The role of dendrin in IgA nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) and its systemic variant IgA vasculitis (IgAV) damage the glomeruli, resulting in proteinuria, hematuria and kidney impairment. Dendrin is a podocyte-specific protein suggested to be involved in the pathogenesis of IgAN. Upon cell injury, dendrin translocates from the slit diaphragm to the nucleus, where it is suggested to induce apoptosis and cytoskeletal changes, resulting in proteinuria and accelerated disease progression in mice. Here we investigated gene and protein expression of dendrin in relation to clinical and histopathological findings to further elucidate its role in IgAN/IgAV. METHODS: Glomerular gene expression was measured using microarray on 30 IgAN/IgAV patients, 5 patients with membranous nephropathy (MN) and 20 deceased kidney donors. Dendrin was spatially evaluated on kidney tissue sections by immunofluorescence (IF) staining (IgAN patients, n = 4; nephrectomized kidneys, n = 3) and semi-quantified by immunogold electron microscopy (IgAN/IgAV patients, n = 21; MN, n = 5; living kidney donors, n = 6). Histopathological grading was performed according to the Oxford and Banff classifications. Clinical data were collected at the time of biopsy and follow-up. RESULTS: Dendrin mRNA levels were higher (P = .01) in IgAN patients compared with MN patients and controls and most prominently in patients with preserved kidney function and fewer chronic histopathological changes. Whereas IF staining did not differ between groups, immunoelectron microscopy revealed that a higher relative nuclear dendrin concentration in IgAN patients was associated with a slower annual progression rate and milder histopathological changes. CONCLUSION: Dendrin messenger RNA levels and relative nuclear protein concentrations are increased and associated with a more benign phenotype and progression in IgAN/IgAV patients.

Plekhh2, a novel podocyte protein downregulated in human focal segmental glomerulosclerosis, is involved in matrix adhesion and actin dynamics.

Pleckstrin homology domain-containing, family H (with MyTH4 domain), member 2 (Plekhh2) is a 1491-residue intracellular protein highly enriched in renal glomerular podocytes for which no function has been ascribed. Analysis of renal biopsies from patients with focal segmental glomerulosclerosis revealed a significant reduction in total podocyte Plekhh2 expression compared to controls. Sequence analysis indicated a putative α-helical coiled-coil segment as the only recognizable domain within the N-terminal half of the polypeptide, while the C-terminal half contains two PH, a MyTH4, and a FERM domain. We identified a phosphatidylinositol-3-phosphate consensus-binding site in the PH1 domain required for Plekhh2 localization to peripheral regions of cell lamellipodia. The N-terminal half of Plekkh2 is not necessary for lamellipodial targeting but mediates self-association. Yeast two-hybrid screening showed that Plekhh2 directly interacts through its FERM domain with the focal adhesion protein Hic-5 and actin. Plekhh2 and Hic-5 coprecipitated and colocalized at the soles of podocyte foot processes in situ and Hic-5 partially relocated from focal adhesions to lamellipodia in Plekhh2-expressing podocytes. In addition, Plekhh2 stabilizes the cortical actin cytoskeleton by attenuating actin depolymerization. Our findings suggest a structural and functional role for Plekhh2 in the podocyte foot processes.

Permeability, ultrastructural changes, and distribution of novel proteins in the glomerular barrier in early puromycin aminonucleoside nephrosis.

BACKGROUND/AIMS: It is still unclear what happens in the glomerulus when proteinuria starts. Using puromycin aminonucleoside nephrosis (PAN) rats, we studied early ultrastructural and permeability changes in relation to the expression of the podocyte-associated molecules nephrin, α-actinin, dendrin, and plekhh2, the last two of which were only recently discovered in podocytes. METHODS: Using immune stainings, semiquantitative measurement was performed under the electron microscope. Permeability was assessed using isolated kidney perfusion with tracers. Possible effects of ACE inhibition were tested. RESULTS: By day 2, some patchy foot process effacement, but no proteinuria, appeared. The amount of nephrin was reduced in both diseased and normal areas. The other proteins showed few changes, which were limited to diseased areas. By day 4, foot process effacement was complete and proteinuria appeared in parallel with signs of size barrier damage. Nephrin decreased further, while dendrin and plekhh2 also decreased but α-actinin remained unchanged. ACE inhibition had no significant protective effect. CONCLUSIONS: PAN glomeruli already showed significant pathology by day 4, despite relatively mild proteinuria. This was preceded by altered nephrin expression, supporting its pivotal role in podocyte morphology. The novel proteins dendrin and plekhh2 were both reduced, suggesting roles in PAN, whereas α-actinin was unchanged.

Neph1 is reduced in primary focal segmental glomerulosclerosis, minimal change nephrotic syndrome, and corresponding experimental animal models of adriamycin-induced nephropathy and puromycin aminonucleoside nephrosis.

BACKGROUND/AIMS: The transmembrane proteins Neph1 and nephrin form a complex in the slit diaphragm (SD) of podocytes. As recent studies indicate an involvement of this complex in the polymerization of the actin cytoskeleton and proteinuria, we wanted to study the subcellular localization of Neph1 in the normal human kidney and its expression in focal segmental glomerulosclerosis (FSGS), minimal change nephrotic syndrome (MCNS), and the corresponding experimental models of Adriamycin-induced nephropathy (ADR) and puromycin aminonucleoside nephrosis (PAN). All these disorders are characterized by substantial foot process effacement (FPE) and proteinuria. MATERIALS AND METHODS: Kidney biopsies from patients with primary FSGS (perihilar type) and MCNS were compared to normal renal tissue. Mouse and rat kidney cortices from days 7 and 14 after Adriamycin injection and days 2 and 4 after puromycin aminonucleoside injection, respectively, were compared to control mouse and rat kidney. Polyclonal antibodies against Neph1 and nephrin were used for immunoelectron microscopy, and semiquantification was performed. RESULTS: We localized Neph1 mainly to, and in close proximity to, the SD. Double staining of Neph1 and nephrin showed the proteins to be in close connection in the SD. The total amount of Neph1 in the podocytes was significantly reduced in FSGS, MCNS, ADR, and PAN. The reduction of Neph1 was also seen in areas with and without FPE. Nephrin was reduced in MCNS and PAN but unchanged in FSGS. CONCLUSION: With nephrin (but not Neph1) unchanged in FSGS, there might be a disruption of the complex and an involvement of Neph1 in its pathogenesis.