Search

Focal segmental glomerulosclerosis (FSGS) may be associated with glomerular epithelial cell (GEC; podocyte) apoptosis due to acquired injury or mutations in [alpha]-actinin-4. This study addresses how FSGS-associated mutant [alpha]-actinin-4 may induce GEC injury, focusing on endoplasmic reticulum (ER) stress and metabolism of mutant [alpha]-actinin-4 via the ubiquitin-proteasome system. In a model of experimental FSGS induced by expression of an [alpha]-actinin-4 K256E transgene in podocytes, we show induction of ER stress, including upregulation of ER chaperones (hip, grp94), phosphorylation of the eukaryotic translation initiation factor-2[alpha] subunit, and induction of the proapoptotic gene C/EBP homologous protein-10 (CHOP). To address mechanisms of ER stress, we studied signaling in cultured GEC and COS cells expressing [alpha]-actinin-4 K256E. Previously, we showed that expression of this [alpha]-actinin-4 mutant in GEC increased apoptosis. In the present study, we show that a-actinin-4 K256E upregulates grp94 and CHOP expression in COS cells and significantly exacerbates induction of bip and CHOP in GEC in the presence of tunicamycin. ER stress was associated with aggregation and ubiquitination of [alpha]-actinin-4 K256E and impairment of the ubiquitin-proteasome system. In addition, [alpha]-actinin-4 K256E exacerbated apoptosis in the context of mild proteasome inhibition. Thus [alpha]-actinin-4 K256E triggers several metabolic abnormalities, which may lead to GEC injury and glomerulosclerosis. apoptosis; endoplasmic reticulum stress; glomerulonephritis; transgenic mice; ubiquitin-proteasome system doi:10.1152/ajprenal.00055.2009.

Expression and activity of the germinal center SLK are increased during kidney development and recovery from renal ischemia-reperfusion injury. SLK promotes apoptosis, in part, via pathway(s) involving apoptosis signal-regulating kinase-1 and p38 mitogen-activated protein kinase. This study addresses the role of p53 as a potential effector of SLK. p53 transactivation was measured after transient transfection of a luciferase reporter plasmid that contains a p53 cis-acting enhancer element. Overexpression of SLK in COS-1 cells and cotransfection of SLK and p53-wild type (wt) cDNAs in glomerular epithelial cells (GECs) stimulated p53 transactivational activity, as measured by a p53 response element-driven luciferase reporter. In GECs, chemical anoxia followed by glucose reexposure (in vitro ischemia-reperfusion) increased p53 reporter activity, and this increase was amplified by overexpression of SLK. Expression of SLK induced p53 phosphorylation on serine (S)-33 and $315. In GECs, cotransfection of SLK with p53-wt, p53-S33A, p53-S315A, or p53-S33A+S315A mutants showed that only the double mutation abolished the SLK-induced increase in p53 reporter activity. SLK-induced stimulation of p53 reporter activity was attenuated by inhibition of JNK. Overexpression of SLK amplified apoptosis induced by subjecting cells to in vitro ischemia-reperfusion injury, while ectopic expression of a dominant negative SLK mutant attenuated the ischemia-reperfusion-induced apoptosis. The p53 transactivation inhibitor pifithrin-a significantly attenuated the amount of apoptosis after ischemia-reperfusion and SLK overexpression. Thus SLK induces p53 phosphorylation and transactivation, which enhances apoptosis after in vitro ischemiareperfusion injury. kidney; ischemia-reperfusion injury; mitogen-activated protein kinases; phosphorylation doi:10.1152/ajprenal.00294.2009.

In experimental membranous nephropathy, complement C5b-9-induced glomerular epithelial cell (GEC) injury leads to morphological changes in GEC and proteinuria, in association with phospholipase [A.sub.2] (PL[A.sub.2]) activation. The present study addresses the role of calcium-independent PL[A.sub.2] (iPL[A.sub.2]) in GEC injury, iPL[A.sub.2][beta] short and iPL[A.sub.2][gamma] were expressed in cultured rat GEC and normal rat glomeruli. To determine whether iPL[A.sub.2] is involved in complement-mediated arachidonic acid (AA) release, GEC were stably transfected with iPL[A.sub.2][gamma] or iPL[A.sub.2][beta] cDNAs (GEC-iPL[A.sub.2][beta]; GEC-iPL[A.sub.2][beta]). Compared with control cells (GEC-Neo), GEC-iPL[A.sub.2][gamma] and GEC-iPL[A.sub.2][beta] demonstrated greater expression of iPL[A.sub.2] proteins and activities. Complement-mediated release of [[sup.3]H]AA was augmented significantly in GEC-iPL[A.sub.2][gamma] compared with GEC-Neo, and the augmented [[sup.3]H]AA release was inhibited by the iPL[A.sub.2]-directed inhibitor bromoenol lactone (BEL). For comparison, overexpression of iPL[A.sub.2][gamma] also amplified [[sup.3]H]AA release after incubation of GEC with [H.sub.2][O.sub.2], or chemical anoxia followed by reexposure to glucose (in vitro ischemia-reperfusion injury). In parallel with release of [[sup.3]H]AA, complement-mediated production of prostaglandin [E.sub.2] was amplified in GEC-iPL[A.sub.2][gamma]. Complement-mediated cytotoxicity was attenuated significantly in GEC-iPL[A.sub.2][gamma] compared with GEC-Neo, and the cytoprotective effect of iPL[A.sub.2][gamma] was reversed by BEL, and in part by indomethacin. Overexpression of iPL[A.sub.2][beta] did not amplify complement-dependent [[sup.3]H]AA release, but nonetheless attenuated complement-mediated cytotoxicity. Thus iPL[A.sub.2][gamma] may be involved in complement-mediated release of AA. Expression of iPL[A.sub.2][gamma] or iPL[A.sub.2][beta] induces cytoprotection against complement-dependent GEC injury. Modulation of iPL[A.sub.2] activity may prove to be a novel approach to reducing GEC injury. iPL[A.sub.2][beta]; iPL[A.sub.2][gamma]; cytoprotection

Ste20-like kinase, SLK, a germinal center kinase found in kidney epithelial cells, signals to promote apoptosis. Expression of SLK mRNA and protein and kinase activity are increased during kidney development and recovery from ischemic acute renal failure. The 3'-untranslated region (3'-UTR) of SLK mRNA contains multiple adenine and uridine-rich elements, suggesting that 3'-UTR may regulate mRNA stability. This was confirmed in COS cell transient transfection studies, which showed that expression of the SLK open-reading frame plus 3'-UTR mRNA was reduced by 35% relative to the open-reading frame alone. To further characterize the SLK-3'-UTR, this nucleotide sequence was subcloned downstream of enhanced green fluorescent protein (EGFP) eDNA. In COS, 293T, and glomerular epithelial cells, expression of EGFP mRNA and protein was markedly reduced in the presence of the SLK-3'-UTR. After transfection and subsequent addition of actinomycin D, EGFP mRNA remained stable in cells for at least 6 h, whereas EGFP-SLK-3'-UTR mRNA decayed with a half-life of ~4 h. A region containing five AUUUA motifs within the SLK-3'-UTR destabilized EGFP mRNA. Deletion of this region from the SLK-3'-UTR, in part, restored mRNA stability. By UV cross-linking and SDS-PAGE, the SLK-3'-UTR bound to protein(s) of ~30 kDa in extracts of COS cells, glomerular epithelial cells, and kidney. Cotransfection of HuR (a RNA binding protein of ~30 kDa) increased the steady-state mRNA level of EGFP-SLK-3'-UTR but not EGFP. Thus the SLK-3'-UTR may interact with kidney RNA-binding proteins to regulate expression of SLK mRNA during kidney development and after ischemic injury. ischemia-reperfusion injury; kidney development; protein kinase; RNA binding proteins

Adhesion of rat glomerular epithelial cells (GEC) to collagen activates focal adhesion kinase (FAK) and the Ras-extracellular signal-regulated kinase (ERK) pathway and supports survival (prevents apoptosis). The present study addresses the relationship between actin organization and the survival phenotype. Parental GEC (adherent to collagen) and GEC stably transfected with constitutively active mutants of mitogen-activated protein kinase kinase (R4F-MEK) or FAK (CD2-FAK) (on plastic) showed ERK activation, low levels of apoptosis, and a cortical distribution of F-actin. Parental GEC adherent to plastic showed increased apoptosis, disorganization of cortical F-actin, and formation of prominent stress fibers. Assembly of cortical F-actin was, at least in part, mediated via ERK. However, disruption of the actin cytoskeleton with cytochalasin D or latrunculin B in parental GEC (on collagen) and in GEC that express R4F-MEK or CD2-FAK (on plastic) decreased ERK activation and increased apoptosis. Expression of a constitutively active RhoA (L63RhoA) induced assembly of cortical F-actin, promoted ERK activation, and supplanted the requirement of collagen for survival. Adhesion of GEC to collagen increased phosphatidylinositol-4, 5-bisphosphate (PIP2). Downregulation or sequestration of PI[P.sub.2] by transfection with an inositol 5'-phosphatase or the plextrin-homology domain of phospholipase C-[delta]1 decreased F-actin content and survival. Moreover, overexpression of wild-type or K256E mutant [alpha]-actinin-4 with increased affinity for F-actin increased apoptosis. These results demonstrate a reciprocal relationship between collageninduced cortical F-actin assembly and collagen-dependent survival signaling, including ERK activation. Appropriate remodeling of the actin cytoskeleton may be necessary for facilitating survival, as both disassembly and excessive crosslinking affect survival adversely. [alpha]-actinin-4; apoptosis; collagen; extracellular signal-regulated kinase; RhoA

Extracellular signals may be transmitted to nuclear or cytoplasmic effectors via the mitogen-activated protein kinases. In the passive Heymann nephritis (PHN) model of membranous nephropathy, complement C5b-9 induces glomerular epithelial cell (GEC) injury, proteinuria, and activation of phospholipases and protein kinases. This study addresses the complement-mediated activation of the extracellular signal-regulated kinase (ERK). C5b-9 induced ERK [threonine.sup.202]/[tyrosine.sup.204] phosphorylation (which correlates with activation) in GEC in culture and PHN in vivo. Expression of a dominant-inhibitory mutant of Ras reduced complement-mediated activation of ERK, but activation was not affected significantly by downregulation of protein kinase C. Complement-induced ERK activation resulted in phosphorylation of cytosolic phospholipase [A.sub.2] and was, in part, responsible for phosphorylation of mitogen-activated protein kinase-associated protein kinase-2, but did not induce phosphorylation of the transcription factor, Elk- 1. Activation of ERK was attenuated by drugs that disassemble the actin cytoskeleton (cytochalasin D, latrunculin B), and these compounds interfered with the activation of ERK by mitogen-activated protein kinase kinase (MEK). Overexpression of a constitutively active RhoA as well as inhibition of Rho-associated kinase blocked complement-mediated ERK activation. Complement cytotoxicity was enhanced after disassembly of the actin cytoskeleton but was unaffected after inhibition of complement-induced ERK activation. However, complement cytotoxicity was enhanced in GEC that stably express constitutively active MEK. Thus complement-induced ERK activation depends on cytoskeletal remodelling and affects the regulation of distinct downstream substrates, while chronic, constitutive ERK activation exacerbates complement-mediated GEC injury. glomerular epithelial cell; inflammation; protein kinases; signal transduction

Cytosolic PL[A.sub.2-[alpha]] (cPL[A.sub.2]) and metabolites of arachidonic acid (AA) are key mediators of complement-dependent glomerular epithelial cell (GEC) injury. Assembly of C5b-9 increases cytosolic [Ca.sup.2+] concentration and results in transactivation of receptor tyrosine kinases and activation of PLC-[gamma]1 and the 1,2-diacylglycerol (DAG)-PKC pathway. [Ca.sup.2+] and PKC are essential for membrane association and increased catalytic activity of cPL[A.sub.2]. This study addresses the role of the actin cytoskeleton in cPL[A.sub.2] activation. Depolymerization of F-actin by cytochalasin D or latrunculin B reduced complement-dependent [[sup.3]H]AA release, as well as the complement-induced increase in cPL[A.sub.2] activity. These effects were due to inhibition of [[sup.3]H]DAG production and PKC activation, implying interference with PLC. Complement-dependent [[sup.3]H]AA release was also reduced by jasplakinolide, a compound that stabilizes F-actin and organizes actin filaments at the cell periphery, and calyculin A, which induces condensation of actin filaments at the plasma membrane. The latter drugs did not affect [[sup.3]H]DAG production, suggesting their inhibitory actions were downstream of PKC. Neither cytnchalasin D, latrunculin B, nor calyculin A affected association of cPL[A.sub.2] with microsomal membranes, and cytochalasin D and latrunculin B did not alter the localization of the endoplasmic reticulum. Stable transfection of constitutively active RhoA induced formation of stress fibers, stabilized F-actin, and attenuated the complement-induced increase in [[sup.3]H]AA. Thus in GEC, cPL[A.sub.2] activation is dependent, in part, on actin remodeling. By regulating complement-mediated activation of cPL[A.sub.2], the actin cytoskeleton may contribute to the pathophysiology of GEC injury. inflammation: lipid mediators; protein kinases; signal transduction

Rat fetal kidney mRNA was analyzed by RT-PCR to identify protein kinases. This screening demonstrated expression of a protein kinase consistent with SK2, a group II germinal center kinase and homolog of human Ste20-like kinase (SLK). SK2 mRNA, protein expression, and kinase activity were increased in rat fetal kidney homogenates (embryonic days 17-21) compared with adult controls. In adult kidneys subjected to cross-clamping of the renal artery, followed by reperfusion, SK2 mRNA, protein expression, and kinase activity were increased compared with untreated contralateral controls. By immunohistochemistry, SK2 expression was evident mainly in the cytoplasm of tubular epithelial cells in fetal and adult kidneys. There was also some expression in developing and mature podocytes, but staining of the interstitium was negative. In cultured renal tubular epithelial cells, SK2 kinase activity was increased after incubation with serum, or alter exposure to chemical anoxia plus reexposure to glucose. Stable overexpression of SLK reduced cell proliferation and increased apoptosis and exacerbated apoptosis and necrosis after chemical anoxia plus reexposure to glucose. Thus SK2 is a renal epithelial protein kinase whose expression and activity are increased during development and recovery from acute renal failure, where tubular epithelial regeneration may recapitulate developmental processes. The actions of SK2 appear to be antiproliferative and may facilitate cell injury. protein kinases; renal development; renal failure; signal transduction

The effect of the adhesion of glomerular epithelial cells (GEC) to extracellular matrix (ECM) on the synthesis of inositol phospholipids was investigated in cultured rat GEC. Measurements of IP3, phosphatidylinositol 4,5-biphosphate and [3H]inositol phosphate masses indicated enhanced GEC adhesion to collagen as compared to the plastic substrate. Sites of GEC adhesion showed increased phospholipase C (PLC) activity and PLC-gamma1 protein levels, indicating that GEC was tyrosine phosphorylated. These results show that phospholipid turnover in GEC is influenced by cell adhesion to the ECM.

The C5b-9 complex causes injury to cells that may cause total death to the cell when mediated by cytosolic phospholipase A. Glomerular epithelial cells which were induced to overexpress cytosolic phospholipase A and then treated with C5b-9 showed signs of injury because they released arachidonic acid and eicosanoids. These cells also had a higher probability of undergoing sublytic cytotoxicity and cytolysis, pointing to a role for C5b-9 in kidney damage.

Ste20-like kinase SLK is critical for embryonic development and may play an important role in wound healing, muscle homeostasis, cell migration, and tumor growth. Mice with podocyte-specific deletion of SLK show albuminuria and damage to podocytes as they age. The present study addressed the role of SLK in glomerular injury. We induced adriamycin nephrosis in 3- to 4-mo-old control and podocyte SLK knockout (KO) mice. Compared with control, SLK deletion exacerbated albuminuria and loss of podocytes, synaptopodin, and podocalyxin. Glomeruli of adriamycintreated SLK KO mice showed diffuse increases in the matrix and sclerosis as well as collapse of the actin cytoskeleton. SLK can phosphorylate ezrin. The complex of phospho-ezrin, Na+/H+ exchanger regulatory factor 2, and podocalyxin in the apical domain of the podocyte is a key determinant of normal podocyte architecture. Deletion of SLK reduced glomerular ezrin and ezrin phosphorylation in adriamycin nephrosis. Also, deletion of SLK reduced the colocalization of ezrin and podocalyxin in the glomerulus. Cultured glomerular epithelial cells with KO of SLK showed reduced ezrin phosphorylation and podocalyxin expression as well as reduced F-actin. Thus, SLK deletion leads to podocyte injury as mice age and exacerbates injury in adriamycin nephrosis. The mechanism may at least in part involve ezrin phosphorylation as well as disruption of the cytoskeleton and podocyte apical membrane structure. [ABSTRACT FROM AUTHOR]

Mutations in α-actinin-4 (actinin-4) result in hereditary focal segmental glomerulosclerosis (FSGS) in humans. Actinin-4 mutants induce podocyte injury because of dysregulation of the cytoskeleton and proteotoxicity. Injury may be associated with endoplasmic reticulum (ER) stress and polyubiquitination of proteins. We assessed if the chemical chaperone 4-phenylbutyrate (4-PBA) can ameliorate the proteotoxicity of an actinin-4 mutant. Actinin-4 K255E, which causes FSGS in humans (K256E in the mouse), showed enhanced ubiquitination, accelerated degradation, aggregate formation, and enhanced association with filamentous (F)-actin in glomerular epithelial cells (GECs). The mutant disrupted ER function and stimulated autophagy. 4-PBA reduced actinin-4 K256E aggregation and its tight association with F-actin. Transgenic mice that express actinin-4 K256E in podocytes develop podocyte injury, proteinuria, and FSGS in association with glomerular ER stress. Treatment of these mice with 4-PBA in the drinking water over a 10-wk period significantly reduced albuminuria and ER stress. Another drug, celastrol, which enhanced expression of ER and cytosolic chaperones in GECs, tended to reduce actinin-4 aggregation but did not decrease the tight association of actinin-4 K256E with F-actin and did not reduce albuminuria in actinin-4 K256E transgenic mice. Thus, chemical chaperones, such as 4-PBA, may represent a novel therapeutic approach to certain hereditary glomerular diseases. [ABSTRACT FROM AUTHOR]

SLK is essential for embryonic development and may play a key role in wound healing, tumor growth, and metastasis. Expression and activation of SLK are increased in kidney development and during recovery from ischemic acute kidney injury. Overexpression of SLK in glomerular epithelial cells/podocytes in vivo induces injury and proteinuria. Conversely, reduced SLK expression leads to abnormalities in cell adhesion, spreading, and motility. Tight regulation of SLK expression thus may be critical for normal renal structure and function. We produced podocyte-specific SLK-knockout mice to address the functional role of SLK in podocytes. Mice with podocyte-specific deletion of SLK showed reduced glomerular SLK expression and activity compared with control. Podocyte-specific deletion of SLK resulted in albuminuria at 4 –5 mo of age in male mice and 8 –9 mo in female mice, which persisted for up to 13 mo. At 11–12 mo, knockout mice showed ultrastructural changes, including focal foot process effacement and microvillous transformation of podocyte plasma membranes. Mean foot process width was approximately twofold greater in knockout mice compared with control. Podocyte number was reduced by 35% in knockout mice compared with control, and expression of nephrin, synaptopodin, and podocalyxin was reduced in knockout mice by 20 –30%. In summary, podocyte-specific deletion of SLK leads to albuminuria, loss of podocytes, and morphological evidence of podocyte injury. Thus, SLK is essential to the maintenance of podocyte integrity as mice age [ABSTRACT FROM AUTHOR]

Kidney cell injury may be associated with protein misfolding and induction of endoplasmic reticulum (ER) stress. Examples include complementinduced glomerular epithelial cell (GEC)/podocyte injury in membranous nephropathy and ischemia-reperfusion injury. Renal cell injury can also result from mutations in integral proteins, which lead to their misfolding and accumulation. Certain nephrin missense mutants misfold, accumulate in the ER, and induce ER stress. We examined if enhancement of ubiquitin-proteasome system function may facilitate proteostasis and confer protection against injury. Ubiquitin-specific protease 14 (Usp14) is reported to retard proteasomal protein degradation. Thus inhibition of Usp14 may enhance degradation of misfolded proteins and attenuate cell injury. In GEC, the reporter proteins GFPu (a "misfolded" protein) and CD3δ (an ER-associated degradation substrate) undergo time-dependent proteasomal degradation. Complement did not affect degradation of CD3δ-yellow fluorescent protein (YFP), but accelerated degradation of GFPu, and the Usp14-directed inhibitor IU1 further accelerated this degradation. Conversely, overexpression of Usp14 reduced degradation of GFPu and CD3δ-YFP. In 293T cells, IU1 did not enhance degradation of disease-associated nephrin missense mutants I171N and S724C, whereas overexpression of Usp14 reduced degradation. IU1 was cytoprotective after injury induced by the ER stressor tunicamycin and in vitro ischemia-reperfusion, but did not affect complement-induced cytotoxicity. In conclusion, Usp14 controls proteasomal degradation of some misfolded proteins. In addition, a Usp14-directed inhibitor reduces cytotoxicity in the context of global protein misfolding during certain types of renal cell injury. [ABSTRACT FROM AUTHOR]

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 A2γ (iPLA2γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA2γ KO mice have demonstrated an important role for iPLA2γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA2γ KO mice to better understand the role of iPLA2γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA2γ 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, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA2γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA2γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria. [ABSTRACT FROM AUTHOR]