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Regulation of the Cl−/[Formula: see text] exchanger pendrin has been suggested to explain the aldosterone paradox. A high-K+ diet has been proposed to downregulate a pendrin-mediated K+-sparing NaCl reabsorption pathway to maximize urinary K+ excretion. Here, we challenged the hypothesis, revealing that the accompanying anion, not K+, drives pendrin expression. Pendrin is downregulated with a high-KCl diet, preventing acidosis, and upregulated with an alkaline-rich high-K+ diet, preventing metabolic alkalosis. Pendrin regulation is prioritized for acid-base balance.

Background: Serum creatinine level, proteinuria, and interstitial fibrosis are predictive of renal prognosis. Fractional excretion of phosphate (FEP)/FGF23 ratio, tubular reabsorption of phosphate (TRP), serum calcification propensity (T50), and Klotho’s serum level are emerging as determinants of poor kidney outcomes in CKD patients. We aimed at analysing the use of FGF23, FEP/FGF23, TRP, T50, and Klotho in predicting the rapid decline of renal function in kidney allograft recipients. Methods: We included 103 kidney allograft recipients in a retrospective study with a prospective follow-up of 4 years. We analysed the predictive values of FGF23, FEP/FGF23, TRP, T50, and Klotho for a rapid decline of renal function defined as a drop of eGFR > 30%. Results: During a follow-up of 4 years, 23 patients displayed a rapid decline of renal function. Tertile of FGF23 (p value = 0.17), FEP/FGF23 (p value = 0.78), TRP (p value = 0.62) and Klotho (p value = 0.31) were not associated with an increased risk of rapid decline of renal function in kidney transplant recipients. The lower tertile of T50 was significantly associated with eGFR decline >30% with a hazard ratio of 3.86 (p = 0.048) and remained significant in multivariable analysis. Conclusion: T50 showed a strong association with a rapid decline of renal function in kidney allograft patients. This study underlines its role as an independent biomarker of loss of kidney function. We found no association between other phosphocalcic markers, such as FGF23, FEP/FGF23, TRP and Klotho, with a rapid decline of renal function in kidney allograft recipients.

Pendrin is an apical Cl−/[Formula: see text] exchanger that provides renal K+-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K+ and limits renal K+ loss by downregulating the epithelial Na+ channel (ENaC). However, with Na+ restriction, the need to reduce ENaC for K+ balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction.

Pendrin is an intercalated cell Cl(−)/ [Formula: see text] exchanger thought to participate in K(+)-sparing NaCl absorption. However, its role in K(+) homeostasis has not been clearly defined. We hypothesized that pendrin-null mice will develop hypokalemia with dietary K(+) restriction. We further hypothesized that pendrin knockout (KO) mice mitigate urinary K(+) loss by downregulating the epithelial Na(+) channel (ENaC). Thus, we examined the role of ENaC in Na(+) and K(+) balance in pendrin KO and wild-type mice following dietary K(+) restriction. To do so, we examined the relationship between Na(+) and K(+) balance and ENaC subunit abundance in K(+)-restricted pendrin-null and wild-type mice that were NaCl restricted or replete. Following a NaCl-replete, K(+)-restricted diet, K(+) balance and serum K(+) were similar in both groups. However, following a Na(+), K(+), and Cl(−)-deficient diet, pendrin KO mice developed hypokalemia from increased K(+) excretion. The fall in serum K(+) observed in K(+)-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. The fall in serum K(+) observed in K(+)-restricted pendrin KO mice was enhanced with ENaC stimulation but eliminated with ENaC inhibition. However, reducing ENaC activity also reduced blood pressure and increased apparent intravascular volume contraction, since KO mice had lower serum Na(+), higher blood urea nitrogen and hemoglobin, greater weight loss, greater metabolic alkalosis, and greater NaCl excretion. We conclude that dietary Na(+) and K(+) restriction induces hypokalemia in pendrin KO mice. Pendrin-null mice limit renal K(+) loss by downregulating ENaC. However, this ENaC downregulation occurs at the expense of intravascular volume. NEW & NOTEWORTHY Pendrin is an apical Cl(−)/ [Formula: see text] exchanger that provides renal K(+)-sparing NaCl absorption. The pendrin-null kidney has an inability to fully conserve K(+) and limits renal K(+) loss by downregulating the epithelial Na(+) channel (ENaC). However, with Na(+) restriction, the need to reduce ENaC for K(+) balance conflicts with the need to stimulate ENaC for intravascular volume. Therefore, NaCl restriction stimulates ENaC less in pendrin-null mice than in wild-type mice, which mitigates their kaliuresis and hypokalemia but exacerbates volume contraction.

The WNK/SPAK “potassium-switch” pathway provides a homeostatic mechanism that activates the sodium chloride cotransporter, NCC, to limit urinary potassium excretion in dietary potassium deficiency at the expense of increasing salt-sensitivity and blood pressure. Although it is well understood that high dietary potassium turns off the switch, and decreases NCC activity, several reports suggest the counter anion in potassium-rich diets may have an influence on the pathway. The goal of the present study was to determine if the chronic response to a high K diet depends on the counter anion. Wild-type C57BL6J mice were randomized to control (1%K+), potassium chloride (KCl: 5%K+), or potassium bicarbonate (KHCO3: 5%K+) diets, and physiological, molecular, and imaging analyses were performed at 4 and 10 days of diet consumption. In contrast to the classic response to high KCl diet, characterized by reduced NCC phosphorylation and membrane localization, NCC phosphorylation and apical localization increased after 10 days on the high KHCO3 diet. This response was paralleled by increased phosphorylation of the downstream potassium-switch kinase, SPAK, which in turn phosphorylates NCC. Immunofluorescence imaging revealed WNK4 bodies were more numerous and larger in the high KHCO3 group compared to the control, consistent with activation of upstream WNK signaling. Urine excretion studies revealed mice on the high KHCO3 retain more Na+ than mice on control or KCl diets, paralleling enhanced NCC activation. Unlike mice on the high KCl diet, mice on the high KHCO3 diet develop and maintain metabolic alkalosis and do not exhibit profound hyperkalemia, coincident with more robust activation of ENaC and ROMK than with the high KCl diet. In summary, these data identify an unexpected NCC activation mechanism that may limit distal sodium delivery to prevent excessive potassium losses when alkalosis super activates potassium secretion in more distal segments. NIDDK, LeDucq Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

This study shows that aquaporin-2 regulation is disrupted by a small fall in plasma potassium levels and the response is influenced by sexual dimorphism in renal potassium handling. The findings provided new insights into the mechanisms by which water balance is altered in dietary potassium deficiency and support defining the disorder as “potassium-dependent nephrogenic diabetes insipidus.”

The association between diabetes insipidus (DI) and chronic dietary K(+) deprivation is well known, but it remains uncertain how the disorder develops and whether it is influenced by the sexual dimorphism in K(+) handling. Here, we determined the plasma K(+) (P(K)) threshold for DI in male and female mice and ascertained if DI is initiated by polydipsia or by a central or nephrogenic defect. C57BL6J mice were randomized to a control diet or to graded reductions in dietary K(+) for 8 days, and kidney function and transporters involved in water balance were characterized. We found that male and female mice develop polyuria and secondary polydipsia. Altered water balance coincided with a decrease in aquaporin-2 (AQP2) phosphorylation and apical localization despite increased levels of the vasopressin surrogate marker copeptin. No change in the protein abundance of urea transporter-A1 was observed. The Na(+)-K(+)-2Cl(−) cotransporter decreased only in males. Desmopressin treatment failed to reverse water diuresis in K(+)-restricted mice. These findings indicate that even a small fall in P(K) is associated with nephrogenic DI (NDI), coincident with the development of altered AQP2 regulation, implicating low P(K) as a causal trigger of NDI. We found that P(K) decreased more in females, and, consequently, females were more prone to develop NDI. Together, these data indicate that AQP2 regulation is disrupted by a small decrease in P(K) and that the response is influenced by sexual dimorphism in K(+) handling. These findings provide new insights into the mechanisms linking water and K(+) balances and support defining the disorder as “potassium-dependent NDI.” NEW & NOTEWORTHY This study shows that aquaporin-2 regulation is disrupted by a small fall in plasma potassium levels and the response is influenced by sexual dimorphism in renal potassium handling. The findings provided new insights into the mechanisms by which water balance is altered in dietary potassium deficiency and support defining the disorder as “potassium-dependent nephrogenic diabetes insipidus.”

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in similar to 12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 +/- 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.

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.

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 full...

Mutations in the CACNA1A gene, encoding the α1 subunit of the voltage-gated calcium channel Ca(V)2.1 (P/Q-type), have been associated with three neurological phenotypes: familial and sporadic hemiplegic migraine type 1 (FHM1, SHM1), episodic ataxia type 2 (EA2), and spinocerebellar ataxia type 6 (SCA6). We report a child with congenital ataxia, abnormal eye movements and developmental delay who presented severe attacks of hemiplegic migraine triggered by minor head traumas and associated with hemispheric swelling and seizures. Progressive cerebellar atrophy was also observed. Remission of the attacks was obtained with acetazolamide. A de novo 3 bp deletion was found in heterozygosity causing loss of a phenylalanine residue at position 1502, in one of the critical transmembrane domains of the protein contributing to the inner part of the pore. We characterized the electrophysiology of this mutant in a Xenopus oocyte in vitro system and showed that it causes gain of function of the channel. The mutant Ca(V)2.1 activates at lower voltage threshold than the wild type. These findings provide further evidence of this molecular mechanism as causative of FHM1 and expand the phenotypic spectrum of CACNA1A mutations with a child exhibiting severe SHM1 and non-episodic ataxia of congenital onset.

Mutations in the phosphoinositide phos- phatase myotubularin (MTM1) results in X-linked myo- tubular/centronuclear myopathy (XLMTM), character- ized by a severe decrease in muscle mass and strength in patients and murine models. However, the molecular mechanism involved in the muscle hypotrophy is un- clear. Here we show that the IGF1R/Akt pathway is affected in Mtm1-deficient murine muscles, character- ized by an increase in IGF1 receptor and Akt levels in both the presymptomatic and symptomatic phases. Moreover, up-regulation of atrogenes was observed in the presymptomatic phase of the myopathy, supporting overactivation of the ubiquitin-proteasome pathway. In parallel, the autophagy machinery was affected as indi- cated by the increase in the number of autophagosomes and of autophagy markers, such as LC3 and P62. However, phosphorylation of FOXO3a and mTOR were abnormal at late but not at early stages of the disease, suggesting that myotubularin acts both up- stream in the IGF1R/Akt pathway and downstream on the balance between the autophagy and ubiquitin-pro- teasome pathways in vivo. Adeno-associated virus-medi- ated delivery of Mtm1 into Mtm1-null muscles rescued muscle mass and normalized the expression levels of IGF1 receptor, the ubiquitin-proteasome pathway, and autophagy markers. These data support the hypothesis that the unbalanced regulation of the ubiquitin protea- some pathway and the autophagy machinery is a pri- mary cause of the XLMTM pathogenesis.—Al-Qusairi, L., Prokic, I., Amoasii, L., Kretz, C., Messaddeq, N., Mandel, J.-L., Laporte, J. Lack of myotubularin (MTM1) leads to muscle hypotrophy through unbal- anced regulation of the autophagy and ubiquitin-pro- teasome pathways. FASEB J. 27, 000 - 000 (2013). www.fasebj.org

Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-linked myotubular myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1 -deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca 2+ release elicited by voltage-clamp depolarizations is strongly depressed in myotubularin-deficient muscle fibers, with myoplasmic Ca 2+ removal and SR Ca 2+ content essentially unaffected. At the molecular level, Mtm1 -deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca 2+ release is responsible for the failure of muscle function in myotubular myopathy.

X-linked myotubular myopathy (XLMTM) is a disease characterized by severe skeletal muscle weakness leading to death during childhood. XLMTM results from mutations in the MTM1 gene, coding for Myotubularin, a phosphoinositide phosphatase believed to play a role in plasma membrane homeostasis. The mechanisms responsible for muscle function impairment in XLMTM are unknown. Here we studied the properties of excitation-contraction coupling in skeletal muscle fibers isolated from a mouse model of the disease. Experiments were performed under silicone-voltage-clamp conditions using indo-1 as Ca2+ indicator. In muscle fibers from 5-week-old MTM1-deficient mice, the amplitude of the voltage-activated Ca2+ transient was strongly reduced. For instance, in response to a 10 ms-long pulse from -80 to +10 mV, the peak Δ[Ca2+] was 0.52 ± 0.1 μM (n=14) in MTM1-KO fibers as compared to 1.4 ± 0.14 μM in WT fibers (n=14). Conversely, the rate of [Ca2+] decay after the end of the pulses was similar in the two strains suggesting overall preserved myoplasmic Ca2+ removal capabilities. The SR calcium content was also found to be unaltered, as estimated from indo-1 signals measured in fibers equilibrated with high intracellular EGTA and in the presence of a SR Ca2+ pump blocker. The reduced amplitude of the Ca2+ transient in MTM1-deficient fibers was associated with a twice reduction in the peak density of the voltage-activated slow Ca2+ current with no apparent concurrent change in the density of intramembrane charge movement. Finally, confocal imaging with di-8-anneps revealed local disruptions in the typical fluorescence banded pattern, indicative of alteration of t-tubule membrane. Overall results unravel a critical role of MTM1 in the proper function of E-C coupling and strongly suggest that defective RyR1-mediated SR Ca2+ release is responsible for the failure of muscle function in myotubular myopathy.