Nephrin is a major component of the glomerular filtration barrier. Mutations in the nephrin gene (NPHS1) are responsible for congenital nephrotic syndrome of the Finnish type (NPHS1). Nephrin was at first thought to be podocyte specific, but recent studies have suggested that nephrin is also expressed in nonrenal tissues such as pancreas and CNS. We studied the expression of nephrin in human and porcine tissues at different stages of development and correlated these findings to clinical characteristics of NPHS1 children. Immunofluorescence staining and Western blotting were used to detect nephrin protein in frozen tissue samples. Polyclonal antibodies against the intracellular part of nephrin were used in these analyses. In situ hybridization was used to detect nephrin mRNA in specimens from normal human subjects and patients with NPHS1. Nephrin protein was not detected in nonrenal tissues obtained from human and porcine fetuses, newborns, and infants. Likewise, nephrin mRNA expression was not observed outside kidney glomerulus in normal or NPHS1 children. The phenotype analysis of NPHS1 children with severe nephrin gene mutations supported the findings in the tissue expression studies and revealed no impairment of the neurologic, testicular, or pancreatic function in a great majority of the patients. The studies suggest that nephrin has no major clinical significance outside the kidney.

Diabetic nephropathy (DN), a major cause of ESRD, is undoubtedly multifactorial and is caused by environmental and genetic factors. To identify a genetic basis for DN susceptibility, we are collecting multiplex DN families in the Caucasian (CA) and African-American (AA) populations for whole genome scanning and candidate gene analysis. A candidate gene search of diabetic sibs discordantly affected, concordantly affected and concordantly unaffected for DN was performed with microsatellite markers in genomic regions suspected to harbor nephropathy susceptibility loci. Regions examined were at human chromosome 10p,10q (orthologous to the rat renal susceptibility Rf-1 locus), and at NPHS1 (nephrin), CD2AP, Wilms tumor (WT1), and NPHS2 (podocin) loci. Linkage analyses were conducted using model-free methods (SIBPAL, S.A.G.E.) for AA, CA, and the combined sample. Allele frequencies and the identity by descent sharing were estimated separately for AA and CA, and race was included as a covariate in the final linkage analysis. To date, we have collected 212 sib pairs from 46 CA and 50 AA families. The average age of diabetes onset was 46.8 yr versus 36.2 yr for CA and 39.5 yr versus 40.2 yr for AA, in males versus females respectively. Genotyping data were available for 106 sib pairs (43 CA, 63 AA) from 27 CA (44% male probands) and 38 AA families (43% male probands). Average AA and CA sibship size was 2.73. Singlepoint and multipoint linkage analyses indicate that marker D10S1654 on chromosome 10p is potentially linked to DN (CA only multipoint P = 4 x 10(-3)). Interestingly, the majority of the linkage evidence derives from the CA sib pairs. We are now adding sib pairs and increasing marker density on chromosome 10. We have excluded linkage with candidate regions for nephrin, CD2AP, WT1, and podocin in this sample. In conjunction with previous reports, our data support evidence for a DN susceptibility locus on chromosome 10.

Study of podocyte biology has been hampered by limitations in available experimental models that both recapitulate the in vivo phenotypes of this cell and can be readily and specifically manipulated at the molecular level. Transgenic manipulation of the podocyte represents one approach that might circumvent these limitations. The purpose of this study was to identify a promoter-enhancer that would direct the expression of transgenes in a podocyte-specific manner. The nephrin (Nphs1) promoter was considered a good candidate for this purpose, because nephrin was thought to be expressed exclusively in podocytes. Two independent BAC clones that contained the murine Nphs1 gene were identified. An 8.3-kb and a 5.4-kb fragment containing the 5' flanking promoter sequence were identified and characterized. Two constructs were generated by placing a bacterial lacZ reporter with a nuclear localization signal under the control of these two DNA fragments. Mice transgenic for both constructs were generated. Using a chemiluminescence assay, beta-galactosidase activity significantly above control was detected only in tissue homogenates of kidneys and brain of transgenic mice. In X-gal stained sections of transgenic adult kidneys, only podocyte nuclei expressed beta-galactosidase. In adult brain examined by tissue sectioning, beta-galactosidase activity was confined to a discrete area in the medulla. Identical patterns of beta-galactosidase expression were observed in multiple transgenic founders, suggesting that the expression pattern observed was independent of the site of transgene integration. The developmental expression of beta-galactosidase in transgenic embryos was also analyzed. Transgenes regulated by this promoter should be useful for studying the biology of gene products that regulate podocyte phenotype and function.

Glomerular podocytes are highly specialized terminally differentiated cells that act as a filtration barrier in the kidney. Mutations in the actin-binding protein, α-actinin 4 (ACTN4), are linked to focal segmental glomerulosclerosis (FSGS), a chronic kidney disease characterized by proteinuria. Aberrant activation of NF-κB pathway in podocytes is implicated in glomerular diseases including proteinuria. We demonstrate here that stable knockdown of ACTN4 in podocytes significantly reduces TNFα-mediated induction of NF-κB target genes, including IL-1β and NPHS1, and activation of an NF-κB-driven reporter without interfering with p65 nuclear translocation. Overexpression of ACTN4 and an actin binding-defective variant increases the reporter activity. In contrast, an FSGS-linked ACTN4 mutant, K255E, which has increased actin binding activity and is predominantly cytoplasmic, fails to potentiate NF-κB activity. Mechanistically, IκBα blocks the association of ACTN4 and p65 in the cytosol. In response to TNFα, both NF-κB subunits p65 and p50 translocate to the nucleus, where they bind and recruit ACTN4 to their targeted promoters, IL-1β and IL-8. Taken together, our data identify ACTN4 as a novel coactivator for NF-κB transcription factors in podocytes. Importantly, this nuclear function of ACTN4 is independent of its actin binding activity in the cytoplasm.

Idiopathic nephrotic syndrome (iNS) with resistance or dependence to steroids is a common disease in children but in spite of an increasing clinical impact its pathogenesis is unknown. We screened for the presence of circulating antibodies against glomerular (podocytes, mesangium) and tubular cells (tubular epithelia) a cohort of 60 children with iNS including 8 patients with a familial trait of iNS or with proven mutation of NPHS1-NPHS2 and 12 with good sensitivity to steroids. Positive sera were found in 8 cases, all belonging to the category without familial trait/molecular defects. The targets of antibodies were characterized with Western blot and MALDI-Mass utilizing beta-hexyl cell extracts separated with two-dimensional electrophoresis. In all cases antibodies of the IgM class were directed against ATP synthase beta chain alone (4 cases) or in combination with actin (3 cases); one child presented IgG against aldose reductase. The clinical picture was nephrotic syndrome with steroid resistance or dependence and variable cyclosporin sensitivity; 3 patients developed end stage renal failure. The basic pathology picture was focal segmental glomerulosclerosis (FSGS) in 4 cases and mesangial proliferative glomerulonephrites with deposition of IgM in 2. Overall, patients with circulating auto-antibodies could not be readely differentiated on clinical grounds with the exception of 3 children who developed positivity for antinuclear antibodies during the follow-up. Affinity-purified IgM from one patient who underwent plasmapheresis for therapeutical pourposes (but not from a normal pool) induced proteinuria in Sprague-Dawley rats and concomitant human IgM deposition within glomeruli. This is the first report of circulating anti-actin/ATP synthase beta chain antibodies in a subset of patients with iNS. Both pathological significance and clinical impact given by the presence of these antibodies and the relationship with other conditions such as lupus-erythematosus, characterized by their presence, must be defined.

OBJECTIVE

The NPHS1 gene product, nephrin, is a crucial component of the glomerular filtration barrier preventing proteinuria and previously assumed to be kidney-specific. The aim of this study was to describe the expression of nephrin mRNA and protein in human pancreas as well as identify the nephrin-expressing cell types.

METHODS

RNA dot blot, reverse transcriptase-polymerase chain reaction, sequencing, immunoblotting and dual immunofluorescence were used for the characterisation of nephrin in the pancreas.

RESULTS

Except for the kidney, the pancreas was found to be the only tissue expressing nephrin as screened with a human tissue RNA dot blot. The expression was verified with reverse transcriptase-polymerase chain reaction and by sequencing nephrin from a human pancreatic complementary DNA library. Nephrin antibody in immunoblot detected a 165,000 M(r) protein in the pancreas. Dual immunofluorescence showed that nephrin was specifically localised in the beta cells of the islets of Langerhans. There was no overlap with glucagon, somatostatin, or the ductal cell marker cytokeratin 19.

CONCLUSIONS

These data show that nephrin is a novel molecule of pancreatic beta cells.

BACKGROUND

Mutations in the NPHS1 and NPHS2 genes are among the main causes of early-onset and familial steroid resistant nephrotic syndrome respectively. This study was carried out to assess the frequencies of mutations in these two genes in a cohort of Pakistani pediatric NS patients.

METHODS

Mutation analysis was carried out by direct sequencing of the NPHS1 and NPHS2 genes in 145 nephrotic syndrome (NS) patients. This cohort included 36 samples of congenital or infantile onset NS cases and 39 samples of familial cases obtained from 30 families.

RESULTS

A total of 7 homozygous (6 novel) mutations were found in the NPHS1 gene and 4 homozygous mutations in the NPHS2 gene. All mutations in the NPHS1 gene were found in the early onset cases. Of these, one patient has a family history of NS. Homozygous p.R229Q mutation in the NPHS2 gene was found in two children with childhood-onset NS.

CONCLUSIONS

Our results show a low prevalence of disease causing mutations in the NPHS1 (22% early onset, 5.5% overall) and NPHS2 (3.3% early onset and 3.4% overall) genes in the Pakistani NS children as compared to the European populations. In contrast to the high frequency of the NPHS2 gene mutations reported for familial SRNS in Europe, no mutation was found in the familial Pakistani cases. To our knowledge, this is the first comprehensive screening of the NPHS1 and NPHS2 gene mutations in sporadic and familial NS cases from South Asia.

BACKGROUND

Podocyte proteins are involved in the pathogenesis of glomerular kidney disease (GKD). However, there is little information on messenger RNA (mRNA) expression patterns of B7-1 and NPHS1 in urinary sediment of patients with GKD. The objective of this study was to analyse the gene expression of B7-1 in urinary sediment and correlate it with the expression of podocyte-specific genes in patients with GKD.

METHODS

Adult patients with proliferative and non-proliferative GKD, proteinuria and stable renal function, were included. A group of healthy subjects was used to determine normal levels of urinary markers and to obtain reference RNA. Biochemical, clinical and experimental procedures included measurement of creatinine level and total urinary protein, renal biopsy, identification of urinary podocytes, gene expression analysis of B7-1, NPHS1, NPHS2 and SyNPO genes and urinary B7-1 protein analysis by enzyme-linked immunosorbent assay.

RESULTS

Between June 2006 and November 2009, 69 patients with GKD (median age: 46 ± 15 years, 64% men) and 14 healthy subjects (median age: 34 ± 12 years, 43% men) were included. In both groups, urinary mRNA levels of B7-1 and NPHS1 were significantly higher in patients with GKD compared to healthy subjects (P = 0.050 and P = 0.008, respectively). Regarding GKD subtypes, patients with focal segmental glomerulosclerosis (FSGS), but not patients with minimal change disease (MCD), had a significantly higher mRNA expression of B7-1 and NPHS1 than healthy subjects (P = 0.012 and P = 0.030, respectively). Patients with MCD had a significantly lower NPHS1 mRNA expression than patients with FSGS (P = 0.012). The B7-1:NPHS1 urinary mRNA ratio was significantly higher in patients with MCD compared with patients with FSGS (P = 0.027).

CONCLUSIONS

mRNA expression analysis of B7-1 and NPHS1 in urinary sediment may be useful to differentiate between different histologic subtypes of GKD, particularly between MCD and FSGS.

The state-of-the-art cultured podocyte is conditionally immortalized by expression of a temperature-sensitive mutant of the SV40 large-T antigen. These cultures proliferate at 33°C and differentiate at 37°C into arborized cells that more closely resemble in vivo podocytes. However, the degree of resemblance remains controversial. In this study, several parameters were measured in podocyte cell lines derived from mouse (JR, KE), human (MS), and rat (HK). In all lines, the quantities of NEPH1 and podocin proteins and NEPH1 and SYNPO mRNAs were comparable to glomeruli, while synaptopodin and nephrin proteins and NPHS1 and NPHS2 mRNAs were <5% of glomerular levels. Expression of Wilms' tumor-1 (WT1) mRNA in mouse lines was comparable to glomeruli, but rat and human lines expressed little WT1. Undifferentiated human and mouse lines had similar proliferation rates that decreased after differentiation, while the rate in rat cells remained constant. The motility of different lines varied as measured by both general motility and wound-healing assays. The toxicity of puromycin aminonucleoside was MS ∼ JR>>) KE, and of doxorubicin was JR ∼ KE>> MS, while HK cells were almost unaffected. Process formation was largely a result of contractile action after formation of lamellipodia. These findings demonstrate dramatic differences in marker expression, response to toxins, and motility between lines of podocytes from different species and even between similarly-derived mouse lines.

BACKGROUND

Infants with congenital nephrotic syndrome (CNS) develop severe nephrotic syndrome that is resistant to medical therapy, and bilateral nephrectomy is recommended toward the end of the first year of life followed by renal replacement therapy. CNS infants in New Zealand have been observed to exhibit a different course to those with the typical Finnish mutation.

METHODS

A database of CNS children at our center was retrospectively examined. All cases diagnosed between 1975 and 2011 were reviewed. Demographic data, clinical features, genetic mutations, treatment, and outcome were extracted from clinical records.

RESULTS

Thirty-five patients with CNS, 23 children of Maori descent, and 12 Caucasians . Fourteen had died of either bacterial sepsis or intracranial thrombosis. Maori children had displayed a highly variable and protracted timeline to end-stage renal disease (ESRD) with median renal survival of 30 years versus 0.7 years in Caucasian patients. Mutation analysis of NPHS1 showed a founder mutation in the Maori population.

CONCLUSIONS

Congenital nephrotic syndrome in New Zealand Maori children exhibit a different clinical course to Caucasian children and have a mutation that was first described in this ethnic group.

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene-rich D19S208-COX7A1 locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain -20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements.

Idiopathic focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome in pediatric and adult patients. Most children with FSGS do not respond to any form of therapy and progress to end-stage renal disease (ESRD). FSGS reoccurs in the transplanted kidney in approximately one third of initial transplants and in a substantially higher percentage of subsequent transplants once FSGS has recurred in an earlier transplant. Thus, FSGS is a disease with substantial morbidity. Over the past several years, the incidence of FSGS in adults and children appears to be increasing, particularly in certain racial groups and ethnic populations. Several recent studies in adult and pediatric patients suggest that the incidence of FSGS is increasing particularly in the black population. In addition, some studies have also demonstrated a more rapid progression of FSGS to ESRD in black patients compared to other ethnic groups. Racial and ethnic background is likely to have a substantial influence on the incidence and progression of FSGS in children and adults. It is likely that specific genes or a combination of genes influence the different clinical manifestations of FSGS in racial and ethnic groups. Genetic mutations in NPHS1 gene, which encodes nephrin, have been found to cause congenital nephrotic syndrome. Genetic mutations in the NPHS2 gene, which encodes podocin, recently have been shown to be strongly associated with a recessive form of steroid-resistant nephrotic syndrome. Mutations in the ACTN4 gene that encodes actinin 4 has also been associated with familial nephrotic syndrome. A role for ACE polymorphisms in the progression of FSGS has been found in some studies. Future investigations to identify polymorphisms that influence the development of FSGS, the progression of FSGS, and the response to therapy will greatly improve understanding of the pathogenesis and management of FSGS.

BACKGROUND

Congenital nephrotic syndrome (CNS), defined as heavy proteinuria, hypoalbuminemia, hyperlipidemia and edema presenting in the first 0-3 months of life, may be caused by congenital syphilis, toxoplasmosis, or congenital viral infections (such as cytomegalovirus). However, the majority of CNS cases are caused by monogenic defects of structural proteins that form the glomerular filtration barrier in the kidneys. Since 1998, an increasing number of genetic defects have been identified for their involvements in the pathogenesis of CNS, including NPHS1, NPHS2, WT1, PLCE1, and LAMB2.

METHODS

We searched databases such as PubMed, Elsevier and Wanfang with the following key words: congenital nephrotic syndrome, proteinuria, infants, neonate, congenital infection, mechanism and treatment; and we selected those publications written in English that we judged to be relevant to the topic of this review.

RESULTS

Based on the data present in the literature, we reviewed the following topics: 1) Infection associated CNS including congenital syphilis, congenital toxoplasmosis, and congenital cytomegalovirus infection; 2) genetic CNS including mutation of NPHS1 (Nephrin), NPHS2 (Podocin), WT1, LAMB2 (Laminin-β2), PLCE1 (NPHS3); 3) Other forms of CNS including maternal systemic lupus erythematosus, mercury poisoning, renal vein thrombosis, neonatal alloimmunization against neutral endopeptidase.

CONCLUSIONS

At present, the main challenge in CNS is to identify the cause of disease for individual patients. To make a definitive diagnosis, with the exclusion of infection-related CNS and maternal-associated disorders, pathology, family history, inheritance mode, and other accompanying congenital malformations are sometimes, but not always, useful indicators for diagnosing genetic CNS. Next-generation sequencing would be a more effective method for diagnosing genetic CNS in some patients, however, there are still some challenges with next-generation sequencing that need to be resolved in the future.

Podocytes are a major component of the glomerular blood filtration barrier, and alterations to the morphology of their unique actin-based foot processes (FP) are a common feature of kidney disease. Adjacent FP are connected by a specialized intercellular junction known as the slit diaphragm (SD), which serves as the ultimate barrier to regulate passage of macromolecules from the blood. While the link between SD dysfunction and reduced filtration selectivity has been recognized for nearly 50 years, our understanding of the underlying molecular circuitry began only 20 years ago, sparked by the identification of NPHS1, encoding the transmembrane protein nephrin. Nephrin not only functions as the core component of the extracellular SD filtration network but also as a signaling scaffold via interactions at its short intracellular region. Phospho-regulation of several conserved tyrosine residues in this region influences signal transduction pathways which control podocyte cell adhesion, shape, and survival, and emerging studies highlight roles for nephrin phospho-dynamics in mechanotransduction and endocytosis. The following review aims to summarize the last 5 years of advancement in our knowledge of how signaling centered at nephrin directs SD barrier formation and function. We further provide insight on promising frontiers in podocyte biology, which have implications for SD signaling in the healthy and diseased kidney.

The sieving of plasma components occurs in the kidney through the glomerular capillary wall. This filter is composed of three layers: endothelium, glomerular basement membrane (GBM), and podocyte foot processes connected by slit diaphragms. Defects in this barrier lead to proteinuria and nephrotic syndrome. Previously, defective GBM was regarded to be responsible for proteinuria. However, recent work on genetic diseases has indicated that podocytes and the slit diaphragm are crucial in restricting protein leakage. Congenital nephrotic syndrome of the Finnish type (NPHS1) is caused by mutations in a novel NPHS1 gene, which encodes for a cell adhesion protein, nephrin. This protein is synthesized by podocytes, and seems to be a major component of the slit diaphragm. In severe NPHS1, lack of nephrin leads to missing slit diaphragm. The role of nephrin in acquired kidney diseases remains unknown. In addition to nephrin, other podocyte proteins (podocin, alpha-actinin-4, CD2AP, FAT) have recently been identified and associated with the development of proteinuria. It seems that the slit diaphragm and its interplay with the podocyte cytoskeleton is critical for the normal sieving process, and defects in one of these components easily lead to proteinuria.

While constant basal levels of macroautophagy/autophagy are a prerequisite to preserve long-lived podocytes at the filtration barrier, MTOR regulates at the same time podocyte size and compensatory hypertrophy. Since MTOR is known to generally suppress autophagy, the apparently independent regulation of these two key pathways of glomerular maintenance remained puzzling. We now report that long-term genetic manipulation of MTOR activity does in fact not influence high basal levels of autophagy in podocytes either in vitro or in vivo. Instead we present data showing that autophagy in podocytes is mainly controlled by AMP-activated protein kinase (AMPK) and ULK1 (unc-51 like kinase 1). Pharmacological inhibition of MTOR further shows that the uncoupling of MTOR activity and autophagy is time dependent. Together, our data reveal a novel and unexpected cell-specific mechanism, which permits concurrent MTOR activity as well as high basal autophagy rates in podocytes. Thus, these data indicate manipulation of the AMPK-ULK1 axis rather than inhibition of MTOR as a promising therapeutic intervention to enhance autophagy and preserve podocyte homeostasis in glomerular diseases.Abbreviations: AICAR: 5-aminoimidazole-4-carboxamide ribonucleotide; AMPK: AMP-activated protein kinase; ATG: autophagy related; BW: body weight; Cq: chloroquine; ER: endoplasmic reticulum; ESRD: end stage renal disease; FACS: fluorescence activated cell sorting; GFP: green fluorescent protein; i.p.: intra peritoneal; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NPHS1: nephrosis 1, nephrin; NPHS2: nephrosis 2, podocin; PLA: proximity-ligation assay; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; RPTOR/RAPTOR: regulatory associated protein of MTOR, complex 1; RFP: red fluorescent protein; TSC1: tuberous sclerosis 1; ULK1: unc-51 like kinase 1.

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.

Congenital nephrotic syndrome of the Finnish type (NPHS1) is associated with the rapid development of glomerular and tubulointerstitial fibrosis. Here we measured morphologic and molecular changes in the peritubular capillaries of the kidney in patients with NPHS1. Immunohistochemical analysis for the endothelial cell marker CD31 showed marked narrowing and a moderate but significant reduction in peritubular capillary density, especially in areas of increased collagen I and alpha-smooth muscle actin content. No evidence of endothelial-mesenchymal transformation was found. There was increased expression (up to 43-fold) of hypoxia inducible factor-1alpha suggesting tubulointerstitial hypoxia. Double-labeling for CD31 and vimentin showed small foci of peritubular capillary loss and tubular cell damage. While the amount of intercellular adhesion molecule-1 was upregulated in endothelial cells, other adhesion molecules were only modestly expressed. Vascular endothelial growth factor expression was reduced by up to half and decreased endothelial progenitor cell marker CD34 expression indicated lack of vascular repair. Our results suggest that hypoxia in the tubulointerstitium caused by hypoperfusion of glomerular and tubulointerstitial capillaries and rarefaction of the latter may be important for the rapid progression of fibrosis in the kidneys of patients with NPHS1.

Nephrotic syndrome (NS) is a renal disease characterized by heavy proteinuria, hypoalbuminemia, edema and hyperlipidemia. Its presentation within the first 3 months of life or in multiple family members suggests an underlying inherited cause. To determine the frequency of inherited NS, 62 cases (representing 49 families with NS) from Saudi Arabia were screened for mutations in NPHS1, NPHS2, LAMB2, PLCE1, CD2AP, MYO1E, WT1, PTPRO and Nei endonuclease VIII-like 1 (NEIL1). We detected likely causative mutations in 25 out of 49 families studied (51%). We found that the most common genetic cause of NS in our cohort was a homozygous mutation in the NPHS2 gene, found in 11 of the 49 families (22%). Mutations in the NPHS1 and PLCE1 genes allowed a molecular genetic diagnosis in 12% and 8% of families, respectively. We detected novel MYO1E mutations in three families (6%). No mutations were found in WT1, PTPRO or NEIL1. The pathogenicity of novel variants was analyzed by in silico tests and by genetic screening of ethnically matched control populations. This is the first report describing the molecular genetics of NS in the Arabian Peninsula.

BACKGROUND

The role of glomerular capillary endothelium in the pathophysiology of nephrotic kidney diseases is poorly known. We analysed the glomerular endothelial lesions in kidneys from patients with congenital nephrotic syndrome of the Finnish type (NPHS1). The disorder is caused by a genetic defect in a major podocyte slit diaphragm protein, nephrin. It manifests as nephrotic syndrome soon after birth and leads to glomerular sclerosis in early childhood.

METHODS

The glomerular capillary and endothelial cell lesions in NPHS1 kidneys nephrectomized at infancy were studied by electron and light microscopy, immunohistochemistry and cytokine antibody array.

RESULTS

Mesangial expansion and capillary obliteration were evident in practically all NPHS1 glomeruli. No thrombus formation was detected by fibrin staining. Electron microscopy revealed endothelial blebs (endotheliosis). The endothelial fenestration and the attachment of endothelial cells to the basement membrane were, however, quite normal. This fits to the abundant expression of a vascular endothelial growth factor (VEGF) and its transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), in NPHS1 glomer- uli. The proliferative activity of the intracapillary cells was modest and no apoptosis was detected. The expression of an endothelial adhesion molecule, intercellular adhesion molecule 1 (ICAM-1) and several chemokines was upregulated in NPHS1 glomeruli as compared to adult control kidneys. The recruitment of leukocytes carrying ligands for the major endothelial adhesion molecules, however, was modest in the mesangial area of NPHS1 glomeruli.

CONCLUSIONS

The findings indicate that the glomerular endothelium is quite resistant to the nephrotic state in NPHS1 kidneys and underscores the importance of mesangial cells in the progression of glomerular sclerosis.

Glomerulosclerosis is a common pathological finding that often progresses to renal failure. The mechanisms of chronic kidney disease progression are not well defined, but may include activation of numerous vasoactive and inflammatory pathways. We hypothesized that podocytes are susceptible to filtered plasma components, including hormones and growth factors that stimulate signaling pathways leading to glomerulosclerosis. Gα12 couples to numerous G-protein-coupled receptors (GPCRs) and regulates multiple epithelial responses, including proliferation, apoptosis, permeability and the actin cytoskeleton. Herein, we report that genetic activation of Gα12 in podocytes leads to time-dependent increases in proteinuria and glomerulosclerosis. To mimic activation of Gα12 pathways, constitutively active Gα12 (QL) was conditionally expressed in podocytes using Nphs2-Cre and LacZ/floxed QLα12 transgenic mice. Some QLα12(LacZ+/Cre+) mice developed proteinuria at 4-6 months, and most were proteinuric by 12 months. Proteinuria increased with age, and by 12-14 months, many demonstrated glomerulosclerosis with ultrastructural changes, including foot process fusion and both mesangial and subendothelial deposits. QLα12(LacZ+/Cre+) mice showed no changes in podocyte number, apoptosis, proliferation or Rho/Src activation. Real-time PCR revealed no significant changes in Nphs1, Nphs2, Cd2ap or Trpc6 expression, but Col4a2 message was increased in younger and older mice, while Col4a5 was decreased in older mice. Confocal microscopy revealed disordered collagen IVα1/2 staining in older mice and loss of α5 without changes in other collagen IV subunits. Taken together, these studies suggest that Gα12 activation promotes glomerular injury without podocyte depletion through a novel mechanism regulating collagen (α)IV expression, and supports the notion that glomerular damage may accrue through persistent GPCR activation in podocytes.

The novel gene NPHS1 is defective in the patients with congenital nephrotic syndrome of the Finnish type (CNF) leading to abnormal expression of the respective protein product nephrin in glomerular cells. CNF patients are treated with early nephrectomy and renal transplantation, but about 20% show recurrence of nephrotic syndrome (NS). We used indirect immunofluorescence microscopy and immunoblotting and an ELISA assay to search for circulating autoantibodies to nephrin, the protein defect in CNF patient kidneys. In serial serum samples gathered before and after recurrence of NS, we show an increased antibody titer to nephrin prior to the NS episode and a subsequent drop in antibody level after its successful treatment and reactivity of the high titer sera with glomeruli in indirect immunofluorescence microscopy as well. The results show that the transplantation treatment introduces a neoantigen inducing production of autoantibodies, which may be pathogenic for perturbation of the function of the glomerular filtration barrier.

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1, NPHS2, CD2AP, and ACTN4, can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or alpha-actinin-4 mRNA by using reconstructed RNA interference vector--psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of alpha-actinin-4, whereas alpha-actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

BACKGROUND

Many factors contribute to the pathogenesis of glomerular proteinuria, but no exact molecular mechanisms are known to date. The recently reported protein nephrin, encoded by the NPHS1 gene, appears to be crucial for the integrity of the glomerular filtration barrier.

METHODS

Immunohistochemistry was used to detect possible changes in glomerular nephrin, and a new proteinuria-associated protein expression was developed in various diagnostic groups of human kidney biopsies.

RESULTS

In normal control kidney, antibodies to intracellular and extracellular nephrin domain showed a typical podocyte pattern of reactivity, while the 18C7 antibody to a normally inaccessible proteinuria-associated epitope was negative. Instead, strong glomerular positivity by 18C7 was seen in membranous glomerulonephropathy, membranoproliferative glomerulonephritis, systemic lupus erythematosus and cryoglobulinemic nephritis, while with antibodies to either intracellular or extracellular nephrin domains, a down-regulation in nephrin expression pattern was shown.

CONCLUSIONS

Unmasking or de novo expression of distinct glomerular proteins may be an important feature reflecting the pathophysiological events in these diseases with altered glomerular permeability, while only mild changes in the slit diaphragm protein nephrin appear to take place.