HIF‐prolyl Hydroxylase‐3 as the downstream pathway of TRPC6 Mediates Hypertensioninduced Renal Injury in 5/6 Ablation/Infarction Model

Abstract

The activation of transient receptor potential canonical 6 (TRPC6) channel participates in the glomerulosclerosis. The TRPC6 also participates in the mechanical or pressure sensation in various cell types, including podocyte. Thus, TRPC6 may be the upstream mediator that transmits the pressure stress into downstream molecular pathways to cause hypertension‐induced renal injury. We previously showed that TRPC6 shRNA blocked the hypertension‐induced renal injury in rats with 5/6 renal ablation/infarction (A/I), a model in which the renal injury is closely associated with blood pressure, suggesting that TRPC6 is the mediator of hypertension‐induced renal injury. However, the signaling pathways downstream of TRPC6 remain unclear. It has been shown that TRPC6 signaling inhibits the activity of HIF‐proly hydroxylase to stabilize hypoxia‐inducible factor (HIF)‐1α, while renal HIF‐1α activation is known to produce chronic renal injury. The present study tested the hypothesis that inhibition of proly hydroxylase (PHD) mediates the injurious effect of activated TRPC6 in the kidney of 5/6 A/I model. Adults male Sprague Dawley rats were divided into shame (S), 5/6 A/I control (C), 5/6 A/I TRPC6 shRNA (T), and 5/6 A/I TRPC6 shRNA+PHD3 shRNA (T+P) groups. The control and shRNA plasmids were transfected via intra‐renal arterial injection using the ultrasound‐microbubble technique. The results showed that the levels of TRPC6 in isolated glomeruli were increased in C group compared with S group and that the increase of TRPC6 was blocked in T and T+P groups, the relative protein levels of TRPC6 by Western blot analysis were 1.0±0.16, 3.7±0.26, 1.3±0.14 and 1.5±0.19 in S, C, T and T+P groups, respectively*. The levels of hydroxylated HIF‐1α (HO‐HIF‐1α), the indicator of PHD activity, were significantly reduced in C group compared with S group; TRPC6 shRNA recovered the levels of HO‐HIF‐1α, whereas PHD3 shRNA blocked the increase of HO‐HIF‐1α by TRPC6 shRNA; the relative protein levels of HO‐HIF‐1α were 1.0±0.06, 0.47±0.03, 0.83±0.09 and 0.31±0.09 in S, C, T and T+P groups, respectively*, suggesting that activated TRPC6 inhibites PHD activities in 5/6 A/I model and that PHD3 shRNA mimics the inhibitory effect of TRPC6 on PHD activity. The shRNA plasmids had no effect on hypertension in 5/6 A/I rats (mean arterial pressure: 153.9 ± 8.1 mmHg in C group vs. 154.7 ± 8.9 and 153.6±14 in T and T+P groups). However, the TRPC6 shRNA‐induced attenuation of albuminuria was eliminated by PHD3 shRNA. The levels of urinary albumin were 0.7±0.11 mg/Kg/24h in S, 35.6±6.7 in C, 4.9±1.5 in T, and 57.9±13.5 in T+P groups, respectively*. The regression slops between the blood pressure and the % of glomerulosclerosis were significantly steeper in C and T+P groups than in T group*, indicating that PHD3 shRNA abolished the protective effect of TRPC6 shRNA in hypertension‐induced glomerular injuries. These data suggested that hypertension‐induced activation of TRPC6 may be the molecular sensor of pressure stress to produce kidney damage associated with hypertension and that PHD3 is the downstream pathway of TRPC6 mediates the damage. * P<0.05Support or Funding InformationNIH grants DK107991 to NL, DK102539 to JR and DK54927 to PLThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.