Kidney-Specific Membrane-Bound Serine Proteases CAP1/Prss8 and CAP3/St14 Affect ENaC Subunit Abundances but Not Its Activity.

The serine proteases CAP1/Prss8 and CAP3/St14 are identified as ENaC channel-activating proteases in vitro, highly suggesting that they are required for proteolytic activation of ENaC in vivo. The present study tested whether CAP3/St14 is relevant for renal proteolytic ENaC activation and affects ENaC-mediated Na+ absorption following Na+ deprivation conditions. CAP3/St14 knockout mice exhibit a significant decrease in CAP1/Prss8 protein expression with altered ENaC subunit and decreased pNCC protein abundances but overall maintain sodium balance. RNAscope-based analyses reveal co-expression of CAP3/St14 and CAP1/Prss8 with alpha ENaC in distal tubules of the cortex from wild-type mice. Double CAP1/Prss8; CAP3/St14-deficiency maintained Na+ and K+ balance on a Na+-deprived diet, restored ENaC subunit protein abundances but showed reduced NCC activity under Na+ deprivation. Overall, our data clearly show that CAP3/St14 is not required for direct proteolytic activation of ENaC but for its protein abundance. Our study reveals a complex regulation of ENaC by these serine proteases on the expression level rather than on its proteolytic activation.

Kidney-Specific CAP1/Prss8-Deficient Mice Maintain ENaC-Mediated Sodium Balance through an Aldosterone Independent Pathway.

The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8cat/+ and Prss8cat/cat), and renal tubule-specific CAP1/Prss8 knockout (Prss8PaxLC1) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the α- or γENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8cat/+ and Prss8cat/cat mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8PaxLC1) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.

GPR182 is an endothelium-specific atypical chemokine receptor that maintains hematopoietic stem cell homeostasis.

G protein-coupled receptor 182 (GPR182) has been shown to be expressed in endothelial cells; however, its ligand and physiological role has remained elusive. We found GPR182 to be expressed in microvascular and lymphatic endothelial cells of most organs and to bind with nanomolar affinity the chemokines CXCL10, CXCL12, and CXCL13. In contrast to conventional chemokine receptors, binding of chemokines to GPR182 did not induce typical downstream signaling processes, including Gq- and Gi-mediated signaling or ß-arrestin recruitment. GPR182 showed relatively high constitutive activity in regard to ß-arrestin recruitment and rapidly internalized in a ligand-independent manner. In constitutive GPR182-deficient mice, as well as after induced endothelium-specific loss of GPR182, we found significant increases in the plasma levels of CXCL10, CXCL12, and CXCL13. Global and induced endothelium-specific GPR182-deficient mice showed a significant decrease in hematopoietic stem cells in the bone marrow as well as increased colony-forming units of hematopoietic progenitors in the blood and the spleen. Our data show that GPR182 is a new atypical chemokine receptor for CXCL10, CXCL12, and CXCL13, which is involved in the regulation of hematopoietic stem cell homeostasis.

Lack of Renal Tubular Glucocorticoid Receptor Decreases the Thiazide-Sensitive Na+/Cl- Cotransporter NCC and Transiently Affects Sodium Handling.

Chronic glucocorticoid infusion impairs NCC activity and induces a non-dipping profile in mice, suggesting that glucocorticoids are essential for daily blood pressure variations. In this paper, we studied mice lacking the renal tubular glucocorticoid receptor (GR) in adulthood (GR knockouts, Nr3c1 Pax8/LC1 ). Upon standard salt diet, Nr3c1 Pax8/LC1 mice grow normally, but show reduced NCC activity despite normal plasma aldosterone levels. Following diet switch to low sodium, Nr3c1 Pax8/LC1 mice exhibit a transient but significant reduction in the activity of NCC and expression of NHE3 and NKCC2 accompanied by significant increased Spak activity. This is followed by transiently increased urinary sodium excretion and higher plasma aldosterone concentrations. Plasma corticosterone levels and 11ßHSD2 mRNA expression and activity in the whole kidney remain unchanged. High salt diet does not affect whole body Na+ and/or K+ balance and NCC activity is not reduced, but leads to a significant increase in diastolic blood pressure dipping in Nr3c1 Pax8/LC1 mice. When high sodium treatment is followed by 48 h of darkness, NCC abundance is reduced in knockout mice although activity is not different. Our data show that upon Na+ restriction renal tubular GR-deficiency transiently affects Na+ handling and transport pathways. Overall, upon standard, low Na+ and high Na+ diet exposure Na+ and K+ balance is maintained as evidenced by normal plasma and urinary Na+ and K+ and aldosterone concentrations.