Does the early aldosterone-induced SGK1 play a role in early Kaliuresis?

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 Sgk1Pax8 / 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, Sgk1Pax8 / 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 Sgk1Pax8 / 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.

Polycystin-1 dependent regulation of polycystin-2 via GRP94, a member of HSP90 family that resides in the endoplasmic reticulum.

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.

Diverging effects of enalapril or eplerenone in primary prevention against doxorubicin-induced cardiotoxicity.

Aims: Clinical 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 results: Acute 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. Conclusion: Enalapril 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.

Drosophila melanogaster positive transcriptional elongation factors regulate metabolic and sex-biased expression in adults.

BACKGROUND: Transcriptional elongation is a generic function, but is also regulated to allow rapid transcription responses. Following relatively long initiation and promoter clearance, RNA polymerase II can pause and then rapidly elongate following recruitment of positive elongation factors. Multiple elongation complexes exist, but the role of specific components in adult Drosophila is underexplored. RESULTS: We conducted RNA-seq experiments to analyze the effect of RNAi knockdown of Suppressor of Triplolethal and lilliputian. We similarly analyzed the effect of expressing a dominant negative Cyclin-dependent kinase 9 allele. We observed that almost half of the genes expressed in adults showed reduced expression, supporting a broad role for the three tested genes in steady-state transcript abundance. Expression profiles following lilliputian and Suppressor of Triplolethal RNAi were nearly identical raising the possibility that they are obligatory co-factors. Genes showing reduced expression due to these RNAi treatments were short and enriched for genes encoding metabolic or enzymatic functions. The dominant-negative Cyclin-dependent kinase 9 profiles showed both overlapping and specific differential expression, suggesting involvement in multiple complexes. We also observed hundreds of genes with sex-biased differential expression following treatment. CONCLUSION: Transcriptional profiles suggest that Lilliputian and Suppressor of Triplolethal are obligatory cofactors in the adult and that they can also function with Cyclin-dependent kinase 9 at a subset of loci. Our results suggest that transcriptional elongation control is especially important for rapidly expressed genes to support digestion and metabolism, many of which have sex-biased function.

Renal Tubular Ubiquitin-Protein Ligase NEDD4-2 Is Required for Renal Adaptation during Long-Term Potassium Depletion.

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.

Renal tubular SGK1 deficiency causes impaired K+ excretion via loss of regulation of NEDD4-2/WNK1 and ENaC.

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 Drosophila Su(var)3-7 gene is required for oogenesis and female fertility, genetically interacts with piwi and aubergine, but impacts only weakly transposon silencing.

Heterochromatin is made of repetitive sequences, mainly transposable elements (TEs), the regulation of which is critical for genome stability. We have analyzed the role of the heterochromatin-associated Su(var)3-7 protein in Drosophila ovaries. We present evidences that Su(var)3-7 is required for correct oogenesis and female fertility. It accumulates in heterochromatic domains of ovarian germline and somatic cells nuclei, where it co-localizes with HP1. Homozygous mutant females display ovaries with frequent degenerating egg-chambers. Absence of Su(var)3-7 in embryos leads to defects in meiosis and first mitotic divisions due to chromatin fragmentation or chromosome loss, showing that Su(var)3-7 is required for genome integrity. Females homozygous for Su(var)3-7 mutations strongly impair repression of P-transposable element induced gonadal dysgenesis but have minor effects on other TEs. Su(var)3-7 mutations reduce piRNA cluster transcription and slightly impact ovarian piRNA production. However, this modest piRNA reduction does not correlate with transposon de-silencing, suggesting that the moderate effect of Su(var)3-7 on some TE repression is not linked to piRNA production. Strikingly, Su(var)3-7 genetically interacts with the piwi and aubergine genes, key components of the piRNA pathway, by strongly impacting female fertility without impairing transposon silencing. These results lead us to propose that the interaction between Su(var)3-7 and piwi or aubergine controls important developmental processes independently of transposon silencing.

Transcriptional activation by GAGA factor is through its direct interaction with dmTAF3.

The GAGA factor (GAF), encoded by the Trithorax like gene (Trl) is a multifunctional protein involved in gene activation, Polycomb-dependent repression, chromatin remodeling and is a component of chromatin domain boundaries. Although first isolated as transcriptional activator of the Drosophila homeotic gene Ultrabithorax (Ubx), the molecular basis of this GAF activity is unknown. Here we show that dmTAF3 (also known as BIP2 and dTAF(II)155), a component of TFIID, interacts directly with GAF. We generated mutations in dmTAF3 and show that, in Trl mutant background, they affect transcription of Ubx leading to enhancement of Ubx phenotype. These results reveal that the gene activation pathway involving GAF is through its direct interaction with dmTAF3.