Jamshid Khoshnoodi

Nephrin strands contribute to a porous slit diaphragm scaffold as revealed by electron tomography

Nephrin is a key functional component of the slit diaphragm, the structurally unresolved molecular filter in renal glomerular capillaries. Abnormal nephrin or its absence results in severe proteinuria and loss of the slit diaphragm. The diaphragm is a thin extracellular membrane spanning the approximately 40-nm-wide filtration slit between podocyte foot processes covering the capillary surface. Using electron tomography, we show that the slit diaphragm comprises a network of winding molecular strands with pores the same size as or smaller than albumin molecules, as demonstrated in humans, rats, and mice. In the network, which is occasionally stratified, immunogold-nephrin antibodies labeled individually detectable globular cross strands, about 35 nm in length, lining the lateral elongated pores. The cross strands, emanating from both sides of the slit, contacted at the slit center but had free distal endings. Shorter strands associated with the cross strands were observed at their base. Immunolabeling of recombinant nephrin molecules on transfected cells and in vitrified solution corroborated the findings in kidney. Nephrin-deficient proteinuric patients with Finnish-type congenital nephrosis and nephrin-knockout mice had only narrow filtration slits that lacked the slit diaphragm network and the 35-nm-long strands but contained shorter molecular structures. The results suggest the direct involvement of nephrin molecules in constituting the macromolecule-retaining slit diaphragm and its pores.

Nephrin Promotes Cell-Cell Adhesion through Homophilic Interactions

Nephrin is a type-1 transmembrane protein and a key component of the podocyte slit diaphragm, the ultimate glomerular plasma filter. Genetic and acquired diseases affecting expression or function of nephrin lead to severe proteinuria and distortion or absence of the slit diaphragm. Here, we showed by using a surface plasmon resonance biosensor that soluble recombinant variants of nephrin, containing the extracellular part of the protein, interact with each other in a specific and concentration-dependent manner. This molecular interaction was increased by twofold in the presence of physiological Ca(2+)concentration, indicating that the binding is not dependent on, but rather promoted by Ca(2+). Furthermore, transfected HEK293 cells and an immortalized mouse podocyte cell line overexpressing full-length human nephrin formed cellular aggregates, with cell-cell contacts staining strongly for nephrin. The distance between plasma membranes at the nephrin-containing contact sites was shown by electron microscopy to be 40 to 50 nm, similar to the width of glomerular slit diaphragm. The cell contacts could be dissociated with antibodies reacting with the first two extracellular Ig-like domains of nephrin. Wild-type HEK293 cells were shown to express slit diaphragm components CD2AP, P-cadherin, FAT, and NEPH1. The results show that nephrin molecules exhibit homophilic interactions that could promote cellular contacts through direct nephrin-nephrin interactions, and that the other slit diaphragm components expressed could contribute to that interaction.

N-Linked Glycosylation Is Critical for the Plasma Membrane Localization of Nephrin

The expression pattern, subcellular localization, and the role of glycosylation of the human nephrin was examined in transfected cells. Stable cell lines, constitutively expressing a full-length human nephrin cDNA construct, were generated from transfected immortalized mouse podocytes (IMP) and a human embryonic kidney cell line (HEK-293). Immunofluorescence confocal microscopy of transfected cells showed plasma membrane localization of the recombinant nephrin. Immunoblotting showed that the recombinant nephrin expressed in transfected cell lines migrated as a double band with a molecular weight of 185 kD. When cells were treated with the N-glycosylation inhibitor, tunicamycin, the molecular weight of nephrin was decreased to a single immunoband of 150 kD, indicating that the shift in the electrophoretic migration of nephrin is due to N-linked carbohydrate moieties. It was further shown that this glycosylation process is highly sensitive to inhibition by tunicamycin, which is a naturally occurring antibiotic, leading to retention of nonglycosylated nephrin molecules in the endoplasmic reticulum. It was concluded that N-glycosylation of nephrin is crucial for its proper folding and thereby plasma membrane localization; therefore, inhibition of this process might be an important factor in the onset of pathogenesis of some acquired glomerular diseases.

Mechanism of Chain Selection in the Assembly of Collagen IV A PROMINENT ROLE FOR THE α2 CHAIN

Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the α1(IV) and α2(IV) NC1 domains. We show that the differential affinity of α2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the α1(IV) NC1 domain, we conclude that the α2(IV) chain plays a regulatory role in directing chain composition in the assembly of (α1)2α2 triple-helical molecule. Detailed crystal structure analysis of the [(α1)2α2]2 NC1 hexamer and sequence alignments of the NC1 domains of all six α-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a β-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.

Congenital nephrotic syndromes

Many acquired and familial renal diseases in man lead to kidney dysfunction and nephrotic syndrome. These diseases share a common pathological fate in the form of glomerular dysfunction and proteinuria. Classification of the disease is difficult because the onset of pathological appearance in congenital nephrotic syndrome (CNS) varies considerably. Recently, classification has been aided by applying molecular genetics to identify genes involved in the pathogenesis of proteinuria. Light has also been shed on the biology and mechanisms of glomerular filtration and the molecular pathogenesis of CNS.

Unraveling the Molecular Make-Up of the Glomerular Podocyte Slit Diaphragm

Recent discoveries in podocyte proteins involved in the renal filtration barrier have shed new light on the ultrastructure of the kidney filter and pathogenesis of proteinuria. The identification of nephrin, a component of the slit diaphragm, and the intracellular slit diaphragm associated proteins CD2AP and podocin has demonstrated the existence of proteins that directly contribute to a functional kidney filter. Mutations in the genes for these three proteins result in proteinuria and nephrotic syndrome, and these proteins are also likely to be involved more generally in the pathomechanisms of proteinuria. This new knowledge has been promoted particularly through the powerful methods of molecular genetics and molecular biology. In this minireview, we present the recent progress in research of the podocyte slit diaphragm.

Monoclonal Antibodies to Human Nephrin

Nephrin is a 180-200-kDa transmembrane protein of the immunoglobulin superfamily. In the kidney, nephrin localizes to the slit diaphragm (SD) between interdigitating podocyte foot processes and mutations in the nephrin gene cause congenital nephrotic syndrome. In addition to this rare genetic disorder, recent reports indicate that nephrin is more generally involved in the pathogenesis of glomerular disease. In this report, we describe production and characterization of mouse monoclonal antibodies to human nephrin, and discuss their applications. Recombinant human nephrin variants were produced in both prokaryotic and eukaryotic expression systems and purified proteins were used in an immunization protocol. A total of 16 antibodies were characterized for their reactivity with the nephrin by using ELISA, Western blots, immunoprecipitation and immunostaining of frozen and formaldehyde-fixed paraffin embedded tissue sections. The antibody epitopes were mapped using a variety of recombinant human nephrin variants. The detailed screening and characterization proved to be essential in order to find the most suitable antibody for each application. These antibodies will find wide use in studies of human nephrin and its involvement in kidney disease.