The serine protease prostasin, encoded by Prss8, has been studied for its role in cancer and epidermal dysfunction. It is expressed in several epithelia, where it is involved in the regulation of multiple substrates, such as the epithelial sodium channel (ENaC), protease-activated receptors (PARs), and extracellular matrix components. However, the physiological role of this serine protease is still unknown. In humans, dysregulation of prostasin expression and activity has been associated with severe preeclampsia and with premature embryonic death in knockout mice. In this study, we analyzed the phenotype of the mouse Prss8−/− embryos at E11.5 and 12.5 prior to their premature death at E13.5, focusing on the yolk sac, the aorta-gonad-mesonephros (AGM), and the fetal liver. In absence of prostasin, we found an overall reduced number of fetal erythrocytes. However, the generation of other cell types was not affected, suggesting that erythropoiesis is impaired in absence of prostasin. We found that terminal differentiation of primitive erythroid cells is defective and that these cells significantly downregulated fetal hemoglobin genes and presented cytoplasmic vacuoles. The impaired primitive erythropoiesis led to anemia and lethality in Prss8−/− embryos. Moreover, the erythroid defect caused the aberrant vascular remodeling of the yolk sac of E12.5 Prss8−/− embryos, which may lead to improper placenta development and preeclampsia.

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NOX2 (nicotinamide adenine dinucleotide phosphate hydrogen [NADPH] oxidase 2), involving the pneumococcal virulence factor PLY (pneumolysin). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of ENaC (epithelial sodium channel) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function-measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans blue dye incorporation in mouse lungs-after infection with pneumococci. PLY-induced hyperpermeability in human lung microvascular endothelial cell monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by colocalization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α knockout (enENaC-α knockout) mice develop increased capillary leak upon intratracheal instillation with PLY or pneumococci, compared with wild-type animals. TIP peptide diminishes capillary leak in Streptococcus pneumoniae-infected wild-type mice, without significantly increasing lung bacterial load. Lung slices from S. pneumoniae-infected enENaC-α knockout mice have significantly increased endothelial NOX2 expression, compared with infected cyclization recombination mice. In conclusion, enENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in acute respiratory distress syndrome, without impairing host defense.

A candidate gene association analysis identified TMPRSS9 as gene for potassium sensitivity in women. To validate this finding, constitutive and conditional Tmprss9 knockout mice were generated and subjected to dietary K+ deprivation and K+ loading. Interestingly, mouse renal Tmprss9 gene expression was similar in both sexes on standard diet but differed when challenged with K+-deprivation or -loading in wildtype (WT) mice. Constitutive deficiency of Tmprss9 was evidenced on a transcriptional level in knockout (KO) mice. Serum Na+ levels were lower in male and female KO mice on low K+ (LKD), while on high K+ (HKD) diet, serum K+ only increased in male KO mice. Upon all diet conditions namely standard diet (SD), LKD and HKD the protein abundances of sodium transporting proteins like the sodium-chloride symporter (NCC), alpha and gamma epithelial sodium channel (ENaC) subunits as well as their ratio of cleaved/full length protein and the sodium-hydrogen exchanger 3 (NHE3) were similar in WT and KO mice and/or showed only minor differences. We propose that in human, TMPRSS9 may function as a sex-specific modifier gene for serum K+ handling in women, whereas in mice, male rather than female Tmprss9 KO retained serum K+ on HKD.

We previously reported that Na+-deprived mice lacking CAP1/Prss8 in kidney tubules maintained epithelial sodium channel-mediated sodium balance albeit persistent hypoaldosteronism, hence indicating an uncoupling from aldosterone production. This further suggested an implication of the serine protease CAP1/Prss8 (prostasin) in the cross talk of the kidney with the adrenal gland that does not express prostasin. When these knockout (Ko) mice were additionally exposed to a high K+ diet, plasma K+ levels and plasma aldosterone concentrations were normalized and no longer different from those of the control mice. The mRNA transcript expression of the adrenal aldosterone synthase Cyp11b2, which was lower in Na+-deprived CAP1/Prss8 Ko animals, was in the normal range. Plasma aldosterone levels were similar to control animals, indicating that K+ rescued the hypoaldosteronism in Na+-deprived CAP1/Prss8 Ko animals. These data suggest that CAP1/Prss8 (prostasin) is implicated in the regulation of aldosterone synthesis or production and that the consequences of CAP1/Prss8 deficiency can be compensated by high dietary K+ supplementation. Prostasin may therefore present a promising regulator of aldosterone production by affecting the adrenal steroidogenic pathway.NEW & NOTEWORTHY We explore the role of the serine protease CAP1/Prss8 in aldosterone synthesis. Described previously as a candidate gene for hypertension, the mechanism by which renal serine protease deficiency is implicated in aldosterone production is still largely unknown. Our findings underscore a role of prostasin in the regulation of aldosterone synthesis. In kidney-specific CAP1/Prss8 knockout mice, K+ supplementation is predominant over Na+ and restores normal aldosterone production proposing new pathways to treat hypo- or hypertension.

Transcellular and paracellular transport are key routes for the transfer of substances between and through cells. Both transport routes are heavily regulated by a myriad of molecular pathways. The epithelial sodium channel (ENaC) transports sodium across the tight epithelia in many different tissues and is primarily composed of three subunits (α, β and γ). A complex array of intrinsic and extrinsic factors, including channel activation by protease cleavage, regulate the expression and function of this evolutionary conserved ion channel. It has been shown by Xenopus oocyte functional experiments that the serine protease TMPRSS2 activates ENaC by cleaving the γ subunit. Our work explores whether TMPRSS2 is also involved in the regulation of ENaC expression and function, with a particular focus on the α subunit of ENaC. TMPRSS2 and αENaC are similarly expressed in most organs and both are highly expressed in the kidney. ENaC is known to localise to the distal convoluted tubule and cortical collecting duct of the nephron in the kidney. When analysing mouse kidney by RNAscope we find that TMPRSS2 is highly expressed in αENaC positive cells. We utilise the mouse cortical collecting duct (mCCD) cell line and CRISPR-Cas9 gene editing to explore this functional relationship further in vitro. TMPRSS2 KO cells reveal that αENaC gene expression is significantly reduced, thus abolishing transepithelial sodium transport. Upon further investigation of these cells by RNA-sequencing, we discover that genes (Epcam, Cldn3 and Cldn7) involved in maintaining the paracellular barrier are downregulated in TMPRSS2 KO cells. Subsequent protein analysis by Western blotting and immunostaining shows that the protein expression of EpCAM, Claudin-3 and Claudin-7 are also reduced in TMPRSS2 KO cells. Taken together, our data indicates that TMPRSS2 is instrumental in regulating ENaC mediated transepithelial transport via αENaC and maintaining the paracellular barrier via the EpCAM/claudin-7 tight junction complex. Swiss NationalFoundation (Grant FNRS 31003A-182478/1) and the National Center of Competence in Research "Kidney.CH," Lausanne, Switzerland (NCCR, N-403-07-23).

The serine protease prostasin, encoded by Prss8, has been studied for its role in cancer and epidermal dysfunction. It is expressed in several epithelia, where it is involved in the regulation of several substrates such as the epithelial sodium channel (ENaC), protease-activated receptors (PARs), and extracellular matrix components. However, the physiological role of this serine protease is still unknown. In human, dysregulation of prostasin expression and activity has been associated with severe pre-eclampsia, and with premature embryonic death in knockout mice. In this study we analyzed the phenotype of the mouse Prss8-/- embryos at E11.5 and 12.5 prior to their premature death at E13.5 focusing on yolk sac, the aorta-gonad-mesonephros (AGM) and fetal liver. In absence of prostasin, we found an overall reduced number of fetal erythrocytes, while reticulocytes count was increased, suggesting a defect in the terminal erythroid differentiation. Indeed, imaging flow cytometry revealed that Prss8-/- fetal liver exhibited a significantly lower number and percentage of BasoE, PolyE and more predominantly of OrthoE. We also assessed the ability of E11.5 AGM-derived cells to form erythroid colonies using a colony-forming assay. Counting revealed significantly less colonies in the Prss8-/- cultures although, the number of hematopoietic stem cells (HSCs) was comparable between the two genotypes. This suggests that in absence of prostasin, erythrocytes specification is impaired. Furthermore, in the yolk sac of E12.5 Prss8-/- embryos an aberrant vascular remodeling was observed. Despite that, comparable numbers of endothelial cells were detected by FACS, suggesting that vasculogenesis is not affected in absence of prostasin and vessel remodeling is impaired likely because of reduced circulating erythrocytes.

In humans, the membrane-bound serine protease prostasin encoded by Prss8 is associated with preeclampsia, a gestational hypertension disorder affecting blood supply of the placenta. Mice deficient in Prss8 resulted in the death of embryos at embryonic day (E) 14.5 and it was characterized by impaired placental labyrinth maturation and vascularization. A pale phenotype was observed in these embryos, suggesting ineffective erythropoiesis. Thus, in this study we analyzed this phenotype further in Prss8-/- embryos at E11.5 and E12.5. We found a reduced number of fetal erythroblasts in placenta, yolk sac and fetal liver of Prss8-/- embryos, while the reticulocyte number was increased, suggesting a defective terminal erythroid differentiation. Further, single-cell RNA sequencing (scRNA-seq) analyses of aorta-gonad-mesonephros (AGM) revealed an upregulation of several ribosomal genes associated with Diamond-Blackfan anemia in erythroid cells of Prss8-/- (KO) embryos. These cells showed a lower capacity to maturate into erythrocytes in vivo and in vitro, despite hematopoietic cells (HSCs) being produced normally. We suggested prostasin influenced erythropoiesis in a cell-extrinsic manner, since Prss8 expression was not detected in erythroid cells but highly expressed in ectoderm-like cells within the AGM. Congruently, while yolk sac-derived cells displayed no erythrocyte maturation defect in vitro, the vascular remodeling of yolk sac in KO embryos was impaired as evidenced by reduced secondary branching likely as a consequence of the reduced blood flow. Our findings unveiled a novel role for this serine protease in terminal maturation of erythrocytes in the fetal liver and open new research avenues for understanding the physiological mechanism of prostasin and its pathological implications. ### Competing Interest Statement The authors have declared no competing interest.

The mouse cortical collecting duct cell line presents a tight epithelium with regulated ion and water transport. The epithelial sodium channel (ENaC) is localized in the apical membrane and constitutes the rate-limiting step for sodium entry, thereby enabling transepithelial transport of sodium ions. The membrane-bound serine protease Tmprss2 is co-expressed with the alpha subunit of ENaC. αENaC gene expression followed the Tmprss2 expression, and the absence of Tmprss2 resulted not only in down-regulation of αENaC gene and protein expression but also in abolished transepithelial sodium transport. In addition, RNA-sequencing analyses unveiled drastic down-regulation of the membrane-bound protease CAP3/St14, the epithelial adhesion molecule EpCAM, and the tight junction proteins claudin-7 and claudin-3 as also confirmed by immunohistochemistry. In summary, our data clearly demonstrate a dual role of Tmprss2 in maintaining not only ENaC-mediated transepithelial but also EpCAM/claudin-7–mediated paracellular barrier; the tight epithelium of the mouse renal mCCD cells becomes leaky. Our working model proposes that Tmprss2 acts via CAP3/St14 on EpCAM/claudin-7 tight junction complexes and through regulating transcription of αENaC on ENaC-mediated sodium transport.

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.

Hypertension is one of the leading causes of premature death in humans and exhibits a complex aetiology including environmental and genetic factors. Mutations within the glucocorticoid receptor (GR) can cause glucocorticoid resistance, which is characterized by several clinical features like hypercortisolism, hypokalaemia, adrenal hyperplasia and hypertension. Altered glucocorticoid receptor signalling further affects sodium and potassium homeostasis as well as blood pressure regulation and cell proliferation and differentiation that influence organ development and function. In salt-sensitive hypertension, excessive renal salt transport and sympathetic nervous system stimulation may occur simultaneously, and, thus, both the mineralocorticoid receptor (MR) and the GR-signalling may be implicated or even act interdependently. This review focuses on identified GR mutations in human primary generalized glucocorticoid resistance (PGGR) patients and their related clinical phenotype with specific emphasis on adrenal gland hyperplasia and hypertension. We compare these findings to mouse and rat mutants harbouring genetically engineered mutations to further dissect the cause and/or the consequence of clinical features which are common or different.

PURPOSE OF REVIEW:This review provides an up-to-date understanding about the regulation of epithelial sodium channel (ENaC) expression and function. In particular, we will focus on its implication in renal Na+ and K+ handling and control of blood pressure using transgenic animal models.RECENT FINDINGS:In kidney, the highly amiloride-sensitive ENaC maintains whole body Na+ homeostasis by modulating Na+ transport via epithelia. This classical role is mostly confirmed using genetically engineered animal models. Recently identified key signaling pathways that regulate ENaC expression and function unveiled some nonclassical and unexpected channel regulatory processes. If aberrant, these dysregulated mechanisms may also result in the development of salt-dependent hypertension.The purpose of this review is to highlight the most recent findings in renal ENaC regulation and function, in considering data obtained from animal models.SUMMARY:Increased ENaC-mediated Na+ transport is a prerequisite for salt-dependent forms of hypertension. To treat salt-sensitive hypertension it is crucial to fully understand the function and regulation of ENaC.

Proteolytic activation of the renal epithelial sodium channel (ENaC) is increased by aldosterone. The aldosterone-sensitive protease remains unidentified. In humans, elevated circulating aldosterone is associated with increased urinary extracellular vesicle (uEVs) excretion of mannan-binding lectin associated serine protease-2 (MASP-2). We hypothesized that MASP-2 is a physiologically relevant ENaC-activating protease. It was confirmed that MASP2 mRNA is abundantly present in liver but not in human and mouse kidneys. Aldosterone-stimulation of murine cortical colleting duct (mCCD) cells did not induce MASP-2 mRNA. In human kidney collecting duct, MASP-2 protein was detected in AQP2-negative/ATP6VB1-positive intercalated cells suggestive of MASP2 protein uptake. Plasma concentration of full-length MASP-2 and the short splice variant MAp19 were not changed in a cross-over intervention study in healthy humans with low (70 mmol/day) versus high (250 mmol/day) Na + intake despite changes in aldosterone. The ratio of MAp19/MASP-2 in plasma was significantly increased with a high Na + diet and the ratio correlated with changes in aldosterone and fractional Na + excretion. MASP-2 was not detected in crude urine or in uEVs. MASP2 activated an amiloride-sensitive current when co-expressed with ENaC in Xenopus oocytes, but not when added to the bath solution. In monolayers of collecting duct M1 cells, MASP2 expression did not increase amiloride-sensitive current and in HEK293 cells, MASP-2 did not affect γENaC cleavage. MASP-2 is neither expressed nor co-localized and co-regulated with ENaC in the human kidney or in urine after low Na + intake. MASP-2 does not mediate physiological ENaC cleavage in low salt/high aldosterone settings.

Proteases are fundamental for a plethora of biological processes, including signalling and tissue remodelling, and dysregulated proteolytic activity can result in pathogenesis. In this review, we focus on a subclass of membrane-bound and soluble proteases that are defined as channel-activating proteases (CAPs), since they induce Na+ ion transport through an autocrine mechanism when co-expressed with the highly amiloride-sensitive epithelial sodium channel (ENaC) in Xenopus oocytes. These experiments first identified CAP1 (channel-activating protease 1, prostasin) followed by CAP2 (channel-activating protease 2, TMPRSS4) and CAP3 (channel-activating protease 3, matriptase) as in vitro mediators of ENaC current. Since then, more serine-, cysteine- and metalloproteases were confirmed as in vitro CAPs that potentially cleave and regulate ENaC, and thus this nomenclature was not further followed, but is accepted as functional term or alias. The precise mechanism of ENaC modulation by proteases has not been fully elucidated. Studies in organ-specific protease knockout models revealed evidence for their role in increasing ENaC activity, although the proteases responsible for ENaC activation are yet to be identified. We summarize recent findings in animal models of these CAPs with respect to their implication in ENaC activation. We discuss the consequences of dysregulated CAPs underlying epithelial phenotypes in pathophysiological conditions, and the role of selected protease inhibitors. We believe that these proteases may present interesting therapeutic targets for diseases with aberrant sodium homoeostasis.

The kidney is strongly dependent on a continuous oxygen supply, and is conversely highly sensitive to hypoxia. Controlled oxygen gradients are essential for renal control of solutes and urine-concentrating mechanisms, which also depend on various hormones including aldosterone. The cortical collecting duct (CCD) is part of the aldosterone-sensitive distal nephron and possesses a key function in fine-tuned distal salt handling. It is well known that aldosterone is consistently decreased upon hypoxia. Furthermore, a recent study reported a hypoxia-dependent down-regulation of sodium currents within CCD cells. We thus investigated the possibility that cells from the cortical collecting duct are responsive to hypoxia, using the mouse cortical collecting duct cell line mCCDcl1 as a model. By analyzing the hypoxia-dependent transcriptome of mCCDcl1 cells, we found a large number of differentially-expressed genes (3086 in total logFC< −1 or >1) following 24 h of hypoxic conditions (0.2% O2). A gene ontology analysis of the differentially-regulated pathways revealed a strong decrease in oxygen-linked processes such as ATP metabolic functions, oxidative phosphorylation, and cellular and aerobic respiration, while pathways associated with hypoxic responses were robustly increased. The most pronounced regulated genes were confirmed by RT-qPCR. The low expression levels of Epas1 under both normoxic and hypoxic conditions suggest that Hif-1α, rather than Hif-2α, mediates the hypoxic response in mCCDcl1 cells. Accordingly, we generated shRNA-mediated Hif-1α knockdown cells and found Hif-1α to be responsible for the hypoxic induction of established hypoxically-induced genes. Interestingly, we could show that following shRNA-mediated knockdown of Esrra, Hif-1α protein levels were unaffected, but the gene expression levels of Egln3 and Serpine1 were significantly reduced, indicating that Esrra might contribute to the hypoxia-mediated expression of these and possibly other genes. Collectively, mCCDcl1 cells display a broad response to hypoxia and represent an adequate cellular model to study additional factors regulating the response to hypoxia.

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.

Dietary treatment is seminal for management of chronic kidney disease (CKD). The aim of our project was to assess the effects of potassium intake on the progression of CKD. We used 2 mouse CKD models to analyze the effects of potassium intake on CKD : the unilateral ureteral obstruction (UUO) and the POD-ATTAC models. POD-ATTAC mice display a podocyte-specific apoptosis after the administration of a chemical inducer. We also studied the effect of mineralocorticoid receptor (MR) using UUO in kidney tubule-specific MR knockout mice. In both UUO and POD-ATTAC mice, high potassium diet increased interstitial fibrosis. High potassium diet also increased the abundance of the extracellular matrix protein fibronectin and decreased the abundance of the epithelial marker Na + -K + ATPase. Consistently, POD-ATTAC mice fed with high potassium diet displayed lower glomerular filtration rate. Spironolactone, a MR antagonist, decreased fibrosis induced by high potassium diet in POD-ATTAC mice. However, kidney tubule-specific MR knockout did not improve the fibrotic lesions induced by UUO under normal or high potassium diets. Macrophages from high potassium-fed POD-ATTAC mice displayed higher mRNA levels of the pro-inflammatory chemokine MCP1. This effect was decreased by spironolactone, suggesting a role of MR signaling in myeloid cells in the pro-fibrotic effect of potassium-rich diet. High potassium intake generates more fibrosis leading to decreased kidney function in experimental CKD. MR signaling plays a pivotal role in this potassium-induced fibrosis. The effect of reducing potassium intake on CKD progression should be assessed in future clinical trials. Translational statement Dietetic approach is a cheap and effective therapy to slow down the development of chronic kidney diseases and kidney fibrosis. Potassium-rich diets are protective against renal and cardiovascular events in the general population, albeit some conflicting data were obtained in patients with chronic kidney disease. We showed that potassium-rich diet accelerates fibrosis development, by enhancing kidney inflammation in two mouse models of chronic kidney disease. These data suggest that potassium-rich diets should not be advised in patients with chronic kidney disease, unless future clinical trials demonstrate any beneficial effect in these patients.

Dietary treatment is seminal for management of chronic kidney disease (CKD). The aim of our project was to assess the effects of potassium intake on the progression of CKD. We used 2 mouse CKD models to analyze the effects of potassium intake on CKD : the unilateral ureteral obstruction (UUO) and the POD-ATTAC models. POD-ATTAC mice display a podocyte-specific apoptosis after the administration of a chemical inducer. We also studied the effect of mineralocorticoid receptor (MR) using UUO in kidney tubule-specific MR knockout mice.In both UUO and POD-ATTAC mice, high potassium diet increased interstitial fibrosis. High potassium diet also increased the abundance of the extracellular matrix protein fibronectin and decreased the abundance of the epithelial marker Na+-K+ ATPase. Consistently, POD-ATTAC mice fed with high potassium diet displayed lower glomerular filtration rate. Spironolactone, a MR antagonist, decreased fibrosis induced by high potassium diet in POD-ATTAC mice. However, kidney tubule-specific MR knockout did not improve the fibrotic lesions induced by UUO under normal or high potassium diets. Macrophages from high potassium-fed POD-ATTAC mice displayed higher mRNA levels of the pro-inflammatory chemokine MCP1. This effect was decreased by spironolactone, suggesting a role of MR signaling in myeloid cells in the pro-fibrotic effect of potassium-rich diet.High potassium intake generates more fibrosis leading to decreased kidney function in experimental CKD. MR signaling plays a pivotal role in this potassium-induced fibrosis. The effect of reducing potassium intake on CKD progression should be assessed in future clinical trials.Translational statement Dietetic approach is a cheap and effective therapy to slow down the development of chronic kidney diseases and kidney fibrosis. Potassium-rich diets are protective against renal and cardiovascular events in the general population, albeit some conflicting data were obtained in patients with chronic kidney disease. We showed that potassium-rich diet accelerates fibrosis development, by enhancing kidney inflammation in two mouse models of chronic kidney disease. These data suggest that potassium-rich diets should not be advised in patients with chronic kidney disease, unless future clinical trials demonstrate any beneficial effect in these patients.### Competing Interest StatementThe authors have declared no competing interest.* AKI : acute kidney injury CKD : chronic kidney disease GFR : glomerular filtration rate UUO : unilateral ureteral obstruction NK : normal potassium diet LK : normal potassium diet HK : high potassium diet midHK : mid-high potassium diet LS : low sodium diet HS : high sodium diet

Significance Statement The hypoxia-inducible factor (HIF) pathway is a key mediator of cellular adaptation to low oxygen tension. The aldosterone-sensitive distal nephron is the site of active and highly ATP-consuming sodium reabsorption, according to the requirement of sodium balance. The authors found that activation of the HIF pathway in cultured principal cells led to decreased amiloride-sensitive current (reflecting decreased epithelial sodium channel [ENaC] activity) and decreased expression of ENaC subunits, whereas HIF silencing led to increased amiloride-sensitive current and expression of ENaC subunits. Hypoxic control mice displayed decreased γENaC, whereas HIF1α knockout mice displayed increased γENaC. These findings suggest that the HIF pathway controls ENaC expression and activity, and may represent a negative feedback mechanism to prevent hypoxia and/or reactive oxygen species–induced cell damage under sustained stimulation of sodium transport. Background Active sodium reabsorption is the major factor influencing renal oxygen consumption and production of reactive oxygen species (ROS). Increased sodium reabsorption uses more oxygen, which may worsen medullary hypoxia and produce more ROS via enhanced mitochondrial ATP synthesis. Both mechanisms may activate the hypoxia-inducible factor (HIF) pathway. Because the collecting duct is exposed to low oxygen pressure and variations of active sodium transport, we assessed whether the HIF pathway controls epithelial sodium channel (ENaC)–dependent sodium transport. Methods We investigated HIF’s effect on ENaC expression in mpkCCDcl4 cells (a model of collecting duct principal cells) using real-time PCR and western blot and ENaC activity by measuring amiloride-sensitive current. We also assessed the effect of hypoxia and sodium intake on abundance of kidney sodium transporters in wild-type and inducible kidney tubule–specific Hif1α knockout mice. Results In cultured cells, activation of the HIF pathway by dimethyloxalylglycine or hypoxia inhibited sodium transport and decreased expression of βENaC and γENaC, as well as of Na,K-ATPase. HIF1α silencing increased βENaC and γENaC expression and stimulated sodium transport. A constitutively active mutant of HIF1α produced the opposite effect. Aldosterone and inhibition of the mitochondrial respiratory chain slowly activated the HIF pathway, suggesting that ROS may also activate HIF. Decreased γENaC abundance induced by hypoxia in normal mice was abolished in Hif1α knockout mice. Similarly, Hif1α knockout led to increased γENaC abundance under high sodium intake. Conclusions This study reveals that γENaC expression and activity are physiologically controlled by the HIF pathway, which may represent a negative feedback mechanism to preserve oxygenation and/or prevent excessive ROS generation under increased sodium transport.