Type 1 Diabetes Results in Significant Purinergic Receptor Remodeling in Podocytes

Abstract

P2 (purinergic) receptors have been proposed to be potential viable targets for the treatment of various cardiovascular and renal disorders. Previous studies in our lab have demonstrated that the predominant purinergic signaling pathway in healthy podocytes is through P2Y1 metabotropic receptors. It was also reported that expression of purinergic receptors in different tissues, including kidney, is dynamic and might be changed under pathological conditions such as diabetic nephropathy. We hypothesized that prolonged type 1 diabetes (T1D) would result in substantial changes to podocyte purinergic calcium signaling and result in a transition from predominantly metabotropic signaling through P2Y1 to increased ionotropic signaling through P2X receptors. To do this we utilized the established T1D model of streptozocin‐injected Dahl Salt Sensitive (SS) rats, and measured intracellular calcium ([Ca2+]i) signaling in podocytes from their freshly isolated glomeruli. We determined that in comparison to non‐diabetic rats, which have a rapid elevation of [Ca2+]i levels in response to ATP (40 μM) that returns to baseline within 2 min, podocytes from T1D rats had reduced peak response amplitudes (0.53 ± 0.04 vs. 0.34±0.02, p<0.001). Furthermore, podocytes of glomeruli isolated from diabetic rats had prolonged sustained elevation of [Ca2+]i level, which was not observed in non‐diabetic rats. Additionally, we used P2 specific antagonists to determine which receptors resulted in the altered calcium response curve in T1D podocytes. We found that pharmacological inhibition of P2X7 and P2X4 (10 μM A438079 and 5‐BDBD, respectively) altered the calcium curve dynamics, but did not significantly reduce the total [Ca2+]i level over a 5 min time period, as determined by area under the curve (AUC) measurements. Similarly, inhibition of P2Y1 (1 μM MRS2500) changed the [Ca2+]i curve response, but did not reduce the AUC. Combining both P2X7 and P2X4 inhibitors reduced the AUC; however, did not completely eliminate podocyte purinergic signaling (51.42 ± 5.52 vs 30.39 ± 5.00). Combined inhibition of P2X7, P2X4, and P2Y1 resulted in the near complete elimination of [Ca2+]i release in response to ATP (AUC, 51.42 ± 5.52 vs 10.57 ± 2.56, p<0.001). These results demonstrate that T1D increases ionotropic purinergic signaling in podocytes; however, does not eliminate metabotropic signaling. These studies establish the potential for targeting both P2X4 and P2X7 for therapeutic intervention in T1D.Support or Funding InformationNational Institutes of Health grants T32 HL134643 and R35 HL135749, and Department of Veteran Affairs I01 BX004024