Salt Reabsorption Research Articles

Regulation of Cl- Electrolyte Permeability in Epithelia by Active Traditional Chinese Medicine Monomers for Diarrhea.

The epithelial layer, lining the inner surface of the mammalian alveolar, kidney, brain and colon, is a typical electrolyte transporting tissue. Large quantities of salt and fluid are actively moved from the mucosal side toward the blood vessel. Transepithelial salt re-absorption in epithelial tissues plays an important role in maintaining fluid homeostasis. In absorptive epithelium, fluid and salt flux is controlled by the machinery mainly composed of epithelial sodium channel, cystic fibrosis transmembrane conductance regulator, Na+-K+-2Cl- cotransporter, Na+/H+ exchanger, and Na+/K+-ATPase. Dysregulation of salt permeability across epithelium contributes to the pathogenesis of organ edema. In numerous ion transporters, epithelial Cl- transportation plays an important role in water secretion across epithelial tissues and regulation of body fluid content. Many traditional Chinese medicines treat diarrhea by regulating the Cl- electrolyte transport. We systematically summarized the recent progress regarding the traditional Chinese medicine on Cl- electrolyte transport in the intestinal epithelial tissues. The pharmaceutical relevance of developing advanced strategies to mitigate edematous disorders is also implicated. In conclusion, the crosstalk between Cl- electrolyte transport and active traditional Chinese medicine monomers may lead to the development of new strategies for diarrhea by manipulating the function and expression of ion channels.

Role of the Ubiquitin Proteasome System in the Regulation of Blood Pressure: A Review.

The kidney and the vasculature play crucial roles in regulating blood pressure. The ubiquitin proteasome system (UPS), a multienzyme process mediating covalent conjugation of the 76-amino acid polypeptide ubiquitin to a substrate protein followed by proteasomal degradation, is involved in multiple cellular processes by regulating protein turnover in various tissues. Increasing evidence demonstrates the roles of UPS in blood pressure regulation. In the kidney, filtered sodium is reabsorbed through diverse sodium transporters and channels along renal tubules, and studies conducted till date have provided insights into the complex molecular network through which ubiquitin ligases modulate sodium transport in different segments. Components of these pathways include ubiquitin ligase neuronal precursor cell-expressed developmentally downregulated 4-2, Cullin-3, and Kelch-like 3. Moreover, accumulating data indicate the roles of UPS in blood vessels, where it modulates nitric oxide bioavailability and vasoconstriction. Cullin-3 not only regulates renal salt reabsorption but also controls vascular tone using different adaptor proteins that target distinct substrates in vascular smooth muscle cells. In endothelial cells, UPS can also contribute to blood pressure regulation by modulating endothelial nitric oxide synthase. In this review, we summarize current knowledge regarding the role of UPS in blood pressure regulation, focusing on renal sodium reabsorption and vascular function.

Calcium-Sensing Receptor and Regulation of WNK Kinases in the Kidney.

The kidney is essential for systemic calcium homeostasis. Urinary calcium excretion can be viewed as an integrative renal response to endocrine and local stimuli. The extracellular calcium-sensing receptor (CaSR) elicits a number of adaptive reactions to increased plasma Ca2+ levels including the control of parathyroid hormone release and regulation of the renal calcium handling. Calcium reabsorption in the distal nephron of the kidney is functionally coupled to sodium transport. Apart from Ca2+ transport systems, CaSR signaling affects relevant distal Na+-(K+)-2Cl− cotransporters, NKCC2 and NCC. NKCC2 and NCC are activated by a kinase cascade comprising with-no-lysine [K] kinases (WNKs) and two homologous Ste20-related kinases, SPAK and OSR1. Gain-of-function mutations within the WNK-SPAK/OSR1-NKCC2/NCC pathway lead to renal salt retention and hypertension, whereas loss-of-function mutations have been associated with salt-losing tubulopathies such as Bartter or Gitelman syndromes. A Bartter-like syndrome has been also described in patients carrying gain-of-function mutations in the CaSR gene. Recent work suggested that CaSR signals via the WNK-SPAK/OSR1 cascade to modulate salt reabsorption along the distal nephron. The review presented here summarizes the latest progress in understanding of functional interactions between CaSR and WNKs and their potential impact on the renal salt handling and blood pressure.

Transient hyponatremia of prematurity caused by mild Bartter syndrome type II: a case report

BackgroundBartter syndrome subtypes are a group of rare renal tubular diseases characterized by impaired salt reabsorption in the tubule, specifically the thick ascending limb of Henle’s loop. Clinically, they are characterized by the association of hypokalemic metabolic alkalosis, hypercalciuria, nephrocalcinosis, increased levels of plasma renin and aldosterone, low blood pressure and vascular resistance to angiotensin II. Bartter syndrome type II is caused by mutations in the renal outer medullary potassium channel (ROMK) gene (KCNJ1), can present in the newborn period and typically requires lifelong therapy.Case presentationWe describe a case of a prematurely born female infant presenting with antenatal polyhydramnios, and postnatal dehydration and hyponatremia. After 7 weeks of sodium supplementation, the patient demonstrated complete resolution of her hyponatremia and developed only transient metabolic alkalosis at 2 months of age but continues to be polyuric and exhibits hypercalciuria, without development of nephrocalcinosis. She was found to have two pathogenic variants in the KCNJ1 gene: a frameshift deletion, p.Glu334Glyfs*35 and a missense variant, p. Pro110Leu. While many features of classic ROMK mutations have resolved, the child does have Bartter syndrome type II and needs prolonged pediatric nephrology follow-up.ConclusionTransient neonatal hyponatremia warrants a multi-system workup and genetic variants of KCNJ1 should be considered.

The WNK signaling pathway and salt-sensitive hypertension

The distal nephron of the kidney has a central role in sodium and fluid homeostasis, and disruption of this homeostasis due to mutations of with-no-lysine kinase 1 (WNK1), WNK4, Kelch-like 3 (KLHL3), or Cullin 3 (CUL3) causes pseudohypoaldosteronism type II (PHAII), an inherited hypertensive disease. WNK1 and WNK4 activate the NaCl cotransporter (NCC) at the distal convoluted tubule through oxidative stress-responsive gene 1 (OSR1)/Ste20-related proline-alanine-rich kinase (SPAK), constituting the WNK-OSR1/SPAK-NCC phosphorylation cascade. The level of WNK protein is regulated through degradation by the CUL3-KLHL3 E3 ligase complex. In the normal state, the activity of WNK signaling in the kidney is physiologically regulated by sodium intake to maintain sodium homeostasis in the body. In patients with PHAII, however, because of the defective degradation of WNK kinases, NCC is constitutively active and not properly suppressed by a high salt diet, leading to abnormally increased salt reabsorption and salt-sensitive hypertension. Importantly, recent studies have demonstrated that potassium intake, insulin, and TNFα are also physiological regulators of WNK signaling, suggesting that they contribute to the salt-sensitive hypertension associated with a low potassium diet, metabolic syndrome, and chronic kidney disease, respectively. Moreover, emerging evidence suggests that WNK signaling also has some unique roles in metabolic, cardiovascular, and immunological organs. Here, we review the recent literature and discuss the molecular mechanisms of the WNK signaling pathway and its potential as a therapeutic target.

A Renal Potassium‐Switch Prioritizes Dietary Potassium Over Sodium, Driving Salt‐Sensitive Hypertension

Reducing dietary salt (NaCl) is well appreciated to lower blood pressure (BP), but a growing body of evidence indicates that increasing dietary potassium (K+) intake plays an equally important role. A ‘renal potassium switch’ that turns on the thiazide‐sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low dietary potassium intake and off in response to high potassium intake has been implicated.Here we test the idea in genetically engineered mice, containing a DCT1‐specific, conditionally active SPAK (CA‐SPAK mice) which phospho‐activates NCC, and locks the switch “on.” BP responses to small, physiologic changes in plasma [K+] (P[K+]) in CA‐SPAK mice were compared to control mice. Dietary potassium content was varied to acutely titrate P[K+] over a physiologic range (3.7mM (LK), 4.4mM (MK), and 5.1mM (HK)). Blood pressure was monitored by telemetry at each P[K+] level in mice consuming control or high salt diet (HNa). At the end of each dietary treatment, the BP response to hydrochlorothiazide (HCTZ) was measured to assess the contribution of NCC to BP.BP decreased by ~10 mmHg when P[K+] increased from 3.7 to 5.1 mM in control mice, coincident with inactivation of NCC. When the switch was on (LK and MK groups), HNa significantly elevated BP but had no effect when the switch was inactivated by HK. HCTZ significantly reduced BP in the LK/HNa and MK/HNa groups but had no effect on BP in the HK/HNa group, further supporting the idea that low potassium‐dependent activation NCC exacerbates the effects of sodium. Studies in CA‐SPAK mice reveal a causal relationship between switch activation and BP responses to sodium and potassium. In contrast to control mice, increasing P[K+] in CA‐SPAK mice had no effect on BP under control salt conditions and failed to blunt the significant hypertensive effects of HNa. HCTZ significantly decreased BP in all CA‐SPAK groups to near control levels, consistent with NCC‐driven salt reabsorption. Thus, locking on the potassium switch prevents the anti‐hypertensive effects of HK. No sex differences were found.In summary, low K+ consumption, common in modern diets, presses the switch pathway to turn on to conserve K+ at the expense of increasing sodium retention, even in the face of high dietary sodium, and this elevates BP. Thus, switch activation can drive salt‐sensitive hypertension.Support or Funding InformationNIH

AMPK phosphorylation of the β1Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na+ channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β1 β1Pix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional β1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates β1Pix in vitro. Among several AMPK phosphorylation sites on β1Pix detected by mass spectrometry, Ser71 was validated as functionally significant. Compared with wild-type β1Pix, overexpression of a phosphorylation-deficient β1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β1Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β1Pix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to β1Pix is critical for the formation of a β1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β1Pix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β1Pix at Ser71, which promotes 14-3-3 interactions with β1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

Genome Engineering Renal Epithelial Cells for Enhanced Volume Transport Function.

Bioengineering an implantable artificial kidney (IAK) will require renal epithelial cells capable of reabsorption of salt and water. We used genome engineering to modify cells for improved Na+/H+ exchange and H2O reabsorption. The non-viral piggyBac transposon system enables genome engineering cells to stably overexpress one or more transgenes simultaneously. We generated epitope-tagged human sodium hydrogen exchanger 3 (NHE3) and aquaporin-1 (AQP1) cDNA expressing piggyBac transposon vectors. Transgene expression was evaluated via western blot and immunofluorescence. Flow cytometry analysis was used to quantitate transporter expression in a library of genome engineered clones. Cell surface biotinylation was used evaluate surface protein localization. Blister formation assays were used to monitor cellular volumetric transport. piggyBac enabled stable transposon integration and overexpression of cumate-inducible NHE3 and/or constitutively expressing AQP1 in cultured renal (MDCK) epithelial cells. Cell surface delivery of NHE3 and AQP1 was confirmed using cell surface biotinylation assays. Flow cytometry of a library of MDCK clones revealed varying expression of AQP1 and NHE3. MDCK cells expressing AQP1 and cumate-inducible NHE3 demonstrated increased volumetric transport. Our results demonstrate that renal epithelial cells an be genome engineered for enhanced volumetric transport that will be needed for an IAK device. Our results lay the foundation for future studies of genome engineering human kidney cells for renal tubule cell therapy.

A novel CLCNKB mutation in a Chinese girl with classic Bartter syndrome: a case report

BackgroundBartter syndrome (BS) is a rare autosomal recessive disorder of salt reabsorption at the thick ascending limb of the Henle loop, characterized by hypokalemia, salt loss, metabolic alkalosis, hyperreninemic hyperaldosteronism with normal blood pressure. BS type III, often known as classic BS (CBS), is caused by loss-of-function mutations in CLCNKB (chloride voltage-gated channel Kb) encoding basolateral ClC-Kb.Case presentationWe reported a 15-year-old CBS patient with a compound heterozygous mutation of CLCNKB gene. She first presented with vomiting, hypokalemic metabolic alkalosis at the age of 4 months, and was clinically diagnosed as CBS. Indomethacin, spironolactone and oral potassium were started from then. During follow-up, the serum electrolyte levels were generally normal, but the patient showed failure to thrive and growth hormone (GH) deficiency was diagnosed. The recombinant human GH therapy was performed, and the growth velocity was improved. When she was 14, severe proteinuria and chronic kidney disease (CKD) were developed. Renal biopsy showed focal segmental glomerulosclerosis (FSGS) with juxtaglomerular apparatus cell hyperplasia, and genetic testing revealed a point deletion of c.1696delG (p. Glu566fs) and a fragment deletion of exon 2–3 deletions in CLCNKB gene. Apart from the CBS, ostium secundum atrial septal defect (ASD) was diagnosed by echocardiography.ConclusionsThis is the first report of this compound heterozygous of CLCNKB gene in BS Children. Our findings contribute to a growing list of CLCNKB mutations associated with CBS. Some recessive mutations can induce CBS in combination with other mutations.

Tacrolimus ameliorates the phenotypes of type 4 Bartter syndrome model mice through activation of sodium–potassium–2 chloride cotransporter and sodium–chloride cotransporter

Type 4 Bartter syndrome (BS) is caused by genetic mutations in barttin, which is coded for by BSND. Barttin serves as the β-subunit of the ClC–K chloride (Cl−) channel, which is widely expressed in distal nephrons. Type 4 BS is characterized by severely impaired reabsorption of salt, which may cause polyuria, hypokalemia, and metabolic alkalosis. Calcineurin inhibitors reportedly induce renal salt retention and hyperkalemia by enhancing the phosphorylation of the sodium (Na+)–potassium (K+)–2Cl− cotransporter (NKCC2) and Na+–Cl− cotransporter (NCC). In addition, we have previously reported that tacrolimus, a calcineurin inhibitor, increases the levels of phosphorylated NCC. In this study, we administered tacrolimus to barttin hypomorphic (Bsndneo/neo) mice, a murine model of type 4 BS that exhibits polyuria, hypokalemia, and metabolic alkalosis. Administration of tacrolimus increased the serum K+ level and suppressed urinary K+ excretion. Furthermore, after treatment with tacrolimus, Bsndneo/neo mice increased levels of phosphorylated NCC and NKCC2. We conclude that tacrolimus partially improves clinical phenotypes of Bsndneo/neo mice, and that calcineurin inhibitors might be effective for treating type 4 BS.

The utility of next generation sequencing in the correct diagnosis of congenital hypochloremic hypokalemic metabolic alkalosis

Persistent hypokalemic hypochloremic metabolic alkalosis represents a heterogeneous group of genetic disorders of which the most common is Bartter syndrome (BS). BS is an inherited renal tubulopathy caused by defective salt reabsorption in the thick ascending loop of Henle, which results in persistent hypokalemic hypochloremic metabolic alkalosis. Here we report a 10-year-old girl of a consanguineous family. She presented prenatally with severe polyhydramnios and distended bowel loops. Thereafter, she displayed failure to thrive and had recurrent admissions due to dehydration episodes associated with diarrhea, and characterized by hypokalemia, hypochloremia and metabolic alkalosis. BS was considered her working diagnosis for several years despite negative genetic analysis of the known genes associated with BS. Whole exome sequencing identified a novel homozygous c.1652delT deleterious frameshift mutation in the SLC26A3 gene, which confirmed the diagnosis of congenital chloride diarrhea (CCD), a rare autosomal recessive disease that mimics biochemically BS. A review of twelve additional reported cases of CCD that were initially misdiagnosed as BS, emphasizes CCD in the differential diagnosis of BS, and highlights the clinical discrepancies between these two entities. Taken together, our report further emphasizes the typical clinical features of CCD, and the importance of next generation sequencing in the diagnosis of syndromes with genetic heterogeneity. We suggest including SLC26A3 in the extended BS targeted gene panels.

Short‐ and Long‐Term Furosemide Treatment Triggers WNK Body Formation in the Rat Distal Nephron

The diuretic furosemide induces water and electrolyte excretion by inhibition of the type 2 Na+, K+, 2Cl−-cotransporter in the thick ascending limb (TAL) of the kidney. By virtue of its rapid onset of action, furosemide is paramount in the management of acute fluid and electrolyte disorders. It is not a preferred agent in conditions such as chronic hypertension, as an induction of salt reabsorption downstream of the TAL occurs in long-term treatment, causing furosemide resistance. This is partly mediated by a kinase cascade located in the distal convoluted tubule (DCT) involving with no lysine (WNK) kinases, which phosphorylate Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Phosphorylated SPAK/OSR1 activate their substrate, the Na+, Cl−-cotransporter (NCC), increasing salt reabsorption. We aimed to examine the effects of furosemide on the distribution pattern of WNKs and SPAK/OSR1 in the DCT. A particular property of WNK and SPAK/OSR1 kinases is that they assemble in discrete cytoplasmic domains (“WNK bodies”) in several other models of NCC activation, especially in hypokalemic settings; we postulated that this effect is inducible by furosemide. Wistar rats were treated with intraperitoneal furosemide for 2 h or daily for 7 d. Kidney sections were examined by a range of microscopic techniques including super-resolution fluorescence microscopy and electron microscopy. In immunogold stainings, SPAK/WNK kinases concentrated in spherical, electron-hypodense, non-membrane-bound structures after 2 h and 7 d treatments. To assess their distribution along the nephron, we performed immunofluorescent stainings of SPAK and marker proteins of the aldosterone-insensitive, early DCT (DCT1) or aldosterone-sensitive distal nephron (ASDN). WNK bodies were similarly enriched in both. In further immunostainings, we detected the active, phosphorylated isoforms of WNKs and SPAK/OSR1 within WNK bodies. In ultrastructural and STED evaluations, we could consistently show an adjacency of WNK bodies to the Golgi apparatus and microtubules (see images below). Our work demonstrates that WNK bodies can form within 2 hours in vivo and that WNK bodies arising as a consequence of furosemide treatment are present in the DCT1 and ASDN. Their consistent association with structures of cellular trafficking (Golgi and microtubules) is intriguing regarding several functional possibilities: the reported enrichment of proteasomes in WNK bodies suggests the centralization of kinase degradation, while the presence of active kinase isoforms in WNK bodies are consistent with the notion that WNK-SPAK signaling may occur in the WNK body environment. Our finding of phosphorylated WNKs and SPAK/OSR1 therein is at odds with parts of the published literature; this discrepancy highlights the need for conclusive studies regarding the phosphorylation status of WNK body components. Support or Funding Information Deutsche Forschungsgemeinschaft (MU 2924/2-1,2 und BA 700/22-1,2; INST 335/596-1 FUGG) WNK bodies in rat DCT after 7 days of furosemide treatment A Electron micrograph of a WNK body (asterisk) with Golgi dictyosomes (arrow) and microtubules (arrowheads) in the neighboring cytoplasm. B Immunofluorescent staining of Golgi protein Gm130 and WNK body component SPAK. SPAK has accumulated in the vicinity of Gm130-positive structures surrounding the nucleus of a DCT cell (dashed line = nuclear membrane). C Immunofluorescent staining of tubulin and WNK body component WNK4. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Intrarenal Metabolite (α‐Ketoglutarate) Signaling Pathway Is Activated by Hnf1α‐Mediated Transcriptional Regulation of OAT10

We recently discovered an unusual pathway that couples alterations in intermediate metabolism to salt retention. We found that thiazide diuretics or genetic loss of NCC phosphorylation (SPAK KO) or hypokalemia with intravascular volume contraction activates the synthesis and secretion of α‐ketoglutarate (αKG) from the proximal tubule (PT). αKG is then delivered in the tubular fluid to the distal nephron where it activates its cognate G‐protein coupled receptor, OxGR1, and this stimulates a salt reabsorption network (Pendrin/NDCBE) as a part of a homeostatic response that limits urinary sodium and potassium loss. Hypokalemia appears to stimulate αKG synthesis, whereas volume contraction stimulates αKG transport through angiotensin II (AngII). In the present study, we explore the mechanism by which αKG secretion is activated.Gene profiling studies identified OAT10 (Slc22a13) as an upregulated gene in each of the hypokalemic volume contraction models. As assessed by quantitative immunofluorescence confocal microscopy, OAT10 localized to the PT, and was increased at the apical membrane in SPAK null and thiazide treated WT mice relative to WT control mice. In LLC‐PK1 cells (porcine PT model), we found AngII (100nM) was sufficient to increase OAT10 transcript abundance, and protein localization at the apical membrane. siRNA mediated knockdown of OAT10 resulted in a proportional decrease in αKG secretion. Taken together, the results indicate that OAT10 is regulated to stimulate αKG secretion in response to hypokalemic volume contraction.Activation of OAT10 gene expression is paralleled by up‐regulation of 7 other PT genes that transport amino‐acid substrates for αKG synthesis. In silico promoter analysis (1500bp from start codon) identified Hnf1α as a candidate transcriptional regulator of all these transport genes. Treating LLC‐PK1 cells with AngII decreased phosphorylated Hnf1α and increased its nuclear localization. Luciferase assays using the porcine and mouse Slc22a13 promoter verified that Hnf1α mediates AngII‐stimulated transcription of OAT10 gene.Collectively these data support OAT10 as a mediator of regulated PT αKG secretion and demonstrate that AngII stimulates Slc22a13 transcription via Hnf1α.Support or Funding InformationSupport was provided to PAW from NIH (DK63049, DK54231, and DK093501) and to PRG from the National Kidney Foundation of Maryland.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

WNK Bodies Develop in the Distal Convoluted Tubule of the Human Kidney in Chronic Hypokalemia

Hypokalemia contributes to the progression of chronic kidney disease and is associated with the distinct histopathological entity of chronic hypokalemic nephropathy. K+ deficiency results in profound alterations of renal epithelial transport. These include an increase of salt reabsorption via the Na+, Cl−-cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes K+ wasting in downstream nephron segments. Within days to hours of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases arise in the murine DCT. In the recent literature, these structures have been termed “WNK bodies”, referring to their essential components, with no lysine (WNK) kinases. We hypothesized that hypokalemia may stimulate WNK body formation in humans. Renal needle biopsies of patients with chronic hypokalemic nephropathy (n=2) and appropriate controls were examined by electron microscopy, histological stains and immunofluorescence. Staining of segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely WNK and Ste20-related proline-alanine-rich (SPAK) kinases. In hypokalemic patients, WNKs and SPAK concentrated in non-membrane bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region and were closely adjacent to microtubules, but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients. Support or Funding Information Deutsche Forschungsgemeinschaft (MU 2924/2-1,2 und BA 700/22-1,2; INST 335/596-1 FUGG) Immunofluorescent staining of SPAK and pS-WNK SPAK and catalytically active WNK isoforms are colocalized in punctate accumulations in the DCT of hypokalemic patients (top); these are absent in controls (bottom). This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of Sodium Glucose Cotransporter 2 Attenuates the Dysregulation of Kelch-Like 3 and NaCl Cotransporter in Obese Diabetic Mice.

Mechanisms underlying the frequent association between salt-sensitive hypertension and type 2 diabetes remain obscure. We previously found that protein kinase C (PKC) activation phosphorylates Kelch-like 3 (KLHL3), an E3 ubiquitin ligase component, at serine 433. We investigated whether impaired KLHL3 activity results in increased renal salt reabsorption via NaCl cotransporter (NCC). We used the db/db diabetes mouse model to explore KLHL3's role in renal salt handling in type 2 diabetes and evaluated mechanisms of KLHL3 dysregulation in cultured cells. We observed PKC activity in the db/db mouse kidney and phosphorylation of serine 433 in KLHL3 (KLHL3S433-P). This modification prevents binding of with-no-lysine (WNK) kinases; however, total KLHL3 levels were decreased, indicating severely impaired KLHL3 activity. This resulted in WNK accumulation, activating NCC in distal convoluted tubules. Ipragliflozin, a sodium glucose cotransporter 2 (SGLT2) inhibitor, lowered PKC activity in distal convoluted tubule cells and reduced KLHL3S433-P and NCC levels, whereas the thiazolidinedione pioglitazone did not, although the two agents similarly reduced in blood glucose levels. We found that, in human embryonic kidney cells expressing KLHL3 and distal convoluted tubule cells, cellular glucose accumulation increased KLHL3S433-P levels through PKC. Finally, the effect of PKC inhibition in the kidney of db/db mice confirmed PKC's causal role in KLHL3S433-P and NCC induction. Dysregulation of KLHL3 is involved in the pathophysiology of type 2 diabetes. These data offer a rationale for use of thiazide in individuals with diabetes and provide insights into the mechanism for cardiorenal protective effects of SGLT2 inhibitors.

Hypertension Associated with Fructose and High Salt: Renal and Sympathetic Mechanisms.

Hypertension is a leading cause of cardiovascular and chronic renal disease. Despite multiple important strides that have been made in our understanding of the etiology of hypertension, the mechanisms remain complex due to multiple factors, including the environment, heredity and diet. This review focuses on dietary contributions, providing evidence for the involvement of elevated fructose and salt consumption that parallels the increased incidence of hypertension worldwide. High fructose loads potentiate salt reabsorption by the kidney, leading to elevation in blood pressure. Several transporters, such as NHE3 and PAT1 are modulated in this milieu and play a crucial role in salt-sensitivity. High fructose ingestion also modulates the renin-angiotensin-aldosterone system. Recent attention has been shifted towards the contribution of the sympathetic nervous system, as clinical trials demonstrated significant reductions in blood pressure following renal sympathetic nerve ablation. New preclinical data demonstrates the activation of the renal sympathetic nerves in fructose-induced salt-sensitive hypertension, and reductions of blood pressure after renal nerve ablation. This review further demonstrates the interplay between sodium handling by the kidney, the renin-angiotensin-aldosterone system, and activation of the renal sympathetic nerves as important mechanisms in fructose and salt-induced hypertension.

Salt-deficient diet exacerbates cystogenesis in ARPKD via epithelial sodium channel (ENaC).

BackgroundAutosomal Recessive Polycystic Kidney Disease (ARPKD) is marked by cyst formation in the renal tubules, primarily in the collecting duct (CD) system, ultimately leading to end-stage renal disease. Patients with PKD are generally advised to restrict their dietary sodium intake. This study was aimed at testing the outcomes of dietary salt manipulation in ARPKD.MethodsPCK/CrljCrlPkhd1pck/CRL (PCK) rats, a model of ARPKD, were fed a normal (0.4% NaCl; NS), high salt (4% NaCl; HS), and sodium-deficient (0.01% NaCl; SD) diets for 8 weeks. Immunohistochemistry, GFR measurements, balance studies, and molecular biology approaches were applied to evaluate the outcomes of the protocol. Renin-angiotensin-aldosterone system (RAAS) levels were assessed using LC-MS/MS, and renal miRNA profiles were studied.FindingsBoth HS and SD diets resulted in an increase in cystogenesis. However, SD diet caused extensive growth of cysts in the renal cortical area, and hypertrophy of the tissue; RAAS components were enhanced in the SD group. We observed a reduction in epithelial Na+ channel (ENaC) expression in the SD group, accompanied with mRNA level increase. miRNA assay revealed that renal miR-9a-5p level was augmented in the SD group; we showed that this miRNA decreases ENaC channel number in CD cells.InterpretationOur data demonstrate a mechanism of ARPKD progression during salt restriction that involves activity of ENaC. We further show that miR-9a-5p potentially implicated in this mechanism and that miR-9a-5p downregulates ENaC in cultured CD cells. Our findings open new therapeutic possibilities and highlight the importance of understanding salt reabsorption in ARPKD.

Molecular mechanisms for the regulation of blood pressure by potassium.

It has been well documented that the amount of potassium in the diet is associated with blood pressure levels in the population: the higher the potassium consumption, the lower the blood pressure and the cardiovascular mortality. In the last few years certain mechanisms for potassium regulation of salt reabsorption in the kidney have been elucidated at the molecular level. In this work we discuss the evidence demonstrating the relationship between potassium intake and blood pressure levels in human populations and in animal models, as well as the experimental data that reveal the effects of potassium on transepithelial Na+ reabsorption in different nephron segments. We also discuss the physiological relevance of K+-induced natriuresis, and finally, we focus on the molecular mechanisms by which extracellular potassium modulates the activity of the renal NaCl cotransporter, which is the mechanism that has been best dissected so far.

Structure-function relationships in the renal NaCl cotransporter (NCC).

The thiazide-sensitive Na+-Cl- cotransporter (NCC) is the major pathway for salt reabsorption in the distal convoluted tubule, serves as a receptor for thiazide-type diuretics, and is involved in inherited diseases associated with abnormal blood pressure. The functional and structural characterization of NCC from different species has led us to gain insights into the structure-function relationships of the cotransporter. Here we present an overview of different studies that had described these properties. Additionally, we report the cloning and characterization of the NCC from the spiny dogfish (Squalus acanthias) kidney (sNCC). The purpose of the present study was to determine the main functional, pharmacological and regulatory properties of sNCC to make a direct comparison with other NCC orthologous. The sNCC cRNA encodes a 1033 amino acid membrane protein, when expressed in Xenopus oocytes, functions as a thiazide-sensitive Na-Cl cotransporter with NCC regulation and thiazide-inhibition properties similar to mammals, rather than to teleosts. However, the Km values for ion transport kinetics are significantly higher than those observed in the mammal species. In summary, we present a review on NCC structure-function relationships with the addition of the sNCC information in order to enrich the NCC cotransporter knowledge.

Patients with hypokalemia develop WNK bodies in the distal convoluted tubule of the kidney.

Hypokalemia contributes to the progression of chronic kidney disease, although a definitive pathophysiological theory to explain this remains to be established. K+ deficiency results in profound alterations in renal epithelial transport. These include an increase in salt reabsorption via the Na+-Cl- cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes electroneutral K+ loss in downstream nephron segments. In experimental conditions of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases have been discovered in the murine DCT and termed "WNK bodies," referring to their component, with no K (lysine) kinases (WNKs). We hypothesized that in humans, WNK bodies occur in hypokalemia as well. Renal needle biopsies of patients with chronic hypokalemic nephropathy and appropriate controls were examined by histological stains and immunofluorescence. Segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely, WNKs and Ste20-related proline-alanine-rich kinase (SPAK). In both patients with hypokalemia, WNKs and SPAK concentrated in non-membrane-bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region close to, but not within, the endoplasmic reticulum. They were closely adjacent to microtubules but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients.