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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.

We investigated the role of the prostaglandin [E.sub.2] (PG[E.sub.2]) E[P.sub.1] receptor in modulating urine concentration as it is expressed along the renal collecting duct where arginine-vasopressin (AVP) exerts its anti-diuretic activity, and in the paraventricular and supraoptic nuclei of the hypothalamus where AVP is synthesized. The urine osmolality of E[P.sub.1]-null mice (E[P.sub.1.sup.-/-]) failed to match levels achieved by wild-type (WT) counterparts upon water deprivation (WD) for 24 h. This difference was reflected by higher plasma osmolality in WD E[P.sub.1.sup.-/-] mice. Along the collecting duct, the induction and subapical to plasma membrane translocation of the aquaporin-2 water channel in WD E[P.sub.1.sup.-/-] mice appeared equivalent to that of WD WT mice as determined by quantitative RT-PCR and immunohistochemistry. However, medullary interstitial osmolalities dropped significantly in E[P.sub.1.sup.-/-] mice following WD. Furthermore, urinary AVP levels of WD E[P.sub.1.sup.-/-] mice were significantly lower than those of WD WT mice. This deficit could be traced back to a blunted induction of hypothalamic AVP mRNA expression in WD E[P.sub.1.sup.-/-] mice as determined by quantitative RT-PCR. Administration of the AVP mimetic [deamino-[Cys.sup.1],D-[Arg.sup.8]]-vasopressin restored a significant proportion of the urine concentrating ability of WD E[P.sub.1.sup.-/-] mice. When mice were water loaded to suppress endogenous AVP production, urine osmolalities increased equally for WT and E[P.sub.1.sup.-/-] mice. These data suggest that PG[E.sub.2] modulates urine concentration by acting at E[P.sub.1] receptors, not in the collecting duct, but within the hypothalamus to promote AVP synthesis in response to acute WD. vasopressin; urine concentration; prostaglandin [E.sub.2]; E[P.sub.1] receptor

Abstract: In experimental glomerulonephritis, inhibition of renal prostaglandin (PG) synthesis by nonsteroidal-anti-inflammatory drugs (NSAIDs) moderates proteinuria, yet can induce harmful effects on renal blood flow and [Na.sup.+]-[K.sup.+]-water balance thereby implicating [...]

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

Martineau, Louis C., Lyne I. McVeigh, Bernard J. Jasmin, and Chris R. J. Kennedy. p38 MAP kinase mediates mechanically induced COX-2 and PG [EP.sub.4] receptor expression in podocytes: implications for the actin cytoskeleton. Am J Physiol Renal Physiol 286: F693-F701, 2004. First published December 9, 2003; 10.1152/ajprenal.00331.2003.--A dynamic cytoskeleton allows podocytes to withstand significant mechanical stress on elevation of intraglomerular capillary pressure ([P.sub.gc]). However, vasoactive hormones, such as prostaglandin [E.sub.2] (PG[E.sub.2]), may challenge the integrity of the actin cytoskeleton, alter podocyte morphology, and compromise glomerular permeability. PG[E.sub.2] synthesis correlates with the onset of proteinuria and increased [P.sub.gc] following reduced nephron mass. We investigated the interplay among mechanical stress, cyclooxygenase (COX), E-prostanoid (EP) receptor expression, and the actin cytoskeleton, using an in vitro model of cell stretch. Immortalized mouse podocytes grown on flexible silicone membranes were cyclically stretched (5% elongation, 0.5 Hz) for 2 h. [EP.sub.4] and COX-2 mRNA increased three-and sevenfold above nonstretched controls, whereas [EP.sub.1] and COX-1 levels were unchanged. Six hours of stretch resulted in a threefold increase in PG[E.sub.2]-stimulated cAMP accumulation, a measure of [EP.sub.4] receptor function, and an increase in COX-2 protein. The stretch-induced effects on COX-2/[EP.sub.4] expression and [EP.sub.4]-induced cAMP production were attributable to p38 MAP kinase, as blockade of this pathway, but not of ERK or JNK, abrogated the response. These stretch-induced changes in expression were transcriptionally dependent as they were actinomycin D sensitive. Finally, we investigated the influence of enhanced [EP.sub.4] signaling on the actin cytoskeleton. Addition of PG[E.sub.2] resulted in actin filament depolymerization observable only in stretched cells. Our results indicate that key components of the eicosanoid pathway are upregulated by mechanically stimulated p38 MAP kinase in podocytes. Enhanced [EP.sub.4] receptor signaling may undermine podocyte cytoskeletal dynamics and thereby compromise filtration barrier function under conditions of increased [P.sub.gc]. eicosanoids; adenosine 3',5'-cyclic monophosphate; mechanical stimulation

Increased glomerular prostaglandin [E.sub.2] (PG[E.sub.2]) production is associated with the progression of diseases such as membranous nephropathy, nephrotic syndrome, and anti-Thy1 nephritis. We investigated the signaling pathways that regulate the synthesis and actions of PG[E.sub.2] in glomerular podocytes. To study its actions, we assessed the ability of PG[E.sub.2] to regulate the production of its own precursor, arachidonic acid (AA), in a mouse podocyte cell line. PG[E.sub.2] dose-dependently reduced phorbol ester (PMA)-mediated AA release. Inhibition of PMA-stimulated AA release by PG[E.sub.2] was found to be cAMP/ PKA-dependent, because PG[E.sub.2] significantly increased levels of this second messenger, whereas the inhibitory actions of PG[E.sub.2] were reversed by PKA inhibition and reproduced by the cAMP-elevating agents forskolin and IBMX. PG[E.sub.2] synthesis in this podocyte cell line increased fourfold at 60 min in response to PMA, coinciding with upregulation of cyclooxygenase (COX)-2 but not COX-1 levels. However, PG[E.sub.2] synthesis was significantly reduced by COX-1-selective inhibition, yet to a lesser extent by COX-2-selective inhibition. Our findings suggest that PMA-stimulated PG[E.sub.2] synthesis in mouse podocytes requires both basal COX-1 activity and induced COX-2 expression, and that PG[E.sub.2] reduces PMA-stimulated AA release in a cAMP/PKA-dependent manner. Such an autocrine regulatory loop might have important consequences for podocyte and glomerular function in the context of renal diseases involving PG[E.sub.2] synthesis. adenosine 3',5'-cyclic monophosphate; cyclooxygenase; E-prostanoid receptors

Excessive production of renal reactive oxygen species (ROS) plays a major role in diabetic kidney disease (DKD). Here, we provide key findings demonstrating the predominant pathological role of the pro-oxidant enzyme NADPH oxidase 5 (NOX5) in DKD, independent of the previously characterized NOX4 pathway. In patients with diabetes, we found increased expression of renal NOX5 in association with enhanced ROS formation and upregulation of ROS-sensitive factors early growth response 1 (EGR-1), protein kinase C-α (PKC-α), and a key metabolic gene involved in redox balance, thioredoxin-interacting protein (TXNIP). In preclinical models of DKD, overexpression of NOX5 in Nox4-deficient mice enhances kidney damage by increasing albuminuria and augmenting renal fibrosis and inflammation via enhanced ROS formation and the modulation of EGR1, TXNIP, ERK1/2, PKC-α, and PKC-ε. In addition, the only first-in-class NOX inhibitor, GKT137831, appears to be ineffective in the presence of NOX5 expression in diabetes. In vitro, silencing of NOX5 in human mesangial cells attenuated upregulation of EGR1, PKC-α, and TXNIP induced by high glucose levels, as well as markers of inflammation (TLR4 and MCP-1) and fibrosis (CTGF and collagens I and III) via reduction in ROS formation. Collectively, these findings identify NOX5 as a superior target in human DKD compared with other NOX isoforms such as NOX4, which may have been overinterpreted in previous rodent studies. [ABSTRACT FROM AUTHOR]

Research suggests that biochemical bradykinin (BK)-stimulated arachidonic acid release involves the catalytic action of enzyme cytosolic phospholipase A2 (cPLA2). It seems that protein kinase C interacts with cPLA2 in the BK pathway. Experiments using rabbit cortical collecting duct epithelial cells are described. Arachidonic acid release data in relation to bradykinin concentration is illustrated.

Conflicting findings concerning the dependence of bradykinin (BK) induced arachidonic acid (AA) release from Madin-Darby canine kidney (MDCK) cells on protein kinase C (PKC) activation were explained by observing the effect of PKC inhibitor, bisindolmaleimide I (BIS) on AA. BIS was found to be incapable of blocking BK-enhanced release of AA, but it abrogated the synergistic effect of short-term exposure of BK to phorbol ester. Moreover, downregulation of PKC reduced BK-induced AA release. These suggested that BK does not require the action of PKC at onset for its ability to release AA.