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Introduction The main cause of acute kidney injury (AKI) is ischemia/ reperfusion (IR). AKI has been associated with chronic kidney disease progression and cardiac alterations. Previous studies showed that in a rat model of kidney IR, mineralocorticoid receptor (MR) antagonism with spironolactone prevents tubular injury and renal dysfunction. Despite their benefits in renal diseases, the current MR antagonists have strong limitations like hyperkalemia, thus motivating the search of novel antagonists with safer profile. Objective Test the efficacy of the non-steroidal MR antagonist BR-4628 against renal injury induced by IR. Methods Twenty male wistar rats were divided in 4 groups: sham-operated (S), bilateral renal ischemia (IR) for 25min, treated with BR-4628 (10mg/kg) either 3 days before IR (B10-pre) or 3 hours after IR (B10-post). All groups were studied 24h after reperfusion. Blood sample was taken for creatinine and urea quantification. The right kidney was processed for molecular studies and the left for histopathological analysis. Results Rats with IR developed renal injury characterized by renal dysfunction, increase in tubular injury markers (Hsp72 and Kim-1), oxidized proteins and pro-inflammatory cytokines. These alterations were absent in the B10-pre and B10-post IR groups. IR induced an increased expression of the M1 macrophage markers iNOS, CCL3, TNF-alpha and MCP-1 (inflammatory phenotype) that was fully prevented with the BR-4628 pre-treatment. Furthermore we observed that in the B10-pre and B10-post groups there was a greater expression of M2 macrophage markers; IL4r, FIZZ1 and Igfr2 (wound healing phenotype), as compared to the non-treated IR group. Conclusion We show for the first time that the non-steroidal BR-4628 MR antagonist is effective to prevent or treat renal injury induced by IR possibly through a mechanism involving macrophage polarization towards the wound healing phenotype.

Introduction Renal ischemia/reperfusion (IR) is an important cause of acute kidney injury (AKI). AKI has been linked with progression to chronic kidney disease and development of cardiac alterations. Previous studies from our laboratory have shown that mineralocorticoid receptor (MR) antagonism with spironolactone prevents tubular injury and renal dysfunction induced by IR. The protection mechanisms remain unclear and whether the blockade of vascular MR is responsible for the protection conferred by MR antagonists in IR remains unexplored. Objective To evaluate the specific contribution of vascular MR in the development of the kidney injury induced by IR. Methods To investigate the contribution of vascular MR we generated two knockout (KO) mouse models. To allow MR inactivation in endothelial cells (MRendoKO mice), floxed MR mice (MRfl/fl) were crossed with mice expressing the Cre recombinase under the Tie2cre promoter. To allow MR inactivation in smooth muscle cells (MRSMCKO mice), MRfl/fl mice were crossed with mice expressing the inducible Cre recombinase under the SMA promoter (MRSMCKO). In these mice, sham surgery or bilateral renal IR for 20min was performed. The animals were studied 24 h after reperfusion. As markers of tubular injury, the mRNA levels of Kim-1 and NGAL were quantified. Results In MRfl/fl mice, IR induced renal dysfunction (plasma creatinine raised from 8.9±0.3 in sham to 33.8±4.8 μmol/L in IR), tubular injury and an increase in mRNA levels of Kim-1 (400-fold) and NGAL (200-fold). The MRendoKO mice displayed similar alterations induced by IR as MRfl/fl mice. In contrast, after renal IR, the MRSMCKO mice presented normal renal function (plasma creatinine was 9.6±0.7 and 14.0±1.9 μmol/L in sham and IR, respectively), absence of histological alterations and a significant reduction in the mRNA levels of Kim-1 and NGAL. We are now testing the effect of MR deficiency in smooth muscle cells in preventing the development of chronic alterations induced by IR. Conclusion We show that the deficiency of the MR expressed in smooth muscle cells protects against the renal injury induced by IR. Our data provides further evidence to support the use MR antagonists as a novel therapeutic approach to prevent acute and chronic consequences of renal IR.

Introduction Acute kidney injury (AKI) is defined as an abrupt decrease (48h) in kidney function. One of the main causes of AKI is ischemia/reperfusion (I/R). AKI is related with high mortality, chronic kidney disease development and cardiac alterations like heart failure and arrhythmias. Mineralocorticoid receptor (MR) antagonism with spironolactone (Sp) prevents tubular injury and renal dysfunction induced by I/R in the rat. Although there is information supporting a role for aldosterone and MR in kidney injury, it remains unexplored the specific role of the MR expressed in the vasculature in mediating the deleterious effects of aldosterone during renal I/R. Objective To study the effect of inducing I/R in mice lacking the MR specifically in the endothelial cells or in the vascular smooth muscle cells. Methods To test if Sp is also able to prevent renal injury induced by I/R in the mice with the C57BL/6 background (same as MR KO mice) we included three groups of mice: 1) Sham, 2) I/R 20 min and 3) I/R 20 min + Sp pre-treatment. We analyzed the presence of renal dysfunction and inflammatory cytokines. In the MR KO mice, we will analyze the effect of MR deficiency after renal I/R in an acute phase (24h) and in chronic kidney disease development (after 4 weeks). In the acute studies the mechanisms that will be explored include: polarization of macrophages, endothelial injury and oxidative stress. In the chronic studies we will test if the wild type or MR knockout mice develop CKD as a consequence of renal I/R. Results Mice underwent renal I/R developed injury characterized by increased serum creatinine and urea levels, urinary Hsp72 and elevation in the mRNA of TNF-alpha and MCP-1 pro-inflammatory cytokines. These alterations were prevented by the Sp pre-treatment. Conclusion The protective effect of Sp against renal I/R that was previously reported in the rat is also observed in the C57BL/6 mice and supports the study of the MR KO mice in the renal I/R setting.