes. In this report, we investigated roles of the retinoic acid receptor (RAR), the retinoid X receptor (RXR) and the vitamin D receptor (VDR) in the regulation of the nephrin gene. Reporter podocytes were treated with agonists and/or antagonists of RAR, RXR or VDR, and activities of the nephrin gene promoter, the retinoic acid response element (RARE) and the vitamin D response element (VDRE) were evaluated. Expression of nephrin in podocytes was up-regulated by ATRA and 1,25(OH)(2)D(3). The nephrin gene promoter was also activated by these agents, which was mediated by RAR and VDR, but unexpectedly, not by RXR. ATRA-triggered, RAR-mediated activation of the nephrin gene promoter was not suppressed by the VDR antagonist. Similarly, ATRA-induced activation of RARE was not inhibited by the VDR antagonist. In contrast, the 1,25(OH)(2)D(3)-triggered, VDR-mediated activation of the nephrin gene promoter was significantly suppressed by the RAR antagonist, but not by RXR antagonists. Interestingly, 1,25(OH)(2)D(3)-induced activation of VDRE was not inhibited by the RAR antagonist. These results suggested selective cooperation of RAR and VDR in the regulation of the nephrin gene, i.e. (1) ATRA induces nephrin gene expression via RAR independently of RXR and VDR and (2) 1,25(OH)(2)D(3) induces nephrin gene expression via selective cooperation of RAR and VDR, which is independent of RXR.

Analysis of the intronic single nucleotide polymorphism rs#466452 of the nephrin gene in patients with diabetic nephropathy

We present the analysis of an intronic polymorphism of the nephrin gene and its relationship to the development of diabetic nephropathy in a study of diabetes type 1 and type 2 patients. The frequency of the single nucleotide polymorphism rs#466452 in the nephrin gene was determined in 231 patients and control subjects. The C/T status of the polymorphism was assessed using restriction enzyme digestions and the nephrin transcript from a kidney biopsy was examined. Association between the polymorphism and clinical parameters was evaluated using multivariate correspondence analysis. A bioinformatics analysis of the single nucleotide polymorphism rs#466452 suggested the appearance of a splicing enhancer sequence in intron 24 of the nephrin gene and a modification of proteins that bind to this sequence. However, no change in the splicing of a nephrin transcript from a renal biopsy was found. No association was found between the polymorphism and diabetes or degree of renal damage in diabetes type 1 or 2 patients. The single nucleotide polymorphism rs#466452 of the nephrin gene seems to be neutral in relation to diabetes and the development of diabetic nephropathy, and does not affect the splicing of a nephrin transcript, in spite of a splicing enhancer site.

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients.

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.

Amelioration of anti-cancer agent adriamycin-induced nephrotic syndrome in rats by Wulingsan (Gorei-San), a blended traditional Chinese herbal medicine

Anti-cancer agent adriamycin (ADR) has demonstrated high anti-tumor efficacy. However, its use in chemotherapy has been limited largely due to its diverse toxicities, including renal toxicity, such as nephrotic syndrome with proteinuria. Podocyte injury leads to glomeruli proteinuria. Wulingsan (WLS) is a blended traditional Chinese herbal medicine specifically used for various kidney diseases. In the present study, we found that a water extract of WLS (480mg/kg, p.o.,×28 days) reduced ADR-induced increase in urine protein excretion, plasma total cholesterol and triglyceride, and decrease in plasma total protein and albumin in rats. Furthermore, the results of electron microscopy demonstrated suppression by WLS of ADR-induced increase in width of foot process, increase in surface density and decrease in volume density. These results suggest that WLS ameliorates ADR-induced proteinuria and podocyte injury. Gene analysis results demonstrated a suppression of renal overexpression of nephrin mRNA and protein by WLS. Radioimmunoassay showed that WLS suppressed ADR-induced increased renal angiotensin II content in rats. Thus our results demonstrate that WLS ameliorates ADR-induced nephrotic syndrome in rats possibly by suppressing ADR-induced hyperactivity of renal renin–angiotensin system to modulate renal nephrin gene expression, thereby protecting podocyte from injury.

Basic science meets clinical medicine: identification of a CD2AP-deficient patient

Recent years have witnessed an explosion of research into the molecular basis of glomerular disease resulting in nephrotic- range urinary protein leak using both human genetics and animal models. Löwik et al. describe the first case report of an early-onset nephrotic syndrome presenting in conjunction with a homozygous CD2AP mutation. These data demonstrate the convergence between basic and clinical approaches and their potential to transform our understanding of the pathogenetic mechanisms underlying human glomerular disease.

Contribution of Proteoglycans Towards the Integrated Functions of Renal Glomerular Capillaries: A Historical Perspective

Since the original colloidal iron tracer study by Farquhar and Palade almost 50 years ago,1 the permeability characteristics of mammalian glomerular capillary wall have been the subject of extensive investigations.2,3 In the ensuing 2 to 3 decades, various studies were performed to delineate whether glomerular basement membrane or the slit diaphragm of the capillary wall is the primary filtration barrier.4,5 Amid this contentious issue, in the mid to late 1970s various clearance and additional tracer studies indicated that the glomerular capillary wall behaves as a size- as well as charge-selective barrier, and the latter biophysical properties were attributed to the anionic sites within the basement membrane that are enriched with sulfated proteoglycans (PGs).6,7,8,9 The article by Harvey et al10 in this issue of The American Journal of Pathology suggests that agrin, a basement sulfated proteoglycan, imparts an electronegative charge to the glomerular basement membrane (GBM), but it may not be responsible for its charge-selective permeability properties. In light of these findings, we deemed it necessary to revisit the tracer work performed during the past few decades and reconcile with recent observations made in various mutant mice keeping in perspective the complex ultrastructure and integrated functions of the glomerular capillary wall.11

Differential Expression of Nephrin According to Glomerular Size in Early Diabetic Kidney Disease

Diabetic nephropathy (DN) is clinically characterized by proteinuria. Many studies tried to demonstrate a relationship between proteinuria and changes in nephrin in various forms of glomerular diseases including DN, but the results are not consistent. Glomerular hypertrophy occurs in DN, yet hypertrophy does not develop in all glomeruli concurrently. For investigation of the differences in nephrin expression according to glomerular size, glomeruli were isolated from 10 control and 10 streptozotocin-induced diabetic rats at 6 wk after the induction of diabetes by a sieving technique using sieves with pore sizes of 250, 150, 125, and 75 microm. Glomeruli then were classified into large glomeruli (LG; on the 125-microm sieve) and small glomeruli (SG; on the 75-microm sieve) groups. Glomerular volumes were determined using an image analyzer, and mRNA and protein expression was determined by real-time PCR and Western blot, respectively. The mean volumes of diabetic LG (1.51 +/- 0.06 x 10(6) microm(3)) and control LG (1.37 +/- 0.05 x 10(6) microm(3)) were significantly higher than those of diabetic SG (0.94 +/- 0.03 x 10(6) microm(3)) and control SG (0.87 +/- 0.03 x 10(6) microm(3); P < 0.01). Nephrin mRNA expression was significantly reduced in the diabetic LG group compared with the diabetic SG and control glomeruli groups (P < 0.05). In contrast, nephrin mRNA expression was significantly higher in the diabetic SG group compared with the diabetic LG and control glomeruli groups (P < 0.05). Even after correction for 18s rRNA and Wilms' tumor-1 mRNA expression, the differences in nephrin mRNA expression remained significant. The expression of nephrin protein showed a similar pattern to the mRNA expression. In conclusion, these data suggest that the nephrin gene is differentially expressed according to glomerular size. Furthermore, more hypertrophied glomeruli with lesser nephrin expression may be responsible for albuminuria in the early stage of DN.

Snail, a transcriptional regulator, represses nephrin expression in glomerular epithelial cells of nephrotic rats

Snail is a DNA-binding molecule that plays a pivotal role in regulating cell adhesion and epithelial to mesenchymal transition. Visceral epithelial cells (podocytes) in kidney glomeruli form a sophisticated cell–cell junction called a slit diaphragm that prevents the loss of plasma protein during ultrafiltration. Nephrin, located in the slit diaphragm and critical for maintaining the integrity of this structure, belongs to the class of cell adhesion molecules of the immunoglobulin super-family. As previously reported, the transcriptional activity of nephrin is a determinant of the integrity of the slit diaphragm in puromycin aminonucleoside (PAN) nephrosis rats. Here, we examined the role of Snail in nephrin expression. In accordance with the downregulation of nephrin in PAN nephrosis rats, Snail was upregulated in vivo and its DNA-binding activity was stimulated in injured podocytes while normal podocytes did not express Snail. An in vitro study demonstrated that Snail bound to E-box motifs in a specific segment of the rat nephrin gene repressed the transcription of nephrin and downregulated nephrin protein. We also found that the expression level of Snail in injured podocytes was regulated by GSK3, which is known to phosphorylate Snail and induce its proteolysis. Pharmacological in vitro and in vivo inhibition studies of GSK3 suggested that GSK3 activity decreased in injured podocytes and this change partially contributed to the decrease in nephrin and increase in Snail and proteinuria. Concordantly, we found that Wnt-2 was upregulated in injured podocytes and activated the Wnt canonical pathway. As the Wnt canonical pathway inactivates GSK3, it is likely that Wnt-2 accounts for the accumulation of Snail in injured podocytes. In conclusion, Snail is a key molecule, which perturbs the integrity of the slit diaphragm through transcriptional repression of nephrin under pathological conditions. Wnt-GSK3 pathway participates in this mechanism.

Recovery and maintenance of nephrin expression in cultured podocytes and identification of HGF as a repressor of nephrin

Cultured podocytes easily lose expression of nephrin. In this report, we developed optimum media for recovery and maintenance of nephrin gene expression in murine podocytes. Using reporter podocytes, we found that activity of the nephrin gene promoter was enhanced by DMEM/F12 or alpha-MEM compared with RPMI-1640. In any of these basal media, addition of 1,25-dihydroxyvitamin D(3), all-trans-retinoic acid or dexamethasone significantly increased activity of the nephrin promoter. The effects of the supplemental components were synergistic, and the maximum activation was achieved by DMEM/F12 supplemented with three agents. This culture medium was designated as vitamin D(3), retinoic acid and dexamethasone-supplemented DMEM/F12 (VRADD). In reporter podocytes that express nephrin, VRADD induced activation of the nephrin gene promoter up to 60-fold. Even in podocytes that have lost nephrin expression during multiple passages, expression of nephrin mRNA was dramatically recovered by VRADD. However, VRADD caused damage of podocytes in prolonged cultures, which was avoided in the absence of dexamethasone (designated as VRAD). VRAD maintained expression of nephrin for extended periods, which was associated with the differentiated phenotype of podocytes. Using the VRAD-primed podocytes, we revealed that expression of nephrin mRNA as well as nephrin promoter activity was suppressed by a putative dedifferentiation factor of podocytes, hepatocyte growth factor.

Transcriptional suppression of nephrin in podocytes by macrophages: Roles of inflammatory cytokines and involvement of the PI3K/Akt pathway

Expression of nephrin, a crucial component of the glomerular slit diaphragm, is downregulated in patients with proteinuric glomerular diseases. Using conditionally immortalized reporter podocytes, we found that bystander macrophages as well as macrophage-derived cytokines IL-1β and TNF-α markedly suppressed activity of the nephrin gene promoter in podocytes. The cytokine-initiated repression was reversible, observed on both basal and inducible expression, independent of Wilms’ tumor suppressor WT1, and caused in part via activation of the phosphatidylinositol-3-kinase/Akt pathway. These results indicated a novel mechanism by which activated macrophages participate in the induction of proteinuria in glomerular diseases.

A pilot study on genetic variation in purine-rich elements in the nephrin gene promoter in type 2 diabetic patients

Diabetic nephropathy (DN) is one of the major complications of type 2 diabetes and is associated with coronary disease. Nephrin, a protein mainly expressed in glomeruli, is decreased in DN and other kidney diseases. Since insulin levels are misregulated in type 2 diabetes, a possible connection between DN and its decreased nephrin expression could be the presence of regulatory elements responsive to insulin in the nephrin gene (NPHS1) promoter region. In this work, using bioinformatic tools, we identified a purine-rich GAGA element in the nephrin gene promoter and conducted a genomic study in search of the presence of polymorphisms in this element and its possible association with DN in type 2 diabetic patients. We amplified and sequenced a 514 bp promoter region of 100 individuals and found no genetic variants in the purine-rich GAGA-box of the nephrin gene promoter between groups of patients with diabetes type 2 with and without renal and coronary complications, control patients without diabetes and healthy controls.

Evaluación de las estrategias terapéuticas en el síndrome nefrótico congénito tipo finlandés

Congenital nephrotic syndrome of the Finnish type (CNFS) is caused by mutations in the nephrin gene. This disease manifests as massive proteinuria, gross peripheral edema, and ascites during the first weeks of life. In the last few years the prognosis has improved due to new treatment strategies: antiproteinuria drugs, intensive nutrition, nephrectomy, dialysis, and renal transplantation. The aim of this study was to determine the impact of these therapeutic measures. We performed a descriptive retrospective epidemiological study of 12 patients diagnosed with CNSF between January 1985 and August 2005. We included patients aged less than 14 years old with massive proteinuria and generalized edema during the neonatal period, a large placenta (> 25 % of birth weight), and normal glomerular filtration rate during the first 6 months of life, in whom other causes of congenital nephrotic syndrome were ruled out. The diagnosis was established after a median period of 17 days (range 6-30). The most commonly used treatments were albumin infusions (91.7 %), angiotensin-converting enzyme inhibitors (66.7 %), and indomethacin (58.3 %). Dialysis was started in 58.3 %, at a median age of 3.76 years (2.81-7.6). The main complication was acute peritonitis (85.7 %). Renal transplantation was performed in 58.3 % of the patients; of these, 71.4 % have normal renal function after a median follow-up of 3.73 years (0.8-6.3). The median plasma albumin level during the pretransplant period was 0.17 g/dL (0.12-0.28). Plasma cholesterol and triglyceride levels decreased significantly after renal transplantation (p = 0.043). Fifty percent of the patients achieved adequate height and weight for their age and gender. Mortality was 33.3 %. Antiproteinuria drugs and intensive nutritional therapy improve clinical control and delay the start of dialysis and renal transplantation, increasing the probability of success.

Screening and identification of substances that regulate nephrin gene expression using engineered reporter podocytes

Downregulation of nephrin in podocytes leads to development of proteinuria in human and experimental kidney diseases. However, little is understood about pathophysiologic substances that regulate nephrin expression. In this report, we established conditionally immortalized reporter podocytes REPON for sensitive, continuous monitoring of nephrin gene expression. A murine podocyte cell line harboring a temperature-sensitive simian virus 40 large T antigen was stably transfected with a gene encoding secreted alkaline phosphatase (SEAP) under the control of the 5.4 or 8.3 kb nephrin gene promoter. The established reporter cells REPON5.4 and REPON8.3 were exposed to various pathophysiologic substances, and culture media were subjected to SEAP assay to identify regulators of nephrin gene expression. Among the bioactive substances tested, three physiological ligands of nuclear receptors including all-trans-retinoic acid, 1,25-dihydroxyvitamin D3, and dexamethasone significantly activated the nephrin gene promoter in a dose-dependent manner. These effects were observed in both REPON5.4 and REPON8.3 and were associated with upregulation of nephrin mRNA. The effects of these substances were synergistic, and the maximum effect was observed by combination of three agents. In contrast, inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha as well as phorbol ester significantly downregulated the activity of the nephrin promoter as well as nephrin gene expression. These results elucidated the bidirectional regulation of nephrin by distinct pathophysiologic substances and may provide molecular bases for explaining how proteinuria is induced under pathologic situations and why some ligands for nuclear receptors have the anti-proteinuric potential.

Transcriptional Regulation of Nephrin Gene by Peroxisome Proliferator–Activated Receptor-γ Agonist

The renoprotective potential of the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) agonist pioglitazone was explored in an immune model of progressive nephropathy, passive Heymann nephritis (PHN), compared with that of an angiotensin II receptor antagonist, taken as standard therapy for renoprotection. PHN rats received orally vehicle, pioglitazone (10 mg/kg twice daily), or candesartan (1 mg/kg twice daily) from months 2 to 8. Pioglitazone reduced proteinuria as effectively as candesartan and limited renal functional and structural changes. Kidneys from untreated PHN rats showed lower nephrin mRNA and protein than controls, both restored by pioglitazone. The effect was seen both early and late during the course of the disease. Whether the antiproteinuric effect of pioglitazone could be due to its effect on nephrin gene transcription also was investigated. HK-2 cells were transfected with plasmids that harbor the luciferase gene under portions (2-kb or 325-bp) of human nephrin gene promoter that contain putative peroxisome proliferator-responsive elements (PPRE) and incubated with pioglitazone (10 muM). Transcriptional activity of luciferase gene was highly increased by pioglitazone, with the strongest expression achieved with the 325-bp fragment. Increase in luciferase activity was prevented by bisphenol A diglycidyl ether, a PPAR-gamma synthetic antagonist. Electrophoretic mobility shift assay experiments showed a direct interaction of PPAR/retinoid X receptor heterodimers to PPRE present in the enhancer region of the nephrin promoter. In conclusion, pioglitazone exerts an antiproteinuric effect in immune-mediated glomerulonephritis as angiotensin II receptor antagonist does. Enhancement of nephrin gene transcription through specific PPRE in its promoter discloses a novel mechanism of renoprotection for PPAR-gamma agonists.

Association of Nephrin Gene Polymorphisms With Type 2 Diabetes in a Japanese Population: The Funagata Study

Nephrin is a major component of the glomerular filtration barrier, and its expression was at first thought to be specific to kidney glomerular podocytes (1). However, it was later found in nonrenal tissues, such as the pancreas (2–4), as well. Several studies (2,3,5) have shown the expression of nephrin in human pancreatic islet cells. Therefore, nephrin may play some roles in the pancreatic islet and, thus, may be involved in the pathophysiology leading to diabetes. We here examined the association of the nephrin gene polymorphisms with type 2 diabetes. The associations of three single nucleotide polymorphisms (SNPs) (C294T, −61C/G, and C2289T) of the nephrin gene with diabetes were examined using two (first and second) sample sets (diabetes, n = 72 and 31, respectively; impaired glucose tolerance [IGT], n = 75 and 77, respectively; and normal glucose tolerance [NGT], n = 227 and 244, respectively) from the cohort population of the Funagata Study, a Japanese community-based study (6). The study conditions were the same as those in our previous report (7). Three SNPs in the nephrin gene (C to T at nucleotide position 294 in exon 3 from the translation intitiation codon ATG (C294T) (JSNP database ID: IMS-JST 035656), C to G in intron 5 at nucleotide position 61 upstream from the intron 5/exon 6 splicing site …

Association of Nephrin Gene Polymorphisms With Type 2 Diabetes in a Japanese Population

Nephrin is a major component of the glomerular filtration barrier, and its expression was at first thought to be specific to kidney glomerular podocytes (1). However, it was later found in nonrenal tissues, such as the pancreas (2–4), as well. Several studies (2,3,5) have shown the expression of nephrin in human pancreatic islet cells. Therefore, nephrin may play some roles in the pancreatic islet and, thus, may be involved in the pathophysiology leading to diabetes. We here examined the association of the nephrin gene polymorphisms with type 2 diabetes. The associations of three single nucleotide polymorphisms (SNPs) (C294T, −61C/G, and C2289T) of the nephrin gene with diabetes were examined using two (first and second) sample sets (diabetes, n = 72 and 31, respectively; impaired glucose tolerance [IGT], n = 75 and 77, respectively; and normal glucose tolerance [NGT], n = 227 and 244, respectively) from the cohort population of the Funagata Study, a Japanese community-based study (6). The study conditions were the same as those in our previous report (7). Three SNPs in the nephrin gene (C to T at nucleotide position 294 in exon 3 from the translation intitiation codon ATG (C294T) (JSNP database ID: IMS-JST 035656), C to G in intron 5 at nucleotide position 61 upstream from the intron 5/exon 6 splicing site …

Muscular dystonia and athetosis in six patients with congenital nephrotic syndrome of the Finnish type (NPHS1)

Congenital nephrotic syndrome of the Finnish type (NPHS1, CNF) is an autosomal recessively inherited disease occurring due to mutations in the nephrin gene (NPHS1). Two main Finnish mutations exist: Fin-major and minor, which both cause a lack of nephrin and absence of the slit diaphragm between the podocytes. This leads to severe proteinuria, nephrotic syndrome and infections, and without dialysis or renal transplantation, death in infancy. Between 1984 and 2003, six (8.6%) of the 70 NPHS1 patients diagnosed at our institution had, in addition to their renal disease, similar neurological symptoms. All six showed a severe dyskinetic cerebral palsy-like syndrome with dystonic features, athetosis and a hearing defect. The neurological symptoms became apparent during their 1st year of life and were diagnosed before 11 months of age. MRI showed increased signal intensity in T2-weighted images in the globus pallidus area. No mitochondrial gene mutations explaining the neurological symptoms were found, nor did external neurological complications explain them when compared with 29 NPHS1 control patients. Four children died at an early age: two during dialysis and two shortly after renal transplantation. Two are still alive with a functioning graft. Both have severe motor defects, but are mentally active and social.

Expression of nephrin by human pancreatic islet endothelial cells

The islet microcirculation has morphological characteristics resembling those of renal glomeruli. Transcription of the nephrin gene, a highly specific barrier protein of the slit diaphragm of podocyte foot processes, has been reported in the pancreas, although its cellular localisation and function remain to be defined. In this study, we purified and characterised microvascular endothelial cells (MECs) isolated from human islets and investigated the expression and distribution of nephrin on these cells. Human islet MECs were extracted and purified using anti-CD105-coated immunomagnetic beads and their endothelial characteristics were confirmed by expression of classical endothelial markers and basal high-level expression of intercellular adhesion molecule-1 and TNF-alpha-inducible vascular cell adhesion molecule-1. Nephrin expression was assessed by immunofluorescence, flow cytometric analysis and western blotting on cell lysates, as well as by RT-PCR. Immunofluorescence studies detected nephrin in a fine, punctate, diffuse pattern on cultured islet MECs, and also in human pancreatic islet sections. In both cases nephrin colocalised with endothelial markers. TNF-alpha treatment induced a marked reduction and redistribution of the protein in one or multiple aggregates. Nephrin expression was confirmed by flow cytometry, western blotting and RT-PCR studies. In contrast, nephrin could not be detected at the protein or mRNA level in human macro- and microvascular cells from other sites. Nephrin is expressed at protein and mRNA levels in islet microendothelium, supporting the hypothesis that islet MECs exhibit distinctive morphological characteristics that indicate functional specialisation of potential pathophysiological importance.

Nephrin expression and three‐dimensional morphogenesis of the Xenopus pronephric glomus

Nephrin (NHPS1) encodes a transmembrane protein of approximately 1,200 amino acids that plays a critical role in podocyte slit-diaphragm formation and the development of functional mammalian glomerular filtration barriers. In humans and mice with congenital defects in the nephrin gene, the glomerular filtration barrier is defective and protein leakage into the kidney filtrate causes a life-threatening proteinuria. This protein also plays an essential role in the formation of the stellate cells of the Drosophila Malpighian tubules. In this report, the sequence and expression of a Xenopus ortholog of nephrin is described using both conventional and novel three-dimensional (3D) visualization methodologies. Xenopus nephrin encodes a protein of 1,238 amino acids and is expressed at high levels in the forming pronephric kidney glomus, the equivalent of the mammalian glomerulus. Expression commences at stage 25 and is specific to the pronephric glomus up until at least tadpole feeding stages. Two-color fluorescent whole-mount in situ analysis of nephrin expression allowed the 3D shape of the glomus to be imaged and contrasted to the pronephric tubules throughout its morphogenesis. Confocal data processing pipelines were established to generate both volumetric and surface models of the developing pronephros, and a Web-based visualization system was used to generate dynamic and manipulable models of the forming nephric organs. This system allows simple on-line morphometric analysis of the developing pronephric components. As in fish embryos, the glomera first form laterally then migrate medially as the pronephros matures. Unlike in the zebrafish, in Xenopus, this migration stops short of complete fusion of the two glomera at the midline, but a nephrin-positive glomeral nexus does form anteriorly and links the two structures from stage 38 onward.