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The mammalian distal nephron is a target of highly effective antihypertensive drugs. Genetic variants that alter its transport activity are also inherited causes of high or low blood pressure, clearly establishing its central role in human blood pressure regulation. Much has been learned during the past 25 years about salt transport along this nephron segment, spurred by the cloning of major transport proteins and the discovery of disease-causing genetic variants. Recognition is increasing that substantial cellular and segmental heterogeneity is present along this segment, with electroneutral sodium transport dominating more proximal segments and electrogenic sodium transport dominating more distal segments. Coupled with recent insights into factors that modulate transport along these segments, we now understand one important mechanism by which dietary potassium intake influences sodium excretion and blood pressure. This finding has solved the aldosterone paradox, by demonstrating how aldosterone can be both kaliuretic, when plasma potassium is elevated, and anti-natriuretic, when extracellular fluid volume is low. However, what also has become clear is that aldosterone itself only stimulates a portion of the mineralocorticoid receptors along this segment, with the others being activated by glucocorticoid hormones instead. These recent insights provide an increasingly clear picture of how this short nephron segment contributes to blood pressure homeostasis and have important implications for hypertension prevention and treatment.

Salt Handling and Blood Pressure Salt intake is associated with blood pressure, but the relationship is complex. This review highlights the interplay among renal salt transport, salt storage in the...

Magnesium is an essential cofactor in many cellular processes, and aberrations in magnesium homeostasis can have life-threatening consequences. The kidney plays a central role in maintaining serum magnesium within a narrow range (0.70-1.10 mmol/L). Along the proximal tubule and thick ascending limb, magnesium reabsorption occurs via paracellular pathways. Members of the claudin family form the magnesium pores in these segments, and also regulate magnesium reabsorption by adjusting the transepithelial voltage that drives it. Along the distal convoluted tubule transcellular reabsorption via heteromeric TRPM6/7 channels predominates, although paracellular reabsorption may also occur. In this segment, the NaCl cotransporter plays a critical role in determining transcellular magnesium reabsorption. Although the general machinery involved in renal magnesium reabsorption has been identified by studying genetic forms of magnesium imbalance, the mechanisms regulating it are poorly understood. This review discusses pathways of renal magnesium reabsorption by different segments of the nephron, emphasizing newer findings that provide insight into regulatory process, and outlining critical unanswered questions.

BackgroundAcute kidney injury (AKI) is associated with mortality in patients hospitalized with COVID-19, however, its incidence, geographic distribution, and temporal trends since the start of the pandemic are understudied.MethodsElectronic health record data were obtained from 53 health systems in the United States (US) in the National COVID Cohort Collaborative (N3C). We selected hospitalized adults diagnosed with COVID-19 between March 6th, 2020, and January 6th, 2022. AKI was determined with serum creatinine (SCr) and diagnosis codes. Time were divided into 16-weeks (P1-6) periods and geographical regions into Northeast, Midwest, South, and West. Multivariable models were used to analyze the risk factors for AKI or mortality.ResultsOut of a total cohort of 306,061, 126,478 (41.0 %) patients had AKI. Among these, 17.9% lacked a diagnosis code but had AKI based on the change in SCr. Similar to patients coded for AKI, these patients had higher mortality compared to those without AKI. The incidence of AKI was highest in P1 (49.3%), reduced in P2 (40.6%), and relatively stable thereafter. Compared to the Midwest, the Northeast, South, and West had higher adjusted AKI incidence in P1, subsequently, the South and West regions continued to have the highest relative incidence. In multivariable models, AKI defined by either SCr or diagnostic code, and the severity of AKI was associated with mortality.ConclusionsUncoded cases of COVID-19-associated AKI are common and associated with mortality. The incidence and distribution of COVID-19-associated AKI have changed since the first wave of the pandemic in the US.

Cullin-RING ligases are a family of E3 ubiquitin ligases that control cellular processes through regulated degradation. Cullin 3 targets with-no-lysine kinase 4 (WNK4), a kinase that activates the Na(+)-Cl(−) cotransporter (NCC), the main pathway for Na(+) reabsorption in the distal convoluted tubule (DCT). Mutations in the cullin 3 gene lead to familial hyperkalemic hypertension by increasing WNK4 abundance. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) regulates the activity of cullin-RING ligases by removing the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Genetic deletion of the catalytically active CSN subunit, Jab1, along the nephron in mice (KS-Jab1(−/−)) led to increased WNK4 abundance; however, NCC abundance was substantially reduced. We hypothesized that the reduction in NCC resulted from a cortical injury that led to hypoplasia of the segment, which counteracted WNK4 activation of NCC. To test this, we studied KS-Jab1(−/−) mice at weekly intervals over a period of 3 wk. The results showed that NCC abundance was unchanged until 3 wk after Jab1 deletion, at which time other DCT-specific proteins were also reduced. The kidney injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin demonstrated kidney injury immediately after Jab1 deletion; however, the damage was initially limited to the medulla. The injury progressed and expanded into the cortex 3 wk after Jab1 deletion coinciding with loss of the DCT. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury that leads to hypoplasia of the DCT. NEW & NOTEWORTHY Cullin 3 (CUL3) targets with-no-lysine-kinase 4 (WNK4), which activates Na(+)-Cl(−) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Renal-specific genetic deletion of the constitutive photomorphogenesis 9 signalosome, an upstream regulator of CUL3, resulted in a reduction of NCC due to DCT hypoplasia, which coincided with cortical kidney injury. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury leading to hypoplasia of the DCT.

Background: MR (mineralocorticoid receptor) antagonists are recommended for patients with resistant hypertension even when circulating aldosterone levels are not high. Although aldosterone activates MR to increase epithelial sodium channel (ENaC) activity, glucocorticoids also activate MR but are metabolized by 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2). 11βHSD2 is expressed at increasing levels from distal convoluted tubule (DCT) through collecting duct. Here, we hypothesized that MR maintains ENaC activity in the DCT2 and early connecting tubule in the absence of aldosterone. Methods: We studied AS (aldosterone synthase)-deficient (AS −/− ) mice, which were backcrossed onto the same C57BL6/J strain as kidney-specific MR knockout (KS-MR −/− ) mice. KS-MR −/− mice were used to compare MR expression and ENaC localization and cleavage with AS −/− mice. Results: MR was highly expressed along DCT2 through the cortical collecting duct (CCD), whereas no 11βHSD2 expression was observed along DCT2. MR signal and apical ENaC localization were clearly reduced along both DCT2 and CCD in KS-MR −/− mice but were fully preserved along DCT2 and were partially reduced along CCD in AS −/− mice. Apical ENaC localization and ENaC currents were fully preserved along DCT2 in AS −/− mice and were not increased along CCD after low salt. AS −/− mice exhibited transient Na + wasting under low-salt diet, but administration of the MR antagonist eplerenone to AS −/− mice led to hyperkalemia and decreased body weight with higher Na + excretion, mimicking the phenotype of MR −/− mice. Conclusions: Our results provide evidence that MR is activated in the absence of aldosterone along DCT2 and partially CCD, suggesting glucocorticoid binding to MR preserves sodium homeostasis along DCT2 in AS −/− mice.

Inhibitors of the main proximal tubular Na-glucose cotransporter (SGLT2) mitigate diabetic glomerular hyperfiltration and have been approved by the United States Food and Drug Administration for slowing the progression of diabetic kidney disease. It has been proposed that SGLT2 inhibitors improve hard renal outcomes by reducing glomerular capillary pressure (PGC) via a tubuloglomerular feedback (TGF) response to a decrease in proximal reabsorption (J(prox)). However, the effect of SGLT2 inhibition on PGC has not been measured. Here, we studied the effects of acute SGLT2 blockade (ertugliflozin) on J(prox) and glomerular hemodynamics in two-period micropuncture experiments using streptozotocin-induced diabetic rats fed high- or low-NaCl diets. PGC was measured by direct capillary puncture or computed from tubular stop-flow pressure (PSF). TGF is intact while measuring PGC directly but rendered inoperative when measuring PSF. Acute SGLT2 inhibitor reduced J(prox) by ∼30%, reduced PGC by 5–8 mmHg, and reduced glomerular filtration rate (GFR) by ∼25% (all P < 0.0001) but had no effect on PSF. The decrease in PGC was larger with the low-NaCl diet (8 vs. 5 mmHg, P = 0.04) where PGC was higher to begin with (54 vs. 50 mmHg, P = 0.003). Greater decreases in PGC corresponded, unexpectedly, to lesser decreases in GFR (P = 0.04). In conclusion, these results confirm expectations that PGC would decline in response to acute SGLT2 inhibition and that a functioning TGF system is required for this. We infer a contribution of postglomerular vasorelaxation to the TGF responses where decreases in PGC were large and decreases in GFR were small. NEW & NOTEWORTHY It has been theorized that Na-glucose cotransporter (SGLT2) blockade slows progression of diabetic kidney disease by reducing physical strain on the glomerulus. This is the first direct measurement of intraglomerular pressure during SGLT2 blockade. Findings confirmed that SGLT2 blockade does reduce glomerular capillary pressure, that this is mediated through tubuloglomerular feedback, and that the tubuloglomerular feedback response to SGLT2 blockade involves preglomerular vasoconstriction and postglomerular vasorelaxation.

Epidemiological and clinical observations have shown that potassium ingestion is inversely correlated with arterial hypertension prevalence and cardiovascular mortality. The higher the dietary potassium, the lower the blood pressure and mortality. This phenomenon is explained, at least in part, by the interaction between salt reabsorption in the distal convoluted tubule (DCT) and potassium secretion in the connecting tubule/collecting duct of the mammalian nephron: In order to achieve adequate K+ secretion levels under certain conditions, salt reabsorption in the DCT must be reduced. Because salt handling by the kidney constitutes the basis for the long‐term regulation of blood pressure, losing salt prevents hypertension. Here, we discuss how the study of inherited diseases in which salt reabsorption in the DCT is affected has revealed the molecular players, including membrane transporters and channels, kinases, and ubiquitin ligases that form the potassium sensing mechanism of the DCT and the processes through which the consequent adjustments in salt reabsorption are achieved.

Cl−-sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl−cotransporter (NCC) in the distal convoluted tubule (DCT). Cl−enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl−channel ClC-K2 or K+-Cl−cotransporter (KCC). While KCC is electroneutral, Cl−exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+channel activity is expected to influence Cl−movement across the basolateral membrane. Although a role for intracellular Cl−in the regulation of WNK and NCC has been established, intracellular Cl−concentrations ([Cl−]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl−]iin DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl−fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl−]iwas ~7 mM. Stimulation of Cl−exit with low-Cl−hypotonic solutions decreased [Cl−]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl−]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl−]i. Blockade of basolateral K+channels (Kir4.1/5.1) with barium significantly increased [Cl−]i. Finally, a decrease in extracellular K+concentration transiently decreased [Cl−]i, whereas raising extracellular K+transiently increased [Cl−]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl−]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl−]iin the DCT.

Expansion of extracellular fluid volume is central to the pathophysiology of heart failure. Increased extracellular fluid leads to elevated intracardiac filling pressures, resulting in a constellation of signs and symptoms of heart failure referred to as congestion. Loop diuretics are one of the cornerstones of treatments for heart failure, but in contrast to other therapies, robust clinical trial evidence to guide the use of diuretics is sparse. A nuanced understanding of renal physiology and diuretic pharmacokinetics is essential for skillful use of diuretics in the management of heart failure in both the inpatient and outpatient settings. Diuretic resistance, defined as an inadequate quantity of natriuresis despite an adequate diuretic regimen, is a major clinical challenge that generally portends a poor prognosis. In this review, the authors discuss the fundamental mechanisms and physiological principles that underlie the use of diuretic therapy and the available data on the optimal use of diuretics.

Potassium disorders are common in patients with kidney disease, particularly in patients with tubular disorders and low glomerular filtration rate. A multidisciplinary group of researchers and clinicians met in October 2018 to identify evidence and address controversies in potassium management. The issues discussed encompassed our latest understanding of the regulation of tubular potassium excretion in health and disease; the relationship of potassium intake to cardiovascular and kidney outcomes, with increasing evidence showing beneficial associations with plant-based diet and data to suggest a paradigm shift from the idea of dietary restriction toward fostering patterns of eating that are associated with better outcomes; the paucity of data on the effect of dietary modification in restoring abnormal serum potassium to the normal range; a novel diagnostic algorithm for hypokalemia that takes into account the ascendency of the clinical context in determining cause, aligning the educational strategy with a practical approach to diagnosis; and therapeutic approaches in managing hyperkalemia when chronic and in the emergency or hospital ward. In sum, we provide here our conference deliberations on potassium homeostasis in health and disease, guidance for evaluation and management of dyskalemias in the context of kidney diseases, and research priorities in each of the above areas.

The calcium-sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiologic conditions, no glucose is delivered to the DCT, and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention.We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice,HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4- and CaSR-dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge.Glycosuria or fructosuria was associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.

BACKGROUND: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) gene cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine (K) Kinase 4 (WNK4), inappropriately activating sterile 20/SPS-1–related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na(+)Cl(–) cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt is unclear. We tested the hypothesis that reduced abundance of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, is mechanistically important. Because JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. METHODS: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3(+/−)/Klhl3(+/−)), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). RESULTS: Renal KLHL3 was higher in Cul3(−/−) mice, but lower in Cul3(−/−/Δ9) mice and in the Cul3(+/−/Δ9) FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3(+/−)/Klhl3(+/−) mice displayed increased WNK4-SPAK activation and phospho-NCC abundance and an FHHt-like phenotype with increased plasma [K(+)] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered the abundance of CUL3 and KLHL3 and increased the abundance of WNK4 and phospho-NCC. CONCLUSIONS: Jab1(−/−) mice and Cul3(+/−)/Klhl3(+/−) mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9–mediated FHHt.

Volume overload, defined as excess total body sodium and water with expansion of extracellular fluid volume, characterizes common disorders such as congestive heart failure, end-stage liver disease, chronic kidney disease, and nephrotic syndrome. Diuretics are the cornerstone of therapy for volume overload and comprise several classes whose mechanisms of action, pharmacokinetics, indications, and adverse effects are essential principles of nephrology. Loop diuretics are typically the first-line treatment in the management of hypervolemia, with additional drug classes indicated in cases of diuretic resistance and electrolyte or acid-base disorders. Separately, clinical trials highlight improved outcomes in some states of volume overload, such as loop diuretics and sodium/glucose cotransporter 2 inhibitors in patients with congestive heart failure. Resistance to diuretics is a frequent, multifactorial clinical challenge that requires creative and physiology-based solutions. In this installment of AJKD's Core Curriculum in Nephrology, we discuss the pharmacology and therapeutic use of diuretics in states of volume overload and strategies to overcome diuretic resistance.

Background and Aims Chronic hypokalemia causes kidney fibrosis with cystic lesions and arterial hypertension. In contrast, potassium-rich diet lowers blood pressure. The acute effects of hypo- and hyperkalemia on heart and kidney are not well understood. Method Wild-type mice were fed with low (LK), normal (NK) and high (HK) potassium diet for 4 and 20 days. Kidneys were examined for site of acute injury, inflammation and fibrosis. Blood analysis of electrolytes and kidney parameters were analyzed. Echocardiography and ECG were used to assess heart function. Further, KCNJ10 knockout mice were used to investigate kidney damage in a genetically induced hypokalemia model. Results Proximal tubule injury as detected by KIM-1+ staining and yH2AX+ DNA-damage was observed after 4 and 20 days of LK diet. Injury was associated with strong Ki-67+ proliferation of proximal tubule cells. No injury was detected in mice on NK and HK diet. After 20 days of LK diet, F4/80+ inflammation and aSMA+ extracellular matrix accumulation, typical for fibrosis, were observed. LK mice developed polyurie, volume depletion, loss of body weight and high BUN. Lower cardiac output and signs of myocardial stress was seen in echocardiography and ECG. Consistent with WT mice on LK diet, KCNJ10 knockout mice developed same pattern of kidney injury. Nine months after deletion of KCNJ10, cysts were observed in the proximal tubule in outer medzulla. Conclusion Acute hypokalemia causes kidney injury and myocardial stress. Cystic lesions originate from late proximal tubule. Hypokalemia should be corrected rapidly to stop progression into kidney fibrosis.

Collective cell migration occurs in many patho-physiological states, including wound healing and invasive cancer growth. The integrity of the expanding epithelial sheets depends on extracellular cues, including cell-cell and cell-matrix interactions. We show that the nano-scale topography of the extracellular matrix underlying epithelial cell layers can strongly affect the speed and morphology of the fronts of the expanding sheet, triggering partial and complete epithelial-mesenchymal transitions (EMTs). We further demonstrate that this behavior depends on the mechano-sensitivity of the transcription regulator YAP and two new YAP-mediated cross-regulating feedback mechanisms: Wilms Tumor-1-YAP-mediated downregulation of E-cadherin, loosening cell-cell contacts, and YAP-TRIO-Merlin mediated regulation of Rho GTPase family proteins, enhancing cell migration. These YAP-dependent feedback loops result in a switch-like change in the signaling and the expression of EMT-related markers, leading to a robust enhancement in invasive cell spread, which may lead to a worsened clinical outcome in renal and other cancers., Reorganisation of the extracellular matrix (ECM) controls processes involving epithelial-mesenchymal transition (EMT). Here, the authors show that EMT occurring in epithelial cells on a fabricated nano-engineered cell adhesion surface is triggered by mechanical cues from the ECM.

Diuretics are among the most commonly prescribed drugs and, although effective, they are often used to treat patients at substantial risk for complications, making it especially important to understand and appreciate their pharmacokinetics and pharmacodynamics (see recent review by Keller and Hann

In the distal kidney tubule, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel (ENaC). Most studies seeking to identify ENaC-regulating aldosterone-induced proteins have used transcriptional profiling of cultured cells. To identify salt-sensitive transcripts in an in vivo model, we used low-NaCl or high-NaCl diet to stimulate or suppress endogenous aldosterone, in combination with magnetic- and fluorescence-activated cell sorting to isolate distal tubule cells from mouse kidney for transcriptional profiling. Of the differentially expressed transcripts, 162 were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, and 161 were more abundant in distal tubule cells isolated from mice fed high-NaCl diet. Enrichment analysis of Gene Ontology biological process terms identified multiple statistically overrepresented pathways among the differentially expressed transcripts that were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, including ion transmembrane transport, regulation of growth, and negative regulation of apoptosis. Analysis of Gene Ontology molecular function terms identified differentially expressed transcription factors, transmembrane transporters, kinases, and G protein-coupled receptors. Finally, comparison with a recently published study of gene expression changes in distal tubule cells in response to administration of aldosterone identified 18 differentially expressed genes in common between the two experiments. When expression of these genes was measured in cortical collecting ducts microdissected from mice fed low-NaCl or high-NaCl diet, eight were differentially expressed. These genes are likely to be regulated directly by aldosterone and may provide insight into aldosterone signaling to ENaC in the distal tubule.

The aim of this mini review is to provide an overview regarding the role of inwardly rectifying potassium channel 4.1 (Kir4.1)/Kir5.1 in regulating renal K+excretion. Deletion of Kir4.1 in the kidney inhibited thiazide-sensitive NaCl cotransporter (NCC) activity in the distal convoluted tubule (DCT) and slightly suppressed Na-K-2Cl cotransporter (NKCC2) function in the thick ascending limb (TAL). Moreover, increased dietary K+intake inhibited, whereas decreased dietary K+intake stimulated, the basolateral potassium channel (a Kir4.1/Kir5.1 heterotetramer) in the DCT. The alteration of basolateral potassium conductance is essential for the effect of dietary K+intake on NCC because deletion of Kir4.1 in the DCT abolished the effect of dietary K+intake on NCC. Since potassium intake-mediated regulation of NCC plays a key role in regulating renal K+excretion and potassium homeostasis, the deletion of Kir4.1 caused severe hypokalemia and metabolic alkalosis under control conditions and even during increased dietary K+intake. Finally, recent studies have suggested that the angiotensin II type 2 receptor (AT2R) and bradykinin-B2 receptor (BK2R) are involved in mediating the effect of high dietary K+intake on Kir4.1/Kir5.1 in the DCT.

IntroductionThere is growing concern about the declining physician-scientist workforce. NIH recently provided a national dashboard describing the biomedical research workforce, but local strategies are needed.MethodsWe used curated local and national data to develop a workforce dashboard.ResultsMany trends at Oregon Health & Science University (OHSU) were similar to those nationally, such as the increasing percentage of Research Project Grant (RPG)-holding PhDs and the aging RPG population, but differences were also apparent. At OHSU, nearly ¾ of physician-scientist RPGs hold MD-only, compared with nationally, where nearly half are MD/PhD. OHSU also lags in the percentage of RPGs held by women physician-scientists.ConclusionsOur analysis also permitted us to gain a more complete picture of research funding that has been done nationally. We used these data to develop a dashboard that allows our institution to develop policies to increase the numbers of physician-scientists. The data generation approaches and dashboard are likely to be useful at other institutions, as well.

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4−/−mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4−/−mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4−/−mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4−/−mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.

Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K(+) concentration ([K(+)]). High extracellular [K(+)] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K(+)] fails to dephosphorylate NCC, suggesting an interplay between NCC-activating and NCC-inactivating switches. Here, we explored the role of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and intracellular Cl(−) concentration in the rapid effects of extracellular K(+) on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in human embryonic kidney cells and ex vivo in kidney slices by high [K(+)]. Acute high-K(+) challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of SPAK puncta. In line with the postulate of interplay between activating and inactivating switches, we found that the “on” switch, represented by with no lysine kinase 4 (WNK4)-SPAK, must be turned off for rapid NCC dephosphorylation by high [K(+)]. Longer-term WNK-SPAK-mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K(+) loading. Although blockade of protein phosphatase (PP)1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, was essential for NCC dephosphorylation. Overall, our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases, with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K(+). NEW & NOTEWORTHY Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K(+) depends on the Cl(−) sensitivity of with no lysine kinase 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.

Background Previous studies have described a ‘renal potassium switch’ within the distal nephron that turns on the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low potassium intake and off in response to high potassium intake. Studies using genetically modified mice indicate that decreased NCC activity is associated with decreased DCT length and mass; increased NCC activity is associated with increased, DCT length and mass. The aim of our study was to test whether dietary potassium intake causes the DCT remodeling physiologically. Methods Male C57Bl/6 mice were provided either control potassium diet or low potassium diet for four weeks and blood and kidneys were harvested. To determine the length of the DCT in three dimensions, we used Ethyl-cinnamate-based optical clearing, combined with whole-mount immunolabeling, confocal microscopy and three-dimensional morphometric analysis. Results Mice on low potassium diet for four weeks were severely hypokalemic (plasma potassium Conclusion Our results indicate that the DCT remodels physiologically to maintain potassium homeostasis. Additional animals are currently being studied.

The thiazide-sensitive Na+-Cl− cotransporter (NCC) is more abundant in kidneys of female subjects than of male subjects. Because morphological remodeling of the distal convoluted tubule (DCT) is dependent on NCC activity, it has been generally assumed that there is a corresponding sexual dimorphism in the structure of the DCT, leading to a larger female DCT. Until now, this has never been directly examined. Here, optical clearing techniques were combined with antibody labeling of DCT segment markers, state-of-the-art high-speed volumetric imaging, and analysis tools to visualize and quantify DCT morphology in male and female mice and study the DCT remodeling response to furosemide. We found an unexpected sex difference in the structure of the DCT. Compared with the male mice, female mice had a shorter DCT, a higher cellular density of NCC, and a greater capacity to elongate in response to loop diuretics. Our study revealed a sexual dimorphism of the DCT. Female mice expressed a greater density of NCC transporters in a shorter structure to protect Na+ balance in the face of greater basal distal Na+ delivery yet have a larger reserve and structural remodeling capacity to adapt to unique physiological stresses. These observations provide insight into mechanisms that may drive sex differences in the therapeutic responses to diuretics.

New treatments, new understanding, and new approaches to translational research are transforming the outlook for patients with kidney diseases. A number of new initiatives dedicated to advancing the field of nephrology-from value-based care to prize competitions-will further improve outcomes of patients with kidney disease. Because of individual nephrologists and kidney organizations in the United States, such as the American Society of Nephrology, the National Kidney Foundation, and the Renal Physicians Association, and international nephrologists and organizations, such as the International Society of Nephrology and the European Renal Association-European Dialysis and Transplant Association, we are beginning to gain traction to invigorate nephrology to meet the pandemic of global kidney diseases. Recognizing the timeliness of this opportunity, the American Society of Nephrology convened a Division Chief Retreat in Dallas, Texas, in June 2019 to address five key issues: (1) asserting the value of nephrology to the health system; (2) productivity and compensation; (3) financial support of faculty's and divisions' educational efforts; (4) faculty recruitment, retention, diversity, and inclusion; and (5) ensuring that fellowship programs prepare trainees to provide high-value nephrology care and enhance attraction of trainees to nephrology. Herein, we highlight the outcomes of these discussions and recommendations to the American Society of Nephrology.

Abstract Eating more potassium may reduce blood pressure and the occurrence of other cardiovascular diseases by actions on various systems, including the vasculature, the sympathetic nervous system, systemic metabolism, and body fluid volume. Among these, the kidney plays a major role in the potassium-rich diet–mediated blood pressure reduction. Purpose of Review To provide an overview of recent discoveries about the mechanisms by which a potassium-rich diet leads to natriuresis. Recent Findings Although the distal convoluted tubule (DCT) is a short part of the nephron that reabsorbs salt, via the sodium-chloride cotransporter (NCC), it is highly sensitive to changes in plasma potassium concentration. Activation or inhibition of NCC raises or lowers blood pressure. Recent work suggests that extracellular potassium concentration is sensed by the DCT via intracellular chloride concentration which regulates WNK kinases in the DCT. Summary High-potassium diet targets NCC in the DCT, resulting in natriuresis and fluid volume reduction, which are protective from hypertension and other cardiovascular problems.

Background: Urinary extracellular vesicles (uEVs) are secreted into urine by cells from the kidneys and urinary tract. Although changes in uEV proteins are used for quantitative assessment of protein levels in the kidney or biomarker discovery, whether they faithfully reflect (patho)physiological changes in the kidney is a matter of debate. Methods: Mass spectrometry was used to compare in an unbiased manner the correlations between protein levels in uEVs and kidney tissue from the same animal. Studies were performed on rats fed a normal or a high K+ diet. Results: Absolute quantification determined a positive correlation (Pearson R=0.46 or 0.45, control or high K+ respectively, p0.9), included exosome markers Tsg101 and Alix. Relative quantification highlighted a monotonic relationship between altered transporter/channel abundances in uEVs and the kidney following dietary K+ manipulation. Analysis of genetic mouse models also revealed correlations between uEVs and kidney. Conclusion: This large-scale unbiased analysis identifies uEV proteins that track the abundance of the parent proteins in the kidney. The data form a novel resource for the kidney community and support the reliability of using uEV protein changes to monitor specific physiological responses and disease mechanisms.

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.