Heikki Ahola

Nephrin localizes at the podocyte filtration slit area and is characteristically spliced in the human kidney

Defects in the newly reported gene NPHS1 in chromosome 19 cause the massive proteinuria of Finnish type congenital nephrotic syndrome (CNF). Together with its gene product, nephrin, NPHS1 is providing new understanding of the pathophysiological mechanisms of glomerular filtration. Here we show the characteristic splicing of NPHS1 mRNA in the normal and CNF kidneys and localize nephrin exclusively in the glomerulus and to the filtration slit area by light and immunoelectron microscopy. These results indicate that nephrin is a new protein of the interpodocyte filtration slit area with a profound role in the pathophysiology of the filtration barrier.

Changes in the Expression of Nephrin Gene and Protein in Experimental Diabetic Nephropathy

Diabetic nephropathy is a major complication of diabetes leading to thickening of the glomerular basement membrane, glomerular hypertrophy, mesangial expansion, and overt renal disease. The pathophysiologic mechanisms of diabetic nephropathy remain poorly understood. Nephrin is a recently found podocyte protein crucial for the interpodocyte slit membrane structure and maintenance of an intact filtration barrier. Here we have assessed the role of nephrin in two widely used animal models of diabetes, the streptozotocin model of the rat and the nonobese diabetic mouse. In both models, the expression levels of nephrin-specific mRNA as determined by real-time quantitative polymerase chain reaction increased up to two-fold during several weeks of follow-up. Immunohistochemical stainings revealed nephrin also more centrally within the glomerular tuft along with its preferential site in podocytes. Interestingly, as detected by immunoblotting, nephrin protein was also found in the urine of streptozotocin-induced rats. We conclude that nephrin is connected to the early changes of diabetic nephropathy and thus may contribute to the loss of glomerular filtration function.

Nephrin TRAP Mice Lack Slit Diaphragms and Show Fibrotic Glomeruli and Cystic Tubular Lesions

The molecular mechanisms maintaining glomerular filtration barrier are under intensive study. This study describes a mutant Nphs1 mouse line generated by gene-trapping. Nephrin, encoded by Nphs1, is a structural protein of interpodocyte filtration slits crucial for formation of primary urine. Nephrin(trap/trap) mutants show characteristic features of proteinuric disease and die soon after birth. Morphologically, fibrotic glomeruli with distorted structures and cystic tubular lesions were observed, but no prominent changes in the branching morphogenesis of the developing collecting ducts could be found. Western blotting and immunohistochemical analyses confirmed the absence of nephrin in nephrin(trap/trap) glomeruli. The immunohistochemical staining showed also that the interaction partner of nephrin, CD2-associated protein (CD2AP), and the slit-diaphragm-associated protein, ZO-1alpha (-), appeared unchanged, whereas the major anionic apical membrane protein of podocytes, podocalyxin, somewhat punctate as compared with the wild-type (wt) and nephrin(wt/trap) stainings. Electron microscopy revealed that >90% of the podocyte foot processes were fused. The remaining interpodocyte junctions lacked slit diaphragms and, instead, showed tight adhering areas. In the heterozygote glomeruli, approximately one third of the foot processes were fused and real-time RT-PCR showed >60% decrease of nephrin-specific transcripts. These results show an effective nephrin gene elimination, resulting in a phenotype that resembles human congenital nephrotic syndrome. Although the nephrin(trap/trap) mice can be used to study the pathophysiology of the disease, the heterozygous mice may provide a useful model to study the gene dose effect of this crucial protein of the glomerular filtration barrier.

Cloning and expression of the rat nephrin homolog

Despite of the increased availability of genetically modified mouse strains, the experimental models in the rat have provided the most widely employed and versatile models for the study of renal pathophysiology and functional genetics. The identification of the human gene mutated in the congenital nephrotic syndrome of the Finnish type (NPHS1) has recently been reported, and its protein product has been termed nephrin. Here we report the molecular cloning and characterization of rat nephrin cDNA. Rat nephrin cDNA has an open reading frame of 3705 bp, shows 82% sequence identity with human nephrin cDNA, and shows characteristic rat-specific splicing variants. The translated nucleotide sequence has 89% sequence identity at the amino acid level. The signal sequence, glycosylation, and cysteine localization patterns are nearly identical to those of human nephrin. As in the human, the rat nephrin transcript is expressed in a tissue-restricted pattern. Antipeptide antibodies raised to the intracellular nephrin-specific domain identified immunoreactivity exclusively within the rat kidney glomerulus by indirect immunofluorescence. Initial results with semiquantitative reverse transcriptase-polymerase chain reaction analysis showed a remarkable down-regulation of nephrin-specific mRNA in the puromycin nephrosis of the rat.

A Novel Protein, Densin, Expressed by Glomerular Podocytes

With the recent molecular findings, the podocyte is emerging as a key cell type involved in glomerular damage, but protein complexes involved remain poorly understood. To systematically search for additional podocyte molecules interacting with nephrin, a key structural molecule of the interpodocyte filtration slit, precipitation of glomerular lysates was set out with anti-nephrin antibodies to identify members of the nephrin-associated protein complex. Proteins of the precipitate were subsequently identified with MALDI-TOF mass analysis. One of the proteins thus obtained showed identity with densin, a protein originally purified from rat forebrain postsynaptic density fraction and so far shown to be highly brain-specific. The expression of densin appeared distinctly in the glomerulus and cultured podocytes by RT-PCR. Immunoblotting studies revealed a specific band of 185 kD in brain and cultured podocytes; in human glomerulus, densin appeared as a 210-kD band. By immunocytochemistry, densin localizes in glomeruli in a podocyte-like pattern. Electron microscopic studies revealed densin localization in the slit diaphragm area. Due to its known involvement in the synaptic organization, maintenance of cell shape and polarity in nerve cells, together with its demonstrated interactions with alpha-actinin-4, densin may share the same functions in podocytes by associating with the nephrin interacting protein complex at the slit diaphragm.

Recurrence of Nephrotic Syndrome after Transplantation in CNF Is due to Autoantibodies to Nephrin

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.

Alternatively Spliced Nephrin in Experimental Glomerular Disease of the Rat

Nephrin is a novel transmembrane protein of kidney glomerular podocytes, which appears crucially important for the maintenance of the glomerular filtration barrier. According to its predicted structure, nephrin has additional roles in cell-cell adhesion and/or signal transduction. We have previously cloned the rat homologue of nephrin and described its alternatively spliced transcripts alpha and beta. In this study we examined the alterations in expression and regulation of particularly the major alternatively spliced nephrin-alpha giving rise to a variant lacking the membrane spanning domain in the puromycin nephrosis of the rat. A down-regulation of up to 78% was observed of the full length mRNA after 10 d of PAN treatment. The expression changes of nephrin-alpha followed closely the expression of the full length mRNA. Interestingly, we also found nephrin protein in urine at the peak proteinuria samples of this model. These results suggest that soluble nephrin variants may be important markers for proteinuric diseases.

Nephrin in human lymphoid tissues

Abstract.When nephrin, the protein product of NPHS1, was cloned, it was proposed to be specific for the kidney glomerular podocytes. Recently, however, new reports have emerged verifying additional nephrin expression sites, particularly the insulin-producing β cells of the pancreas, as well as the central nervous system. In this study, we demonstrate nephrin expression in lymphoid tissues, specifically the tonsil, adenoid and lymph node. Nephrin mRNA expression levels were 4-fold higher in tonsils and adenoids than in thymus or B lymphocytes, and 20-fold higher than in T lymphocytes or monocytes, as shown by quantitative RT-PCR analysis. Anti-nephrin antibodies recognised a specific 165-kDa band in lysates of tonsil and adenoid. In immunofluorescence and immunohistochemichal stainings of adenoid and lymph node sections, nephrin-positive cells were detected in the germinal centres of the lymphoid follicles in a staining pattern typical for interdigitating cells. These results indicate a definite and additional presence of nephrin in lymphoid tissue.

Interactions between Mitochondrial Proteins and Lipid Peroxidation Products in the Maintenance of the Glomerular Filtration Barrier in the in vitro Perfused Kidney

Background: The fourth complex of the mitochondrial respiratory chain, cytochrome-c oxidase (CytC) consists of thirteen both mitochondrially and nuclearly encoded subunits, which are differently regulated in proteinuric kidneys. The effect of mitochondrial involvement on proteinuria is not known. Methods: We set up an in vitro kidney perfusion model to study the direct effect of inhibitors of the mitochondrial respiratory chain, rotenone and antimycin A, on the glomerular filtration barrier by using immunohistochemistry and Northern blotting and quantitating the resulting proteinuria. Results: Rapid onset of proteinuria and characteristic changes in CytC subunits were seen in the perfused kidneys. Urinary protein excretion increased significantly in the rotenone- and antimycin-A-treated groups during perfusion. Downregulation of CytC subunits I and IV was similarly found in the groups treated with rotenone and antimycin A, while increases in the lipid peroxidation (LPO) products malondialdehyde and 4-hydroxynonenal which reflect mitochondrial damage, were observed. Conclusions: These data show rapid changes in mitochondrial proteins and induction of proteinuria in response to exposure to mitochondrial inhibitors. Together with the concomitant increase in local LPO products, these results suggest that continuous maintenance of a proper energy balance is important to maintain the glomerular filtration barrier.