Nox 4 Null Mutation Attenuates Podocyte Calcium Regulation Dysfunction and Kidney Damage in Streptozotocin‐Treated Dahl Salt‐Sensitive Rats
Authors: Blass Gregory , Ilatovskaya Daria , Winsor Kristen , Levchenko Vladislav , Palygin Oleg , Cowley Allen W. , Staruschenko Alexander 
Published: November 2023

The progressive deterioration of kidney function observed in diabetic nephropathy is thought to, in part, result from the depletion of glomerular podocytes. The loss of these cells, among other reasons, can be attributed to the dysregulation of their intracellular calcium signaling. A variety of stimuli can induce excessive calcium influx in podocytes, and reactive oxygen species (ROS) has been shown to be one of the major ones. Specifically, renal tissue ROS is largely produced by the NADPH oxidase Nox4, which is abundantly expressed in the kidney and was found upregulated in models of diabetes. We hypothesized here that Nox4 contributes to the podocyte depletion in type 1 diabetes by potentiating podocyte [Ca2+]i dysfunction. Here we utilized the Dahl Salt‐Sensitive (SS) rat with a null mutation of the Nox4 gene (Nox4−/−). We described recently that an i.p. injection of streptozotocin (STZ, 75 mg/kg) into SS rats not only resulted in type 1 diabetes, but also diabetic nephropathy and podocytes damage (Ilatovskaya et al., 2015). We confirmed here that 11 weeks post injection was sufficient for STZ‐treated SS rats (STZ‐SS) to develop essential hallmarks of diabetes (such as hyperglycemia, excessive urination, and microalbuminuria). STZ‐treated Nox4−/− rats (STZ‐Nox4) had significantly attenuated albuminuria, glomeruli injury score, and percent tubule cast formation compared to STZ‐SS rats (p<0.05). To test the role of Nox4 in calcium‐related podocyte damage, we assessed the basal [Ca2+]i in podocytes of freshly isolated glomeruli by ex vivo confocal calcium imaging. STZ‐Nox4 podocytes had significantly lower basal [Ca2+]i, (p<0.001) than STZ‐SS podocytes where, consistent with our previously published data, intracellular [Ca2+]i was dramatically elevated. We reported earlier that Ang II activates calcium channels in podocytes, and this response is significantly enhanced in STZ‐SS rats. Here we applied enzymatic biosensors to test a role of Nox4 in H2O2 production in glomeruli. Our data revealed that Nox4−/− glomeruli have a dramatic reduction in Ang II‐induced H2O2 production. Patch clamp analysis revealed an acute TRPC channel open probability increase in murine podocytes elicited by H2O2. Additionally, a dose‐dependent increase in podocyte extracellular Ca2+ influx was observed from H2O2 applications (2.5 μM to 300 μM; EC50=56 μM). Interestingly, lower concentrations of H2O2 caused a transient [Ca2+]i peak, whereas doses of 50 μM and higher evoked a long‐term [Ca2+]i elevation attributed to apoptotic effects. This produced an incentive to assess whether Nox4 deficiency could be protective in the studied model of type 1 diabetes. To test this, we performed live ratiometric calcium imaging and acutely treated podocytes of freshly isolated glomeruli with 10 μM Ang II. STZ‐SS podocytes had a significant potentiation of the Ang II‐induced total [Ca2+]i response (p<0.05) that was significantly attenuated in STZ‐Nox4 podocytes. We conclude based on these findings that Ang II–induced H2O2 production by Nox4 may contribute to the podocyte loss in type 1 diabetes by potentiating their calcium signaling dysfunction.

Support or Funding Information

Supported by NIH R01HL122662 and American Diabetes Association 1‐15‐BS‐172