Urinary K + potassium excretion rapidly increases after a potassium‐rich meal. The early aldosterone‐induced sgk1 gene (encoding serum and glucocorticoid‐induced kinase 1), activates potassium clearance, but the role of this kinase in the early activation of K + secretion has not been clearly defined. Here, we challenged inducible renal‐tubule‐specific Sgk1 Pax8 / LC1 knockout mice with an acute high‐potassium load (HK:5%K + ) and compared the physiological and molecular responses to control mice. We observe that urinary excretion after a K + load over the first 3 h is not dependent on SGK1 but is coincident with the rapid dephosphorylation of the Na + ,Cl − ‐cotransporter (NCC) to increase distal salt delivery. Molecular analyses indicate that whereas SGK1‐mediated phosphorylation of the ubiquitin‐protein ligase NEDD4‐2 begins to increase by 3h, SGK1‐dependent proteolytic activation of ENaC only becomes detectable after 6 h of HK intake. Consistent with SGK1‐dependent ENaC activation via inhibition of NEDD4‐2‐mediated ubiquitylation, Sgk1 Pax8 / LC1 mice are unable to efficiently inhibit NEDD4‐2 or increase ENaC cleavage after 6 h of HK. Nevertheless, no defect in acute K + balance was detected in the mutant mice after 6 h of HK. Moreover, we found that Sgk1 Pax8 / LC1 mice reduce NCC phosphorylation and NCC‐mediated salt absorption to a greater extent than control mice after a K + load, promoting increased amiloride‐sensitive Na + ‐reabsorption via ENaC to maintain adequate kaliuresis. Together, these data indicate that: (a) during the early 3 h of HK intake, K + excretion is SGK1‐independent even under an extreme K + challenge, (b) shortly after, SGK1 inhibits NEDD4‐2 and activates ENaC to stimulate K + ‐secretion, (c) SGK1‐dependent phosphorylation of NCC occurs, acting more likely as a brake pedal to prevent excessive K + loss.

Autosomal dominant polycystic kidney disease is a common inherited renal disorder that results from mutations in either PKD1 or PKD2, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively. Downregulation or overexpression of PKD1 or PKD2 in mouse models results in renal cyst formation, suggesting that the quantity of PC1 and PC2 needs to be maintained within a tight functional window to prevent cystogenesis. Here we show that enhanced PC2 expression is a common feature of PKD1 mutant tissues, in part due to an increase in Pkd2 mRNA. However, our data also suggest that more effective protein folding contributes to the augmented levels of PC2. We demonstrate that the unfolded protein response is activated in Pkd1 knockout kidneys and in Pkd1 mutant cells and that this is coupled with increased levels of GRP94, an endoplasmic reticulum protein that is a member of the HSP90 family of chaperones. GRP94 was found to physically interact with PC2 and depletion or chemical inhibition of GRP94 led to a decrease in PC2, suggesting that GRP94 serves as its chaperone. Moreover, GRP94 is acetylated and binds to histone deacetylase 6 (HDAC6), a known deacetylase and activator of HSP90 proteins. Inhibition of HDAC6 decreased PC2 suggesting that HDAC6 and GRP94 work together to regulate PC2 levels. Lastly, we showed that inhibition of GRP94 prevents cAMP-induced cyst formation in vitro. Taken together our data uncovered a novel HDAC6-GRP94-related axis that likely participates in maintaining elevated PC2 levels in Pkd1 mutant cells.

AimsClinical studies suggest beneficial effects of renin-angiotensin system blockade for prevention of left ventricular (LV) dysfunction after chemotherapy. However, the efficacy of this strategy as primary prevention has been poorly studied. This study aimed at identifying the pathophysiological mechanisms by which mineralocorticoid receptor antagonism (MRA) or angiotensin converting enzyme inhibition (ACEi) provide protection against doxorubicin-induced cardiotoxicity (DIC) in mouse models of acute and chronic toxicity.Methods and resultsAcute DIC was induced by a single injection of Dox at 15 mg/kg and chronic DIC applied 5 injections of Dox at 4 mg/kg/week. MRA was achieved using eplerenone or cardiomyocyte-specific ablation of the MR gene in transgenic mice and ACEi using enalapril. Drugs were provided with the first dose of Dox and applied until the end of the study. In both model of DIC, Dox induced cardiac atrophy with decreased LV volume, reduced cardiomyocyte cell size, and cardiac dysfunction. In the acute model, neither MRA nor ACEi protected against these manifestations of DIC. In the chronic model, concomitant treatment with eplerenone did not protect against DIC and drastically increased plasma aldosterone levels and cardiac levels of angiotensin II type 1 receptor and of connective tissue growth factor (CTGF), as observed in acute DIC. Enalapril treatment in the chronic model, however, protected against cardiac dysfunction and cardiomyocyte atrophy and was associated with increased activation of the PI3K/AKT/mTOR pathway along with normal levels of CTGF.ConclusionEnalapril and eplerenone disparately impact on cellular signalling in DIC. Eplerenone, on top of Dox treatment was not protective and associated with increased levels of plasma aldosterone and of cardiac CTGF. In contrast, we show that primary prevention with enalapril preserves LV morphology and function in a clinically relevant model of chronic DIC, with increased stimulation of the PI3K/AKT/mTOR axis and normal CTGF levels suggesting potential therapeutic implications.

Adaptation of the organism to potassium (K+) deficiency requires precise coordination among organs involved in K+ homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K+ retention is not well understood. Here, we investigated the role of ubiquitin-protein ligase NEDD4-2, which negatively regulates the epithelial sodium channel (ENaC), Na+/Cl- cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K+ restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 knockout (Nedd4LPax8/LC1 ) mice exhibited severe hypokalemia and urinary K+ wasting. Notably, expression of the ROMK K+ channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. However, K+ restriction also enhanced ENaC expression in Nedd4LPax8/LC1 mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K+ wasting. Moreover, K+ restriction increased WNK1 and WNK4 expression and enhanced SPAK-mediated NCC phosphorylation in Nedd4LPax8/LC1 mice, with no change in total NCC. We propose a mechanism in which NEDD4-2 deficiency exacerbates hypokalemia during dietary K+ restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K+ conservation through direct and indirect effects on ENaC, distal nephron K+ channels, and WNK signaling.

The stimulation of postprandial K(+) clearance involves aldosterone-independent and -dependent mechanisms. In this context, serum- and glucocorticoid-induced kinase (SGK)1, a ubiquitously expressed kinase, is one of the primary aldosterone-induced proteins in the aldosterone-sensitive distal nephron. Germline inactivation of SGK1 suggests that this kinase is fundamental for K(+) excretion under conditions of K(+) load, but the specific role of renal SGK1 remains elusive. To avoid compensatory mechanisms that may occur during nephrogenesis, we used inducible, nephron-specific Sgk1(Pax8/LC1) mice to assess the role of renal tubular SGK1 in K(+) regulation. Under a standard diet, these animals exhibited normal K(+) handling. When challenged by a high-K(+) diet, they developed severe hyperkalemia accompanied by a defect in K(+) excretion. Molecular analysis revealed reduced neural precursor cell expressed developmentally downregulated protein (NEDD)4-2 phosphorylation and total expression. γ-Epithelial Na(+) channel (ENaC) expression and α/γENaC proteolytic processing were also decreased in mutant mice. Moreover, with no lysine kinase (WNK)1, which displayed in control mice punctuate staining in the distal convoluted tubule and diffuse distribution in the connecting tubule/cortical colleting duct, was diffused in the distal convoluted tubule and less expressed in the connecting tubule/collecting duct of Sgk(Pax8/LC1) mice. Moreover, Ste20-related proline/alanine-rich kinase phosphorylation, and Na(+)-Cl(-) cotransporter phosphorylation/apical localization were reduced in mutant mice. Consistent with the altered WNK1 expression, increased renal outer medullary K(+) channel apical localization was observed. In conclusion, our data suggest that renal tubular SGK1 is important in the regulation of K(+) excretion via the control of NEDD4-2, WNK1, and ENaC.

The adaptation to K + deficiency requires high coordination between organs involved in K + homeostasis including kidney, muscle and liver. However, how the kidney performs functional and molecular changes to ensure K + retention is not well understood. Here, we show that the ubiquitin‐protein ligase NEDD4‐2, known as a negative regulator of ENaC, NCC and more recently WNK1, is essential for the adaptation of the kidney to potassium deficiency. Our analyses revealed that the induciblerenal‐tubular NEDD4‐2 (NEDD4‐2 Pax8/LC1 ) mice have normal K + homeostasis when challenged by high K + (HKD) or low K + (LKD)diet for one week. Upon longer exposure to LKD (2 weeks), mutant mice exhibited hypokalemia together with increased urinary K + loss. Under the sehypokalemic conditions, increase of the large, Ca 2+ ‐activated K + channel BK and decrease of ROMK protein levels and membrane localization are observed in NEDD4‐2 Pax8/LC1 mice. SPAK‐mediated NCC phosphorylation was enhanced in the knock‐out animals with no change in total NCC. Moreover, the mutant mice showed increased WNK1 signaling in the CNT and the CD, which may explain these deregulations. In addition, ENaC expression and activity were both enhanced in the mutant mice. Interestingly, IP injection of Benzamil, anENaC inhibitor, restored K + loss in NEDD4‐2 Pax8/LC1 mice. Altogether, our data strongly suggest that NEDD4‐2plays an essential role in K + retention via coordination of K + channels including BK and ROMK on the one hand and of Na + transporters including ENaC and NCC on the other hand. Support or Funding Information Swiss National Science Foundation grant 310030_159735 (to OS), Swiss Kidney.ch NCCR (to OS), National Institutes of Health grant DK098145 (to ARS). LAQ was supported by a fellowship of the Marie Curie Co‐funding IKPP (International Fellowship Program on Integrative Kidney Physiology and Pathophysiology).

Dietary K+ load results in hyperkalemia, with consequent aldosterone release in order to stimulate K+ secretion in the distal nephron. The molecular mechanisms of this regulation are still not fully elucidated. Here, we aim to identify the role of the aldosterone induced SGK1 kinase and its target, the ubiquitin‐protein ligase NEDD4‐2 in the regulation of short and long term K+ secretion. To avoid the compensatory mechanisms which may mask the role of SGK1 and Nedd4‐2 during kidney development, we employed the previously described inducible nephron specific SGK1‐KO and Nedd4‐2‐KO mouse models in which a deletion of the target gene in renal tubules was observed. Our results indicate that short‐term K+ regulation (30 min to 2h) is not altered in SGK1 KO mice. However, these animals exhibit 35% decrease in urinary K+ excretion after 2 days of exposure to high K+ diet (5%) leading to hyperkalemia. Molecular analysis of WT and SGK1 KO mice after 2 days of HK diet revealed that the cleavage and the membrane localization of ENaC alpha and gamma subunits are decreased in the KO mice. On the same line, Nedd4‐2 phosphorylation was also decreased suggesting that NEDD4‐2 mediated internalization of ENaC is more active in mutant mice. Interestingly, when Nedd4‐2 KO mice were challenged by low K+ diet (<0.1%), they showed an abnormal ability to handle K+ deprivation. More specifically, they exhibit hypokalemia and urinary K+ loss likely mediated by increased ENaC activity as demonstrated by the increased membrane localization of alpha and gamma ENaC subunits. In conclusion, our data suggest that SGK1 and its target NEDD4‐2 control K+ homeostasis through the regulation of ENaC membrane localization

Transcription by RNA polymerase II is regulated, in part, by the positive transcription elongation factor b (P-TEFb), which promotes transition from abortive to productive elongation. The Drosophila P-TEFb complex is composed of the Cdk9 kinase and a cyclin partner, CyclinT or CyclinK. To investigate the physiological role of P-TEFb, we generated transgenic flies allowing the conditional expression of wild-type or mutant Cdk9, alone or together with CyclinT or CyclinK. We found that the two P-TEFb complexes have similar binding pattern on chromosomes and are recruited to transcriptionally active loci. By expressing a dominant-negative form of Cdk9 in specific tissues, we showed that P-TEFb function is required for endoreplication of larval tissues, for proper differentiation of imaginal discs and for oogenesis. We demonstrated that the two cyclin subunits have non-redundant activities in vivo, and that P-TEFb containing CyclinT, but not CyclinK, activates transcription when tethered to promoter.