ACE N‐domain regulates high‐glucose mediated interleukin‐1β production by renal epithelial cells
Authors: Giani Jorge Fernando , Bernstein Ellen A. , Eriguchi Masahiro , Gonzalez‐Villalobos Romer A. , Bernstein Kenneth E. 
Published: November 2023

Background and aimDiabetic nephropathy is characterized by a sub‐clinical inflammatory state that injures renal parenchyma. This process prevents renal compensation leading to salt and water retention and salt‐sensitive hypertension. Here, we hypothesize that ACE catalytic activity regulates interleukin (IL)‐1β production by renal tubular epithelial cells in response to a high‐ glucose stress. This cytokine contributes to the inflammatory response that underlies salt sensitivity and regulates epithelial sodium channels (ENaC) during diabetes.MethodsTo study this, we isolated proximal tubular (PT) epithelial cells from wild‐type (WT) and mice lacking either the functional N‐domain (NKO) or the C‐domain (CKO) of ACE. These cells were exposed to low (5 mM) or high (30 mM) glucose for 24 hours. The culture media was assessed for cytokines by ELISA and transferred to cortical collecting duct (M1) cells to evaluate the expression of ENaC α subunit by Western blot.ResultsHigh‐glucose stress induced WT PT cells to release significant amounts of IL‐1β (from 2.6±0.3 to 35±3 pg/ml, p<0.001; n=3–6). Notably, when WT PT cells were exposed to the high‐ glucose media in the presence of an ACE inhibitor (lisinopril), IL‐1β levels were significantly reduced (from 35±3 to 19±3 pg/ml, p<0.01). In contrast, AT1 receptor blockade by losartan did not change the amount of IL‐1β produced by WT PT cells. PT cells did not release TNFα or IL‐6 in response to high glucose. To determine which ACE domain is associated with IL‐1β production, NKO and CKO PT cells were exposed to high glucose. Strikingly, NKO PT cells release lower amounts of IL‐1β when exposed to high glucose compared to WT (10±1 vs. 31±3 pg/ml, p<0.01, n=3–4). No differences were observed between WT and CKO PT cells. These data suggest that the catalytic activity of the ACE N‐domain is a key contributor to IL‐1β production by PT cells. To study whether the culture media obtained from PT cells exposed to high glucose can modify the expression of αENaC in M1 cells, the conditioned media from PT cells was transferred to a co‐culture of M1 cells and thioglycolate‐elicited peritoneal macrophages (4:1 ratio). The high‐glucose media obtained from WT PT increased αENaC expression compared to a low‐glucose media (1.5±0.2‐fold increase; p<0.05; n=3). However, conditioned media obtained from WT PT cells in the presence of lisinopril did not modify αENaC abundance. The high‐glucose conditioned media from WT PT cells failed to modify the expression of αENaC in M1 cells in the absence of macrophages.ConclusionsOur data indicate that: 1) PT cells can sense and respond to high glucose by secreting IL‐1β, 2) the synthesis of IL‐1β is increased by the catalytic activity of the ACE N‐ domain independently from AT1 receptor activation, and 3) high‐glucose conditioned media from WT PT cells can modify the expression of αENaC in M1 cells co‐cultured with macrophages. Thus, IL‐1β may mediate a potential communication between early and distal portions of the nephron during high glucose stress. This communication is mediated by macrophages.Support or Funding InformationThis study was supported by a postdoctoral fellowship from AHA 15POST22520015 and a AHA Scientist Development Grant 16SDG30130015 to JFG, NIH grants R03DK101592 to RAGV, R01HL110353, R21AI114965, R01DK098382 to KEB,