The Role of TRPC6 Channels in Glomerular Capillary Endothelial Cell Injury Induced by Mechanic Stretch and High Glucose
Authors: Wang Zhen , do Carmo Jussara M. , Aberdein Nicola , Fang Taolin , Hall John E. 
Published: November 2023

Transient receptor potential C6 (TRPC6) is a non–voltage‐gated Ca2+ entry channel that responds to neurohormonal and mechanical stimuli and is widely expressed in kidney cells, including endothelium and podocytes of the glomerulus. Previous studies suggest that prolonged TRPC6 activation may increase Ca2+ influx and cause glomerular injury. Our studies in type 2 diabetic rats suggest that mechanical stretch induced by high blood pressure may interact synergistically with hyperglycemia to promote glomerular injury. However, the role of TRPC6 channels in glomerular injury and their relationship to mechanical stretch in hypertension, especially in the presence of diabetes, is still unclear. In this study we examined whether TRPC3/6 channels are excessively activated and lead to cellular injury in glomerular endothelial cells exposed to elevated glucose to mimic diabetes and cyclic stretch to mimic moderate hypertension. Primary mouse glomerular capillary endothelial cells were cultured in DMED media containing 100 mg/dL of D‐glucose on flexible‐bottomed membranes in culture wells. A Flexcell tension system was used to produce cyclic stretch (0.5 sec of stretch and 0.5 sec of relaxation, 60 cycles per minute) for 24 hrs with vacuum pressure of −15 kPa, which induced an average 20% radius elongation of the culture membranes. Cells were studied under four different conditions: 1) stretch with different levels of glucose (100 or 500 mg/dL of D‐glucose); 2) different glucose without stretch; 3) stretch with different glucose and TRPC3/6 inhibitor (GSK‐417651A, 10mg/ml); and 4) different glucose plus TRPC3/6 inhibitor. After 24 hrs, calpain activity, a calcium‐dependent protease, was measured in cell lysates by fluorometric assay and cytochrome C expression was measured by western blot as an indicator of apoptosis. In addition, reactive oxygen species (ROS) generation was assessed by MitoSOX staining in cells submitted to stretch with high glucose and when combined with TRPC3/6 inhibitor. Calpain activity increased significantly when media glucose increased from 100 to 500 mg/dL and rose further after application of 20% stretch for 24 hrs (64.0±2.0 vs 30.2±3.3 Unit in 100mg/dL glucose and 77.6±15.2 vs 54.9±6.6 Unit in 500mg/dL glucose when compared with cells without stretch). TRPC3/6 inhibition decreased calpain activity in stretched cells (37.9±5.6 vs 64.0±2.0 Unit in 100mg/dL glucose and 39.3±3.1 vs 77.6±15.2 Unit in 500mg/dL glucose compared to the stretched cells without treatment). Cyclic stretch significantly increased cytochrome C expression and this change was prevented by TRPC3/6 inhibition. Cyclic stretch with high glucose also increased ROS generation and this effect that was blocked by inhibition of TRPC3/6. These findings suggest that mechanic stretch of endothelial cells exposed to high glucose may impair Ca2+ homeostasis and increase ROS generation to induce cell apoptosis. These effects may be mediated by stretch‐induced TRPC3/6 channel activation.

Support or Funding Information

Supported by NHLBI‐PO1HL51971, and NIGMS P20GM10435776