Renal Tubular IL‐1β Induces Salt Sensitivity in Diabetes by Activating Renal Macrophages

Abstract

We recently showed that interleukin (IL)‐6 induces salt‐sensitivity in diabetic mice by dysregulating the epithelial sodium channel (ENaC). After 4 weeks on a high salt diet, male db/db mice displayed salt sensitivity, the accumulation of proinflammatory (CD80+) macrophages, and higher levels of IL‐6 and IL‐1β in the kidney compared to db/+ controls. However, the origin of this inflammatory response is unknown. We hypothesize that, during diabetes, renal tubular epithelial cells release IL‐1β which activates renal macrophages to produce IL‐6 and impair kidney function. Immunofluorescence analysis showed that the higher IL‐1β expression of db/db mice is exclusively localized in the renal tubules. Flow cytometry analysis of renal cells showed that db/db mice have higher IL‐1β expression in tubular (E‐cadherin+) cells (21 ± 2 vs. 8 ± 3 % of E‐cadherin+ cells, P<0.001) compared to db/+ controls. To evaluate whether high glucose triggers IL‐1β synthesis in tubular cells, a primary culture of renal tubular epithelial cells from wild‐type (WT) mice was exposed to either a low (LG, 5mM) or high (HG, 15mM) glucose media. HG‐treated cells released higher levels of IL‐1β (28 ± 12 vs. 8 ± 4 pg/ml, P<0.01) and displayed higher expression of NLRP3 (4 ± 1‐fold increase, P<0.001) and Caspase‐1 (2.1 ± 0.2‐fold increase, P<0.001) compared to LG‐treated cells. These are major components of the NLRP3 inflammasome that synthesizes IL‐1β. No equivalent effect was observed using mannitol. To evaluate whether tubular cells can modify macrophage phenotype through IL‐1β, we co‐cultured tubular cells with bone marrow‐derived macrophages. Flow cytometry revealed that HG‐treated tubular cells induced a pro‐inflammatory polarization of macrophages not observed with LG‐treated cells (50 ± 4 vs. 15 ± 2 CD80+ macrophages, P<0.001). This effect was blunted when macrophages were obtained from IL‐1 receptor KO mice (IL1RKO) confirming a role of IL‐1β. To evaluate whether IL‐1β‐mediated macrophage activation plays a role in vivo, 2‐mo‐old diabetic db/db mice (n=6) were transplanted with a bone marrow of IL1RKO mice. These mice, named db/db‐IL1RKO, cannot respond to IL‐1β. db/db receiving wild‐type (WT) bone marrow (db/db‐WT) were used as control. No differences in body weight and hyperglycemia were observed after transplantation. At 7 months, mice were exposed to high salt diet for 4 weeks. Compared to db/db‐WT, db/db‐IL1RKO did not develop salt‐sensitivity (blood pressure was 110 ± 7 vs. 122 ± 9 mmHg, P<0.05), have less renal CD80+ macrophages, (21 ± 4 vs. 54 ± 5 CD80+ macrophages, P<0.01), less renal IL‐6 (138 ± 35 vs. 263 ± 28 pg/mg kidney, P<0.05), and lower ENaC activity (65% decrease by amiloride test, P<0.05) despite an accumulation of tubular IL‐1β. In conclusion, our data show that tubular epithelial cells are a major source of IL‐1β under high glucose conditions. This cytokine promotes a pro‐inflammatory phenotype of macrophages resulting in higher renal IL‐6 levels which increases ENaC activity leading to salt sensitivity.