Inducible Deletion of the Prostaglandin EP3 Receptor in Kidney Tubules of Male and Female Mice Has No Major Effect on Water Homeostasis.

The prostaglandin E2 (PGE2) receptor EP3 has been detected in the thick ascending limb (TAL) and the collecting duct of the kidney, where its actions are proposed to inhibit water reabsorption. However, EP3 is also expressed in other cell types, including vascular endothelial cells. The aim here was to determine the contribution of EP3 in renal water handling in male and female adult mice by phenotyping a novel mouse model with doxycycline-dependent deletion of EP3 throughout the kidney tubule (EP3-/- mice). RNAscope demonstrated that EP3 was highly expressed in the cortical and medullary TAL of adult mice. Compared to controls EP3 mRNA expression was reduced by >80% in whole kidney (RT-qPCR) and non-detectable (RNAscope) in renal tubules of EP3-/- mice. Under basal conditions, there were no significant differences in control and EP3-/- mice of both genders in food and water intake, bodyweight, urinary output or clinical biochemistries. No differences were detectable between genotypes in handling of an acute water load, or in their response to the vasopressin analogue dDAVP. No differences in water handling were observed when PGE2 production was enhanced using a 1% NaCl load. Expression of proteins involved in kidney water handling were not different between genotypes. This study demonstrates that renal tubular EP3 is not essential for body fluid homeostasis in males or females, even when PGE2 levels are high. The mouse model is a novel tool for examining the role of EP3 in kidney function independently of potential developmental abnormalities or systemic effects.

The Effect of Thiazide Diuretics on Urinary Prostaglandin E2 Excretion and Serum Sodium in the General Population.

CONTEXT: Thiazide-induced hyponatremia is one of the most common forms of hyponatremia, but its pathogenesis is incompletely understood. Recent clinical data suggest links with prostaglandin E2 (PGE2) and a single nucleotide polymorphism (SNP) in the prostaglandin transporter gene (SLCO2A1), but it is unknown if these findings also apply to the general population. OBJECTIVE: To study the associations between serum sodium, thiazide diuretics, urinary excretions of PGE2 and its metabolite (PGEM), and the rs34550074 SNP in SLCO2A1 in the general population. DESIGN: Prospective population-based cohort study (Rotterdam Study). SETTING: General population. PARTICIPANTS: 2,178 participants (65% female, age 64 ± 8 years). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Serum sodium levels. RESULTS: Higher urinary PGE2 excretion was associated with lower serum sodium: difference in serum sodium for each two-fold higher PGE2 -0.19 mmol/l (95%CI -0.31 to -0.06), PGEM -0.29 mmol/l (95%CI -0.41 to -0.17). This association was stronger in thiazide users (per two-fold higher PGE2 -0.73 vs. -0.12 mmol/l and PGEM -0.6 vs. -0.25 mmol/l, p for interaction < 0.05 for both). A propensity score matching analysis of thiazide vs. non-thiazide users yielded similar results. The SNP rs34550074 was not associated with lower serum sodium or higher urinary PGE2 or PGEM excretion in thiazide or non-thiazide users. CONCLUSIONS: Serum sodium is lower in people with higher urinary PGE2 and PGEM excretion and this association is stronger in thiazide users. This suggests that PGE2-mediated water reabsorption regulates serum sodium, which is relevant for the pathogenesis of hyponatremia in general and thiazide-induced hyponatremia in specific.

Regulation of the water channel aquaporin-2 by cullin E3 ubiquitin ligases.

Aquaporin 2 (AQP2) is a vasopressin (VP)-regulated water channel in the renal collecting duct. Phosphorylation and ubiquitylation of AQP2 play an essential role in controlling the cellular abundance of AQP2 and its accumulation on the plasma membrane in response to VP. Cullin-RING ubiquitin ligases (CRLs) are multisubunit E3 ligases involved in ubiquitylation and degradation of their target proteins, eight of which are expressed in the collecting duct. Here, we used an established cell model of the collecting duct (mpkCCD14 cells) to study the role of cullins in modulating AQP2. Western blotting identified Cul-1 to Cul-5 in mpkCCD14 cells. Treatment of cells for 4 h with a pan-cullin inhibitor (MLN4924) decreased AQP2 abundance, prevented a VP-induced reduction in AQP2 Ser261 phosphorylation, and attenuated VP-induced plasma membrane accumulation of AQP2 relative to the vehicle. AQP2 ubiquitylation levels were significantly higher after MLN4924 treatment compared with controls, and they remained higher despite VP treatment. Cullin inhibition increased ERK1/2 activity, a kinase that regulates AQP2 Ser261 phosphorylation, and VP-induced reductions in ERK1/2 phosphorylation were absent during MLN4924 treatment. Furthermore, the greater Ser261 phosphorylation and reduction in AQP2 abundance during MLN4924 treatment were attenuated during ERK1/2 inhibition. MLN4924 increased intracellular calcium levels via calcium release-activated calcium channels, inhibition of which abolished MLN4924 effects on Ser261 phosphorylation and AQP2 abundance. In conclusion, CRLs play a vital role in mediating some of the effects of VP to increase AQP2 plasma membrane accumulation and AQP2 abundance. Whether modulation of cullin activity can contribute to body water homeostasis requires further studies.NEW & NOTEWORTHY Aquaporin 2 (AQP2) is essential for body water homeostasis and is regulated by the antidiuretic hormone vasopressin. The posttranslational modification ubiquitylation is a key regulator of AQP2 abundance and plasma membrane localization. Here we demonstrate that cullin-RING E3 ligases play a vital role in mediating some of the effects of vasopressin to increase AQP2 abundance and plasma membrane accumulation. The results suggest that manipulating cullin activity could be a novel strategy to alter kidney water handling.

Potassium-Switch Signaling Pathway Dictates Acute Blood Pressure Response to Dietary Potassium.

BACKGROUND: Potassium (K+)-deficient diets, typical of modern processed foods, increase blood pressure (BP) and NaCl sensitivity. A K+-dependent signaling pathway in the kidney distal convoluted tubule, coined the K+ switch, that couples extracellular K+ sensing to activation of the thiazide-sensitive NaCl cotransporter (NCC) and NaCl retention has been implicated, but causality has not been established. METHODS: To test the hypothesis that small, physiological changes in plasma K+ (PK+) are translated to BP through the switch pathway, a genetic approach was used to activate the downstream switch kinase, SPAK (SPS1-related proline/alanine-rich kinase), within the distal convoluted tubule. The CA-SPAK (constitutively active SPS1-related proline/alanine-rich kinase mice) were compared with control mice over a 4-day PK+ titration (3.8-5.1 mmol) induced by changes in dietary K+. Arterial BP was monitored using radiotelemetry, and renal function measurements, NCC abundance, phosphorylation, and activity were made. RESULTS: As PK+ decreased in control mice, BP progressively increased and became sensitive to dietary NaCl and hydrochlorothiazide, coincident with increased NCC phosphorylation and urinary sodium retention. By contrast, BP in CA-SPAK mice was elevated, resistant to the PK+ titration, and sensitive to hydrochlorothiazide and salt at all PK+ levels, concomitant with sustained and elevated urinary sodium retention and NCC phosphorylation and activity. Thus, genetically locking the switch on drives NaCl sensitivity and prevents the response of BP to potassium. CONCLUSIONS: Low K+, common in modern ultraprocessed diets, presses the K+-switch pathway to turn on NCC activity, increasing sodium retention, BP, and salt sensitivity.

SAM68 directs STING signaling to apoptosis in macrophages.

DNA is a danger signal sensed by cGAS to engage signaling through STING to activate innate immune functions. The best-studied downstream responses to STING activation include expression of type I interferon and inflammatory genes, but STING also activates other pathways, including apoptosis. Here, we report that STING-dependent induction of apoptosis in macrophages occurs through the intrinsic mitochondrial pathway and is mediated via IRF3 but acts independently of gene transcription. By intersecting four mass spectrometry datasets, we identify SAM68 as crucial for the induction of apoptosis downstream of STING activation. SAM68 is essential for the full activation of apoptosis. Still, it is not required for STING-mediated activation of IFN expression or activation of NF-κB. Mechanistic studies reveal that protein trafficking is required and involves SAM68 recruitment to STING upon activation, with the two proteins associating at the Golgi or a post-Golgi compartment. Collectively, our work identifies SAM68 as a STING-interacting protein enabling induction of apoptosis through this DNA-activated innate immune pathway.

Urinary Prostaglandin E2 Excretion and the Risk of Cardiovascular and Kidney Disease.

BACKGROUND: Inhibition of prostaglandin synthesis by nonsteroidal anti-inflammatory drugs is associated with cardiovascular mortality and kidney disease. This study hypothesizes that urinary prostaglandin E2 (PGE2) and PGE2 metabolite (PGEM) excretions are markers of cardiovascular and kidney health, because they reflect both systemic and kidney-derived PGE2 production. METHODS AND RESULTS: PGE2 and PGEM were measured in spot urine samples from 2291 participants (≥55 years old) of the population-based Rotterdam Study. Urinary PGE2 and PGEM excretions were analyzed using linear regression analyses to identify cross-sectional associations with cardiovascular risk factors and baseline estimated glomerular filtration rate (eGFR). Longitudinal associations with cardiovascular mortality and kidney outcomes (eGFR <60 or <45 mL/min per 1.73 m2 and the composite outcome 40% eGFR loss or kidney failure) were assessed with Cox regression. Urinary PGE2 and PGEM excretions were higher with increasing age, lower eGFR, smoking, diabetes, and albuminuria. A 2-fold higher urinary PGE2 and PGEM excretion was associated with a higher risk of cardiovascular mortality (28 825 patient-years; 160 events; PGE2 hazard ratio [HR], 1.27, [95% CI, 1.06-1.54]; PGEM HR, 1.36 [95% CI, 1.10-1.67]). Higher PGE2 excretions were also associated with a higher risk of incident eGFR <60 mL/min per 1.73 m2 (31 530 person-years; 691 events; HR, 1.13 [95% CI, 1.02-1.25]) with similar HRs for the other kidney outcomes. CONCLUSIONS: Urinary PGE2 and PGEM excretions are novel markers for the presence and progression of cardiovascular and kidney disease. Future studies should address whether these associations are causal and can be targeted to improve cardiovascular and kidney outcomes.

Trypanosoma brucei Invariant Surface Glycoprotein 75 Is an Immunoglobulin Fc Receptor Inhibiting Complement Activation and Antibody-Mediated Cellular Phagocytosis.

Various subspecies of the unicellular parasite Trypanosoma brucei cause sleeping sickness, a neglected tropical disease affecting millions of individuals and domestic animals. Immune evasion mechanisms play a pivotal role in parasite survival within the host and enable the parasite to establish a chronic infection. In particular, the rapid switching of variant surface glycoproteins covering a large proportion of the parasite's surface enables the parasite to avoid clearance by the adaptive immune system of the host. In this article, we present the crystal structure and discover an immune-evasive function of the extracellular region of the T. brucei invariant surface gp75 (ISG75). Structural analysis determined that the ISG75 ectodomain is organized as a globular head domain and a long slender coiled-coil domain. Subsequent ligand screening and binding analysis determined that the head domain of ISG75 confers interaction with the Fc region of all subclasses of human IgG. Importantly, the ISG75-IgG interaction strongly inhibits both activation of the classical complement pathway and Ab-dependent cellular phagocytosis by competing with C1q and host cell FcγR CD32. Our data reveal a novel immune evasion mechanism of T. brucei, with ISG75 able to inactivate the activities of Abs recognizing the parasite surface proteins.

Guidelines on antibody use in physiology research.

Antibodies are one of the most used reagents in scientific laboratories and are critical components for a multitude of experiments in physiology research. Over the past decade, concerns about many biological methods, including those that use antibodies, have arisen as several laboratories were unable to reproduce the scientific data obtained in other laboratories. The lack of reproducibility could be largely attributed to inadequate reporting of detailed methods, no or limited verification by authors, and the production and use of unvalidated antibodies. The goal of this guideline article is to review best practices concerning commonly used techniques involving antibodies, including immunoblotting, immunohistochemistry, and flow cytometry. Awareness and integration of best practices will increase the rigor and reproducibility of these techniques and elevate the quality of physiology research.

Metabolic Communication by SGLT2 Inhibition.

BACKGROUND: SGLT2 (sodium-glucose cotransporter 2) inhibitors (SGLT2i) can protect the kidneys and heart, but the underlying mechanism remains poorly understood. METHODS: To gain insights on primary effects of SGLT2i that are not confounded by pathophysiologic processes or are secondary to improvement by SGLT2i, we performed an in-depth proteomics, phosphoproteomics, and metabolomics analysis by integrating signatures from multiple metabolic organs and body fluids after 1 week of SGLT2i treatment of nondiabetic as well as diabetic mice with early and uncomplicated hyperglycemia. RESULTS: Kidneys of nondiabetic mice reacted most strongly to SGLT2i in terms of proteomic reconfiguration, including evidence for less early proximal tubule glucotoxicity and a broad downregulation of the apical uptake transport machinery (including sodium, glucose, urate, purine bases, and amino acids), supported by mouse and human SGLT2 interactome studies. SGLT2i affected heart and liver signaling, but more reactive organs included the white adipose tissue, showing more lipolysis, and, particularly, the gut microbiome, with a lower relative abundance of bacteria taxa capable of fermenting phenylalanine and tryptophan to cardiovascular uremic toxins, resulting in lower plasma levels of these compounds (including p-cresol sulfate). SGLT2i was detectable in murine stool samples and its addition to human stool microbiota fermentation recapitulated some murine microbiome findings, suggesting direct inhibition of fermentation of aromatic amino acids and tryptophan. In mice lacking SGLT2 and in patients with decompensated heart failure or diabetes, the SGLT2i likewise reduced circulating p-cresol sulfate, and p-cresol impaired contractility and rhythm in human induced pluripotent stem cell-derived engineered heart tissue. CONCLUSIONS: SGLT2i reduced microbiome formation of uremic toxins such as p-cresol sulfate and thereby their body exposure and need for renal detoxification, which, combined with direct kidney effects of SGLT2i, including less proximal tubule glucotoxicity and a broad downregulation of apical transporters (including sodium, amino acid, and urate uptake), provides a metabolic foundation for kidney and cardiovascular protection.

Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure.

Consumption of low dietary potassium, common with ultraprocessed foods, activates the thiazide-sensitive sodium chloride cotransporter (NCC) via the with no (K) lysine kinase/STE20/SPS1-related proline-alanine-rich protein kinase (WNK/SPAK) pathway to induce salt retention and elevate blood pressure (BP). However, it remains unclear how high-potassium "DASH-like" diets (dietary approaches to stop hypertension) inactivate the cotransporter and whether this decreases BP. A transcriptomics screen identified Ppp1Ca, encoding PP1A, as a potassium-upregulated gene, and its negative regulator Ppp1r1a, as a potassium-suppressed gene in the kidney. PP1A directly binds to and dephosphorylates NCC when extracellular potassium is elevated. Using mice genetically engineered to constitutively activate the NCC-regulatory kinase SPAK and thereby eliminate the effects of the WNK/SPAK kinase cascade, we confirmed that PP1A dephosphorylated NCC directly in a potassium-regulated manner. Prior adaptation to a high-potassium diet was required to maximally dephosphorylate NCC and lower BP in constitutively active SPAK mice, and this was associated with potassium-dependent suppression of Ppp1r1a and dephosphorylation of its cognate protein, inhibitory subunit 1 (I1). In conclusion, potassium-dependent activation of PP1A and inhibition of I1 drove NCC dephosphorylation, providing a mechanism to explain how high dietary K+ lowers BP. Shifting signaling of PP1A in favor of activation of WNK/SPAK may provide an improved therapeutic approach for treating salt-sensitive hypertension.

Prostaglandin E2, Osmoregulation, and Disease Progression in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND: Prostaglandin E2 (PGE2) plays a physiological role in osmoregulation, a process that is affected early in autosomal dominant polycystic kidney disease (ADPKD). PGE2 has also been implicated in the pathogenesis of ADPKD in preclinical models, but human data are limited. Here, we hypothesized that urinary PGE2 excretion is associated with impaired osmoregulation, disease severity, and disease progression in human ADPKD. METHODS: Urinary excretions of PGE2 and its metabolite (PGEM) were measured in a prospective cohort of patients with ADPKD. The associations between urinary PGE2 and PGEM excretions, markers of osmoregulation, eGFR and height-adjusted total kidney volume were assessed using linear regression models. Cox regression and linear mixed models were used for the longitudinal analysis of the associations between urinary PGE2 and PGEM excretions and disease progression defined as 40% eGFR loss or kidney failure, and change in eGFR over time. In two intervention studies, we quantified the effect of starting tolvaptan and adding hydrochlorothiazide to tolvaptan on urinary PGE2 and PGEM excretions. RESULTS: In 562 patients with ADPKD (61% female, eGFR 63±28 ml/min per 1.73 m 2 ), higher urinary PGE2 or PGEM excretions were independently associated with higher plasma copeptin, lower urine osmolality, lower eGFR, and greater total kidney volume. Participants with higher baseline urinary PGE2 and PGEM excretions had a higher risk of 40% eGFR loss or kidney failure (hazard ratio, 1.28; 95% confidence interval [CI], 1.13 to 1.46 and hazard ratio, 1.50; 95% CI, 1.26 to 1.80 per two-fold higher urinary PGE2 or PGEM excretions) and a faster change in eGFR over time (-0.39 [95% CI, -0.59 to -0.20] and -0.53 [95% CI, -0.75 to -0.31] ml/min per 1.73 m 2 per year). In the intervention studies, urinary PGEM excretion was higher after starting tolvaptan, while urinary PGE2 excretion was higher after adding hydrochlorothiazide to tolvaptan. CONCLUSIONS: Higher urinary PGE2 and PGEM excretions in patients with ADPKD are associated with impaired osmoregulation, disease severity, and progression.

Randomized Trial on the Effect of Oral Potassium Chloride Supplementation on the Thiazide-Sensitive Sodium Chloride Cotransporter in Healthy Adults.

Introduction: The putative "renal-K switch" mechanism links dietary potassium intake with sodium retention and involves activation of the sodium chloride (NaCl) cotransporter (NCC) in the distal convoluted tubule in response to low potassium intake, and suppression in response to high potassium intake. This study examined NCC abundance and phosphorylation (phosphorylated NCC [pNCC]) in urinary extracellular vesicles (uEVs) isolated from healthy adults on a high sodium diet to determine tubular responses to alteration in potassium chloride (KCl) intake. Methods: Healthy adults maintained on a high sodium (∼4.5 g [200 mmol]/d) low potassium (∼2.3 g [60 mmol]/d) diet underwent a 5-day run-in period followed by a crossover study, with 5-day supplementary KCl (active phase, Span-K 3 tablets (potassium 24 mmol) thrice daily) or 5-day placebo administrated in random order and separated by 2-day washout. Ambulatory blood pressure (BP) and biochemistries were assessed, and uEVs were analyzed by western blotting. Results: Among the 18 participants who met analysis criteria, supplementary KCl administration (vs. placebo) was associated with markedly higher levels of plasma potassium and 24-hour urine excretion of potassium, chloride, and aldosterone. KCl supplementation was associated with lower uEV levels of NCC (median fold change (KCl/Placebo) = 0.74 [0.30-1.69], P < 0.01) and pNCC (fold change (KCl/Placebo) = 0.81 [0.19-1.75], P < 0.05). Plasma potassium inversely correlated with uEV NCC (R2 = 0.11, P = 0.05). Conclusions: The lower NCC and pNCC in uEVs in response to oral KCl supplementation provide evidence to support the hypothesis of a functional "renal-K switch" in healthy human subjects.

The Influence of Age, Sex, and Socioeconomic Status on Glycemic Control Among People With Type 1 and Type 2 Diabetes in Canada: Patient-Led Longitudinal Retrospective Cross-sectional Study With Multiple Time Points of Measurement.

BACKGROUND: Clinical guidelines for most adults with diabetes recommend maintaining hemoglobin A1c (HbA1c) levels ≤7% (≤53 mmol/mol) to avoid microvascular and macrovascular complications. People with diabetes of different ages, sexes, and socioeconomic statuses may differ in their ease of attaining this goal. OBJECTIVE: As a team of people with diabetes, researchers, and health professionals, we aimed to explore patterns in HbA1c results among people with type 1 or type 2 diabetes in Canada. Our research question was identified by people living with diabetes. METHODS: In this patient-led retrospective cross-sectional study with multiple time points of measurement, we used generalized estimating equations to analyze the associations of age, sex, and socioeconomic status with 947,543 HbA1c results collected from 2010 to 2019 among 90,770 people living with type 1 or type 2 diabetes in Canada and housed in the Canadian National Diabetes Repository. People living with diabetes reviewed and interpreted the results. RESULTS: HbA1c results ≤7.0% represented 30.5% (male people living with type 1 diabetes), 21% (female people living with type 1 diabetes), 55% (male people living with type 2 diabetes) and 59% (female people living with type 2 diabetes) of results in each subcategory. We observed higher HbA1c values during adolescence, and for people living with type 2 diabetes, among people living in lower income areas. Among those with type 1 diabetes, female people tended to have lower HbA1c levels than male people during childbearing years but higher HbA1c levels than male people during menopausal years. Team members living with diabetes confirmed that the patterns we observed reflected their own life courses and suggested that these results be communicated to health professionals and other stakeholders to improve the treatment for people living with diabetes. CONCLUSIONS: A substantial proportion of people with diabetes in Canada may need additional support to reach or maintain the guideline-recommended glycemic control goals. Blood sugar management goals may be particularly challenging for people going through adolescence or menopause or those living with fewer financial resources. Health professionals should be aware of the challenging nature of glycemic management, and policy makers in Canada should provide more support for people with diabetes to live healthy lives.

Association of ISVsa3 with Multidrug Resistance in Salmonella enterica Isolates from Cattle (Bos taurus).

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.

Dissociation of sodium-chloride cotransporter expression and blood pressure during chronic high dietary potassium supplementation.

Dietary potassium (K+) supplementation is associated with a lowering effect in blood pressure (BP), but not all studies agree. Here, we examined the effects of short- and long-term K+ supplementation on BP in mice, whether differences depend on the accompanying anion or the sodium (Na+) intake and molecular alterations in the kidney that may underlie BP changes. Relative to the control diet, BP was higher in mice fed a high NaCl (1.57% Na+) diet for 7 weeks or fed a K+-free diet for 2 weeks. BP was highest on a K+-free/high NaCl diet. Commensurate with increased abundance and phosphorylation of the thiazide sensitive sodium-chloride-cotransporter (NCC) on the K+-free/high NaCl diet, BP returned to normal with thiazides. Three weeks of a high K+ diet (5% K+) increased BP (predominantly during the night) independently of dietary Na+ or anion intake. Conversely, 4 days of KCl feeding reduced BP. Both feeding periods resulted in lower NCC levels but in increased levels of cleaved (active) α and γ subunits of the epithelial Na+ channel ENaC. The elevated BP after chronic K+ feeding was reduced by amiloride but not thiazide. Our results suggest that dietary K+ has an optimal threshold where it may be most effective for cardiovascular health.

In Primary Aldosteronism Acute Potassium Chloride Supplementation Suppresses Abundance and Phosphorylation of the Sodium-Chloride Cotransporter.

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 <4.0 mmol/L, patients were given sufficient oral potassium chloride (KCl) over 24 hours to achieve as close to 4.0 mmol/L as possible. Clinical chemistries were assessed, and urinary extracellular vesicles (uEVs) were examined to investigate effects on NCC. Results: Among 21 patients with PA who received a median total dose of 6.0 g (2.4-16.8 g) of KCl, increases were observed in plasma potassium (from 3.4 to 4.0 mmol/L; P<0.001), aldosterone (from 305 to 558 pmol/L; P=0.01), and renin (from 1.2 to 2.5 mIU/L; P<0.001), whereas decreases were detected in uEV levels of NCC (median fold change(post/basal) [FC]=0.71 [0.09-1.99]; P=0.02), pT60-NCC (FC=0.84 [0.06-1.66]; P=0.05), and pT55/60-NCC (FC=0.67 [0.08-2.42]; P=0.02). By contrast, in 10 patients with PA who did not receive KCl, there were no apparent changes in plasma potassium, NCC abundance, and phosphorylation status, but increases were observed in plasma aldosterone (from 178 to 418 pmol/L; P=0.006) and renin (from 2.0 to 3.0 mU/L; P=0.009). Plasma potassium correlated inversely with uEV levels of NCC (R 2=0.11; P=0.01), pT60-NCC (R 2=0.11; P=0.01), and pT55/60-NCC (R 2=0.11; P=0.01). Conclusions: Acute oral KCl loading replenished plasma potassium in patients with PA and suppressed NCC abundance and phosphorylation, despite a significant rise in plasma aldosterone. This supports the view that potassium supplementation in humans with PA overrides the aldosterone stimulatory effect on NCC. The increased plasma aldosterone in patients with PA without KCl supplementation may be due to aldosterone response to posture challenge.

The nuclear factor of activated T cells 5 (NFAT5) contributes to the renal corticomedullary differences in gene expression.

The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype.