Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB132

Poster Communications

Mineralocorticoid receptor deficiency in endothelial cells does not aggravate renal fibrosis during unilateral ureteral obstruction in mice

N. O. Lorenzen1, J. Stubbe1, H. Dimke1, K. Madsen1,2

1. Department of Cardiovascular and Renal Research, University of Southern Denmark, Odense C, Denmark. 2. Department of Pathology, Odense University Hospital, Odense, Denmark.

Mineralocorticoid receptors (MR) are involved in inflammation and end-stage organ damage such as renal fibrosis in chronic kidney diseases (CKD) independent of their role in blood pressure regulation. Capillary rarefaction is a hallmark feature in CKD, correlating with declining renal function and development of renal fibrosis. MR is expressed in endothelial cells of the renal microvasculature, and we tested the hypothesis that loss of MR signaling in the endothelium attenuates the development of renal fibrosis through improved renal microvascular integrity. Mice with conditional knock out (KO) of MR in endothelial cells (EC) were generated by crossing mice with a floxed exon 3 in the Nr3c2 gene encoding the MR with mice expressing cre recombinase driven by the Tie-2 promoter. Conditional EC-MR KO mice and littermate controls were anaesthetized using a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg) and unilateral ureteral obstruction (UUO) or sham surgery was performed. 7 days after surgery, animals were sacrificed and kidneys snap frozen in liquid nitrogen or fixed by systemic perfusion with 4% paraformaldehyde with kidney tissue embedded in paraffin for morphological analyses. Renal fibrosis was examined by quantitative determination of extracellular matrix markers by quantitative PCR. The density of renal microvessels was investigated by immunohistochemical staining of the endothelial cell marker CD34. Results showed no significant changes in body weight between genotypes. Kidney/body weight ratio of the obstructed kidneys was significantly increased in both EC-MR KO mice and littermate controls compared to sham operated controls (n=9). Moreover, kidney/body weight ratio of the contralateral kidney showed a strong tendency towards an increase in both genotypes but this did not reach statistical significance. Quantitative determination of extracellular matrix markers showed a significant increase of collagen I, collagen III, fibronectin, alpha-smooth muscle cell actin and fibroblast-specific protein 1 mRNA expression in both EC-MR KO mice and littermate controls after 7 days UUO. No difference was detected in between genotypes. No difference in renal microvessel density was observed in sham operated EC-MR KO mice and littermate controls. After 7 days UUO, capillary rarefaction was evident but no difference between genotypes was observed. Since Tie-2 is also expressed in a subgroup of macrophages, MR expression was determined in primary cultures of bone marrow derived macrophages (BMDM) isolated from both EC-MR KO mice and littermate controls. No significant difference in MR mRNA expression was detected between genotypes. In conclusion, MR signaling in endothelial cells does not play a role for renal fibrogenesis including capillary rarefaction in the UUO model of CKD.

Where applicable, experiments conform with Society ethical requirements