Effect of bradykinin on renal mesangial cell proliferation and extracellular matrix secretion.

Recent studies have found that bradykinin (BK) plays a role in delaying glomerulosclerosis, although the mechanism of this phenomenon remains unclear. Mesangial cell proliferation (MCP) and extracellular matrix (ECM) secretion are important mechanisms for glomerulosclerosis. This study investigated the impact of BK on the platelet-derived growth factor (PDGF)-induced proliferation of mesangial cells, and evaluated its correlations with the extracellular signal-related kinase (ERK) signaling pathway. The results showed that on its own, 10-1000 mg/L BK promoted MCP and ECM secretion and induced ERK phosphorylation. However, BK administration after PDGF pre-incubation inhibited PDGF-induced MCP, ECM secretion, and ERK phosphorylation. The BK B2 receptor-specific antagonist, HOE-140, and tyrosine phosphatase inhibitor (OV) effectively blocked the function of BK. In summary, these results demonstrated that BK has a bidirectional effect on MCP and ECM secretion: when used alone, it promoted effects on these phenomena, but these effects were inhibited when combined with PDGF. This suggests that the role of BK might be achieved through inhibiting activation of the PDGF-induced ERK1/2 pathway.

The effect of hypoxia and reoxygenation in the response of mesangial cells to angiotensin II in vitro / Efeito da hipóxia e reoxigenação na resposta à angiotensina II em células mesangiais in vitro

INTRODUCTION: Mesangial cells (MC) may be involved in the glomerular alterations induced by ischemia/reperfusion injury. OBJECTIVE: To evaluate the response of immortalized MC (IMC) to 30 minutes of hypoxia followed by reoxygenation periods of 30 minutes (H/R30) or 24 hours (H/R24). METHODS: The intracellular calcium concentration ([Ca+2]i) was measured before (baseline) and after adding angiotensin II (AII, 10-5 M) in the presence and absence of glybenclamide (K ATP channel blocker). We estimated the level of intracellular ATP, nitric oxide (NO) and PGE2. RESULTS: ATP concentration decreased after hypoxia and increased after reoxygenation. Hypoxia and H/R induced increases in basal [Ca+2]i. AII induced increases in [Ca+2]i in normoxia (97 ± 9%), hypoxia (72 ± 10%) or HR30 (85 ± 17%) groups, but there was a decrease in the response to AII in group H/R24 since the elevation in [Ca+2]i was significantly lower than in control (61 ± 10%, p < 0.05). Glybenclamide did not modify this response. It was observed a significant increase in NO generation after 24 hours of reoxygenation, but no difference in PGE2 production was observed. Data suggest that H/R injury is characterized by increased basal [Ca+2]i and by an impairment in the response of cells to AII. Results suggest that the relative insensibility to AII may be at least in part mediated by NO but not by prostaglandins or vasodilator K ATP channels. CONCLUSION: H/R caused dysfunction in IMC characterized by increases in basal [Ca+2]i during hypoxia and reduction in the functional response to AII during reoxygenation.

INTRODUÇÃO: Células mesangiais (CM) podem estar envolvidas na lesão glomerular induzida por hipoxia/reperfusão (H/R). OBJETIVO: Avaliar a resposta de CM imortalizadas (CMI) à hipoxia por 30 minutos seguida de reoxigenação por 30 minutos (H/R30) ou 24 horas (H/R24). MÉTODOS: Concentração de cálcio intracelular ([Ca+2]i) foi avaliada antes (basal) e após a adição de angiotensina II (AII, 10-5 M), na presença e na ausência de glibenclamida (bloqueador de canais K ATP). Foram estimados o nível de ATP intracelular, de óxido nítrico (NO) e de PGE2. RESULTADOS: Nível de ATP diminuiu após hipóxia e aumentou após a reoxigenação. H/R induziu aumento na [Ca+2]i basal. A AII elevou a [Ca+2]i nas condições de normoxia (97 ± 9%), hipoxia (72 ± 10%) ou HR30 (85 ± 17%), porém no grupo H/R24, houve diminuição significativa na resposta à AII, uma vez que a elevação da [Ca+2]i foi mais baixa do que no controle (61 ± 10%, p < 0,05). Glibenclamida não alterou esta resposta. Houve um aumento significativo na geração de NO após 24 horas de reoxigenação, mas não foi observada nenhuma diferença na produção de PGE2. Os dados indicam que a injuria celular causada pela hipoxia/reoxigenação é caracterizada pelo aumento na [Ca+2]i basal e por uma diminuição na reatividade celular à AII. Resultados sugerem que a insensibilidade ao agonista constritor pode ser pelo menos em parte, mediada pelo NO, mas não pelas prostaglandinas ou por canais K ATP. CONCLUSÃO: H/R resultou em disfunção das CMI, caracterizada pelo aumento na [Ca+2]i basal durante a hipóxia e redução da resposta funcional a AII durante a reoxigenação.

The effect of hypoxia and reoxygenation in the response of mesangial cells to angiotensin II in vitro.

INTRODUCTION: Mesangial cells (MC) may be involved in the glomerular alterations induced by ischemia/reperfusion injury. OBJECTIVE: To evaluate the response of immortalized MC (IMC) to 30 minutes of hypoxia followed by reoxygenation periods of 30 minutes (H/R30) or 24 hours (H/R24). METHODS: The intracellular calcium concentration ([Ca+2]i) was measured before (baseline) and after adding angiotensin II (AII, 10-5 M) in the presence and absence of glybenclamide (K ATP channel blocker). We estimated the level of intracellular ATP, nitric oxide (NO) and PGE2. RESULTS: ATP concentration decreased after hypoxia and increased after reoxygenation. Hypoxia and H/R induced increases in basal [Ca+2]i. AII induced increases in [Ca+2]i in normoxia (97 ± 9%), hypoxia (72 ± 10%) or HR30 (85 ± 17%) groups, but there was a decrease in the response to AII in group H/R24 since the elevation in [Ca+2]i was significantly lower than in control (61 ± 10%, p < 0.05). Glybenclamide did not modify this response. It was observed a significant increase in NO generation after 24 hours of reoxygenation, but no difference in PGE2 production was observed. Data suggest that H/R injury is characterized by increased basal [Ca+2]i and by an impairment in the response of cells to AII. Results suggest that the relative insensibility to AII may be at least in part mediated by NO but not by prostaglandins or vasodilator K ATP channels. CONCLUSION: H/R caused dysfunction in IMC characterized by increases in basal [Ca+2]i during hypoxia and reduction in the functional response to AII during reoxygenation.

Soluble uric acid increases intracellular calcium through an angiotensin II-dependent mechanism in immortalized human mesangial cells.

Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6-10 mg/dL) for 24-144 h. In further experiments, ihMCs were treated with UA (6-10 mg/dL) for 12 and 24 h simultaneously with losartan (10(-7) mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca(2+)](i), and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.

Regulation of glucose uptake in mesangial cells stimulated by high glucose: role of angiotensin II and insulin.

Mesangial cells (MCs) play a central role in the pathogenesis of diabetic nephropathy (DN). MC dysfunction arises from excessive glucose uptake through insulin-independent glucose transporter (GLUT1). The role of the insulin-dependent transporter (GLUT4) remains unknown. This study evaluated the effect of high glucose on GLUT1, GLUT4, and fibronectin expression levels. Glucose uptake was determined in the absence and presence of insulin. Angiotensin II has been implicated as a mediator of MC abnormalities in DN, and its effects on the GLUTs expression were evaluated in the presence of losartan. MCs were exposed to normal (NG, 10 mM) or high (HG, 30 mM) glucose for 1, 4, 12, 24, and 72 hrs. Glucose uptake was elevated from 1 hr up to 24 hrs of HG, but returned to NG levels after 72 hrs. HG induced an early (1-, 4-, and 12-hrs) rise in GLUT1 expression, returning to NG levels after 72 hrs, whereas GLUT4 was overexpressed at later timepoints (24 and 72 hrs). HG during 4 hrs induced a 40% rise in glucose uptake, which was unaffected by insulin. In contrast, after 72 hrs, glucose uptake was increased by 50%, only under insulin stimulus. Losartan blunted the effects of HG on GLUT1, GLUT4, and fibronectin expression and on glucose uptake. Results suggest that MCs can be highly susceptible to the HG environment since they uptake glucose in both an insulin-independent and insulin-dependent manner. The beneficial effects of angiotensin II inhibition in DN may also involve a decrease in the rate of glucose uptake by MCs.

Escherichia coli lipopolysaccharide inhibits renin activity in human mesangial cells.

Hyperactivation of systemic renin-angiotensin system (RAS) during sepsis is well documented. However, the behavior of intrarenal RAS in the context of endotoxemia is yet to be defined. The present study evaluates the direct effect of Escherichia coli lipopolysaccharide (LPS) on immortalized human mesangial cell (HMC) RAS. Quiescent HMC were incubated with vehicle or LPS (1-100 microg/ml), and levels of angiotensin I and II (Ang I and II) and their metabolites were analyzed by high-performance liquid chromatography. In addition, angiotensin-converting enzyme (ACE) and renin activity were also investigated. Cell lysate and extracellular medium levels of Ang II were rapidly reduced (1 h) in a time- and concentration-dependent manner, reaching a significant -9 fold-change (P<0.001) after 3 h of LPS incubation. Similar results were obtained for Ang I levels (-3 fold-change, P<0.001). We ruled out Ang I and II degradation, as levels of their metabolic fragments were also significantly decreased by LPS. ACE activity was slightly increased following LPS incubation. On the other hand, renin activity was significantly inhibited, as Ang I concentration elevation following exogenous angiotensinogen administration was blunted by LPS (-60% vs vehicle, P<0.001). Renin and angiotensinogen protein levels were not affected by LPS according to Western blot analysis. Taken together, these data demonstrate for the first time that LPS significantly downregulates HMC RAS through inhibition of renin or renin-like activity. These findings are potentially related to the development of and/or recovery from acute renal failure in the context of sepsis.