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

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.