Aaro Miettinen

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.

Involvement of Lipid Rafts in Nephrin Phosphorylation and Organization of the Glomerular Slit Diaphragm

NPHS1 has recently been identified as the gene whose mutations cause congenital nephrotic syndrome of the Finnish type. The respective gene product nephrin is a transmembrane protein expressed in glomerular podocytes and primarily localized to the glomerular slit diaphragm. This interpodocyte junction functions in the glomerular filtration by restricting the passage of plasma proteins into the urinary space in a size-selective manner. The functional role of nephrin in this filtration process is so far not very well understood. In this study, we show that nephrin associates in an oligomerized form with signaling microdomains, also known as lipid rafts, and that these localize to the slit diaphragm. We also show that the nephrin-containing rafts can be immunoisolated with the 27A antibody recognizing a podocyte-specific 9-O-acetylated GD3 ganglioside. In a previous study it has been shown that the in vivo injection of this antibody leads to morphological changes of the filtration slits resembling foot process effacement. Here, we report that, in this model of foot process effacement, nephrin dislocates to the apical pole of the narrowed filtration slits and also that it is tyrosine phosphorylated. We suggest that lipid rafts are important in the spatial organization of the glomerular slit diaphragm under physiological and pathological conditions.

Organ-Specific Autoimmunity and HLA-DR Antigens as Markers for β-Cell Destruction in Patients With Type II Diabetes

Islet cell antibodies (ICAs), thyrogastric antibodies, and HLA-DR antigens were determined in 204 patients with type II (non-insulin-dependent) diabetes controlled with diet and/or oral hypoglycemic agents (NIR) and in 108 age-matched patients who required insulin to control their hyperglycemia (IR). β-Cell function measured as C-peptide response to glucagon was evaluated in relation to the presence of ICAs and HLA-DR antigens. The IR patients differed from the NIR patients with respect to higher frequency of ICAs (P < .001), thyroid antibodies (P < .02), and the HLA antigen DR4 (P < .02). The highest frequency of ICAs and thyroid antibodies was observed in female insulin-treated subjects (51.2 and 46.4%). Patients who were heterozygous for HLA-DR3/DR4 showed significantly higher frequency of ICAs (P < .01) and complement-fixing ICAs (P < .001) than patients without the heterozygous form DR3/DR4. Neither the presence of ICA alone nor DR3/DR4 alone was associated with a significant impairment of β-cell function. However, when both ICA and DR3/DR4 were present in a diabetic individual, β-cell function was markedly impaired (P < .001), suggesting that both genetic and autoimmune factors are necessary to facilitate the process leading to β-cell destruction of the patients. Our findings suggest that type II diabetes is a heterogeneous disorder including at least two major subgroups, which can be further characterized by HLA-DR antigens and organ-specific antibodies

In vitro segregation of the metanephric nephron

Abstract In vitro segregation of the metanephric nephron was examined using three probes for the main segments: fluorochrome-conjugated wheat germ agglutinin (WGA) binding to the glomerular epithelial surface, an antiserum against the brush-border antigens (BB) of the proximal tubules, and an antiserum against the Tamm-Horsfall glycoprotein (TH) of the distal tubules. In vivo , these markers appeared sequentially on Days 13 to 15. The same sequence was obtained in experimental recombinants of the metanephric mesenchyme and its inductor. When the inductor was removed after a 24 hr initial transfilter contact with the mesenchyme, segregation was similarly observed after subculture of the isolated mesenchyme. Hence, the sequential, multiphase differentiation of the nephron is initiated during a short induction period.

Podocalyxin in Rat Platelets and Megakaryocytes

Podocalyxin is a membrane protein of rat podocytes and endothelial cells. It has not been described in other cell types, and no amino acid or DNA sequence data are available about it. Here we show that podocalyxin antigens are present in rat platelets and megakaryocytes. In resting platelets, the antigens are mainly intracellular but become surface exposed after thrombin stimulation, as shown by immunofluorescence and flow cytometry. By Western blotting, platelet podocalyxin has an apparent Mr of 140,000. Cytocentrifuge slides of rat bone marrow show that anti-podocalyxin antibodies recognize large polyploid cells also expressing CD62P, indicating that the cells are megakaryocytes. From a rat glomerular cDNA library we isolated a clone covering the carboxyl-terminal nucleotides of rat podocalyxin. Its putative transmembrane or intracellular domains are 100% or >93% identical, respectively, with the human and rabbit podocalyxin-like proteins. The truncated extracellular domain extends to include two of the four conserved cysteines shared by podocalyxin-like proteins. By Northern blotting, a 5.5-kb renal cortical transcript is seen. By in situ hybridization, cRNA probes recognize podocytes, endothelial cells, and megakaryocytes, and by reverse transcription polymerase chain reaction, platelets are shown to contain podocalyxin mRNA. Our results show that rat podocalyxin is a homologue of the previously cloned podocalyxin-like proteins and suggest that also in mammals podocalyxin has a role in hematopoiesis, as previously shown in the chicken.

Induction of brush border antigens of the proximal tubule in the developing kidney.

Abstract Brush border (BB) antigens of renal tubules were studied in the developing mouse metanephros by indirect immunofluorescence technique. Rabbit antiserum reacting with the plasma membrane of the brush border microvilli of the proximal tubules was used. The antigens appeared in renal tubules some 30 hr after the formation of the lumen both in fetal mesenchymes in vivo and in mesenchymes induced by the spinal cord in vitro . Using the BB antigens as markers for the tissue-specific biosynthetic events during renal differentiation, two types of tubules could be distinguished in vivo and in vitro . Thus, though some of them stained intensely with the antiserum, other well-developed tubules not reacting with the antibodies were also constantly seen. In addition, structures resembling glomeruli developed in vitro . The results suggest that induction of three main segments of the nephron also takes place in cultured mesenchymes. This differentiation, extending over a period of 4 days, was programmed during the initial 24-hr induction.

Organogenesis in a defined medium supplemented with transferrin

Organ culture of embryonic tissues provides means to analyse complex morphogenetic processes. Differentiation of the metanephrogenic mesenchyme into the epithelium of the nephron is know to occur in vitro. We have now formulated a synthetic medium which permits induction of the nephrogenic mesenchyme and its differentiation into the main segments of the nephron, the proximal and the distal tubules and primitive glomeruli. The medium consists of a basal medium supplemented with a single factor, transferrin. In the absence of transferrin differentiation failed to occur. None of the other factors tested (EGF, FGF, insulin, Pedersen fetuin) could replace transferrin.

Podocalyxin in human haematopoietic cells

Podocalyxin‐like protein (PCLP) is a sialomucin‐type membrane protein structurally related to CD34 and endoglycan. It was first described in glomerular podocytes and endothelial cells. In mice, PCLP is present in haemangioblasts, and in both chicken and mice it is a marker of early haematopoietic stem cells and lineage‐restricted haematopoietic progenitors. Its expression decreases during differentiation of haematopoietic cells. Of mature blood cells, only chicken and rat thrombocytes express PCLP protein. PCLP expression in human haematopoietic cells has not been studied. Here we demonstrate PCLP mRNA in human CD34+ cells, in lineage committed erythroid, megakaryocyte and myeloid progenitors, in K562 leukaemia cells, and in peripheral blood leucocytes. The mRNA expression level was higher in developing cells than in mature leucocytes. By Northern blotting and cDNA sequencing, the haematopoietic and renal PCLP mRNAs were identical. Of the mobilized CD34+ cells, 28% (mean; range 14–61%) expressed PCLP protein and the majority of PCLP+ cells were CD117+. Almost all of the K562 cells expressed PCLP protein. Surprisingly, PCLP protein was not detected in any mature blood cells. These results suggest that human PCLP may be a valuable marker for a subset of haematopoietic stem cells.

The glomerular mesangium: studies of its developmental origin and markers in vivo and in vitro

Cultured mesangial cells are widely used to explore their role in kidney glomerular functions, but methods to reliably identify these cells in vivo and in vitro are lacking. Furthermore, the proposed relationship of mesangial cells to e.g. fibroblasts and smooth muscle cells has not been systematically studied. Here we wanted to search for markers of practical use also in identifying cultured mesangial cells, and to apply these markers in a study of the origin of glomerular mesangium. No epitopes specific for only mesangial cells could be identified, and no evidence of their true relationship with neural or lymphocytic lineages could be found. Findings with the variety of markers used suggest that mesangial cells may be indistinguishable from smooth muscle cells and fibroblasts. A panel of antibodies, including those against Thy 1.1, smooth muscle actin, desmin, cellular fibronectin and pi integrin al and a5 chains, and Wistaria floribunda (WFA) and Ricinus communis (RCA I) lectins, were found useful for mesangial cell detection in vivo and in vitro. The origin of glomerular mesangial cells could not be conclusively determined, although the results indirectly suggest that mesangial cells together with endothelial cells migrate to the glomerulus from the outside.

Simultaneous anti-glomerular basement membrane and membranous glomerulonephritis: Case report and literature review

A 20-year-old male experienced a sore throat, fever, and lumbar pain. Examination revealed haematuria, proteinuria, and transiently impaired renal function. Renal biopsy revealed minor mesangial widening and small cellular crescents in 20% of the glomeruli under the light microscope, whereas immunofluorescence showed bright, linear staining of IgG along the glomerular basement membrane (GBM). Ultrastructural analysis showed minute subepithelial deposits analogous to early membranous glomerulonephritis (MGN). Anti-GBM antibodies were detected in the patients serum. These findings were suggestive of simultaneous anti-GBM and immune complex glomerulonephritis in a patient with a mild, reversible renal illness.