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Diabetic nephropathy (DN) is the leading cause of chronic kidney disease needing renal replacement therapy in the modern world. Therapy for this condition continues to be suboptimal warranting te need for newer therapeutic strategies., Recent clinical observations indicate the therapeutic benefit of combining endothelin receptor blockers (ERB) with those of angiotensin (ARB). However, the precise mechanisms by which such a combination provides greater therapeutic benefit is currently unknown. The purpose of this study is to examine what signaling pathways are significantly affected by this combination that would support the clinically observed therapeutic synergy.
To verify our hypothesis, we used Human glomerular endothelial cells (HGEC) and rat mesangial cells (RMC) in culture. They were grown in DMEM media either under control low glucose (5mM) or high glucose (25 mM) concentration to simulate diabetic milieu. The cells were further divided into four groups depending on exposure to Losartan (LOS 10uM) or Darusentan (DAR 10uM) or a combination (DAR+LOS) or control (none). The cells were exposed to these agents for a maximum of 48 hours. The cells were then separated from the flask, homogenized and the cellular precipitate was used to examine the expression of several cytokines involved in signaling by Western blotting.
Our observations show that in HGEC (and not in RMC) the NOS 3 expression was significantly increased in cells exposed to DOR or the combination of DAR and LOS compared to LOS. We also observed a decrease in CTGF (Connective tissue growth factor) expression in HGEC and RMC exposed to both agents and with more pronounced effects in DAR group in HG conditions. Furthermore, PLCB1 expression was increased in both RMC and HGEC particularly by DAR under HG exposure.
Conclusions
In cultured renal glomerular endothelial cells, exposure to high glucose caused decreased expression of eNOS which mediates constitutive NO production. Treatment with LOS and DAR and especially DAR increased NO production in these cells. NO has salutary benefits in slowing the progression of DN. CTGF is a profibrotic factor that mediates glomerular and interstitial fibrosis in DN. In both RMC and HGEC, treatment with DAR and LOS decreased the expression of CTGF. Furthermore, we have seen an increase in PLCB1 expression with exposure to both agents but more profoundly with DAR. PLCB1 improves insulin sensitivity acting through AKT signaling. Given the differences between LOS and DAR in the magnitude of impact on the signaling mechanisms and possible differences in downstream events, one could envision how the combination could have synergistic effects. By modulating renal NO production, profibrotic mediators and insulin sensitivity, DAR and LOS possibly complement their effects to offer enhanced renoprotection.