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The Na +/H + exchanger isoform 3 (NHE3) facilitates Na + reabsorption and H + secretion by the kidneys. Despite stronger NHE3 abundance in the thick ascending limb (TAL) compared to the S1 and S2 segments of the proximal tubule, the role of NHE3 in the TAL is poorly understood. To investigate the role of NHE3 in the TAL, we generated and phenotyped TAL-specific NHE3 knockout mice (NHE3 TAL-KO). Compared to control mice, NHE3 TAL-KO mice did not show significant differences in body weight, blood pH or plasma Na +, K + or Cl - levels. Fluid intake trended to be higher and urine osmolality was significantly lower in NHE3 TAL-KO mice. Despite a similar GFR, NHE3 TAL-KO mice had a greater urinary K +/creatinine ratio. One proposed role of NHE3 relates to furosemide-induced urinary acidification. Acute bolus treatment with furosemide under anesthesia did not result in differences in the dose dependence of urinary flow rate, Cl - excretion or maximal urinary acidification between genotypes; however, in contrast to control mice, urinary pH returned immediately towards baseline levels in NHE3 TAL-KO mice. Chronic furosemide treatment reduced urine osmolality similarly in both genotypes but metabolic alkalosis, hypokalemia and calciuresis were absent in NHE3 TAL-KO mice. Compared to vehicle, chronic furosemide treatment in control mice resulted in greater NKCC2 and lower Npt2a abundances, effects that were absent in NHE3 TAL-KO mice. In summary, NHE3 in the TAL plays a role for the sustained acidification effect of furosemide. Consistent with this, long-term treatment with furosemide did not result in metabolic alkalosis in NHE3 TAL-KO mice.

The sodium-potassium-chloride transporter NKCC1 (SLC12A2) performs Na+-dependent Cl− and K+ ion uptake across plasma membranes. NKCC1 is important for regulating e.g. cell volume, hearing, blood pressure, and chloride gradients defining GABAergic and glycinergic signaling in brain. Here, we present a 2.6 Å resolution cryo-electron microscopy (cryo-EM) structure of human NKCC1 in the substrate-loaded (Na+, K+, 2 Cl−) and inward-facing conformation adopting an occluded state that has also been observed for the SLC6 type transporters MhsT and LeuT. Cl− binding at the Cl1 site together with the nearby K+ ion provide a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl− ion binding at the Cl2 site seems to undertake a structural role similar to a conserved glutamate of SLC6 transporters and may allow for chloride-sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations we describe the Na+ binding site and a putative Na+ release pathway along transmembrane helix 5. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

The thiazide-sensitive sodium-chloride-cotransporter (NCC) in the kidney distal convoluted tubule (DCT) plays an essential role in sodium and potassium homeostasis. Here, we demonstrate that NCC activity is increased by the β2-adrenoceptor agonist salbutamol, a drug prevalently used to treat asthma. Relative to β1-adrenergic receptors, the β2-adrenergic receptors were greatly enriched in mouse DCT cells. In mice, administration of salbutamol increased NCC phosphorylation (indicating increased activity) within 30 minutes but also caused hypokalemia, which also increases NCC phosphorylation. In ex vivo kidney slices and isolated tubules, salbutamol increased NCC phosphorylation in the pharmacologically relevant range of 0.01-10 μM, an effect observed after 15 minutes and maintained at 60 minutes. Inhibition of the inwardly rectifying potassium channel (Kir) 4.1 or the downstream with-no-lysine kinases (WNKs) and STE20/SPS1-related proline alanine-rich kinase (SPAK) pathway greatly attenuated, but did not prevent, salbutamol-induced NCC phosphorylation. Salbutamol increased cAMP in tubules, kidney slices and mpkDCT cells (model of DCT). Phosphoproteomics indicated that protein phosphatase 1 (PP1) was a key upstream regulator of salbutamol effects. A role for PP1 and the PP1 inhibitor 1 (I1) was confirmed in tubules using inhibitors of PP1 or kidney slices from I1 knockout mice. On normal and high salt diets, salbutamol infusion increased systolic blood pressure, but this increase was normalized by thiazide suggesting a role for NCC. Thus, β2-adrenergic receptor signaling modulates NCC activity via I1/PP1 and WNK-dependent pathways, and chronic salbutamol administration may be a risk factor for hypertension.

BACKGROUND: Elevated abundance of sodium-chloride cotransporter (NCC) and phosphorylated NCC (pNCC) are potential markers of primary aldosteronism (PA), but these effects may be driven by hypokalemia. METHODS: We measured plasma potassium in patients with PA. If potassium was

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5

To review recent developments in urinary extracellular vesicles (uEVs) to study kidney physiology and disease.Proteomic analysis in rats showed significant correlations between kidney and uEV protein abundances. Consistent with uEV biogenesis, these correlations were stronger for membrane-associated proteins than for e.g. soluble kinases or E3 ubiquitin ligases. When challenged with a high potassium diet, the physiologically predicted protein changes occurred both in kidney and uEVs, suggesting that analysis of uEVs might be utilized as a proxy or even replacement for tissue analysis. Although kidney-uEV correlations are more difficult to obtain in humans, analysis of uEV cargo from patients with inherited tubulopathies or with primary aldosteronism were also consistent with the predicted changes at the tissue level. The kidney appears to be the main source of uEVs, with a recent study showing that nephron mass determines uEV excretion rate. Therefore, a measure of nephron mass should be included for between-subject comparisons.The overall good correlation between kidney and uEV protein abundances renders uEVs an attractive noninvasive source of biomarkers for studying kidney physiology or disease. However, differences in per-protein kidney-uEV correlations and per-person uEV excretion rates should be considered in uEV biomarker studies.

BACKGROUND: Impaired mineral ion metabolism is a hallmark of CKD-metabolic bone disorder. It can lead to pathologic vascular calcification and is associated with an increased risk of cardiovascular mortality. Loss of calcium-sensing receptor (CaSR) expression in vascular smooth muscle cells exacerbates vascular calcification in vitro. Conversely, vascular calcification can be reduced by calcimimetics, which function as allosteric activators of CaSR. METHODS: To determine the role of the CaSR in vascular calcification, we characterized mice with targeted Casr gene knockout in vascular smooth muscle cells ( SM22α CaSR Δflox/Δflox ). RESULTS: Vascular smooth muscle cells cultured from the knockout (KO) mice calcified more readily than those from control (wild-type) mice in vitro. However, mice did not show ectopic calcifications in vivo but they did display a profound mineral ion imbalance. Specifically, KO mice exhibited hypercalcemia, hypercalciuria, hyperphosphaturia, and osteopenia, with elevated circulating fibroblast growth factor 23 (FGF23), calcitriol (1,25-D 3), and parathyroid hormone levels. Renal tubular α-Klotho protein expression was increased in KO mice but vascular α-Klotho protein expression was not. Altered CaSR expression in the kidney or the parathyroid glands could not account for the observed phenotype of the KO mice. CONCLUSIONS: These results suggest that, in addition to CaSR's established role in the parathyroid-kidney-bone axis, expression of CaSR in vascular smooth muscle cells directly contributes to total body mineral ion homeostasis.

BACKGROUND: Diabetic nephropathy (DN) is a serious complication of diabetes and a common cause of end stage renal failure. Insulin-like growth factor (IGF)-signaling has been implicated in DN, but is mechanistically poorly understood. Here, we assessed the activity of the metalloproteinase PAPP-A, an activator of IGF activity, and its possible interaction with the endogenous PAPP-A inhibitors stanniocalcin (STC)-1 and -2 in the mammalian kidney under normal and hyperglycemic conditions.METHODS AND RESULTS: Immunohistochemistry demonstrated that PAPP-A, its proteolytic substrate IGF binding protein-4, STC1 and STC2 are present in the human kidney. Endogenous inhibited complexes of PAPP-A (PAPP-A:STC1 and PAPP-A:STC2) were demonstrated in media conditioned by human mesangial cells (HMCs), suggesting that PAPP-A activity is regulated by the STCs in kidney tissue. A method for the selective detection of active PAPP-A in tissue was developed and a significant increase in glomerular active PAPP-A in human diabetic kidney relative to normal was observed. In DN patients, the estimated glomerular filtration rate correlated with PAPP-A activity. In diabetic mice, glomerular growth was reduced when PAPP-A activity was antagonized by adeno-associated virus-mediated overexpression of STC2.CONCLUSION: We propose that PAPP-A activity in renal tissue is precisely balanced by STC1 and STC2. An imbalance in this equilibrium causing increased PAPP-A enzymatic activity potentially contributes to the development of DN, and thus, therapeutic targeting of PAPP-A activity may represent a novel strategy for its treatment.

The hormone aldosterone is essential for maintaining K+ and Na+ balance and controlling blood pressure. Aldosterone has different effects if it is secreted due to hypovolemia or hyperkalemia. The kidney distal convoluted tubule (DCT) is believed to play a central role in mediating the differential responses to aldosterone. To determine the alterations in the DCT that may be responsible for these effects, male mice with green fluorescent protein expression specifically in the DCT were maintained on diets containing low NaCl (hypovolemic state) or high potassium citrate (hyperkalemic state) for 4 days, and DCT cells were isolated using fluorescence-activated cell sorting. This pure population of DCT cells was subjected to analysis by liquid chromatography-coupled tandem mass spectrometry. Over 3,000 proteins were identified in the DCT, creating the first proteome of the mouse DCT. Of the identified proteins, 210 proteins were altered in abundance following a low-NaCl diet and 625 proteins following the high-K+ diet. Many of these changes were not detectable by analyzing whole kidney samples from the same animals. When comparing responses to high-K+ versus low-Na+ diets, protein translation, chaperone-mediated protein folding, and protein ubiquitylation were likely to be significantly altered in the DCT subsequent to a high-K+ diet. In conclusion, this study defines an in vivo protein landscape of the DCT in male mice following either a low-NaCl or a high-K+ diet and acts as an essential resource for the kidney research community. The hormone aldosterone is essential for maintaining K+ and Na+ balance and controlling blood pressure. Aldosterone has different effects if it is secreted due to hypovolemia or hyperkalemia. The kidney distal convoluted tubule (DCT) is believed to play a central role in mediating the differential responses to aldosterone. To determine the alterations in the DCT that may be responsible for these effects, male mice with green fluorescent protein expression specifically in the DCT were maintained on diets containing low NaCl (hypovolemic state) or high potassium citrate (hyperkalemic state) for 4 days, and DCT cells were isolated using fluorescence-activated cell sorting. This pure population of DCT cells was subjected to analysis by liquid chromatography- coupled tandem mass spectrometry. Over 3,000 proteins were identified in the DCT, creating the first proteome of the mouse DCT. Of the identified proteins, 210 proteins were altered in abundance following a low-NaCl diet and 625 proteins following the high-K+ diet. Many of these changes were not detectable by analyzing whole kidney samples from the same animals. When comparing responses to high-K+ versus low-Na+ diets, protein translation, chaperone-mediated protein folding, and protein ubiquitylation were likely to be significantly altered in the DCT subsequent to a high-K+ diet. In conclusion, this study defines an in vivo protein landscape of the DCT in male mice following either a low-NaCl or a high-K+ diet and acts as an essential resource for the kidney research community.

Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.

Salmonella enterica is, globally, an important cause of human illness with beef being a significant attributable source. In the human patient, systemic Salmonella infection requires antibiotic therapy, and when strains are multidrug resistant (MDR), no effective treatment may be available. MDR in bacteria is often associated with the presence of mobile genetic elements (MGE) that mediate horizontal spread of antimicrobial resistance (AMR) genes. In this study, we sought to determine the potential relationship of MDR in bovine Salmonella isolates with MGE. The present study involved 111 bovine Salmonella isolates obtained collectively from specimens derived from healthy cattle or their environments at Midwestern U.S. feedyards (2000–2001, n = 19), or specimens from sick cattle submitted to the Nebraska Veterinary Diagnostic Center (2010–2020, n = 92). Phenotypically, 33/111 isolates (29.7%) were MDR (resistant to ≥3 drug classes). Based on whole-genome sequencing (WGS; n = 41) and PCR (n = 111), a MDR phenotype was strongly associated (OR = 186; p < 0.0001) with carriage of ISVsa3, an IS91-like Family transposase. In all 41 isolates analyzed by WGS ((31 MDR and 10 non-MDR (resistant to 0–2 antibiotic classes)), MDR genes were associated with carriage of ISVsa3, most often on an IncC type plasmid carrying blaCMY-2. The typical arrangement was floR, tet(A), aph(6)-Id, aph(3″)-Ib, and sul2 flanked by ISVsa3. These results suggest that AMR genes in MDR S. enterica isolates of cattle are frequently associated with ISVsa3 and carried on IncC plasmids. Further research is needed to better understand the role of ISVsa3 in dissemination of MDR Salmonella strains.

Background Sodium chloride (NaCl) loading and volume expansion suppress the renin-angiotensin-aldosterone system to reduce renal tubular reabsorption of NaCl and water, but effects on the sodium-chloride cotransporter (NCC) and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. Methods We used urinary extracellular vesicles (uEVs) as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 patients with hypertension who had repeatedly raised aldosterone/renin ratios undergoing infusion of 2 L of 0.9% saline over 4 hours. Results When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change [FC]=0.70); pendrin (FC=0.84); AQP2 (FC=0.62); and uEV markers, including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52), and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC; FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, whereas the significant decrease in AQP2 persisted (FC=0.62). Conclusion We conclude that (1) decreases in NCC and pNCC, induced by acute NaCl loading and volume expansion, may be due to diluted post-test urines; (2) the lack of change of NCC and pNCC when corrected for ALIX, despite a fall in plasma aldosterone, may be due to the lack of change in plasma K+; and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion. Background Sodium chloride (NaCl) loading and volume expansion suppress the renin-angiotensin-aldosterone system to reduce renal tubular reabsorption of NaCl and water, but effects on the sodium-chloride cotransporter (NCC) and relevant renal transmembrane proteins that are responsible for this modulation in humans are less well investigated. Methods We used urinary extracellular vesicles (uEVs) as an indirect readout to assess renal transmembrane proteins involved in NaCl and water homeostasis in 44 patients with hypertension who had repeatedly raised aldosterone/renin ratios undergoing infusion of 2 L of 0.9% saline over 4 hours. Results When measured by mass spectrometry in 13 patients, significant decreases were observed in NCC (median fold change [FC]=0.70); pendrin (FC=0.84); AQP2 (FC=0.62); and uEV markers, including ALIX (FC=0.65) and TSG101 (FC=0.66). Immunoblotting reproduced the reduction in NCC (FC=0.54), AQP2 (FC=0.42), ALIX (FC=0.52), and TSG101 (FC=0.55) in the remaining 31 patients, and demonstrated a significant decrease in phosphorylated NCC (pNCC; FC=0.49). However, after correction for ALIX, the reductions in NCC (FC=0.90) and pNCC (FC=1.00) were no longer apparent, whereas the significant decrease in AQP2 persisted (FC=0.62). Conclusion We conclude that (1) decreases in NCC and pNCC, induced by acute NaCl loading and volume expansion, may be due to diluted post-test urines; (2) the lack of change of NCC and pNCC when corrected for ALIX, despite a fall in plasma aldosterone, may be due to the lack of change in plasma K+; and (3) the decrease in AQP2 may be due to a decrease in vasopressin in response to volume expansion.

The Na+/H+ exchanger 3 (NHE3) mediates Na+ (re)absorption in the intestine and the kidney, thereby playing a key role in fluid homeostasis and blood pressure (BP) regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule, where the majority of renal Na+ reabsorption (60% to 75%) occurs. Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Study in kidney-specific (proximal tubule [PT] and thick ascending limb [TAL]) NHE3 knockout mice has reported a rather mild phenotype in terms of Na+ homeostasis and no phenotype for acid-base homeostasis (Kidney International; 2017, 92:397-414). NHE3 is also substantially expressed in the TAL and our data point to a novel role in this nephron segment for urinary concentrating ability. To determine the physiological role of NHE3 in the TAL for salt homeostasis, urinary concentrating ability and BP regulation, we generated a novel tamoxifen (Tam)-inducible TAL-specific NHE3 knockout (NHE3TAL-KO) mice by crossing floxed NHE3 (Control, Con) with UmodCreERT2 mice. Immunofluorescent staining showed that application of Tam (3 days, 67 mg/kg via oral gavage) selectively deleted NHE3 from TAL but not PT. Before Tam, in Con and NHE3TAL-KO mice had comparable food (4.9±0.1 vs 5.3±0.2 g/(day*30g)) and water intake (5.6±0.3 vs 5.9±0.4 g/(day*30g)), urinary osmolality (2435±107.4 vs 2220±111.9 mOsm/kg), urinary pH (6.02±0.06 vs 6.02±0.06), urinary Na+/creatinine (46.3±6.2 vs 52.0±5.2 mmol/mmol) and K+/creatinine (109.0±9.5 vs 97.2±9.2 mmol/mmol). Two weeks after Tam, compared to Con mice, NHE3TAL-KO mice showed lower urinary osmolality (2224±90.1 vs 1826±92.8 mOsm/kg, P

Transmembrane potassium (K) gradients are key determinants of membrane potential that can modulate action potentials, control muscle contractility, and influence ion channel and transporter activity. Daily K intake is normally equal to the amount of K in the entire extracellular fluid (ECF) creating a critical challenge - how to maintain ECF [K] and membrane potential in a narrow range during feast and famine. Adaptations to maintain ECF [K] include sensing the K intake, sensing ECF [K] vs. desired set-point and activating mediators that regulate K distribution between ECF and ICF, and regulate renal K excretion. In this focused review, we discuss the basis of these adaptions, including (1) potential mechanisms for rapid feedforward signaling to kidney and muscle after a meal (before a rise in ECF [K]), (2) how skeletal muscles sense and respond to changes in ECF [K], (3) effects of K on aldosterone biosynthesis, and (4) how the kidney responds to changes in ECF [K] to modify K excretion. The concepts of sexual dimorphisms in renal K handling adaptation are introduced, and the molecular mechanisms that can account for the benefits of a K-rich diet to maintain cardiovascular health are discussed. Although the big picture of K homeostasis is becoming more clear, we also highlight significant pieces of the puzzle that remain to be solved, including knowledge gaps in our understanding of initiating signals, sensors and their connection to homeostatic adjustments of ECF [K].

This article challenges both contemporary and classic urban theory by analyzing the historical case of coastal Ecuador. Working from primary and secondary sources, I track the urbanization of coastal Ecuador during the long nineteenth century, when cacao exports determined not only the economic wellbeing of the city of Guayaquil, but of the entire tropical lowland region. I argue that this extended urban geography was both experienced and practiced as an unbounded economic and cultural region. As the value of cacao exports skyrocketed, capitalists in the city invested in infrastructural projects and financial instruments, divorcing money-making from cacao production. After the Gran Incendio (great fire) of 1896, the city was rebuilt according to the ideals of modern liberal planning that further separated the city from the country symbolically, despite their continued material interconnection. This work suggests that long histories of capitalist urbanization provide material and theoretical support for critiquing bounded urban theory both past and present, by moving beyond the city and highlighting the processes undergirding spatial production under capitalist social relations. Likewise, this historical case study argues that city-centrism, rather than being constituted epistemologically, was tied to liberal notions of the urban based on nineteenth-century ideologies of modernization and progress.

In primary aldosteronism (PA), the occurrence of K+ loss and hypertension suggest alterations in renal tubular transport, but the molecular basis of these alterations in humans is unclear. In this study, urinary extracellular vesicles (uEVs) isolated from patients undergoing fludrocortisone suppression testing (FST, as a means of confirming or excluding PA) were analyzed using mass spectrometry-based proteomics to determine the combined effects of an aldosterone analogue, NaCl and KCl supplementation on renal tubular protein abundance. Of quantified proteins, the Cl−/HCO3− exchanger pendrin decreased by a median 37% [−15, 57] (P < 0.01) and the potassium channel ROMK increased by a median 31% [−10, 85] (P < 0.01) during FST among 10 PA subjects. The trends remained, but to a lesser degree, in two subjects cured of PA by unilateral adrenalectomy. In PA subjects, plasma K+ increased from median 3.6 to 4.2 mM (P < 0.01) and 24 h urine K+ from 101 to 202 mmol (P < 0.01), while 24 h urine Na+/K+ decreased from 2.3 to 0.8 (P < 0.01). At baseline, pendrin negatively correlated with plasma K+ (P < 0.05) and positively correlated with plasma aldosterone (P < 0.01). There were no clear correlations between Δ pendrin (Δ = D4–D0) and changes in blood or urine variables, and no correlations between ROMK in any of the blood or urine variables either at baseline or during FST. We conclude that oral co-administration of mineralocorticoid and KCl in PA patients is associated with reduced pendrin and enhanced ROMK in uEVs. Pendrin reduction during FST suggests that the suppressive effects of oral KCl may outweigh pendrin upregulation by mineralocorticoids.

In mammals, conservation of body water is critical for survival and is dependent on the kidneys’ ability to minimize water loss in the urine during periods of water deprivation. The collecting duct water channel aquaporin-2 (AQP2) plays an essential role in this homeostatic response by facilitating water reabsorption along osmotic gradients. The ability to increase the levels of AQP2 in the apical plasma membrane following an increase in plasma osmolality is a rate-limiting step in water reabsorption, a process that is tightly regulated by the antidiuretic hormone arginine vasopressin (AVP). In this review, the focus is on the role of the carboxyl-terminus of AQP2 as a key regulatory point for AQP2 trafficking. We provide an overview of AQP2 structure, disease-causing mutations in the AQP2 carboxyl-terminus, the role of posttranslational modifications such as phosphorylation and ubiquitylation in the tail domain, and their implications for balanced trafficking of AQP2. Finally, we discuss how various modifications of the AQP2 tail facilitate selective protein-protein interactions that modulate the AQP2 trafficking mechanism.

The sodium–hydrogen exchanger isoform 3 (NHE3, SLC9A3) is abundantly expressed in the gastrointestinal tract and is proposed to play essential roles in Na+ and fluid absorption as well as acid–base homeostasis. Mutations in the SLC9A3 gene can cause congenital sodium diarrhea (CSD). However, understanding the precise role of intestinal NHE3 has been severely hampered due to the lack of a suitable animal model. To navigate this problem and better understand the role of intestinal NHE3, we generated a tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout mouse model (NHE3IEC-KO). Before tamoxifen administration, the phenotype and blood parameters of NHE3IEC-KO were unremarkable compared with control mice. After tamoxifen administration, NHE3IEC-KO mice have undetectable levels of NHE3 in the intestine. NHE3IEC-KO mice develop watery, alkaline diarrhea in combination with a swollen small intestine, cecum and colon. The persistent diarrhea results in higher fluid intake. After 3 weeks, NHE3IEC-KO mice show a ∼25% mortality rate. The contribution of intestinal NHE3 to acid–base and Na+ homeostasis under normal conditions becomes evident in NHE3IEC-KO mice that have metabolic acidosis, lower blood bicarbonate levels, hyponatremia and hyperkalemia associated with drastically elevated plasma aldosterone levels. These results demonstrate that intestinal NHE3 has a significant contribution to acid–base, Na+ and volume homeostasis, and lack of intestinal NHE3 has consequences on intestinal structural integrity. This mouse model mimics and explains the phenotype of individuals with CSD carrying SLC9A3 mutations.

Daily dietary potassium (K+) intake may be as large as the extracellular K+pool. To avoid acute hyperkalemia, rapid removal of K+from the extracellular space is essential. This is achieved by translocating K+into cells and increasing urinary K+excretion. Emerging data now indicate that the renal thiazide-sensitive NaCl cotransporter (NCC) is critically involved in this homeostatic kaliuretic response. This suggests that the early distal convoluted tubule (DCT) is a K+sensor that can modify sodium (Na+) delivery to downstream segments to promote or limit K+secretion. K+sensing is mediated by the basolateral K+channels Kir4.1/5.1, a capacity that the DCT likely shares with other nephron segments. Thus, next to K+-induced aldosterone secretion, K+sensing by renal epithelial cells represents a second feedback mechanism to control K+balance. NCC’s role in K+homeostasis has both physiological and pathophysiological implications. During hypovolemia, NCC activation by the renin-angiotensin system stimulates Na+reabsorption while preventing K+secretion. Conversely, NCC inactivation by high dietary K+intake maximizes kaliuresis and limits Na+retention, despite high aldosterone levels. NCC activation by a low-K+diet contributes to salt-sensitive hypertension. K+-induced natriuresis through NCC offers a novel explanation for the antihypertensive effects of a high-K+diet. A possible role for K+in chronic kidney disease is also emerging, as epidemiological data reveal associations between higher urinary K+excretion and improved renal outcomes. This comprehensive review will embed these novel insights on NCC regulation into existing concepts of K+homeostasis in health and disease.

AIMS: The kidneys play a major role in maintaining Pi homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal-specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen-inducible intestinal epithelial cell-specific NHE3 knockout (NHE3IEC-KO ) mice.METHODS: Mice underwent dietary Pi challenges, and hormones as well as urinary/plasma Pi were determined. Intestinal 33 P uptake studies were conducted in vivo to compare the effects of tenapanor and NHE3IEC-KO . Ex vivo Pi transport was measured in everted gut sacs and brush border membrane vesicles. Intestinal and renal protein expression of Pi transporters were determined.RESULTS: On the control diet, NHE3IEC-KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice. Everted gut sacs and brush border membrane vesicles showed enhanced Pi uptake associated with increased Npt2b expression in NHE3IEC-KO mice. Acute oral Pi loading resulted in higher plasma Pi in NHE3IEC-KO mice. Tenapanor inhibited intestinal 33 P uptake acutely but then led to hyper-absorption at later time points compared to vehicle. In response to high dietary Pi , plasma Pi and FGF23 increased to higher levels in NHE3IEC-KO mice which was associated with greater Npt2b expression. Reduced renal Npt2c and a trend for reduced Npt2a expression were unable to correct for higher plasma Pi .CONCLUSION: Intestinal NHE3 has a significant contribution to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC-KO mice show enhanced, rather than reduced, intestinal Pi uptake.