Paolo Menè

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.

Upregulation of Mesangial Growth Factor and Extracellular Matrix Synthesis by Advanced Glycation End Products Via a Receptor-Mediated Mechanism

Enhanced advanced glycosylation end product (AGE) formation has been shown to participate in the patho-genesis of diabetes-induced glomerular injury by mediating the increased extracellular matrix (ECM) deposition and altered cell growth and turnover leading to mesangial expansion. These effects could be exerted via an AGE-receptor-mediated upregulation of growth factors, such as the IGFs and transforming growth factor-β (TGF-β). We tested this hypothesis in human and rat mesangial cells grown on nonglycated or native bovine serum albumin (BSA), glycated BSA with AGE formation (BSA-AGE), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM), in the presence or absence of an antibody, α-p60, directed against the p60/OST protein named AGE-receptor 1 (AGE-R1), or normal control (pre-immune) serum. The mRNA and/or protein levels of IGF-I, IGF-II, IGF receptors, IGF binding proteins (IGFBPs), TGF-β1 and the ECM components fibronectin, laminin, and collagen IV were measured, together with cell proliferation. Both human and rat mesangial cells grown on BSA-AGE showed increased IGF-I and total and bioac-tive TGF-β medium levels and enhanced IGF-I, IGF-II, and TGF-β1 gene expression, compared with cells grown on BSA, whereas total IGFBP and IGFBP-3 medium content, IGF receptor density and affinity, and IGF-I receptor transcripts were unchanged. Moreover, cells grown on BSA-AGE showed increased ECM protein and mRNA levels versus cells cultured on BSA, whereas cell proliferation was unchanged in human mesangial cells and slightly reduced in rat mesangial cells. Growing cells on BSA-AM did not affect any of the measured parameters. Co-incubation of BSA-AGE with anti-AGE-R1, but not with pre-immune serum, prevented AGE-induced increases in IGF-I, TGF-β1, and ECM production or gene expression; anti-AGE-R1 also reduced growth factor and matrix synthesis in cells grown on BSA. These results demonstrate that mesangial IGF and TGF-β1 synthesis is upregulated by AGE-modified proteins through an AGE-receptor-mediated mechanism. The parallelism with increased ECM production raises the speculation that the enhanced synthesis of these growth factors resulting from advanced nonen-zymatic glycation participates in the pathogenesis of hyperglycemia-induced mesangial expansion.

Mechanisms of glucose-enhanced extracellular matrix accumulation in rat glomerular mesangial cells

In view of the importance of mesangial extracellular matrix (ECM) accumulation in the pathogenesis of diabetic glomerulosclerosis, we investigated 1) the effects of high glucose on ECM production by rat glomerular mesangial cells in culture (study A) and 2) the mechanisms underlying these effects, particularly the role of high sugar levels irrespective of intracellular metabolism (study B1) and of excess glucose disposal via the polyol pathway and associated biochemical alterations (study B2). Cells were cultured for 4 weeks, through six to eight passages, under the experimental conditions indicated below and, at each passage, the levels of fibronectin (FN), laminin (LAM), and collagen types I (C-I), III (C-III), IV (C-IV), and VI (C-VI) in media and cell extracts were quantified by an enzyme immunoassay. In study A, medium and cell content of matrix were assessed, together with [3H]leucine and [3H]thymidine incorporation into monolayers, polyol, fructose, and myo-inositol levels and the cytosolic redox state, in cells grown in high (30 mM) D-glucose or iso-osmolar mannitol versus cells cultured in normal (5.5 mM) D-glucose. FN, LAM, C-IV, and C-VI accumulation, but not C-I and C-III accumulation, was increased by 30 mM glucose, but not by iso-osmolar mannitol, when compared with 5.5 mM glucose, starting at week 2 and, except for C-VI, persisting throughout the remaining 2 weeks, whereas no change was observed in the measured indexes of total protein synthesis and DNA synthesis/cell proliferation. At any time point, polyol levels were increased, whereas myo-inositol was reduced by high glucose; in cells grown under elevated glucose concentrations, the lactate/pyruvate (L/P) ratio, an index of the cytosolic redox state, progressively increased. In study B1, the effects of high D-glucose were compared with those of iso-osmolar concentrations of sugars that are partly or not metabolized but are capable of inducing nonenzymatic glycosylation, such as D-galactose and L-glucose, and of mannitol, which does not enter the cell. Both D-galactose and L-glucose, but not mannitol, partly mimicked D-glucose-induced ECM overproduction. Although D-galactose is metabolized via the polyol pathway and alters the cytosolic redox state, ECM changes induced by high galactose were not prevented by the use of an aldose reductase inhibitor (ARI), Alcon 1576 (14 μM). In study B2, agents interfering with intracellular metabolism of excess glucose via the polyol pathway (14 μM Alcon 1576) and associated changes in myo-inositol metabolism (1 mM myo-inositol) and cytosolic redox state (1 mM sodium pyruvate, which corrects glucose-induced polyol pathway-dependent increased NADH/NAD+) were added to cells cultured in 30 and 5.5 mM D-glucose. Alcon 1576 inhibited polyol pathway activity with decreasing efficacy during the 4-week period, whereas myo-inositol and pyruvate produced complete and persistent prevention of reduced myo-inositol levels and increased L/P ratio, respectively. High glucose-induced ECM overproduction was transiently reduced by pyruvate and, to a lesser extent, by the ARI and myo-inositol. These results suggest that 1) high glucose selectively increases accumulation of basement membrane components and 2) multiple mechanisms seem to be operating in the pathogenesis of glucose-induced ECM overproduction, including elevated sugar levels per se, possibly via nonenzymatic glycosylation, and to a lesser extent, intracellular glucose metabolism via the polyol pathway and associated changes in myo-inositol metabolism and cytosolic redox state.

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.

Uric acid: bystander or culprit in hypertension and progressive renal disease?

In humans, uric acid is the main urinary metabolite of purines. Serum levels are higher compared with other mammalians. Uric acid is an antioxidant and perhaps helps to control blood pressure during a low Na+ diet through stimulation of the renin–angiotensin system. Serum uric acid is also considered a marker of tubular reabsorption and ‘effective’ circulating blood volume. Moreover, hyperuricemia seems to be a cofactor in Na+-sensitive hypertension, a marker and possibly itself responsible for microvascular damage through stimulation of the renin–angiotensin system, inhibition of endothelial nitric oxide, and proliferative effects on vascular smooth muscle. As fructose-rich diets increase uric acid levels, hyperuricemia may also play a role in the metabolic syndrome, triggering insulin resistance and hypertension. A number of studies on rats rendered hyperuricemic by administration of uricase inhibitors have recently confirmed induction of arterial hypertension and microvascular injury, particularly in the remnant kidney or in cyclosporine-induced renal fibrosis.

High glucose level inhibits capacitative Ca2 + influx in cultured rat mesangial cells by a protein kinase C-dependent mechanism

Summary In cultured mesangial cells (MC), capacitative Ca2 + influx via store-operated channels (SOC) is potentiated by agents that release Ca2 + from intracellular stores, and inhibited by protein kinase C (PKC). Cells grown under high glucose conditions, as a model of the diabetic microenvironment, display reduced Ca2 + signalling in response to vasoconstrictors, probably due to downregulation by elevated PKC activity. Since SOC might be relevant to this phenomenon, we assessed Ca2 + influx by microfluorometry of fura-2-loaded rat MC cultured for 5 days in normal (5.5 mmol/l, NG) or high glucose (30 mmol/l, HG). The addition of 1–10 mmol/l Ca2 + to NG cells equilibrated in Ca2 + -free media induced an immediate Ca2 + influx with a free cytosolic Ca2 + ([Ca2 + ]i) plateau of 155 ± 50 and 318 ± 114 nmol/l, respectively. Basal influx was reduced to 88 ± 8 and 145 ± 17 nmol/l [Ca2 + ]i (1–10 mmol/l Ca2 + , p < 0.01) by 30 mmol/l d-glucose. This effect of HG was confirmed by Mn2 + quenching of fura-2, indicating reduced entry of divalent cations via the capacitative pathway. Equimolar l-glucose had no effect on Ca2 + influx, consistent with a non-osmotic mechanism. Arginine vasopressin (10 μmol/l) elicited weaker release of stored Ca2 + and subsequent influx in HG cells (191 ± 33 vs 153 ± 24 nmol/l, 400 ± 76 vs 260 ± 33 nmol/l, 1–10 mmol/l Ca2 + , NG/HG, p < 0.05). To examine the involvement of PKC in the effect of HG on capacitative Ca2 + influx, the enzyme was activated or downregulated by treatment with 0.1 μmol/l phorbol myristate acetate (PMA) for 3 min or 24 h, respectively. PMA acutely inhibited Ca2 + influx in NG cells, while PKC downregulation restored it in HG cells. Similarly, the PKC inhibitors staurosporin or H-7 normalized SOC activity in HG cells. In summary, impairment of Ca2 + influx via SOC by HG is one mechanism of the reduced MC [Ca2 + ]i responsiveness to vasoconstrictors. This event is mediated by PKC and may contribute to the glomerular haemodynamic changes in the initial stages of diabetes mellitus. [Diabetologia (1997) 40: 521–527]

Right Ventricular Dysfunction in Patients with End-Stage Renal Disease

Background: While chronic dialysis treatment has been suggested to increase pulmonary pressure values, right ventricular dysfunction (RVD) is a major cause of death in patients with end-stage renal disease. We investigated the impact of different dialysis treatments on right ventricular function. Methods: We examined 220 subjects grouped as follows: healthy controls (n = 100), peritoneal dialysis (PD; n = 26), hemodialysis (HD) with radial arteriovenous fistula (AVF; n = 62), and HD with brachial AVF (n = 32). Echocardiography including tissue Doppler imaging (TDI) of the right ventricle was performed in all patients. Results: Pulmonary pressure values progressively rose from controls across the 3 dialysis groups (21.7 ± 6.8, 29.7 ± 6.7, 37.9 ± 6.7 and 40.8 ± 6.6 mm Hg, respectively; p < 0.001). TDI indices of right ventricular function were more impaired in HD patients, particularly in those with brachial AVF. RVD, assessed by TDI myocardial performance index, was higher in HD patients compared with PD patients (71.3 vs. 34.6%, p < 0.001). Moreover, the prevalence of RVD further increased in patients with brachial AVF compared with the radial access (90.6 vs. 61.3%, p < 0.001). Conclusions: Compared to DP, HD increases the risk of RVD, particularly in the presence of brachial AVF. TDI may detect early functional failure of the right ventricle in HD patients.

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.

Endothelin-1 activates the phosphoinositide cascade in cultured glomerular mesangial cells

Summary The newly described peptide, endothelin-1 (ET-1), causes profound vasoconstriction, but the pathways of transmembrane signaling remain unclear. We demonstrate that in glomerular mesangial cells, smooth muscle-like vascular pericytes, ET-1 elevates intracellular Ca2+ ([Ca2+]i) by activating the phosphoinositide cascade. ET-1 increased [Ca2+]i in two distinct kinetic patterns. Concentrations of 0.1–10.0 pM ET-1 caused a slow but sustained increase in [Ca2+]i that was insensitive to voltage-gated Ca2+ channel blockade but dependent on extracellular Ca2+. In contrast, ET-1 ≥0. 1 nM evoked a rapid, transient increase in [Ca2+]i followed by a lesser, sustained increase. Only the sustained increment of [Ca2+]i required extracellular Ca2+, but both phases were unaffected by Ca2+ channel blockade. The transient increase in [Ca2+]i resulted from activation of phosphoinositide-specific phospholipase C to release inositol trisphosphate (IPs), which mobilizes Ca2+ from intracellular stores. ET-1 also stimulated amiloride-inhibitable Na+/H+ exchange, causing cytosolic alkalinization. Thus, the phosphoinositide cascade probably mediates some biological functions of ET-1, including possibly contraction via pharmacomechanical coupling.

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells.

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura‐2 responded to exogenous platelet‐activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF‐induced [CA2+ ]i transients consisted of a dose‐dependent phasic peak response followed by a sustained tonic phase of increased [Ca24 ]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+ ]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+ ]i : a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor‐operated channels. Lyso‐PAF had no effect on basal [Ca2+ ]i, and the PAF‐receptor antagonist L652,731 selectively inhibited responses to PAF. PAF‐stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium‐mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF‐induced [Ca2+ ]i transient, suggesting a regulatory role for PKC in PAF‐activation of mesangial cells. An increase in [Ca2+ ]i, as a result of receptor‐linked activation of phospholipase C, may mediate PAF‐induced hemodynamic and inflammatory events in renal glomeruli.— Kester, M.; Menè, P.; Dubyak, G. R.; Dunn, M. J. Elevation of cytosolic free calcium by platelet‐activating factor in cultured rat mesangial cells. FASEB J. 1: 215‐219; 1987.