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.

Cell-specific regulation of type II phospholipase A2 expression in rat mesangial cells.

IL-1 stimulates mesangial cells to synthesize specific proteins, including a non-pancreatic (Type II) phospholipase A2 (PLA2). We have studied the regulation of PLA2 by proinflammatory mediators, implicated in the pathogenesis of glomerulonephritis, and have assessed whether the activation of second messenger systems modulates or mimics PLA2 gene expression by cytokines. IL-1 alpha and beta, TNF alpha, and LPS, but not serum, IL-2, or PDGF, potently induce PLA2 mRNA, and enzyme expression. IL-1-stimulated mesangial cells express a 1.0 kB PLA2 mRNA transcript that is induced in a dose- and time-dependent manner. IL-1-stimulated increases in steady-state PLA2 mRNA abundance result from a moderate increase in PLA2 transcription rate that is amplified by the prolonged persistence of the transcript. Forskolin and dibutyryl cAMP potentiate IL-1-induced PLA2 mRNA and enzyme expression, but have no effect in the absence of cytokine. 12-tetradecanoyl phorbol 13-acetate, sn-1, 2-dioctanoyl glycerol or 1-oleoyl-2-acetyl-sn-glycerol fail to induce PLA2 expression or to alter the effect of IL-1 when coincubated with the cytokine. In contrast, serum deprivation for 24 h specifically enhances IL-1-stimulated PLA2. Genistein potentiates PLA2 mRNA expression in cells exposed to both IL-1 and serum. The inhibitory effect of serum on IL-1-induced PLA2 mRNA abundance is reproduced by PDGF but not dexamethasone. These data demonstrate that the signaling pathways directly engaged by IL-1 to induce PLA2 expression in mesangial cells interact with several second messenger systems in a cell-specific manner. We speculate that IL-1 induces specialized changes in mesangial cell structure and function through direct activation of a transcription factor(s), that result in induction of a specific gene set.

Histamine modulates contraction and cyclic nucleotides in cultured rat mesangial cells. Differential effects mediated by histamine H1 and H2 receptors.

Histamine influences the glomerular microcirculation and modulates immune-inflammatory responses. In the rat kidney, histamine is synthesized by glomeruli and stimulates cyclic nucleotide production specifically in glomeruli. We investigated the in vitro effect of histamine on cyclic nucleotide accumulation in rat cultured glomerular mesangial and epithelial cells. Histamine stimulated cyclic AMP (cAMP) accumulation in cultured mesangial cells (64.0 +/- 22.1 to 511.4 +/- 86.6 pmol/mg protein, n = 9) but had no effect on cAMP accumulation in epithelial cells. This effect was dose-dependent and time-dependent. Stimulation of cAMP accumulation occurred in the range of 5 X 10(-6) M-10(-4) M histamine with a half maximal stimulatory effect of 2 X 10(-5) M. Initial stimulation was noted by 30 s, and maximum stimulation was observed at 5 min. The H2 antagonist cimetidine (10(-4) M) abolished the stimulatory effect of histamine (10(-4) M), while equimolar concentrations of the H1 antagonist diphenhydramine had no significant effect on cAMP accumulation. Moreover, the specific H2 agonist dimaprit, but not the H1 agonist 2-pyridylethylamine, stimulated cAMP accumulation. Histamine had no effect on cAMP accumulation in epithelial cells or on cyclic guanosine monophosphate accumulation in epithelial or mesangial cells. Since the in vivo infusion of histamine reduces ultrafiltration coefficient and since mesangial cell contraction is thought to be responsible for the reduction in the ultrafiltration coefficient, we examined the effect of histamine on the contractile property of mesangial cells. Histamine (5 X 10(-6)-10(-4) M) contracted mesangial cells, and the H1 antagonist diphenhydramine (10(-4) M) but not the H2 antagonist cimetidine (10(-4) M) prevented histamine (10(-4) M) induced contraction. In addition, the H1 agonist 2-pyridylethylamine, but not the H2 agonist dimaprit, contracted mesangial cells. Histamine and its specific agonists and antagonists induced contraction of isolated glomeruli as assessed by glomerular planar surface area in a manner parallel to their effect on mesangial cells. Cinnarizine (10(-5) M), a Ca++ channel blocker, or Ca++, Mg++-free medium prevented histamine (10(-4) M) induced mesangial cell and glomerular contraction. Thus, histamine enhances cAMP accumulation specifically in mesangial cells via an H2 receptor. In contrast, histamine contracts mesangial cells and glomeruli via an H1 receptor, an effect that is dependent on extracellular Ca++ entry. These findings show that histamine potentially influences intraglomerular hemodynamics via effects on mesangial cell contraction. Moreover, our findings considered with the in vivo observation that histamine reduces kf via and H1 receptor provide further support of the hypothesis that mesangial cell contraction regulates the glomerular capillary surface area available for filtration. Our studies also show that this contractile effect of histamine is dependent on extracellular calcium. The presence of a cAMP system sensitive to histamine may have major implications in the pathogenesis of inflammatory glomerulopathies. Mesangial cells possess characteristics similar to circulating and tissue immune effector cells, including lysosomal enzyme release, oxygen radical production, and release of a number of immunomodulatory factors. Histamine and cAMP have been shown to modulate such characteristics of inflammatory cells. It is therefore conceivable that histamine, via its interaction with H2 receptors and subsequent generation cAMP, may have profound effects on such properties of mesangial cells, suggesting that this autacoid may modulate not only glomerular hemodynamics but also immune, inflammatory responses within the glomerulus.

Interleukin-1 generates transmembrane signals from phospholipids through novel pathways in cultured rat mesangial cells.

Although IL-1 stimulates cellular responses in both lymphoid and nonlymphoid cells, the second messengers by which IL-1 activates cells are unknown. Recombinant IL-1 alpha (rIL-1) is a comitogen for glomerular mesangial cells. Using this model we explored potential transmembrane signals by which IL-1 stimulates cellular responses. Certain mitogens hydrolyze inositol phospholipids by phospholipase C to generate 1,2-diacylglycerol, a cofactor for protein kinase C, and inositol (1,4,5)-trisphosphate, which mobilizes intracellular calcium. rIL-1 induced a peak increase in [3H]1,2-diacylglycerol formation at 1 min. Production of 1,2-diacylglycerol often parallels the generation of phosphatidic acid; however, rIL-1 stimulated [32P]phosphatidate formation only after 60 min. rIL-1 did not change the inositol phosphate or cytosolic free calcium concentrations, demonstrating that rIL-1 does not activate an inositol phospholipid-specific phospholipase C. [3H]Phosphorylethanolamine, but not [3H]phosphorylserine or [3H]phosphorylcholine, was maximally elevated at 1 min in mesangial cells incubated with rIL-1. Radioactivity incorporated into phosphatidylethanolamine but not phosphatidylcholine was also decreased in IL-1-stimulated mesangial cells compared with control at 1 min. These data suggest that rIL-1 activates a phospholipase C predominantly linked to phosphatidylethanolamine. In contrast to other mitogens, rIL-1 did not alter intracellular pH. Both 12-0-tetradecanoyl-phorbol-13-acetate, a homologue of 1,2-diacylglycerol, and phosphatidate but not phosphatidylcholine in the presence of 0.5% fetal bovine serum stimulated mesangial cell proliferation. rIL-1-induced cellular activation may be mediated, at least in part, by phospholipid-derived second messengers generated through novel pathways.

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.

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.

Expression of functional interleukin 2 receptors by peripheral blood monocytes from patients with active pulmonary tuberculosis.

Peripheral blood monocytes from patients with active tuberculosis are "activated" by a number of criteria, including selective depression of T-lymphocyte responses to the mycobacterial antigen, tuberculin-purified protein derivative (PPD). We studied monocytes from patients with tuberculosis and healthy skin test reactive controls for expression and function of IL 2 receptors (IL 2R). Depletion of adherent monocytes increased the IL 2 activity of supernatants of PPD-stimulated T cell cultures from patients by 30-fold. When cultured with purified IL 2, adherent cells from the patients depleted IL 2 activity by 66%. Monocytes from patients displayed IL 2R on their surface as evidenced by anti-Tac reactivity, and released soluble IL 2R into the medium during culture. The release of soluble IL 2R was augmented when monocytes were cultured with PPD. Finally, freshly isolated adherent monocytes from patients but not healthy individuals expressed the gene encoding Tac protein. Thus, blood monocytes from patients with tuberculosis express functional IL 2R constitutively. This property may be important in the immunoregulatory and effector function of mononuclear phagocytes during tuberculosis.

Monoclonal antibody characterization of a chymotrypsin-like molecule on neutrophil membrane associated with cellular activation.

Monoclonal antibody 1-15 (Ab 1-15), is a murine anti-human neutrophil (PMN) IgG1 that inhibits PMN effector responses to N-formyl-met-leu-phe (FMLP) and phorbol myristate acetate. In this study, the effects of Ab 1-15 on PMN membrane-related functions were characterized: Ab 1-15 inhibited PMN superoxide (O-2) response to FMLP by 60% (P less than 0.005) without effect on the onset or duration of O-2 production. This inhibition of O-2 response was associated with a significant inhibition of PMN chymotrypsin-like, but not trypsin-like, protease activity. Cell fractionation studies indicated the presence of an Ab 1-15 inhibitable, chymotryptic neutral protease activity in PMN membranes. In studies of Ab 1-15 effects on membrane-related second messenger pathways, Ab 1-15 augmented both FMLP- and isoproterenol-induced intracellular cAMP accumulation, whereas alpha-chymotrypsin decreased PMN cAMP response to these stimuli. Our data suggest that the function-inhibiting, anti-PMN monoclonal Ab 1-15 defines a PMN chymotryptic enzyme on the membrane surface that is involved in regulation of two membrane-related functions, O-2 generation and cAMP generation.

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.

Endothelin stimulates phospholipase C, Na+/H+ exchange, c-fos expression, and mitogenesis in rat mesangial cells.

A recently described peptide hormone, endothelin, is a potent vasoconstrictor, but it is unclear whether endothelin has other biological actions. These experiments extend the range of biological actions of endothelin to stimulation of mitogenesis. Endothelin at low concentrations (0.1-10 nM) induced mitogenesis by quiescent rat glomerular mesangial cells in culture. Mitogenesis induced by endothelin was accompanied by activation of phospholipase C with increased inositol phosphate turnover and increments of intracellular [Ca2+]. Endothelin also activated Na+/H+ exchange, causing cytosolic alkalinization, and enhanced transcription of the c-fos protooncogene, additional biochemical signals closely linked to proliferation. In addition to being a vasoconstrictor, endothelin thus also functions as a mitogen, presumably through activation of phospholipase C.

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.

Regulation of prostaglandin endoperoxide synthase gene expression in cultured rat mesangial cells: induction by serum via a protein kinase-C-dependent mechanism.

Recent evidence suggests that the steady state level of prostaglandin (PG) endoperoxide synthase (PES) might regulate long term increases in PG synthesis, but the mechanism(s) regulating PES gene expression remains unclear. The present experiments investigated the regulation of PES expression in quiescent mesangial cells treated with serum. Serum induced time-dependent increases in PES steady state mRNA, protein level, and enzyme activity. Both the kinetics and cycloheximide sensitivity of PES mRNA induction suggest that PES was induced as a member of the delayed-early gene set. Beta-adrenergic-mediated cAMP accumulation was similar in Go and cycling cells, suggesting that the increase in PES expression did not reflect global up-regulation of signaling cascades in serum-treated cells. Three lines of evidence suggest that protein kinase-C (PKC) mediates serum-stimulated PES gene expression: 1) 12-tetradecanoyl phorbol 13-acetate mimicked the serum response; 2) PES gene expression by serum was attenuated in cells depleted of PKC; and 3) a PKC inhibitor, sangivamycin, blocked serum-stimulated PES gene induction. In addition, the ability of dexamethasone to block serum-induced PES enzyme activity without affecting the increase in PES mRNA points to posttranscriptional mechanisms of regulation. In summary, we conclude that in glomerular mesangial cells the PES gene is inducible, not constitutive, and that expression of this gene in Go cells is induced by serum and results in chronic elevations of PGE2. These findings are consistent with the hypothesis that differential regulation of PES gene expression might play a role in diverse cellular responses, such as mitogenesis and inflammation.

Effects of sulindac and indomethacin on renal prostaglandin synthesis.

We compared the effects of sulindac and indomethacin, the effects of two nonsteroidal anti-inflammatory drugs, on renal prostaglandin synthesis and renal function. Sulindac, 200 mg twice daily, indomethacin, 25 mg four times a day, or placebo were taken by 15 normal female subjects (five in each of three treatment groups). Indomethacin decreased renal excretion of prostaglandins PGE2, PGF2 alpha, and 6-keto-PGF1 alpha, but sulindac and placebo had no effect on renal prostaglandin excretion. Concomitant with the reduction of renal prostaglandin synthesis in the indomethacin group, sodium and chloride excretion decreased; sulindac and placebo had no discernible effects on urine electrolytes. Extrarenal cyclooxygenase activity, as assessed by platelet thromboxane beta 2 release, was inhibited by both sulindac and indomethacin. Plasma renin activity and plasma aldosterone levels fell in all groups as a result of positive sodium balance, but the decrements of aldosterone were greater after indomethacin and sulindac. None of the treatments altered glomerular filtration rate or renal plasma flow in these normal women. We conclude that in normal women renal prostaglandin synthesis and prostaglandin-dependent tubular functions such as Na and Cl reabsorption are relatively unaffected by doses of sulindac (200 mg twice daily) that inhibit nonrenal cyclooxygenase. This may reflect the capacity of oxidative enzymes in the kidney to convert the active sulfide metabolite of sulindac to the inactive prodrug sulindac sulfoxide.

Effects of nonsteroidal anti-inflammatory drugs in healthy subjects.

Nonsteroidal anti-inflammatory drugs inhibit cyclo-oxygenase activity and thereby reduce prostaglandin synthesis. Studies in humans have used these cyclo-oxygenase inhibitors to examine the role of prostaglandins in controlling renal function. Although short-term studies have demonstrated reductions in effective renal plasma flow, glomerular filtration rate, urinary sodium excretion, and plasma renin activity, long-term administration of nonsteroidal anti-inflammatory drugs does not result in significant or clinically important changes in renal function in normal human subjects. If healthy volunteers are placed on low-sodium diets or treated with diuretics, both renal hemodynamics and salt and water excretion can become prostaglandin-dependent. Studies in normal subjects suggest that sulindac, a nonsteroidal anti-inflammatory drug that undergoes biotransformation in the kidney, does not inhibit renal prostaglandin synthesis or urinary sodium excretion under basal conditions but may impair furosemide-stimulated prostaglandin synthesis and changes in renal function. Doses of sulindac that spare basal renal cyclo-oxygenase do inhibit extrarenal cyclo-oxygenase. The mechanism responsible for this biochemical selectivity of sulindac is not related to a differential sensitivity of the renal cyclo-oxygenase to the active metabolite of sulindac, sulindac sulfide. Sulindac sulfide, in concentrations as low as 1 nM, was equipotent to indomethacin as an inhibitor of prostaglandin E2 synthesis in primary cultures of three renal cell lines. Appropriate clinical use of all nonsteroidal anti-inflammatory drugs, including sulindac, requires careful consideration of risk factors that predispose to nephrotoxicity and careful monitoring when administered to patients at risk.

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.

IL-1 alpha increases arachidonyl-CoA: lysophospholipid acyltransferase activity and stimulates [3H]arachidonate incorporation into phospholipids in rat mesangial cells.

The proinflammatory cytokine interleukin-1 alpha is a potent stimulus of prostaglandin synthesis. We have previously shown that IL-1 amplifies mesangial cell prostaglandin synthesis by inducing synthesis of a non-pancreatic phospholipase A2. Phospholipase A2 activation results in the formation of lysophospholipids and free fatty acids. We now investigate the effects of IL-1 alpha on reacylation of lysophospholipids. Incubations with IL-1 alpha for 24 hours significantly stimulated mesangial cell [3H]arachidonic acid incorporation but not [3H]oleic acid incorporation into phosphatidylinositol and phosphatidylethanolamine. Lysophospholipid acyltransferase activity was measured in vitro. Cytokine treatment increased enzyme activity when lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylinositol were used as exogenous substrates. We conclude that IL-1 promotes cellular phospholipid remodeling by stimulating the deacylation and reacylation of phospholipids.