Hyperglycemia-induced Renal P2X7 Receptor Activation Enhances Diabetes-related Injury

Abstract

Diabetes is a leading cause of renal disease. Glomerular mesangial expansion and fibrosis are hallmarks of diabetic nephropathy and this is thought to be promoted by infiltration of circulating macrophages. Monocyte chemoattractant protein-1 (MCP-1) has been shown to attract macrophages in kidney diseases. P2X7 receptors (P2X7R) are highly expressed on macrophages and are essential components of pro-inflammatory signaling in multiple tissues. Here we show that in diabetic patients, renal P2X7R expression is associated with severe mesangial expansion, impaired glomerular filtration (≤40ml/min/1.73sq.m.), and increased interstitial fibrosis. P2X7R activation enhanced the release of MCP-1 in human mesangial cells cultured under high glucose conditions. In mice, P2X7R-deficiency prevented glomerular macrophage attraction and collagen IV deposition; however, the more severe interstitial inflammation and fibrosis often seen in human diabetic kidney diseases was not modelled. Finally, we demonstrate that a P2X7R inhibitor (AZ11657312) can reduce renal macrophage accrual following the establishment of hyperglycemia in a model of diabetic nephropathy. Collectively these data suggest that P2X7R activation may contribute to the high prevalence of kidney disease found in diabetics.

Keywords: CKD; Cytokine; Glucose; P2; Purine; Renal.

Fig. 1

Renal P2X7R in human diabetic nephropathy. Patient biopsies expressing (a) P2X7R protein and (b) CD68 + monocytes in glomeruli indicated with black arrows. In the most severe cases (c) P2X7R staining and (d) CD68 + staining of the interstitium overlapped in regions of inflammation. Estimated glomerular filtration rate (eGFR) (e), fibrosis (f) and mesangial expansion (g) in patients with interstitial (DNInt = 7), glomerular (DNGlom = 5) P2X7R immunoreactivity compared to thin basement membrane (TBM = 3) controls subjects. *P < 0.05, **P < 0.01 DNint vs. TBM. a, b × 200 mag c, d × 100 mag.

Fig. 2

Glomerular mesangial P2X7R and MCP-1. Activation of P2X7R with BzATP increased MCP-1 secretion from pHMCs under high glucose conditions (a). P2X7R protein expression in mesangial cells under normal and high glucose conditions (b). Effect of a P2X7R inhibitor (A438079) on MCP-1 secretion (c). Subtraction of normal glucose MCP-1 release highlighted the absolute magnitude of MCP-1 reduction by P2X7R blockade alone across 9 separate experiments (d). *P < 0.05, **P < 0.01, ***P < 0.001 vs baseline; †P < 0.05, ††P < 0.01, †††P < 0.01 versus 30 mM D pHMC; ‡‡‡‡P < 0.0001, 4 mM D + BzATP vs. 30 mM D + BzATP.

Fig. 3

P2X7R-deficient mice and STZ-induced glomerular macrophage accrual. Change in blood glucose following STZ injections (a). Renal P2X7 mRNA levels (b). Glomerular P2X7R staining in WT (bi) and P2X7R-deficient mice (bii). Glomerular macrophage accumulation following STZ (c). Glomerular CD68 staining in WT (ci) and P2X7R-deficient mice (cii). Glomerular type IV collagen deposition increased following STZ (d). Immunolocalization of lomerular collagen IV staining in WT (di) and P2X7R-deficient mice (dii). *P < 0.05, **P < 0.01, ***P < 0.001. WT: wild-type. STZ: streptozotocin. KO: P2X7R-deficient mice. b–d × 200 mag.

Fig. 4

Effect of P2X7R inhibitor on blood glucose levels and kidney function. Blood glucose measured at week 12 (a). Serum creatinine concentration in terminal serum (b) and albumin excretion rate (AER) (c). *P < 0.05 UNx STZ groups vs. Sham or UNx Veh. ****P < 0.0001 UNx STZ groups vs. Sham or UNx Veh. Measurements were made at 12 weeks.

Fig. 5

Immunolocalization of P2X7R in rat kidney. Normal Wistar Han kidney collected during UNx (a). Kidney tissue from a week 12 vehicle-injected UNx Wistar Han rat (b). Representative P2X7R staining in a UNx STZ Veh rat kidney at week 12 (c). P2X7R staining in and STZ injected UNx rat where anti-P2X7 antibody was pre-absorbed with its conjugate peptide (1:1) to demonstrate antibody specificity (d). a–d × 200 mag.

Fig. 6

Effect of P2X7R inhibitor on diabetic renal macrophage accrual. Interstitial macrophage abundance (a). Renal interstitial CD68 staining, indicated by black arrows, in sham-treated rat kidney with minimal macrophage staining (b), UNx STZ Drug vehicle-treated diabetic rat kidney with interstitial macrophage accumulation (c), Unx STZ rat treated with 10 mg/kg P2X7Ri (d), and 50 mg/kg P2X7Ri-treated rat kidney (e). Glomerular macrophage abundance (f). Measurements were made at 12 weeks. *P < 0.05 UNx STZ groups vs. Sham or UNx Veh NS; not significant. c–f × 200 mag.