Essential Involvement of Purinergic Signaling in Calcium Handling in the Podocytes of Type 2 Diabetic Rats

Abstract

Purinergic signaling (initiated via interaction of ATP with P2 receptors) is one of the major triggers of intracellular calcium influx in the epithelial cells of the glomeruli ‐ podocytes. Excessive elevation of intracellular calcium concentration in these cells results in their loss, subsequent damage of the glomeruli filtration barrier and, eventually, proteinuria. Growing evidence suggests that the activation of extracellular ATP signaling may play a crucial role in the development of diabetic nephropathy. The goal of this study was to characterize purinergic signaling in the podocytes of Type 2 Diabetic (T2D) rats. Here we used 12 week old and aged (40–60 weeks old) Wistar (control), Goto‐Kakizaki (GK) and Type 2 Diabetic Nephropathy (T2DN) rats. The GK rat is a non‐obese Wistar substrain which develops T2D early in life. T2DN rat (created by an introgression of the mitochondria and some passenger loci from the Fawn Hooded Hypertensive (FHH) rat into the background of GK rat) is a model of T2D accompanied with renal histologic abnormalities characteristic for diabetic nephropathy (DN). Using real‐time confocal microscopy we measured acute effects of ATP on intracellular calcium dynamics in the podocytes of glomeruli freshly isolated from these rat strains. We have shown that basal intracellular calcium concentration was within normal range in Wistar and GK podocytes (150.1 ± 10.8 and 174.7 ± 29.7 nM, respectively), whereas in T2DN rats it was pathologically elevated up to 232.9 ± 20.1 nM. Effect of ATP in Wistar rat podocytes (in the range of 1 to 200 μM) was dose dependent and similar to what we previously reported for Sprague Dawley rats. However, ATP‐triggered calcium influx in GK rats was substantially enhanced with dose‐response curve shifted towards stronger activation of calcium influx with lower concentrations of ATP. Importantly, the response to ATP was further increased in T2DN compared to GK rat podocytes, and was found to be dose‐independent within the same ATP concentration range. The same phenomenon was observed in aged (approximately 12 month old) GK and T2DN animals. Furthermore, experiments in calcium‐free solutions and studies using alpha, beta‐methylene‐ATP revealed a major role of extracellular calcium influx as distinct from intracellular depot depletion, as well as a likely involvement of the ionotropic P2X receptors, or calcium channels activated by P2Y‐evoked metabotropic GPCR cascades. Western blotting revealed a significantly higher expression of P2X7 receptors in the cortex of T2DN and GK strains compared to Wistar rats, and a decrease in P2Y1 expression. Thus, our data indicate that in T2DN podocytes ATP‐triggered calcium influx is enhanced compared to control animals with even low concentrations of ATP causing activation of calcium flux and augmented intracellular calcium concentration, correspondingly. This mechanism might be an important determinant of podocyte injury resulting from pathological remodeling from metabotropic P2Y to ionotropic P2X receptors occurring in DN.Support or Funding InformationSupported by ASN Ben J. Lipps fellowship, ADA 1‐15‐BS‐172, NIH DK105160 and HL122662.