#5554 ROLE OF THE TUBULAR EPITHELIAL CELL NLRP3 INFLAMMASOME DURING HYPERGLYCEMIA AND GLUCOSURIA IN DIABETES Free

Diabetes is a prominent risk factor for the progression of CKD because hyperglycemia and glucose filtration increase the hemodynamic and metabolic workload to the nephrons, which imposes adaptive mechanisms. Tubules adapt to diabetes by enlargement and epithelial cell hypertrophy but an exceeding metabolic workload promotes oxidative stress, cytokine signaling and tubular epithelial (TEC) cell demise leading to tubular atrophy and interstitial fibrosis. Several studies propose a role of the NLRP3 inflammasome in tubulointerstitial inflammation in diabetic and non-diabetic kidney disease. The NLRP3 inflammasome is a sensor of cellular damage responsible for regulate the IL-1β and IL-18 production, playing important role during the progression of CKD. However, the role of NLRP3 in diabetes-induced tubular adaptation is unknown.

scRNAseq analysis showed previously that the TECs do not express NLRP3 or inflammasome components. However, a group of patients diagnosed with Cryopyrin-Associated Autoinflammatory Syndromes, have a mutation that overexpress NLRP3, which is associated with chronic and systemic inflammation. To investigate the role of NLRP3 on tubular cells under diabetes conditions, we generated a mouse line by crossing mice bearing LoxP-target A350V allele with Pax8-driven Cre transgenic mice. Animals carrying the A350V allele have overexpression of NLRP3 in tubular cells. The combination of unilateral nephrectomy and treatment with multiple low doses of streptozotocin (60 mg/Kg) accelerate the kidney injury caused by hyperglycaemia. Kidney function, histology and gene expression were evaluated at the end of 16 weeks of diabetes. In vitro experiments carried out using TECs isolated from Pax8-A350V, and stimulated with LPS (1µg/ml) and/or ATP (20 mM).

In silico analysis showed TECs do not express NLRP3 or inflammasome components. A350V-TECs under inflammasome activation did not produce IL-1β or IL-18. However, the Nlrp3 overexpression upregulated kidney injury markers, as well IL-6 and TNF. Moreover, the increase in kidney inflammation was associated with significative upregulation of fibrosis markers (Tgfb1, Asma, and Fn1) and extracellular molecules (Ctgf, AnxaII, and Chd2). Diabetic mice carrying the A350V mutation did not have significative differences in blood glucose, albuminuria, or body weight when compared with diabetic control mice. The overexpression of NLRP3 in tubular cells was associated with significative reduction in GFR and BUN when compared with controls mice, even though no differences in tubular injury markers observed. Furthermore, Pax8-A350V diabetic mice expressed significantly more Nlrp3 and Il1b than control group. Although, no differences observed in cryopyrin staining.

Our data suggest the NLRP3-associated damage in tubular epithelial cells occurs in a non-canonical manner.