Background Loss of glomerular podocytes is an indicator of diabetic kidney disease (DKD). The damage to these cells has been attributed in part to elevated intrarenal oxidative stress. The primary source of the renal reactive oxygen species, particularly H₂O₂, is NADPH oxidase 4 (NOX4). We hypothesized that NOX4-derived H₂O₂ contributes to podocyte damage in DKD via elevation of podocyte calcium.

Methods We used Dahl salt-sensitive (SS) rats with a null mutation for the Nox4 gene (SS^Nox4−/−) and mice with knockout of the nonselective calcium channel TRPC6 or double knockout of TRPC5 and TRPC6. We performed whole animal studies and used biosensor measurements, electron microscopy, electrophysiology, and live calcium imaging experiments to evaluate the contribution of this pathway to the physiology of the podocytes in freshly isolated glomeruli.

Results Upon induction of type 1 diabetes with streptozotocin, SS^Nox4−/− rats exhibited significantly lower basal intracellular Ca²⁺ levels in podocytes and less DKD-associated damage than SS rats did. Furthermore, the angiotensin II–elicited calcium flux was blunted in glomeruli isolated from diabetic SS^Nox4−/− rats compared with that in glomeruli from diabetic SS rats. H₂O₂ stimulated TRPC-dependent calcium influx in podocytes from wild-type mice, but this influx was blunted in podocytes from Trpc6-knockout mice and, in a similar manner, in podocytes from Trpc5/6 double-knockout mice. Finally, electron microscopy revealed that podocytes of glomeruli isolated from Trpc6-knockout or Trpc5/6 double-knockout mice were protected from damage induced by H₂O₂ to the same extent.

Conclusions These data reveal a novel signaling mechanism involving NOX4 and TRPC6 in podocytes that could be pharmacologically targeted to abate the development of DKD.

背景肾小球足细胞的丢失是糖尿病性肾脏疾病（DKD）的指标。这些细胞的损伤部分归因于肾内氧化应激的升高。肾脏活性氧物质（尤其是H ₂ O _2）的主要来源是NADPH氧化酶4（NOX4）。我们假设NOX4衍生的H ₂ O ₂通过增加足细胞钙而导致DKD中足细胞损伤。

方法我们使用了Nohl4基因无效突变的Dahl盐敏感性（SS）大鼠（SS ^Nox4-/-）和非选择性钙通道TRPC6敲除或TRPC5和TRPC6双重敲除的小鼠。我们进行了整个动物研究，并使用生物传感器测量，电子显微镜，电生理学和实时钙成像实验来评估该途径对新鲜分离的肾小球足细胞生理学的贡献。

结果用链脲佐菌素诱导1型糖尿病后，SS ^Nox4-/-大鼠的足细胞基础细胞内Ca ²⁺水平显着降低，而与DKD相关的损伤较少。此外，与从糖尿病SS大鼠肾小球分离出的肾小球相比，从糖尿病SS ^Nox4-/-大鼠分离出的肾小球中血管紧张素II引起的钙通量变钝。H ₂ O ₂刺激了野生型小鼠足细胞中依赖TRPC的钙内流，但是这种流入在Trpc 6基因敲除小鼠的足细胞中以及以类似的方式在Trpc足细胞中均受到抑制5/6双敲除小鼠。最后，电子显微镜检查显示，从Trpc 6敲除小鼠或Trpc 5/6双敲除小鼠中分离出的肾小球足细胞在相同程度上受到了H ₂ O ₂诱导的损害的保护。

结论这些数据揭示了在足细胞中涉及NOX4和TRPC6的新型信号传导机制，该机制可在药理学上靶向减轻DKD的发展。