The mineralocorticoid receptor (MR) regulates ENaC but not NCC in mice with random MR deletion

Aldosterone binds to the mineralocorticoid receptor (MR) and increases renal Na+ reabsorption via up-regulation of the epithelial Na+ channel (ENaC) and the Na+-K+- ATPase in the collecting system (CS) and possibly also via the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). However, whether aldosterone directly regulates NCC via MR, or indirectly through systemic alterations remains controversial. We used mice with deletion of MR in ~20% of renal tubule cells (MR/X mice), in which MR-positive (MRwt) and -negative (MRko) cells can be studied sideby- side in the same physiological context. Adult MR/X mice showed similar mRNA and protein levels of renal ion transport proteins to control mice. In MR/X mice, no differences in NCC abundance and phosphorylation was seen between MRwt and MRko cells and dietary Na+ restriction up-regulated NCC to similar extent in both groups of cells. In contrast, MRko cells in the CS did not show any detectable alpha- ENaC abundance or apical targeting of ENaC neither on control diet nor in response to dietary Na+ restriction. Furthermore, Na+-K+-ATPase expression was unaffected in MRko cells of the DCT, while it was lost in MRko cells of the CS. In conclusion, MR is crucial for ENaC and Na+-K+-ATPase regulation in the CS, but is dispensable for NCC and Na+-K+-ATPase regulation in the DCT

Protein Phosphatase 1α enhances renal aldosterone signaling via mineralocorticoid receptor stabilization

Stimulation of the mineralocorticoid receptor (MR) by aldosterone controls several physiological parameters including blood pressure, inflammation or metabolism. We previously showed that MR turnover constitutes a crucial regulatory step in the responses of renal epithelial cells to aldosterone. Here, we identified Protein Phosphatase 1 alpha (PP1α), as a novel cytoplasmic binding partner of MR that promotes the receptor activity. The RT-PCR expression mapping of PP1α reveals a high expression in the kidney, particularly in the distal part of the nephron. At the molecular level, we demonstrate that PP1α inhibits the ubiquitin ligase Mdm2 by dephosphorylation, preventing its interaction with MR. This results in the accumulation of the receptor due to reduction of its proteasomal degradation and consequently a greater aldosterone-induced Na+ uptake by renal cells. Thus, our findings describe an original mechanism involving a phosphatase in the regulation of aldosterone signaling and provide new and important insights into the molecular mechanism underlying the MR turnover

Collecting system–specific deletion of Kcnj10 predisposes for thiazide- and low-potassium diet–induced hypokalemia

The basolateral potassium channel KCNJ10 (Kir4.1), is expressed in the renal distal convoluted tubule and controls the activity of the thiazide-sensitive sodium chloride cotransporter. Loss-of-function mutations of KCNJ10 cause EAST/SeSAME syndrome with salt wasting and severe hypokalemia. KCNJ10 is also expressed in the principal cells of the collecting system. However, its pathophysiological role in this segment has not been studied in detail. To address this, we generated the mouse model AQP2cre:Kcnj10flox/flox with a deletion of Kcnj10 specifically in the collecting system (collecting system-Kcnj10-knockout). Collecting system-Kcnj10-knockout mice responded normally to standard and high potassium diet. However, this knockout exhibited a higher kaliuresis and lower plasma potassium than control mice when treated with thiazide diuretics. Likewise, collecting systemKcnj10-knockout displayed an inadequately high kaliuresis and renal sodium retention upon dietary potassium restriction. In this condition, these knockout mice became hypokalemic due to insufficient downregulation of the epithelial sodium channel (ENaC) and the renal outer medullary potassium channel (ROMK) in the collecting system. Consistently, the phenotype of collecting system-Kcnj10-knockout was fully abrogated by ENaC inhibition with amiloride and ameliorated by genetic inactivation of ROMK in the collecting system. Thus, KCNJ10 in the collecting system contributes to the renal control of potassium homeostasis by regulating ENaC and ROMK. Hence, impaired KCNJ10 function in the collecting system predisposes for thiazide and low potassium diet-induced hypokalemia and likely contributes to the pathophysiology of renal potassium loss in EAST/SeSAME syndrome

GDF15 Suppresses Lymphoproliferation and Humoral Autoimmunity in a Murine Model of Systemic Lupus Erythematosus

Growth and differentiation factor 15 (GDF15), a divergent member of the transforming growth factor-beta superfamily, has been associated with acute and chronic inflammatory conditions including autoimmune disease, i.e., type I diabetes and rheumatoid arthritis. Still, its role in systemic autoimmune disease remains elusive. Thus, we studied GDF15-deficient animals in Fas-receptor intact (C57BL/6) or deficient (C57BL/6(lpr/lpr)) backgrounds. Further, lupus nephritis (LN) microdissected kidney biopsy specimens were analyzed to assess the involvement of GDF15 in human disease. GDF15-deficiency in lupus-prone mice promoted lymphoproliferation, T-, B- and plasma cell-expansion, a type I interferon signature, and increased serum levels of anti-DNA autoantibodies. Accelerated systemic inflammation was found in association with a relatively mild renal phenotype. Splenocytes of phenotypically overall-normal Gdf15-/- C57BL/6 and lupus-prone C57BL/6(lpr/lpr) mice displayed increased in vitro lymphoproliferative responses or interferon-dependent transcription factor induction in response to the toll-like-receptor (TLR)-9 ligand CpG, or the TLR-7 ligand Imiquimod, respectively. In human LN, GDF15 expression was downregulated whereas type I interferon expression was upregulated in glomerular- and tubular-compartments versus living donor controls. These findings demonstrate that GDF15 regulates lupus-like autoimmunity by suppressing lymphocyte-proliferation and -activation. Further, the data indicate a negative regulatory role for GDF15 on TLR-7 and -9 driven type I interferon signaling in effector cells of the innate immune system

BEX1 Is Differentially Expressed in Aldosterone-Producing Adenomas and Protects Human Adrenocortical Cells From Ferroptosis

Aldosterone-producing adenomas (APAs) are a major cause of primary aldosteronism. Somatic mutations in ion channels and transporters drive the aldosterone overproduction in the majority of APAs with mutations in the KCNJ5 G protein-coupled potassium channel predominating in most reported populations. Our objective was to gain insight into biological mechanisms of APA tumorigenesis by comparing transcriptomes of APAs of distinct sizes by mRNA sequencing analysis (9 APAs with adenoma diameter ≥30 mm versus 12 APAs ≤10 mm). Genes with significantly altered expression levels between these 2 groups were identified in APAs with no mutation detected (348 genes) and with a KCNJ5 mutation (155 genes). We validated the differential expression of 10 genes with a known function related to cell death and proliferation in an expanded sample set of 71 APAs by real-time quantitative polymerase chain reaction (58 macro-APAs, diameter ≥10 mm; 13 micro-APAs, diameter <10 mm). We focused on BEX1 that was upregulated in micro-APAs relative to macro-APAs (2.76-fold, P<0.001) and compared with paired adrenal cortex (3.85-fold, P<0.05), and showed a linear negative correlation with APA diameter in the no mutation detected group (r=−0.501, P=0.007). Compared with control cells, stable expression of BEX1 in human adrenocortical cells did not alter cell cycle progression or sensitivity to apoptosis but conferred protection from ferroptosis (P<0.01), a form of regulated cell death, measured by flow cytometry. Taken together, these findings demonstrate that BEX1 promotes cell survival in adrenal cells by mediating the inhibition of ferroptosis and suggest a function for BEX1 in the pathogenesis of APAs