Fig. 11.

Comparison of low-dose and high-dose Hydralazine administration in experimental kidney fibrosis and CKD patients. (A) Representative photomicrographs of Masson's trichrome-stained (MTS), αSMA or Fsp1 immunolabeled kidney sections from mice receiving either vehicle buffer PBS, low-dose (5 mg/kg/day) or high-dose Hydralazine (50 mg/kg/day). The panel shows fibrotic kidney sections from mice challenged with UUO at indicated time points after ureteral obstruction. As compared to control mice receiving vehicle buffer PBS, only treatment with low-dose Hydralazine attenuated renal fibrogenesis and fibroblast accumulation. In contrast, high-dose Hydralazine failed to attenuate kidney fibrosis in this mouse model (scale bars: 50 µm). (B–D) The time course summarizes average values at the indicated time points of each group (n = 6 in each group, data are presented as means ± s.e.m. *p < 0.05, values of p were calculated respective to vehicle-treated UUO mice). (E, F) Rasal1 promoter methylation was analyzed by MeDIP in mice challenged with unilateral ureteral obstruction (UUO) and treated with either vehicle buffer PBS, 5 mg/kg/day (low-dose), or 50 mg/kg/day Hydralazine (high-dose), Rasal1 mRNA expression levels were analyzed by qRT-PCR. As compared to vehicle-treated UUO mice, both concentrations of Hydralazine were capable to normalize Rasal1 promoter methylation and mRNA expression (n = 6 in each group, experiments were done in triplicate, data are presented as means ± s.e.m. *p < 0.05, values of p were calculated respective to vehicle-treated UUO mice). (G) As determined by SDS-Page of total kidney protein lysates and subsequent immunoblotting, we found levels of Hif1α markedly increased in kidneys of mice that received high-dose Hydralazine at 50 mg/kg/day, but not in kidneys of mice which had received Hydralazine at 5 mg/kg/day (n = 3 in each group). (H) Compared to hypertensive patients without Dihydralazine medication (n = 4), low-dose Dihydralazine medication was associated with attenuation of CKD progression indicated by blunted rise of serum creatinine from baseline over time (n = 3). In contrast, high doses of Dihydralazine failed to attenuate CKD progression (n = 5, data are presented as scatter dot blots with lines at means. *p < 0.05, n.s. no significance, values of p were calculated respective to patients without Dihydralazine medication).