Fig 2: Expression of P2 receptors in renal cortex of old ratsWestern blot images and corresponding summary graphs showing densitometric analysis of P2Y1 (A), P2X4 and P2X7 (B) protein expression levels in renal cortex of the aged (>48-weeks-old) Wistar, GK and T2DN rats. All Western blot values were normalized to loading control (β-actin or Gapdh). Significance (p < 0.05) is indicated by ∗, Tukey's multiple-comparison adjustment was applied. In the box plot graphs, the box represents ± SE, and a line inside the box marks the median. The whiskers are ± SD.

Fig 3: Effects of P2 receptor inhibition on [Ca2+ ]i handling in T1D(A) Summarized ATP (40 μM) response curves from podocytes in the presences of a P2 antagonist (black) versus T1D (gray, no inhibitor) or non-diabetic (light gray). Shown are effects of pre-incubation with 1 μM MRS2500 (P2Y1 antagonist), 10 μM A438079 (P2X7 antagonist), 10 μM 5-BDBD (P2X4 inhibitor), both P2X receptor antagonists, and with antagonists targeting all 3 P2 receptors.(B) Summarized [Ca2+]i transient as a percentage of T1D [Ca2+]i response in the presence of P2Y1, P2X4 and P2X7 antagonists from curves shown in (A). Significance (p < 0.05) is indicated by ∗, One-way ANOVA with Dunnett's multiple-comparison adjustment was applied. Values shown in B are mean ± SEM.

Fig 4: Expression of P2 receptors in renal cortex of young rats(A) Western blot images and corresponding summary graphs showing densitometry of metabotropic (depicted in brown) P2Y1, P2Y2 and P2Y12 protein expression levels in renal cortex of 12-weeks-old Wistar, GK and T2DN rats.(B) Western blot images and corresponding summary graphs showing densitometric analysis of ionotropic (depicted in blue) P2X4 and P2X7 protein expression levels in renal cortex of 12-weeks-old Wistar, GK and T2DN rats. All Western blot values were normalized to loading control (β-actin). Significance (p < 0.05) is indicated by ∗, Tukey's multiple-comparison adjustment was applied. In the box plot graphs, the box represents ± SE, and a line inside the box marks the median. The whiskers are ± SD.

Fig 5: Nitric oxide (NO) potentiates adenosine triphosphate (ATP) release from glia and promotes neuron death in situ through a mechanism that involves glial connexin-43 (Cx43) hemichannels. (A) ATP release from myenteric ganglia after stimulation of glial P2Y1 receptors (P2Y1Rs) with adenosine 5′-[β-thio]diphosphate trilithium salt (ADPβS, 100 μM) alone or in the presence of the NO donor propylamine propylamine NONOate (PAPA NONOate, n = 4; *P < .05, unpaired t test). (B) Mean packing density of HuC/D-immunoreactive myenteric neurons after direct glial P2Y1R stimulation with adenosine diphosphate (ADP) and inhibition of inducible nitric oxide synthase (iNOS, 1400W; 10 μM) or P2X7 receptors (P2X7Rs, A740003; 10 μM). Inhibition of iNOS or P2X7Rs protects against P2Y1R-driven neuron death (*P ≤ .01, analysis of variance [ANOVA] as compared to ADP; n = 3–4 animals). (C) Mean packing density of myenteric neurons after application of BzATP or NO-modifying drugs in wild-type (bottom bars) or iNOS-knockout mice (iNOS−/−, top two green bars). Inhibition (1400W; 10 μM) or ablation (iNOS−/−) of iNOS protects against P2X7R-driven neuron death. The NO donor PAPA NONOate (100 μM) drives neuron death to an equal extent as BzATP but the combination is not additive. Like BzATP, PAPA NONOate–driven neuron death requires iNOS (blocked by 1400W and in iNOS−/− mice) and Cx43 hemichannel opening (blocked by 43Gap26). *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001, ANOVA as compared to BzATP; n = 3–11 animals.