The disease Familial Hyperkalemic Hypertension (FHHt; also known as Gordon Syndrome) is caused by aberrant accumulation of WNK4 activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-SPAK-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We utilized Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3-/-) or Jab1 (DCT-Jab1-/-) only in DCT and examined the mice after short- and long-term deletion. Short-term, DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, SPAK, and pNCC abundance. However, neither model demonstrated changes in plasma K+, Cl-, or TCO2, even though no injury was present. Long-term DCT-Jab1-/- mice showed significantly lower NCC and parvalbumin abundance, and higher abundance of kidney injury molecule 1 (KIM-1), a marker of proximal tubule injury. No injury, or reduction in NCC or parvalbumin were observed in long-term DCT-Cul3-/- mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1-/- mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter\'s syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

Although hyponatremia and salt wasting are common in patients with HIV/AIDS, the understanding of their contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the distal tubules and on the expression level of the Slc12a3 gene, encoding the sodium-chloride cotransporter (which is responsible for sodium reabsorption in distal nephron segments), single-nucleus RNA sequencing was performed on kidney cortices from three wild-type (WT) and three Vpr transgenic (Vpr Tg) mice. The results show that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05); in Vpr Tg mice, Slc12a3 expression was not significantly different in DCT cells. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT mice (P < 0.01). Immunohistochemistry revealed fewer Slc12a3+Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis between Vpr Tg and WT samples in the DCT cluster showed down-regulation of the Ier3 gene, which is an inhibitor of apoptosis. The in vitro knockdown of Ier3 by siRNA transfection induced apoptosis in mouse DCT cells. These observations suggest that the salt-wasting effect of Vpr in Vpr Tg mice is likely mediated by Ier3 down-regulation in DCT1 cells and loss of Slc12a3+Pvalb+ DCT1 segments.

Dietary potassium deficiency causes stimulation of sodium reabsorption leading to an increased risk in blood pressure elevation. The distal convoluted tubule (DCT) is the main rheostat linking plasma K+ levels to the activity of the Na-Cl cotransporter (NCC). This occurs through basolateral membrane potential sensing by inwardly rectifying K+ channels (Kir4.1/5.1); decrease in intracellular Cl-; activation of WNK4 and interaction and phosphorylation of STE20/SPS1-related proline/alanine-rich kinase (SPAK); binding of calcium-binding protein 39 (cab39) adaptor protein to SPAK, leading to its trafficking to the apical membrane; and SPAK binding, phosphorylation, and activation of NCC. As kidney-specific with-no-lysine kinase 1 (WNK1) isoform (KS-WNK1) is another participant in this pathway, we examined its function in NCC regulation. We eliminated KS-WNK1 specifically in the DCT and demonstrated increased expression of WNK4 and long WNK1 (L-WNK1) and increased phosphorylation of NCC. As in other KS-WNK1 models, the mice were not hyperkalemic. Although wild-type mice under low-dietary K+ conditions demonstrated increased NCC phosphorylation, the phosphorylation levels of the transporter, already high in KS-WNK1, did not change under the low-K+ diet. Thus, in the absence of KS-WNK1, the transporter lost its sensitivity to low plasma K+. We also show that under low K+ conditions, in the absence of KS-WNK1, there was no formation of WNK bodies. These bodies were observed in adjacent segments, not affected by the targeting of KS-WNK1. As our data are overall consistent with those of the global KS-WNK1 knockout, they indicate that the DCT is the predominant segment affecting the salt transport regulated by KS-WNK1.NEW & NOTEWORTHY In this paper, we show that KS-WNK1 is a critical component of the distal convoluted tubule (DCT) K+ switch pathway. Its deletion results in an inability of the DCT to sense changes in plasma potassium. Absence of KS-WNK1 leads to abnormally high levels of WNK4 and L-WNK1 in the DCT, resulting in increased Na-Cl phosphorylation and function. Our data are consistent with KS-WNK1 targeting WNK4 and L-WNK1 to degradation.

Renal tubules are featured with copious mitochondria and robust transport activity. Mutations in mitochondrial genes cause congenital renal tubulopathies, and changes in transport activity affect mitochondrial morphology, suggesting mitochondrial function and transport activity are tightly coupled. Current methods of using bulk kidney tissues or cultured cells to study mitochondrial bioenergetics are limited. Here, we optimized an extracellular flux analysis (EFA) to study mitochondrial respiration and energy metabolism using microdissected mouse renal tubule segments. EFA detects mitochondrial respiration and glycolysis by measuring oxygen consumption and extracellular acidification rates, respectively. We show that both measurements positively correlate with sample sizes of a few centimeter-length renal tubules. The thick ascending limbs (TALs) and distal convoluted tubules (DCTs) critically utilize glucose/pyruvate as energy substrates, whereas proximal tubules (PTs) are significantly much less so. Acute inhibition of TALs\' transport activity by ouabain treatment reduces basal and ATP-linked mitochondrial respiration. Chronic inhibition of transport activity by 2-week furosemide treatment or deletion of with-no-lysine kinase 4 (Wnk4) decreases maximal mitochondrial capacity. In addition, chronic inhibition downregulates mitochondrial DNA mass and mitochondrial length/density in TALs and DCTs. Conversely, gain-of-function Wnk4 mutation increases maximal mitochondrial capacity and mitochondrial length/density without increasing mitochondrial DNA mass. In conclusion, EFA is a sensitive and reliable method to investigate mitochondrial functions in isolated renal tubules. Transport activity tightly regulates mitochondrial bioenergetics and biogenesis to meet the energy demand in renal tubules. The system allows future investigation into whether and how mitochondria contribute to tubular remodeling adapted to changes in transport activity.

Renal diseases associated with hypomagnesemia are a complex and diverse group of tubulopathies caused by mutations in genes encoding proteins that are expressed in the thick ascending limb of the loop of Henle and in the distal convoluted tubule. In this paper, we review the initial description, the clinical expressiveness and etiology of four of the first hypomagnesemic tubulopathies described: type 3 Bartter and Gitelman diseases, Autosomal recessive hypomagnesemia with secondary hypocalcemia and Familial hypomagnesemia with hypercalciuria and nephrocalcinosis. The basic biochemical patterns observed in renal tubular hypomagnesemias and the modalities of transport and interaction that occur between the transporters involved in the reabsorption of magnesium in the distal convoluted tubule are described below. Finally, the recent report of a new renal disease with hypomagnesemia, type 2 hypomagnesemia with secondary hypocalcemia caused by reduced TRPM7 channel activity is described.

Kidney-specific with-no-lysine kinase 1 (KS-WNK1) is an isoform of WNK1 kinase that is predominantly found in the distal convoluted tubule of the kidney. The precise physiological function of KS-WNK1 remains unclear. Some studies have suggested that it could play a role in regulating potassium renal excretion by modulating the activity of the Na+-Cl- cotransporter (NCC). However, changes in the potassium diet from normal to high failed to reveal a role for KS-WNK1, but under a normal-potassium diet, the expression of KS-WNK1 is negligible. It is only detectable when mice are exposed to a low-potassium diet. In this study, we investigated the role of KS-WNK1 in regulating potassium excretion under extreme changes in potassium intake. After following a zero-potassium diet (0KD) for 10 days, KS-WNK1-/- mice had lower plasma levels of K+ and Cl- while exhibiting higher urinary excretion of Na+, Cl-, and K+ compared with KS-WNK1+/+ mice. After 10 days of 0KD or normal-potassium diet (NKD), all mice were challenged with a high-potassium diet (HKD). Plasma K+ levels markedly increased after the HKD challenge only in mice previously fed with 0KD, regardless of genotype. KSWNK1+/+ mice adapt better to HKD challenge than KS-WNK1-/- mice after a potassium-retaining state. The difference in the phosphorylated NCC-to-NCC ratio between KS-WNK1+/+ and KS-WNK1-/- mice after 0KD and HKD indicates a role for KS-WNK1 in both NCC phosphorylation and dephosphorylation. These observations show that KS-WNK1 helps the distal convoluted tubule to respond to extreme changes in potassium intake, such as those occurring in wildlife.NEW & NOTEWORTHY The findings of this study demonstrate that kidney-specific with-no-lysine kinase 1 plays a role in regulating urinary electrolyte excretion during extreme changes in potassium intake, such as those occurring in wildlife.  .

Vasopressin regulates water homeostasis via the V2 receptor in the kidney at least in part through protein kinase A (PKA) activation. Vasopressin, through an unknown pathway, upregulates the activity and phosphorylation of Na+-Cl- cotransporter (NCC) and Na+-K+-2Cl- cotransporter 2 (NKCC2) by Ste20-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), which are regulated by the with-no-lysine kinase (WNK) family. Phosphorylation of WNK4 at PKA consensus motifs may be involved. Inhibitor 1 (I1), a protein phosphatase 1 (PP1) inhibitor, may also play a role. In human embryonic kidney (HEK)-293 cells, we assessed the phosphorylation of WNK4, SPAK, NCC, or NKCC2 in response to forskolin or desmopressin. WNK4 and cotransporter phosphorylation were studied in desmopressin-infused WNK4-/- mice and in tubule suspensions. In HEK-293 cells, only wild-type WNK4 but not WNK1, WNK3, or a WNK4 mutant lacking PKA phosphorylation motifs could upregulate SPAK or cotransporter phosphorylation in response to forskolin or desmopressin. I1 transfection maximized SPAK phosphorylation in response to forskolin in the presence of WNK4 but not of mutant WNK4 lacking PP1 regulation. We observed direct PP1 regulation of NKCC2 dephosphorylation but not of NCC or SPAK in the absence of WNK4. WNK4-/- mice with desmopressin treatment did not increase SPAK/OSR1, NCC, or NKCC2 phosphorylation. In stimulated tubule suspensions from WNK4-/- mice, upregulation of pNKCC2 was reduced, whereas upregulation of SPAK phosphorylation was absent. These findings suggest that WNK4 is a central node in which kinase and phosphatase signaling converge to connect cAMP signaling to the SPAK/OSR1-NCC/NKCC2 pathway.NEW & NOTEWORTHY With-no-lysine kinases regulate the phosphorylation and activity of the Na+-Cl- and Na+-K+-2Cl- cotransporters. This pathway is modulated by arginine vasopressin (AVP). However, the link between AVP and WNK signaling remains unknown. Here, we show that AVP activates WNK4 through increased phosphorylation at putative protein kinase A-regulated sites and decreases its dephosphorylation by protein phosphatase 1. This work increases our understanding of the signaling pathways mediating AVP actions in the kidney.

Hyponatremia and salt wasting is a common occurance in patients with HIV/AIDS, however, the understanding of its contributing factors is limited. HIV viral protein R (Vpr) contributes to HIV-associated nephropathy. To investigate the effects of Vpr on the expression level of the Slc12a3 gene, encoding the Na-Cl cotransporter, which is responsible for sodium reabsorption in distal nephron segments, we performed single-nucleus RNA sequencing of kidney cortices from three wild-type (WT) and three Vpr-transgenic (Vpr Tg) mice. The results showed that the percentage of distal convoluted tubule (DCT) cells was significantly lower in Vpr Tg mice compared with WT mice (P < 0.05), and that in Vpr Tg mice, Slc12a3 expression was not different in DCT cell cluster. The Pvalb+ DCT1 subcluster had fewer cells in Vpr Tg mice compared with WT (P < 0.01). Immunohistochemistry demonstrated fewer Slc12a3+ Pvalb+ DCT1 segments in Vpr Tg mice. Differential gene expression analysis comparing Vpr Tg and WT in the DCT cluster showed Ier3, an inhibitor of apoptosis, to be the most downregulated gene. These observations demonstrate that the salt-wasting effect of Vpr in Vpr Tg mice is mediated by loss of Slc12a3+ Pvalb+ DCT1 segments via apoptosis dysregulation.

The activity of the Na+-Cl- cotransporter (NCC) in the distal convoluted tubule (DCT) is finely tuned by phosphorylation networks involving serine/threonine kinases and phosphatases. While much attention has been paid to the With-No-lysine (K) kinase (WNK)- STE20-related Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive kinase 1 (OSR1) signaling pathway, there remain many unanswered questions regarding phosphatase-mediated modulation of NCC and its interactors. The phosphatases shown to regulate NCC\'s activity, directly or indirectly, are protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), calcineurin (CN), and protein phosphatase 4 (PP4). PP1 has been suggested to directly dephosphorylate WNK4, SPAK, and NCC. This phosphatase increases its abundance and activity when extracellular K+ is increased, which leads to distinct inhibitory mechanisms towards NCC. Inhibitor-1 (I1), oppositely, inhibits PP1 when phosphorylated by protein kinase A (PKA). CN inhibitors, like tacrolimus and cyclosporin A, increase NCC phosphorylation, giving an explanation to the Familial Hyperkalemic Hypertension-like syndrome that affects some patients treated with these drugs. CN inhibitors can prevent high K+-induced dephosphorylation of NCC. CN can also dephosphorylate and activate Kelch-like protein 3 (KLHL3), thus decreasing WNK abundance. PP2A and PP4 have been shown in in vitro models to regulate NCC or its upstream activators. However, no studies in native kidneys or tubules have been performed to test their physiological role in NCC regulation. This review focuses on these dephosphorylation mediators and the transduction mechanisms possibly involved in physiological states that require of the modulation of the dephosphorylation rate of NCC.