Kunimasa Yan

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.

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.

Defective Trafficking of Nephrin Missense Mutants Rescued by a Chemical Chaperone

The nephrin gene (NPHS1) is mutated in congenital nephrotic syndrome of the Finnish type. Most mutations found in non-Finnish patients are missense mutations. The most common consequence of missense mutations in congenital nephrotic syndrome is a defect in intracellular transport and retention of the mutant proteins in the endoplasmic reticulum (ER), possibly as a result of misfolding and unfavored conformation. Because sodium 4-phenylbutyrate has been shown to function as a chemical chaperone and to correct the cellular trafficking of several mislocalized or misfolded mutant plasma membrane proteins, the effects of this compound on the missense mutants identified in patients with congenital nephrotic syndrome of the Finnish type were investigated. This study was performed using human embryonic kidney 293 cells stably expressing wild-type or missense nephrin mutants trapped in the ER. Immunofluorescence microscopy and cell surface biotinylation showed that treatment with sodium 4-phenylbutyrate rescued several of the missense mutants from the ER to the cell surface. All of the rescued mutants were found to be able to interact with Neph1. Furthermore, their tyrosine phosphorylation was rapidly induced by clustering with anti-nephrin antibodies, suggesting that the rescued mutants may be functionally intact.

Characterization of the interactions of the nephrin intracellular domain.

Nephrin is a signalling cell–cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3‐kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein–protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3‐kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S‐transferase–nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell–cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.

The Increase of Memory T Cell Subsets in Children with Idiopathic Nephrotic Syndrome

Two-color and three-color flow cytometry was carried out to determine whether the memory T cells (CD45RO+ T cells) play a major role in lymphocyte dysfunction of 26 children with idiopathic nephrotic syndrome (INS). The INS patients were divided into three groups: (1) 10 patients who were not receiving glucocorticoid hormone (GCH) and were suffering from acute nephrotic state were referred to as N1; (2) 8 patients who were in remission maintained by GCH therapy alone were referred to as N2; (3) 8 patients who were free of GCH therapy for at least 4 months were referred to as N3. Group N1 demonstrated a significant increase in the percentage of CD45RO+CD4+ T cells and CD45RO+CD8+ T cells (p < 0.05) compared with 11 controls, and these subsets were noted to have a tendency to decrease to control levels in groups N2 and N3. Furthermore, interleukin-2 receptor-α expressed subsets in CD45RO+CD4+ T cells (CD45RO+CD4+CD25+ T cells) were also increased only in group N1 (p < 0.02). A similar tendency of absolute counts was observed in these subsets. These results suggest that activated memory T cells reflect lymphocyte dysfunction at initial onset or relapse in INS children.

Expression of galectin-1, a new component of slit diaphragm, is altered in minimal change nephrotic syndrome

Nephrin is an essential structural component of the glomerular slit diaphragm (SD), a highly organized intercellular junction that constitutes the ultrafiltration barrier of the kidney. Recent studies have identified two additional nephrin-interacting SD proteins (NEPH1 and NEPH2), suggesting that the zipper-like pattern of the SD is formed through complex intra- and intermolecular interactions of these proteins. However, the complexity of the SD structure suggests that additional SD components remain to be discovered. In this study, we identified galectin-1 (Gal-1) as a new component of the SD, binding to the ectodomain of nephrin. Using dual-immunofluorescence and confocal microscopy and dual-immunoelectron microscopy, we found Gal-1 colocalizing with the ectodomain of nephrin at the SD in normal human kidney. By immunoprecipitation and surface plasmon resonance, we confirmed a direct molecular interaction between Gal-1 and nephrin. Moreover, recombinant Gal-1 induced tyrosine phosphorylation of the cytoplasmic domain of nephrin and activation of the extracellular signal-regulated kinase 1/2 in podocytes. We also showed that podocytes are a major site of biosynthesis of Gal-1 in the glomerulus and that the normal expression patterns and levels of Gal-1 are altered in patients with minimal change nephrotic syndrome. Finally, in puromycin aminonucleoside-induced rat nephrosis, an apparent reduction in the levels of Gal-1 and nephrin around the onset of heavy proteinuria was also revealed. Our data present Gal-1 as a new extracellular ligand of nephrin localized at the glomerular SD, and provide further insight into the complex molecular organization, interaction, and structure of the SD, which is an active site of intracellular signaling necessary for podocyte function.

Charge Selective Function in Childhood Glomerular Diseases

The charge selectivity (CS) function in human renal disease has not been unequivocally demonstrated to date. However, the clearance ratio of IgA to IgG may be theoretically useful in estimating CS in humans, since IgA and IgG have similar sizes and tertiary structures, but distinct isoelectric points (3.5–5.5 [IgA] and 4.5–9.0 [IgG]), and Stokes-Einstein radius: 61 Å (IgA) and 49–60 Å (IgG). Two-dimensional electrophoresis with the following immunoblotting revealed that the considerably anionic portion (isoelectric points [pI] <4.0) of IgA, visible in serum, was absent in the urine in steroid-sensitive nephrotic syndrome (SSNS) but present in the same during IgA nephropathy (IgAN) and membranoproliferative glomerulonephritis (MPGN). A latex assay revealed the CS index (CSI) was significantly low in patients with podocyte disease (group A), including SSNS, focal and segmental glomerulosclerosis (FSGS) and Finnish-type congenital nephrotic syndrome (FCNS), but high in those with Alport syndrome (AS), IgAN, Henoch-Schönlein purpura nephritis (HSPN), and MPGN (group B). The linear regression analysis of the IgA size selectivity index (IgA SSI; clearance ratio of IgA to transferrin) and SSI (clearance ratio of IgG to transferrin), which represents the clearance ratio of IgA to IgG referring to the transferrin clearance, revealed the influence of the charge more accurately. Indeed, the slope of the regression lines of IgA SSI (y) to SSI (x) were concluded to be y = 0.39x (group A) and y = 1.05x (group B), respectively. These results suggested that the charge selective barrier among podocyte diseases (group A) is preserved to some degree, but lost in cases of nephritis and AS (group B).

Variable expression of podocyte-related markers in the glomeruloid bodies in Wilms tumor

Several podocyte‐related markers are organized to express in glomerular differentiation. However, whether expression of them is virtually synchronized and a reliable indicator of the state of differentiation is unknown. The present study investigated, by immunohistochemistry, the divergent expression of several podocyte markers in the improperly differentiated glomeruloid bodies from four cases of Wilms tumors. The glomeruloid bodies were classified into immature (IGB) or mature forms (MGB) based on morphology and epithelial features. Podocytes in IGB expressed WT1, synaptopodin, podocalyxin, and nephrin, and their expression was stronger in MGB. In contrast, Pax2 was strong in IGB and diminished in MGB. p27 was first expressed in MGB. The expression pattern in each molecule mimics normal glomerulogenesis. Podocytes in MGB showed persistent expression of bcl‐2 and cytokeratin with synaptopodin, podocalyxin, and nephrin by serial section, a finding unusual for normal glomerulogenesis. Moreover, parietal cells in MGB also occasionally expressed these podocyte markers. The ultrastructure revealed that podocytes in MGB showed tight junctions without foot process formations, which indicated incomplete differentiation. These results suggest that a set of podocyte differentiation markers are occasionally diversely expressed, and raise the possibility that expression of these markers is insufficient to determine the state of terminal differentiation in podocytes.

Reevaluation of glomerular charge selective protein-sieving function

Recently, disorders of the slit diaphragm have been considered as major causes of proteinuria in renal disease and the charge barrier function of the glomerular capillary wall has been given less attention. We evaluated the charge selectivity index (CSI) in 40 patients with podocyte disease (PD), 75 with chronic glomerulonephritis (CGN), and 8 with Dent disease, to reexamine the charge barrier function. We evaluated CSI in Dent disease because the urinary protein profile in Dent disease was assumed to be a concentrate of a normal glomerular filtrate. CSI was defined as the renal clearance ratio between IgA and IgG. CSI values (mean ± SD) in the CGN and PD groups and in Dent disease were 1.12 ± 0.25, 0.42 ± 0.31, and 0.16 ± 0.06, respectively, suggesting that the charge barrier function was defective in the CGN group and of reduced capacity in the PD group. The results suggest that functional interactions between the slit diaphragm and the glomerular basement membrane exist, and that a slit diaphragm disorder is accompanied by a decrease in the charge barrier function in PD, as argued by the conventional hypothesis.

Regulation of the C3 production by gamma interferon from peripheral blood T cells in patients with membranoproliferative glomerulonephritis and poststreptococcal acute glomerulonephritis.

This study assessed the regulatory role of peripheral blood T cells in the C3 production in patients with poststreptococcal glomerulonephritis and branoproliferative glomerulonephritis. Peripheral blood T cells from patients at various stages of disease were cultured and the supernatants tested for gamma interferon (IFN-gamma) content and the capacity to stimulate C3 production by HuH-7 cells. Supernatants from patients with membranoproliferative glomerulonephritis and from convalescent patients with poststreptococcal glomerulonephritis significantly stimulated the C3 production; the degree of stimulation correlated with the IFN-gamma content of the supernatants. Similar results were obtained using recombinant IFN-gamma. In both cases, the effect was blocked by the addition of anti-IFN-gamma monoclonal antibody to the cultures. Interleukin 2 and interleukin 6 levels in supernatants from T cell cultures of patients and controls were essentially the same. In summary, IFN-gamma plays a regulatory role in C3 production by human hepatoma cell lines.