HIV infection changes glomerular podocyte cytoskeletal composition and results in distinct cellular mechanical properties.

In addition to forming the selective filtration barrier for the renal glomerulus, podocytes maintain glomerular capillary architecture by opposing distending hemodynamic forces. To understand the relationship of cytoskeletal properties and the mechanical characteristics of podocytes, we studied filamin expression and distribution and measured cell membrane deformability in conditionally immortalized wild-type (WT) mouse podocytes, and in podocytes derived from a mouse model of HIV-associated nephropathy (HIVAN). In the WT cells, filamin and F-actin were localized at the periphery and in prominent stress fibers. In the HIVAN cells, filamin expression was reduced, and stress fibers were sparse. In a microaspiration assay, HIVAN cells ruptured under minimal negative pressure. Atomic force microscopy demonstrated that the WT cells had a stiffness of 17 kPa, whereas the value for the HIVAN cells was 4 kPa. These results demonstrate that the mechanical properties of WT and HIVAN podocytes are markedly different in a manner that is consistent with differences in the composition and arrangement of their cytoskeletons. The mechanical properties of the WT podocytes suggest that these cells can better maintain capillary integrity than the HIVAN podocytes and implicate pathological assembly of the cytoskeleton as a mechanism of HIVAN.

The NHE1 Na+/H+ exchanger regulates cell survival by activating and targeting ezrin to specific plasma membrane domains.

NHE1 is a ubiquitously expressed Na+/H+ exchanger, which is important for vital cell functions. Using in vivo models of kidney podocyte injury and renal tubular epithelial cell (RTC) culture systems, we previously demonstrated that NHE1 defends against apoptosis by a mechanism involving ezrin binding to the NHE1 cytoplasmic domain. We now extend the NHE1 role to diabetic mouse models and refine the mechanism of NHE1-dependent ezrin activation. Streptozotocin induced diabetes resulted in greater azotemia, albuminuria and tubulointerstitial pathology in NHE1-deficient swe/swe compared to wild-type control mice. Increased RTC apoptosis was noted in swe/swe mice, suggesting that loss of NHE1 function leads to tubular atrophy, which predicts kidney disease progression. In vitro, proximal RTC derived from swe/swe mice also underwent increased apoptosis in response to staurosporine or a hypertonic environment. Activated ezrin normally resides in the apical domain of the proximal RTC, while NHE1 is a basolateral protein. After NHE1 activation by intracellular acidification or extracellular hypertonicity, confocal immunofluorescence microscopy in polarized LLC-PK1 cells demonstrated transient ezrin localization to lateral membrane domains, where it is positioned to interact with NHE1. We conclude that cell stresses promote NHE1-ezrin interaction, which activate cell survival pathways to prevent apoptosis in diabetic and non-diabetic kidney diseases.

Genetic and genomic approaches to glomerulosclerosis.

Chronic kidney disease (CKD) is common, progressive and expensive to manage. Although modifiable risk factors can be treated and outcomes improved, CKD remains a chronic disease with excessive morbidity and mortality. The completion of the human genome sequence and the advent of methodologies to define gene function provide new opportunities to manage and treat patients with CKD and other chronic diseases. Despite the lack of clear correspondence between genotype and phenotype and an obvious Mendelian inheritance pattern, CKD susceptibility has a genetic basis. In this review, we focus on recent studies of familial focal segmental glomerulosclerosis and the discoveries that have resulted from both genetic and genomic approaches used to understand its pathogenesis. Key slit diaphragm proteins were discovered using linkage analyses of these rare causes of glomerulosclerosis and subsequent work has characterized slit diaphragm function in health and disease. Podocyte dysfunction is now recognized as a key contributor to the functional and histologic derangements that characterize glomerular dysfunction in many common causes of CKD. In aggregate, these studies provide a paradigm for approaches to better define mechanisms of CKD and to identify novel therapeutic targets.

Activation of EphA receptor tyrosine kinase inhibits the Ras/MAPK pathway.

Interactions between Eph receptor tyrosine kinases (RTKs) and membrane-anchored ephrin ligands critically regulate axon pathfinding and development of the cardiovascular system, as well as migration of neural cells. Similar to other RTKs, ligand-activated Eph kinases recruit multiple signalling and adaptor proteins, several of which are involved in growth regulation. However, in contrast to other RTKs, activation of Eph receptors fails to promote cell proliferation or to transform rodent fibroblasts, indicating that Eph kinases may initiate signalling pathways that are distinct from those transmitted by other RTKs. Here we show that stimulation of endogenous EphA kinases with ephrin-A1 potently inhibits the Ras/MAPK cascade in a range of cell types, and attenuates activation of mitogen-activated protein kinase (MAPK) by receptors for platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). In prostatic epithelial cells and endothelial cells, but not fibroblasts, treatment with ephrin-A1 inhibits cell proliferation. Our results identify EphA kinases as negative regulators of the Ras/MAPK pathway that exert anti-mitogenic functions in a cell-type-specific manner.

A family-based strategy to identify genes for diabetic nephropathy.

Diabetic nephropathy (DN) clusters in families and specific ethnic groups, suggesting a genetic basis of disease transmission. Identification of DN susceptibility loci should reveal new therapeutic targets but requires accurate phenotyping. A powerful family-based strategy, which is novel to the pursuit of nephropathy genes in type 2 diabetes, is being used to collect a sample for candidate gene and genome scan analyses. Sib pairs that include DN index cases plus (1) sibs concordant for type 2 diabetes and DN (affected sib pairs [ASPs]) and (2) sibs concordant for type 2 diabetes but discordant for DN (discordant sib pairs [DSPs]) are targeted specifically for recruitment. Type 2 diabetes and DN phenotype criteria for index cases include diabetes onset after 38 years of age, duration 10 years or longer, no initial insulin treatment, diabetic retinopathy, end-stage renal disease (ESRD), and history of nephrotic proteinuria. ESRD patients were screened by questionnaire and medical record review (n = 2114). Of 666 patients with ESRD secondary to DN, 227 had a family history of ESRD, 150 had a living diabetic sib, and 124 families were enrolled. Sixty-five families, with 86 diabetic relative pairs (69 sibs, 17 children), have been completely phenotyped. If nephropathy in diabetic sibs is defined as albuminuria greater than 0.3 g/24 h, 31 ASPs and 26 DSPs (diabetic sib with albuminuria <0.3 g/24 h) were identified. Applying more stringent criteria, only 12 ASPs (sib with diabetes >10 years, diabetic retinopathy, and nephrotic proteinuria) and 9 DSPs (sib with diabetes >10 years and normal urine albumin excretion) were identified. Extrapolating from the number of subjects recruited using stringent phenotyping criteria, nearly 10,000 ESRD patients are required for screening to achieve adequate statistical power for linkage analysis (80% power to detect locus-specific relative risk of 2.2 at a lod score of 3.0). Careful phenotyping requires a large recruitment effort but is necessary to reduce population heterogeneity, a strategy that increases the likelihood of identifying DN loci.

Inaccuracy of clinical phenotyping parameters for hypertensive nephrosclerosis.

BACKGROUND: Multiple studies suggest that hypertension-induced end-stage renal disease (ESRD) is heritable. Identification of nephropathy susceptibility genes absolutely requires accurate phenotyping, but the clinical hypertensive nephrosclerosis (HN) phenotype is poorly characterized. We hypothesized that many patients with HN as the indicated cause of ESRD on the Health Care Financing Administration (HCFA) 2728 form, fail to satisfy stringent HN phenotyping criteria. METHODS: Since renal biopsy documentation of HN is uncommon, clinical parameters for HN phenotype were applied: family history of hypertension, left ventricular hypertrophy, proteinuria <0.5 g/day, and hypertension preceding renal dysfunction (Schlessinger et al., 1994) or urine protein:creatinine (prot:creat) ratio <2.0 and no evidence of other renal diseases (AASK Trial Group, 1997). RESULTS: ESRD patients (n=607, 73% African American, 25% Caucasian) were enrolled in a study to identify HN susceptibility genes. HN was the most common cause of ESRD according to HCFA 2728 forms (37% prevalence). Phenotyping of randomly selected patients with HN from the total cohort revealed that 4/100 subjects satisfied the Schlessinger criteria, and 28/91 African Americans met AASK criteria for HN. From these figures, the adjusted prevalence of HN was only 1.5-13.5%. Of patients that could not be phenotyped for HN, 14 were misdiagnosed, 14 had urine prot:creat >2.0, and insufficient data were available in the remainder. Four patients underwent renal biopsy, but histology from only one was consistent with HN. If the HN phenotype definitions are revised to exclude 'hypertension preceding renal dysfunction', or proteinuria limits, then 44/100 and 39/91 patients respectively satisfy clinical phenotyping parameters for HN. CONCLUSIONS: (i) We provide the strongest evidence to date that HN is less frequent in an ESRD population than commonly assumed if strict clinical criteria are used; many patients clinically diagnosed with HN may have undetected, treatable renal disease from other causes; (ii) relaxing HN phenotype criteria may erroneously include patients with glomerular diseases and secondary hypertension; (iii) reliance on HCFA 2728 diagnoses will confound identification of HN susceptibility genes; (iv) to attain adequate statistical power for genotype analysis, rigorous HN phenotyping will require screening an extremely large number of patients, which can be reasonably accomplished only in a multi-centre trial design.

High-flux hemodialysis without hemoperfusion is effective in acute valproic acid overdose.

OBJECTIVE: To report a case of valproic acid overdose treated successfully with high-flux hemodialysis without the addition of charcoal hemoperfusion. CASE SUMMARY: A 25-year-old white woman with a history of multiple suicide attempts and schizophrenia presented after ingesting an unknown amount of valproic acid. She became comatose and developed hypotension and lactic acidosis as valproic acid concentrations increased to > 1200 micrograms/mL (therapeutic concentration 50-100). High-flux hemodialysis was performed for four hours; the calculated elimination rate constant (kel) during the procedure was 0.2522 h-1 with a half-life (t1/2) of 2.74 hours compared with posthemodialysis kel of 0.0296 h-1 and t1/2 of 23.41 hours, suggesting that high-flux hemodialysis effectively eliminates valproic acid. The patient's hemodynamic status and mental function improved in conjunction with the acute reduction in valproic acid concentrations. Her subsequent hospital course was complicated only by transient thrombocytopenia. DISCUSSION: Most literature reports of valproic acid overdose have described the use of charcoal hemoperfusion alone or in combination with hemodialysis to accelerate valproic acid clearance at toxic concentrations. However, the pharmacokinetic properties of valproic acid indicate that hemodialysis alone would be effective therapy for an acute valproic acid overdose. CONCLUSIONS: We suggest that toxic concentrations of valproic acid can be effectively reduced with high-flux hemodialysis without the addition of charcoal hemoperfusion and its attendant risks.

Serum C-peptide concentrations poorly phenotype type 2 diabetic end-stage renal disease patients.

BACKGROUND: A homogeneous patient population is necessary to identify genetic factors that regulate complex disease pathogenesis. In this study, we evaluated clinical and biochemical phenotyping criteria for type 2 diabetes in end-stage renal disease (ESRD) probands of families in which nephropathy is clustered. C-peptide concentrations accurately discriminate type 1 from type 2 diabetic patients with normal renal function, but have not been extensively evaluated in ESRD patients. We hypothesized that C-peptide concentrations may not accurately reflect insulin synthesis in ESRD subjects, since the kidney is the major site of C-peptide catabolism and would poorly correlate with accepted clinical criteria used to classify diabetics as types 1 and 2. METHODS: Consenting diabetic ESRD patients (N = 341) from northeastern Ohio were enrolled. Clinical history was obtained by questionnaire, and predialysis blood samples were collected for C-peptide levels from subjects with at least one living diabetic sibling (N = 127, 48% males, 59% African Americans). RESULTS: Using clinical criteria, 79% of the study population were categorized as type 1 (10%) or type 2 diabetics (69%), while 21% of diabetic ESRD patients could not be classified. In contrast, 98% of the patients were classified as type 2 diabetics when stratified by C-peptide concentrations using criteria derived from the Diabetes Control and Complications Trial Research Group (DCCT) and UREMIDIAB studies. Categorization was concordant in only 70% of ESRD probands when C-peptide concentration and clinical classification algorithms were compared. Using clinical phenotyping criteria as the standard for comparison, C-peptide concentrations classified diabetic ESRD patients with 100% sensitivity, but only 5% specificity. The mean C-peptide concentrations were similar in diabetic ESRD patients (3.2 +/- 1.9 nmol/L) and nondiabetic ESRD subjects (3.5 +/- 1.7 nmol/L, N = 30, P = NS), but were 2.5-fold higher compared with diabetic siblings (1.3 +/- 0.7 nmol/L, N = 30, P < 0.05) with normal renal function and were indistinguishable between type 1 and type 2 diabetics. Although 10% of the diabetic ESRD study population was classified as type 1 diabetics using clinical criteria, only 1.5% of these patients had C-peptide levels less than 0.20 nmol/L, the standard cut-off used to discriminate type 1 from type 2 diabetes in patients with normal renal function. However, the criteria of C-peptide concentrations> 0.50 nmol/L and diabetes onset in patients who are more than 38 years old identify type 2 diabetes with a 97% positive predictive value in our ESRD population. CONCLUSIONS: Accepted clinical criteria, used to discriminate type 1 and type 2 diabetes, failed to classify a significant proportion of diabetic ESRD patients. In contrast to previous reports, C-peptide levels were elevated in the majority of type 1 ESRD diabetic patients and did not improve the power of clinical parameters to separate them from type 2 diabetic or nondiabetic ESRD subjects. Accurate classification of diabetic ESRD patients for genetic epidemiological studies requires both clinical and biochemical criteria, which may differ from norms used in diabetic populations with normal renal function.

Use of serial analysis of gene expression to generate kidney expression libraries.

Chronic renal disease initiation and progression remain incompletely understood. Genome-wide expression monitoring should clarify mechanisms that cause progressive renal disease by determining how clusters of genes coordinately change their activity. Serial analysis of gene expression (SAGE) is a technique of expression profiling, which permits simultaneous, comparative, and quantitative analysis of gene-specific, 9- to 13-bp sequence tags. Using SAGE, we have constructed a tag expression library from ROP-+/+ mouse kidney. Tag sequences were sorted by abundance, and identity was determined by sequence homology searching. Analyses of 3,868 tags yielded 1,453 unique kidney transcripts. Forty-two percent of these transcripts matched mRNA sequence entries with known function, 35% of the transcripts corresponded to expressed sequence tag (EST) entries or cloned genes, whose function has not been established, and 23% represented unidentified genes. Previously characterized transcripts were clustered into functional groups, and those encoding metabolic enzymes, plasma membrane proteins (transporters/receptors), and ribosomal proteins were most abundant (39, 14, and 12% of known transcripts, respectively). The most common, kidney-specific transcripts were kidney androgen-regulated protein (4% of all transcripts), sodium-phosphate cotransporter (0.3%), renal cytochrome P-450 (0.3%), parathyroid hormone receptor (0.1%), and kidney-specific cadherin (0.1%). Comprehensively characterizing and contrasting gene expression patterns in normal and diseased kidneys will provide an alternative strategy to identify candidate pathways, which regulate nephropathy susceptibility and progression, and novel targets for therapeutic intervention.

Genetic susceptibility to end-stage renal disease.

New methods have been developed to uncover the genotypes that result in complex diseases. End-stage renal disease is a complex disease, without a simple correspondence between genotype and phenotype. Both population-based and family-based epidemiological studies and analysis of model organisms suggest that the pathogenesis of end-stage renal disease may have a genetic component. A number of studies have analyzed candidate nephropathy genes with little success, but recently several well-designed studies of multiplex families with diabetic nephropathy have identified candidate nephropathy susceptibility loci. To date, kidney disease-oriented research has focused on effector mechanisms responsible for the initiation and progression of chronic renal disease. However, because end-stage renal disease is a complex disease, interruption of a single effector pathway is unlikely to result in significant therapeutic benefit. Further understanding of the pathogenesis of kidney disease and the development of new kidney disease therapies will require continued application of genetic and genomic tools to kidney disease research.

The IL-1 receptor and Rho directly associate to drive cell activation in inflammation.

IL-1-stimulated mesenchymal cells model molecular mechanisms of inflammation. Binding of IL-1 to the type I IL-1 receptor (IL-1R) clusters a multi-subunit signaling complex at focal adhesion complexes. Since Rho family GTPases coordinately organize actin cytoskeleton and signaling to regulate cell phenotype, we hypothesized that the IL-1R signaling complex contained these G proteins. IL-1 stimulated actin stress fiber formation in serum-starved HeLa cells in a Rho-dependent manner and rapidly activated nucleotide exchange on RhoA. Glutathione S-transferase (GST) fusion proteins, containing either the full-length IL-1R cytosolic domain (GST-IL-1Rcd) or the terminal 68 amino acids of IL-1R required for IL-1-dependent signal transduction, specifically coprecipitated both RhoA and Rac-1, but not p21(ras), from Triton-soluble HeLa cell extracts. In whole cells, a small-molecular-weight G protein coimmunoprecipitated by anti-IL-1R antibody was a substrate for C3 transferase, which specifically ADP-ribosylates Rho GTPases. Constitutively activated RhoA, loaded with [gamma-32P]GTP, directly interacted with GST-IL-1Rcd in a filter-binding assay. The IL-1Rcd-RhoA interaction was functionally important, since a dominant inhibitory mutant of RhoA prevented IL-1Rcd-directed transcriptional activation of the IL-6 gene. Consistent with our previous data demonstrating that IL-1R-associated myelin basic protein (MBP) kinases are necessary for IL-1-directed gene expression, cellular incorporation of C3 transferase inhibited IL-1R-associated MBP kinase activity both in solution and in gel kinase assays. In summary, IL-1 activated RhoA, which was physically associated with IL-1Rcd and necessary for activation of cytosolic nuclear signaling pathways. These findings suggest that IL-1-stimulated, Rho-dependent cytoskeletal reorganization may cluster signaling molecules in specific architectures that are necessary for persistent cell activation in chronic inflammatory disease.

Interleukin-1 stimulates Jun N-terminal/stress-activated protein kinase by an arachidonate-dependent mechanism in mesangial cells.

BACKGROUND: We have studied interleukin-1 (IL-1)-stimulated signals and gene expression in mesangial cells (MCs) to identify molecular mechanisms of MC activation, a process characteristic of glomerular inflammation. The JNK1 pathway has been implicated in cell fate decisions, and IL-1 stimulates the Jun N-terminal/stress-activated protein kinases (JNK1/SAPK). However, early postreceptor mechanisms by which IL-1 activates these enzymes remain unclear. Free arachidonic acid (AA) activates several protein kinases, and because IL-1 rapidly stimulates phospholipase A2 (PLA2) activity release AA, IL-1-induced activation of JNK1/SAPK may be mediated by AA release. METHODS: MCs were grown from collagenase-treated glomeruli, and JNK/SAPK activity in MC lysates was determined using an immunocomplex kinase assay. RESULT: Treatment of MCs with IL-1 alpha induced a time-dependent increase in JNK1/SAPK kinase activity, assessed by phosphorylation of the activating transcription factor-2 (ATF-2). Using similar incubation conditions, IL-1 also increased [3H]AA release from MCs. Pretreatment of MCs with aristolochic acid, a PLA2 inhibitor, concordantly reduced IL-1-regulated [3H]AA release and JNK1/SAPK activity, suggesting that cytosolic AA in part mediates IL-1-induced JNK1/SAPK activation. Addition of AA stimulated JNK1/SAPK activity in a time- and concentration-dependent manner. This effect was AA specific, as only AA and its precursor linoleic acid stimulated JNK1/SAPK activity. Other fatty acids failed to activate JNK1/SAPK. Pretreatment of MCs with specific inhibitors of AA oxidation by cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase had no effect on either IL-1- or AA-induced JNK1/SAPK activation. Furthermore, stimulation of MCs with the exogenous cyclooxygenase-, lipoxygenase-, phosphodiesterase-, and epoxygenase-derived arachidonate metabolites, in contrast to AA itself, did not activate JNK1/SAPK. CONCLUSION: We conclude that IL-1-stimulated AA release, in part, mediates stimulation of JNK1/SAPK activity and that AA activates JNK1/SAPK by a mechanism that does not require enzymatic oxygenation. JNK1 signaling pathway components may provide molecular switches that mediate structural rearrangements and biochemical processes characteristic of MC activation and could provide a novel target(s) for therapeutic intervention.

Management of tumor lysis syndrome with standard continuous arteriovenous hemodialysis: case report and a review of the literature.

Tumor lysis syndrome (TLS) is a critical illness with few treatment options. This report describes the clinical course of a patient with non-Hodgkin's lymphoma, who developed TLS and required renal replacement therapy. Institution of the standard therapeutic approach, intermittent hemodialysis, was not possible because of persistent hypotension. Instead, the patient was treated with a short course of continuous arteriovenous hemofiltration (CAVH) and conventional continuous arteriovenous hemodialysis (CAVHD) with dialysate flow rate of 1 L/h), which resulted in effective control of serum uric acid, potassium, urea nitrogen, phosphorus, and extracellular fluid volume. This case is in distinction to a previous report of TLS treatment with CAVHD using 4 L/h dialysate flow rate. We conclude that continuous renal replacement therapies with standard dialysate flow rates and replacement volumes should be considered for the treatment of TLS, particulary if the syndrome is accompanied by hypotension.

Cytosolic domain of the type I interleukin-1 receptor spontaneously recruits signaling molecules to activate a proinflammatory gene.

Immediate postreceptor events activated by IL-1-IL-1R interaction remain undefined. We have initiated studies to identify candidate signal transducers that associate with the cytosolic domain (cd) of the IL-1R. Immunocomplex kinase assays demonstrated an IL-1-activated myelin basic protein kinase activity that coprecipitated with the IL-1R from rat mesangial, mouse EL-4, and HeLa cells. Using glutathione-S-transferase (GST) fusion proteins, HeLa cell lysates next were assayed for kinases that associated with IL-1R cytoplasmic sequences. A GST-IL-1R fusion protein containing the entire cd (amino acids 369-569; GST-IL-1Rcd) recruited a kinase activity in the absence and presence of IL-1 stimulation. In contrast, a GST-IL-1R membrane-proximal region mutant (amino acids 369-501; GST-IL-1RcdDelta), which lacks COOH-terminal amino acid residues required for nuclear factor-kappaB activation, poorly phosphorylated MBP. In gel, kinase assays demonstrated 63-, 83-, and 100-kD kinases that specifically coprecipitated with the HeLa IL-1R and the GST-IL-1Rcd, but not GST-IL-1RcdDelta. 35S-labeled proteins, with Mrs identical to the kinase activities, stably associated with GST-IL-1Rcd. Transient transfection assays of 293 cells were used to evaluate the functional significance of these findings. Simply increasing IL-1cd expression in 293 cells stimulated 5'-IL-6 flanking region-regulated CAT activity threefold above control, an effect blocked by the kinase inhibitors staurosporine and calphostin C. In summary, we have identified two previously unrecognized 63- and 83-kD kinases as well as a protein with an Mr similar to the recently cloned IL-1R-associated kinase, all of which associate spontaneously with the IL-1Rcd. Ectopic IL-1Rcd expression was sufficient to trigger cellular activation, suggesting that the extracellular domain of the intact receptor represses signal transduction until IL-1 is bound. Given that the IL-1Rcd signaling domain has been conserved in a functionally diverse group of transmembrane receptors, further characterization of this signaling process may define novel molecular mechanisms controlling cellular function and differentiation.

PDGF and IL-1 induce and activate specific protein kinase C isoforms in mesangial cells.

In vitro and in vivo data suggest a remarkable plasticity in the differentiated phenotype of intrinsic glomerular cells, which after injury express new structures and functions. We have shown that a protein kinase C (PKC) isoform, beta II, is expressed in diseased but not normal glomeruli. Since intrarenal cytokine synthesis has been implicated in the pathogenesis of progressive glomerular injury, we have hypothesized that these mediators induce a change in isoform profile. To test this hypothesis in vitro, we have determined whether platelet-derived growth factor (PDGF) and interleukin-1 (IL-1) alter the expression or activation of PKC isoforms in cultured mesangial cells (MCs). By immunoblot and ribonuclease (RNase) protection assays, both PDGF and IL-1 induce as early as 2 h de novo synthesis of PKC-beta II. Since MCs constitutively express PKC-alpha, -beta I, and -zeta, we also determined whether IL-1 or PDGF alter the activity of these isoforms. PDGF maximally induced translocation of PKC-alpha (10 min), -beta I (90 min), -epsilon (120 min), and -zeta (120 min) from the cytosolic to the membrane fraction. IL-1, in contrast, did not alter the distribution of alpha, beta I, or epsilon at any time measured but did induce PKC-zeta translocation. These data suggest inflammatory mediators regulate PKC isoform activity in diseased glomeruli both by de novo synthesis of unexpressed isoforms and by activation of constitutively expressed PKC isoforms.

Glucocorticoid uncoupling of antiogensin II-dependent phospholipase C activation in rat vascular smooth muscle cells.

Vascular tone is maintained by both angiotensin II (Ang II) and glucocorticoids, but the effect of glucocorticoids on Ang II function in vascular smooth muscle cells (VSMC) is unclear. To determine the direct influence of glucocorticoids on VSMC Ang II receptor function, the effects of dexamethasone on Ang II receptor binding, Ang II-induced phospholipase C (PLC) activation, and Ang II-dependent cell growth were studied in cultured rat VSMC. Dexamethasone caused concentration- and time-dependent increases in Ang II binding which were prevented by glucocorticoid receptor inhibition with RU 38486. Dexamethasone-induced enhancement of Ang II binding resulted from increased AT1 receptors, as indicated by Northern blot analysis and competitive binding assays. Despite causing increased Ang II receptor number, dexamethasone preincubation prevented Ang II-induced PLC activation, as indicated by phosphatidylinositol 4,5-bisphosphate degradation and inositol trisphosphate formation. When PLC activity was directly measured in VSMC soluble and membrane fractions, Ang II receptor activation caused decreased soluble and increased membrane PLC activity, consistent with the interpretation that Ang II caused cytosol-to-membrane PLC translocation. The effect of Ang II on PLC translocation was prevented by dexamethasone preincubation. Finally, prolonged incubation with dexamethasone and Ang II had additive effects on VSMC hypertrophy. In conclusion, glucocorticoids directly altered Ang II function in VSMC by causing increased Ang II receptor number, Ang II receptor/PLC uncoupling, and enhanced Ang II-dependent hypertrophy.

Endothelin-1 stimulates cytosolic phospholipase A2 in Chinese hamster ovary cells stably expressing the human ETA or ETB receptor subtype.

The ability of endothelin-1 (ET-1) to activate cytosolic phospholipase A2 (cPLA2) has been studied in Chinese Hamster ovary (CHO) cells stably expressing either the human ETA (CHO/ETA) or the human ETB (CHO/ETB) receptor subtype. ET-1 dose-dependently increased a dithiothreitol-insensitive cPLA2 activity in both cell types. In CHO/ETA cells, BQ-123, an ETA-selective antagonist, completely blocked ET-1-induced cPLA2 stimulation. In CHO/ETB cells, the ET-1 response was mimicked by 4AlaET-1 which could be blocked partially by PD 145065, a nonselective antagonist of ETA and ETB. As expected, ET-1 stimulated PGE2 synthesis in CHO cells transfected with ET receptors. We conclude that ET-1 can stimulate cPLA2 via both the human ETA and ETB receptor subtype.

Interleukin 1 alpha causes rapid activation of cytosolic phospholipase A2 by phosphorylation in rat mesangial cells.

We have shown previously that interleukin 1 (IL-1) stimulates eicosanoid production in glomerular mesangial cells (MC) by de novo synthesis of a 14-kD, group II phospholipase A2 (PLA2). IL-1-stimulated prostaglandin E2 synthesis precedes expression of this enzyme, suggesting that another PLA2 isoform must be more rapidly activated. In the presence but not absence of calcium inophore, [3H]arachidonate release is increased significantly as early as 5 min after addition of IL-1, and IL-1 concurrently stimulates a Ca(2+)-dependent phospholipase activity, which was characterized as the cytosolic form of PLA2 (cPLA2). IL-1 does not alter either cPLA2 mRNA expression or mass in serum-stimulated MC, suggesting that cPLA2 activity is increased by a posttranslational modification. IL-1 treatment for 30 min doubles 32P incorporation into immunoprecipitable cPLA2 protein, concordant with the increase in enzyme activity. Immunoblot analysis of extracts derived from IL-1-treated (30 min) cells demonstrates a decreased mobility of cPLA2, and treatment of MC lysates with acid phosphatase significantly reduces cytokine-activated cPLA2 activity, further indicating that IL-1 stimulates phosphorylation of the enzyme. IL-1 treatment (24 h) of serum-deprived MC doubled cPLA2 mRNA, protein, and activity. In summary, IL-1 increases cPLA2 activity in a biphasic, time-dependent manner both by posttranslational modification and de novo synthesis. We consider cPLA2 activation a key step in IL-1-stimulated synthesis of pro-inflammatory, lipid mediators, and an integral event in the phenotypic responses induced in target cells by this cytokine.