Genetic ablation of calcium-independent phospholipase A2γ induces glomerular injury in mice

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA(2)γ KO mice have demonstrated an important role for iPLA(2)γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA(2)γ KO mice to better understand the role of iPLA(2)γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA(2)γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA(2)γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA(2)γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA(2)γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria

Regulation of Ste20-like kinase, SLK, activity: Dimerization and activation segment phosphorylation

The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development,cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival.Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpressionof SLK was shown to induce apoptosis in cultured glomerular epithelial cells(GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consistsof a N-terminal catalytic domain and an extensive C-terminal domain, which containscoiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerizationof the SLK catalytic domain enhanced autophosphorylation of SLK at T183 andS189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as amodel SLK substrate (to address exogenous kinase activity), we demonstrate that dimerizedSLK 1±373 or full-length SLK can effectively induce activation-specific phosphorylationof ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation,and showed a greater reduction in ezrin phosphorylation. Mutations in thecoiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantlyreduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. [...

Coiled-coil domain mutations reduce SLK activity.

<p>A-C) COS-1 cells were transiently transfected with WT or mutants of full-length HA-SLK (Untr, untransfected control). A and B) After 48 h, lysates were immunoblotted with anti-pT183, anti-HA or anti-SLK antibodies. A) Representative immunoblots. B) Densitometric quantification. pT183 values were adjusted for HA-SLK expression, and background values (T183A/S189A mutant) were subtracted. T183 phosphorylation was evident in SLK WT and to a lesser extent in the coiled-coil domain mutants (I848G, L986G and L986G/I989G), although differences did not reach statistical significance. The minor T183 phosphorylation in Untr and the T183A/S189A mutant most likely reflects endogenous SLK. 5 experiments. C) COS-1 cells were transiently transfected as above. After 48 h, lysates were immunoprecipitated with anti-HA antibody (+), or nonimmune IgG (control; -). Immune complexes were then immunoblotted with anti-pT183 or anti-HA antibodies. T183 phosphorylation was absent in the T183A/S189A mutant, but was evident in the WT and coiled-coil domain mutants. Representative immunoblots. D) COS-1 cells were transiently transfected with WT or mutants of full-length HA-SLK, as indicated, plus GFP-ezrin (Untr, untransfected control). After 48 h, lysates were immunoblotted with anti-SLK, anti-pERM, anti-GFP, anti-ezrin, or anti-actin antibodies. SLK mutants reduced ezrin phosphorylation. D) Representative immunoblots. E) Densitometric quantification. E) *P<0.0005 WT vs I848G, P<0.03 WT vs L986G, P<0.0001 WT vs K63R. 6 experiments performed in duplicate.</p

Role of the Ste20‐like kinase SLK in podocyte adhesion

Abstract SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte‐specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins—paxillin, vinculin, and talin‐1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin‐1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin‐induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin‐1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin‐1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease