Alpha-actinin-4 and CLP36 protein deficiencies contribute to podocyte defects in multiple human glomerulopathies.

Genetic alterations of α-actinin-4 can cause podocyte injury through multiple mechanisms. Although a mechanism involving gain-of-α-actinin-4 function was well described and is responsible for a dominantly inherited form of human focal segmental glomerulosclerosis (FSGS), evidence supporting mechanisms involving loss-of-α-actinin-4 function in human glomerular diseases remains elusive. Here we show that α-actinin-4 deficiency occurs in multiple human primary glomerulopathies including sporadic FSGS, minimal change disease, and IgA nephropathy. Furthermore, we identify a close correlation between the levels of α-actinin-4 and CLP36, which form a complex in normal podocytes, in human glomerular diseases. siRNA-mediated depletion of α-actinin-4 in human podocytes resulted in a marked reduction of the CLP36 level. Additionally, two FSGS-associated α-actinin-4 mutations (R310Q and Q348R) inhibited the complex formation between α-actinin-4 and CLP36. Inhibition of the α-actinin-4-CLP36 complex, like loss of α-actinin-4, markedly reduced the level of CLP36 in podocytes. Finally, reduction of the CLP36 level or disruption of the α-actinin-4-CLP36 complex significantly inhibited RhoA activity and generation of traction force in podocytes. Our studies reveal a critical role of the α-actinin-4-CLP36 complex in podocytes and provide an explanation as to how α-actinin-4 deficiency or mutations found in human patients could contribute to podocyte defects and glomerular failure through a loss-of-function mechanism.

Integrin-linked kinase in renal disease: connecting cell-matrix interaction to the cytoskeleton.

PURPOSE OF REVIEW: Cellular functions like proliferation, differentiation, migration, morphogenesis and apoptosis are modulated by the extracellular matrix. Integrins are the prototypic heterodimeric transmembrane matrix receptors with competing affinities for individual extracellular matrix ligands. The intracellular integrin domain clusters cytoplasmic proteins into focal adhesion plaques for bidirectional (outside-in and inside-out) signalling. Integrin-linked kinase organizes the connections of the extracellular matrix via integrins to the cytoskeleton and is involved in adhesion plaque signalling. In this review, an introduction of integrin-linked kinase structure and function is followed by a summary of our current understanding of integrin-linked kinase in renal disease with special focus on glomerular cell-matrix interaction. RECENT FINDINGS: Differential regulation of integrin-linked kinase has been observed during the pathogenesis of glomerular disease and tubulo-interstitial fibrosis. In outside-in signalling integrin-linked kinase mediates the response of renal cells to alterations in matrix and growth factor environments. Inside-out signalling transduces inflammatory and oxidative stress responses into decreased matrix attachment. Downstream signalling of integrin-linked kinase activates the Wnt pathway with a switch towards a proliferative, mesenchymal phenotype. In concert with interacting molecules integrin-linked kinase influences the actin cytoskeleton, resulting in shape change and focal adhesion dysfunction observed in podocyte failure and tubulo-interstitial fibrosis. SUMMARY: Integrin-linked kinase has emerged as a key player at the interface between extracellular matrix, integrins, actin-based cytoskeleton and cellular phenotype in kidney diseases. Future studies focusing on interacting molecules and modification of integrin-linked kinase function in vivo will better define the role of cell matrix signalling in progressive renal failure.

Functional consequences of integrin-linked kinase activation in podocyte damage.

BACKGROUND: The delicate foot process architecture of glomerular podocytes critically depends on integrin mediated cell-glomerular basement membrane (GBM) interaction. Integrin signaling via the integrin-linked kinase (ILK) is activated in podocyte damage and associated with considerable podocyte phenotype alterations. ILK has been shown to regulate cell fate via nuclear interaction of beta-catenin with lymphoid enhancer factor (LEF-1) transcription factors. The aim of this study was to elucidate the molecular mechanisms of ILK dependant phenotype regulation in podocytes. METHODS: ILK function was evaluated in conditionally immortalized murine glomerular epithelial cells using overexpression of ILK and a small molecule ILK inhibitor in puromycin/adriamycin-induced podocyte damage in vitro and in vivo. RESULTS: Kinase active, but not mutant ILK induced translocation of beta-catenin to the cell nucleus, de novo expression of LEF-1, and nuclear colocalization of beta-catenin and LEF-1. The role of ILK signaling in podocyte damage was evaluated using puromycin, an agent known to cause selective proteinuria and to increase ILK activity. The small molecular ILK inhibitor MC-5 blocked puromycin-induced nuclear translocation of beta-catenin, podocyte detachment, cell proliferation, and repression of the slit membrane molecules P-cadherin and CD2ap. In vivo activation of the beta-catenin pathway could be shown by nuclear colocalization of beta-catenin with WT-1 in adriamycin nephropathy. CONCLUSION: ILK regulates podocyte cell matrix interaction, proliferation, and slit membrane gene expression in podocyte damage. As this pathway is amendable to pharmacologic intervention, further detailed studies of in vivo ILK function in glomerular disease appear justified.

Glomerular podocytes possess the synaptic vesicle molecule Rab3A and its specific effector rabphilin-3a.

Several recent studies have focused on similarities between glomerular podocytes and neurons because the two cells share a specialized cytoskeletal organization and several expression-restricted proteins, such as nephrin and synaptopodin. In neurons, the small guanosine triphosphatase Rab3A and its effector rabphilin-3A form a complex required for the correct docking of synaptic vesicles to their target membrane. Because rabphilin-3A binds in neurons to cytoskeletal proteins also important for podocyte homeostasis, and the complex rabphilin-3A-Rab3A has been demonstrated in neurons and neuroendocrine cells, the aim of our work was to investigate their possible expression and regulation in podocytes. Normal kidneys from mouse, rat, and human were studied by immunohistochemistry, Western blotting, and reverse transcriptase-polymerase chain reaction to evaluate the expression of Rab3A and rabphilin-3A. Double-staining immunohistochemistry and immunogold electron microscopy were then used to precisely localize the two proteins at the cellular and subcellular levels. Rab-3A and rabphilin-3A regulations in disease were then analyzed in growth hormone-transgenic mice, a well established model of focal and segmental glomerulosclerosis, and in human biopsies from proteinuric patients. Our results demonstrated that rabphilin-3A and Rab3A are present in normal mouse, rat, and human kidneys, with an exclusively glomerular expression and a comma-like pattern of positivity along the glomerular capillary wall, suggestive for podocyte staining. Co-localization of both molecules with synaptopodin confirmed their presence in podocytes. By immunogold electron microscopy both proteins were found around vesicles contained in podocyte foot processes. Their expression was increased in growth hormone-transgenic mice compared to their wild-type counterpart, and in a subset of biopsies from proteinuric patients. Our data, demonstrating the presence of two synaptic proteins in podocytes, further supports similarities between cytoskeletal and vesicular organization of podocytes and neurons. The altered expression observed in mouse and human proteinuric diseases suggests a possible role for these molecules in glomerulopathies.