Publications by authors named "Paul A Welling"

66 Publications

Critical role of the mineralocorticoid receptor in aldosterone-dependent and aldosterone-independent regulation of ENaC in the distal nephron.

Am J Physiol Renal Physiol 2021 Jul 12. Epub 2021 Jul 12.

Institut für Zelluläre und Molekulare Physiologie, grid.5330.5, Friedrich-Alexander-University of Erlangen-Nuremberg), Germany.

The epithelial sodium channel (ENaC) constitutes the rate-limiting step for sodium absorption in the aldosterone-sensitive distal nephron (ASDN) comprising the late distal convoluted tubule (DCT2), the connecting tubule (CNT) and the collecting duct. Previously, we demonstrated that ENaC activity in the DCT2/CNT transition zone is constitutively high and independent of aldosterone, in contrast to its aldosterone dependence in the late CNT and initial cortical collecting duct (CNT/CCD). The mineralocorticoid receptor (MR) is expressed in the entire ASDN. Its activation by glucocorticoids is prevented through 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) abundantly expressed in the late but probably not the early part of ASDN. We hypothesized that ENaC function in the early part of the ASDN is aldosterone-independent but may depend on MR activated by glucocorticoids due to low 11β-HSD2 abundance. To test this hypothesis, we used doxycycline-inducible nephron-specific MR-deficient mice (MR KO). Whole-cell ENaC currents were investigated in isolated nephron fragments from DCT2/CNT or CNT/CCD transition zones using the patch-clamp technique. ENaC activity was detectable in CNT/CCD of control mice but absent or barely detectable in the majority of CNT/CCD preparations from MR KO mice. Importantly, ENaC currents in DCT2/CNT were greatly reduced in MR KO mice compared to ENaC currents in DCT2/CNT of control mice. Immunofluorescence for 11β-HSD2 was abundant in CCD, less prominent in CNT and very low in DCT2. We conclude that MR is critically important for maintaining aldosterone-independent ENaC activity in DCT2/CNT. Aldosterone-independent MR activation is probably mediated by glucocorticoids due to low expression of 11β-HSD2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00139.2021DOI Listing
July 2021

Long-term kidney function and survival in recipients of allografts from living kidney donors with hypertension: a national cohort study.

Transpl Int 2021 Jun 15. Epub 2021 Jun 15.

Department of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

Allografts from living kidney donors with hypertension may carry subclinical kidney disease from the donor to the recipient and, thus, lead to adverse recipient outcomes. We examined eGFR trajectories and all-cause allograft failure in recipients from donors with versus without hypertension, using mixed-linear and Cox regression models stratified by donor age. We studied a US cohort from 1/1/2005 to 6/30/2017; 49 990 recipients of allografts from younger (<50 years old) donors including 597 with donor hypertension and 21 130 recipients of allografts from older (≥50 years old) donors including 1441 with donor hypertension. Donor hypertension was defined as documented predonation use of antihypertensive therapy. Among recipients from younger donors with versus without hypertension, the annual eGFR decline was -1.03 versus -0.53 ml/min/m (P = 0.002); 13-year allograft survival was 49.7% vs. 59.0% (adjusted allograft failure hazard ratio [aHR] 1.23; 95% CI 1.05-1.43; P = 0.009). Among recipients from older donors with versus without hypertension, the annual eGFR decline was -0.67 versus -0.66 ml/min/m (P = 0.9); 13-year allograft survival was 48.6% versus 52.6% (aHR 1.05; 95% CI 0.94-1.17; P = 0.4). In secondary analyses, our inferences remained similar for risk of death-censored allograft failure and mortality. Hypertension in younger, but not older, living kidney donors is associated with worse recipient outcomes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tri.13947DOI Listing
June 2021

Rapid development of vasopressin resistance in dietary K deficiency.

Am J Physiol Renal Physiol 2021 05 22;320(5):F748-F760. Epub 2021 Mar 22.

Departments of Medicine, Nephrology, and Physiology, Johns Hopkins University Medical School, Baltimore, Maryland.

The association between diabetes insipidus (DI) and chronic dietary K deprivation is well known, but it remains uncertain how the disorder develops and whether it is influenced by the sexual dimorphism in K handling. Here, we determined the plasma K (P) threshold for DI in male and female mice and ascertained if DI is initiated by polydipsia or by a central or nephrogenic defect. C57BL6J mice were randomized to a control diet or to graded reductions in dietary K for 8 days, and kidney function and transporters involved in water balance were characterized. We found that male and female mice develop polyuria and secondary polydipsia. Altered water balance coincided with a decrease in aquaporin-2 (AQP2) phosphorylation and apical localization despite increased levels of the vasopressin surrogate marker copeptin. No change in the protein abundance of urea transporter-A1 was observed. The Na-K-2Cl cotransporter decreased only in males. Desmopressin treatment failed to reverse water diuresis in K-restricted mice. These findings indicate that even a small fall in P is associated with nephrogenic DI (NDI), coincident with the development of altered AQP2 regulation, implicating low P as a causal trigger of NDI. We found that P decreased more in females, and, consequently, females were more prone to develop NDI. Together, these data indicate that AQP2 regulation is disrupted by a small decrease in P and that the response is influenced by sexual dimorphism in K handling. These findings provide new insights into the mechanisms linking water and K balances and support defining the disorder as "potassium-dependent NDI." This study shows that aquaporin-2 regulation is disrupted by a small fall in plasma potassium levels and the response is influenced by sexual dimorphism in renal potassium handling. The findings provided new insights into the mechanisms by which water balance is altered in dietary potassium deficiency and support defining the disorder as "potassium-dependent nephrogenic diabetes insipidus."
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00655.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8174811PMC
May 2021

Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.

Am J Physiol Renal Physiol 2020 11 14;319(5):F754-F764. Epub 2020 Sep 14.

Division of Nephrology, Department of Medicine, and Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland.

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

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00441.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7789988PMC
November 2020

Mutation affecting the conserved acidic WNK1 motif causes inherited hyperkalemic hyperchloremic acidosis.

J Clin Invest 2020 12;130(12):6379-6394

Université de Paris, INSERM, PARCC, F-75006, Paris, France.

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3-Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK's cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values"Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine-rich kinase-Na+-Cl- cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI94171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685730PMC
December 2020

Authors' Reply.

J Am Soc Nephrol 2020 08 1;31(8):1918-1919. Epub 2020 Jul 1.

Nephrology Division, University of Virginia, Charlottesville, Virginia.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2020060799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460906PMC
August 2020

GDNF drives rapid tubule morphogenesis in a novel 3D model for ADPKD.

J Cell Sci 2020 07 16;133(14). Epub 2020 Jul 16.

University of Maryland School of Medicine, Department of Physiology, Baltimore, MD 21201, USA

Cystogenesis is a morphological consequence of numerous genetic diseases of the epithelium. In the kidney, the pathogenic mechanisms underlying the program of altered cell and tubule morphology are obscured by secondary effects of cyst expansion. Here, we developed a new 3D tubuloid system to isolate the rapid changes in protein localization and gene expression that correlate with altered cell and tubule morphology during cyst initiation. Mouse renal tubule fragments were pulsed with a cell differentiation cocktail including glial-derived neurotrophic factor (GDNF) to yield collecting duct-like tubuloid structures with appropriate polarity, primary cilia, and gene expression. Using the 3D tubuloid model with an inducible knockout system allowed the tracking of morphological, protein, and genetic changes during cyst formation. Within hours of inactivation of and loss of polycystin-2, we observed significant progression in tubuloid to cyst morphology that correlated with 35 differentially expressed genes, many related to cell junctions, matrix interactions, and cell morphology previously implicated in cystogenesis.This article has an associated First Person interview with the first author of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.249557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375472PMC
July 2020

Acute Kidney Injury in COVID-19: Emerging Evidence of a Distinct Pathophysiology.

J Am Soc Nephrol 2020 07 4;31(7):1380-1383. Epub 2020 May 4.

Division of Nephrology, and Center for Immunity, Inflammation and Regenerative Medicine, University of Virginia, Charlottesville, Virginia

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2020040419DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350999PMC
July 2020

Rigor before speculation in COVID-19 therapy.

Am J Physiol Lung Cell Mol Physiol 2020 05;318(5):L1027-L1028

Department of Medicine, Duke University School of Medicine, and Durham Veterans Affairs Medical Centers, Durham, North Carolina.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00152.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7203572PMC
May 2020

Identification and characterization of alternative STK39 transcripts within human and mouse kidneys reveals species-specific regulation of blood pressure.

Physiol Rep 2020 02;8(4):e14379

Division of Endocrinology, Diabetes and Nutrition, University of Maryland School of Medicine, Baltimore, MD, USA.

STK39 encodes a serine threonine kinase, SPAK, which is part of a multi-kinase network that determines renal Na reabsorption and blood pressure (BP) through regulation of sodium-chloride co-transporters in the kidney. Variants within STK39 are associated with susceptibility to essential hypertension, and constitutively active SPAK mice are hypertensive and hyperkalemic, similar to familial hyperkalemic hyperkalemia in humans. SPAK null mice are hypotensive and mimic Gitelman syndrome, a rare monogenic salt wasting human disorder. Mice exhibit nephron segment-specific expression of full length SPAK and N-terminally truncated SPAK isoforms (SPAK2 and KS-SPAK) with impaired kinase function. SPAK2 and KS-SPAK function to inhibit phosphorylation of cation co-transporters by full length SPAK. However, the existence of orthologous SPAK2 or KS-SPAK within the human kidney, and the role of such SPAK isoforms in nephron segment-specific regulation of Na reabsorption, still have not been determined. In this study, we examined both human and mouse kidney transcriptomes to uncover novel transcriptional regulation of STK39. We established that humans also express STK39 transcript isoforms similar to those found in mice but differ in abundance and are transcribed from human-specific promoters. In summary, STK39 undergoes species-specific transcriptional regulation, resulting in differentially expressed alternative transcripts that have implications for the design and testing of novel SPAK-targeting antihypertensive medications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.14379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048380PMC
February 2020

NBCe1-A is required for the renal ammonia and K response to hypokalemia.

Am J Physiol Renal Physiol 2020 02 16;318(2):F402-F421. Epub 2019 Dec 16.

Division of Nephrology, Hypertension and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida.

Hypokalemia increases ammonia excretion and decreases K excretion. The present study examined the role of the proximal tubule protein NBCe1-A in these responses. We studied mice with Na-bicarbonate cotransporter electrogenic, isoform 1, splice variant A (NBCe1-A) deletion [knockout (KO) mice] and their wild-type (WT) littermates were provided either K control or K-free diet. We also used tissue sections to determine the effect of extracellular ammonia on NaCl cotransporter (NCC) phosphorylation. The K-free diet significantly increased proximal tubule NBCe1-A and ammonia excretion in WT mice, and NBCe1-A deletion blunted the ammonia excretion response. NBCe1-A deletion inhibited the ammoniagenic/ammonia recycling enzyme response in the cortical proximal tubule (PT), where NBCe1-A is present in WT mice. In the outer medulla, where NBCe1-A is not present, the PT ammonia metabolism response was accentuated by NBCe1-A deletion. KO mice developed more severe hypokalemia and had greater urinary K excretion during the K-free diet than did WT mice. This was associated with blunting of the hypokalemia-induced change in NCC phosphorylation. NBCe1-A KO mice have systemic metabolic acidosis, but experimentally induced metabolic acidosis did not alter NCC phosphorylation. Although KO mice have impaired ammonia metabolism, experiments in tissue sections showed that lack of ammonia does impair NCC phosphorylation. Finally, urinary aldosterone was greater in KO mice than in WT mice, but neither expression of epithelial Na channel α-, β-, and γ-subunits nor of H-K-ATPase α- or α-subunits correlated with changes in urinary K. We conclude that NBCe1-A is critical for the effect of diet-induced hypokalemia to increase cortical proximal tubule ammonia generation and for the expected decrease in urinary K excretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00481.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7052652PMC
February 2020

The single-cell transcriptomic landscape of early human diabetic nephropathy.

Proc Natl Acad Sci U S A 2019 09 10;116(39):19619-19625. Epub 2019 Sep 10.

Division of Nephrology, Department of Medicine, Washington University in St. Louis, St. Louis, MO 63110;

Diabetic nephropathy is characterized by damage to both the glomerulus and tubulointerstitium, but relatively little is known about accompanying cell-specific changes in gene expression. We performed unbiased single-nucleus RNA sequencing (snRNA-seq) on cryopreserved human diabetic kidney samples to generate 23,980 single-nucleus transcriptomes from 3 control and 3 early diabetic nephropathy samples. All major cell types of the kidney were represented in the final dataset. Side-by-side comparison demonstrated cell-type-specific changes in gene expression that are important for ion transport, angiogenesis, and immune cell activation. In particular, we show that the diabetic thick ascending limb, late distal convoluted tubule, and principal cells all adopt a gene expression signature consistent with increased potassium secretion, including alterations in Na/K-ATPase, , mineralocorticoid receptor, and expression, as well as decreased paracellular calcium and magnesium reabsorption. We also identify strong angiogenic signatures in glomerular cell types, proximal convoluted tubule, distal convoluted tubule, and principal cells. Taken together, these results suggest that increased potassium secretion and angiogenic signaling represent early kidney responses in human diabetic nephropathy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1908706116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6765272PMC
September 2019

Optical Clearing in the Kidney Reveals Potassium-Mediated Tubule Remodeling.

Cell Rep 2018 12;25(10):2668-2675.e3

Division of Nephrology and Hypertension, Oregon Health & Science University, Portland, OR 97239, USA; Fondation LeDucq Transatlantic Networks of Excellence, Paris 75116, France; Renal Section, Veterans Affairs Portland Health Care System, Portland, OR 97239, USA.

Distal nephron remodeling contributes to the pathophysiology of many clinically relevant scenarios, including diuretic resistance and certain Mendelian disorders of blood pressure. However, constitutive genetic disruptions are likely to have substantial developmental effects in this segment, and whether tubule remodeling upon physiological stimuli is a normal homeostatic mechanism is not known. Since the distal nephron acts as a potassium sensor, we assessed proliferation and tubule length in three dimensions upon dietary or inducible genetic manipulation by using optical clearing of adult mouse kidneys, whole-mount immunolabeling, and advanced light microscopy. We show that dietary potassium restriction leads promptly to proliferation of various nephron segments, including the distal convoluted tubule, whereas disruption of the potassium sensor Kir4.1 causes atrophy, despite ambient hypokalemia. These results provide proof that kidney tubules adapt rapidly to diet and indicate the power of clearing approaches to assess cell number and tubule length in healthy and diseased kidney.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2018.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339512PMC
December 2018

Autosomal dominant PKD gets an atomic map.

Authors:
Paul A Welling

Nat Rev Nephrol 2018 12;14(12):725-726

University of Maryland Medical School, Baltimore, MD, USA.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41581-018-0066-7DOI Listing
December 2018

Golgin-97 Targets Ectopically Expressed Inward Rectifying Potassium Channel, Kir2.1, to the -Golgi Network in COS-7 Cells.

Front Physiol 2018 3;9:1070. Epub 2018 Aug 3.

Department of Physiology, Maryland Center for Kidney Discovery, School of Medicine, University of Maryland, Baltimore, Baltimore, MD, United States.

The inward rectifying potassium channel, Kir2.1, is selected as cargo at the -Golgi network (TGN) for export to the cell surface through a unique signal-dependent interaction with the AP1 clathrin-adaptor, but it is unknown how the channel is targeted at earlier stages in the secretory pathway for traffic to the TGN. Here we explore a mechanism. A systematic screen of Golgi tethers identified Golgin-97 as a Kir2.1 binding partner. protein-interaction studies revealed the interaction is direct, occurring between the GRIP domain of Golgin-97 and the cytoplasmic domain of Kir2.1. Imaging and interaction studies in COS-7 cells suggest that Golgi-97 binds to the channel en route through the Golgi. RNA interference-mediated knockdown of Golgin-97 prevented exit of Kir2.1 from the Golgi. These observations identify Golgin-97 as a Kir2.1 binding partner that is required for targeting the channel to the TGN. Based on our studies in COS-7 cells, we propose Golgi-97 facilitates formation of AP1-dependent export carriers for Kir2.1 by coupling anterograde delivery of Kir2.1 with retrograde recycling of AP-1 containing endosomes to the TGN.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fphys.2018.01070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085455PMC
August 2018

Renal sodium and magnesium reabsorption are not coupled in a mouse model of Gordon syndrome.

Physiol Rep 2018 07;6(14):e13728

Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, Nijmegen, The Netherlands.

Active reabsorption of magnesium (Mg ) in the distal convoluted tubule (DCT) of the kidney is crucial for maintaining Mg homeostasis. Impaired activity of the Na -Cl -cotransporter (NCC) has been associated with hypermagnesiuria and hypomagnesemia, while increased activity of NCC, as observed in patients with Gordon syndrome, is not associated with alterations in Mg balance. To further elucidate the possible interrelationship between NCC activity and renal Mg handling, plasma Mg levels and urinary excretion of sodium (Na ) and Mg were measured in a mouse model of Gordon syndrome. In this model, DCT1-specific expression of a constitutively active mutant form of the NCC-phosphorylating kinase, SPAK (CA-SPAK), increases NCC activity and hydrochlorothiazide (HCTZ)-sensitive Na reabsorption. These mice were normomagnesemic and HCTZ administration comparably reduced plasma Mg levels in CA-SPAK mice and control littermates. As inferred by the initial response to HCTZ, CA-SPAK mice exhibited greater NCC-dependent Na reabsorption together with decreased Mg reabsorption, compared to controls. Following prolonged HCTZ administration (4 days), CA-SPAK mice exhibited higher urinary Mg excretion, while urinary Na excretion decreased to levels observed in control animals. Surprisingly, CA-SPAK mice had unaltered renal expression of Trpm6, encoding the Mg -permeable channel TRPM6, or other magnesiotropic genes. In conclusion, CA-SPAK mice exhibit normomagnesemia, despite increased NCC activity and Na reabsorption. Thus, Mg reabsorption is not coupled to increased thiazide-sensitive Na reabsorption, suggesting a similar process explains normomagnesemia in Gordon syndrome. Further research is required to unravel the molecular underpinnings of this phenomenon and the more pronounced Mg excretion after prolonged HCTZ administration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.13728DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054696PMC
July 2018

WNKs on the Fly.

Authors:
Paul A Welling

J Am Soc Nephrol 2018 05 12;29(5):1347-1349. Epub 2018 Apr 12.

Department of Physiology, Maryland Center for Kidney Discovery, University of Maryland Medical School, Baltimore, Maryland

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2018030318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967773PMC
May 2018

Mechanism of Hyperkalemia-Induced Metabolic Acidosis.

J Am Soc Nephrol 2018 05 26;29(5):1411-1425. Epub 2018 Feb 26.

Division of Nephrology, Hypertension and Renal Transplantation, University of Florida College of Medicine, Gainesville, Florida;

Hyperkalemia in association with metabolic acidosis that are out of proportion to changes in glomerular filtration rate defines type 4 renal tubular acidosis (RTA), the most common RTA observed, but the molecular mechanisms underlying the associated metabolic acidosis are incompletely understood. We sought to determine whether hyperkalemia directly causes metabolic acidosis and, if so, the mechanisms through which this occurs. We studied a genetic model of hyperkalemia that results from early distal convoluted tubule (DCT)-specific overexpression of constitutively active Ste20/SPS1-related proline-alanine-rich kinase (DCT-CA-SPAK). DCT-CA-SPAK mice developed hyperkalemia in association with metabolic acidosis and suppressed ammonia excretion; however, titratable acid excretion and urine pH were unchanged compared with those in wild-type mice. Abnormal ammonia excretion in DCT-CA-SPAK mice associated with decreased proximal tubule expression of the ammonia-generating enzymes phosphate-dependent glutaminase and phosphoenolpyruvate carboxykinase and overexpression of the ammonia-recycling enzyme glutamine synthetase. These mice also had decreased expression of the ammonia transporter family member Rhcg and decreased apical polarization of H-ATPase in the inner stripe of the outer medullary collecting duct. Correcting the hyperkalemia by treatment with hydrochlorothiazide corrected the metabolic acidosis, increased ammonia excretion, and normalized ammoniagenic enzyme and Rhcg expression in DCT-CA-SPAK mice. In wild-type mice, induction of hyperkalemia by administration of the epithelial sodium channel blocker benzamil caused hyperkalemia and suppressed ammonia excretion. Hyperkalemia decreases proximal tubule ammonia generation and collecting duct ammonia transport, leading to impaired ammonia excretion that causes metabolic acidosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2017111163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967781PMC
May 2018

α-Ketoglutarate stimulates pendrin-dependent Cl absorption in the mouse CCD through protein kinase C.

Am J Physiol Renal Physiol 2018 07 7;315(1):F7-F15. Epub 2018 Feb 7.

Department of Medicine, Emory University School of Medicine , Atlanta, Georgia.

α-Ketoglutarate (α-KG) is a citric acid cycle intermediate and a glutamine catabolism product. It is also the natural ligand of 2-oxoglutarate receptor 1 (OXGR1), a G protein-coupled receptor expressed on the apical membrane of intercalated cells. In the cortical collecting duct (CCD), Cl/[Formula: see text] exchange increases upon α-KG binding to the OXGR1. To determine the signaling pathway(s) by which α-KG stimulates Cl absorption, we examined α-KG-stimulated Cl absorption in isolated perfused mouse CCDs. α-KG increased electroneutral Cl absorption in CCDs from wild-type mice but had no effect on Cl absorption in pendrin knockout mice. Because G protein-coupled receptors activate PKC, we hypothesized that α-KG stimulates Cl absorption through PKC. If so, PKC agonists should mimic, whereas PKC inhibitors should abolish, α-KG-stimulated Cl absorption. Like α-KG, PKC agonist (phorbol-12,13-dibutyrate, 500 nM) application increased Cl absorption in wild-type but not in pendrin null CCDs. Moreover, PKC inhibitors (2.5 mM GF109203X and 20 nM calphostin C), Ca chelators (BAPTA, 10-20 μM), or PKC-α or -δ gene ablation eliminated α-KG-stimulated Cl absorption. We have shown that STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) gene ablation increases urinary α-KG excretion, renal pendrin abundance, and CCD Cl absorption. However, in SPAK null CCDs, Cl absorption was not activated further by luminal α-KG application nor was Cl absorption reduced with the PKC inhibitor GF109203 . Thus SPAK gene ablation likely acts through a PKC-independent pathway to produce a chronic adaptive increase in pendrin function. In conclusion, α-KG stimulates pendrin-dependent Cl/[Formula: see text] exchange through a mechanism dependent on PKC and Ca that involves PKC-α and PKC-δ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00576.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6087786PMC
July 2018

The endosomal trafficking factors CORVET and ESCRT suppress plasma membrane residence of the renal outer medullary potassium channel (ROMK).

J Biol Chem 2018 03 8;293(9):3201-3217. Epub 2018 Jan 8.

From the Departments of Biological Sciences and

Protein trafficking can act as the primary regulatory mechanism for ion channels with high open probabilities, such as the enal uter edullary (ROMK) channel. ROMK, also known as Kir1.1 (KCNJ1), is the major route for potassium secretion into the pro-urine and plays an indispensable role in regulating serum potassium and urinary concentrations. However, the cellular machinery that regulates ROMK trafficking has not been fully defined. To identify regulators of the cell-surface population of ROMK, we expressed a pH-insensitive version of the channel in the budding yeast We determined that ROMK primarily resides in the endoplasmic reticulum (ER), as it does in mammalian cells, and is subject to ER-associated degradation (ERAD). However, sufficient ROMK levels on the plasma membrane rescued growth on low-potassium medium of yeast cells lacking endogenous potassium channels. Next, we aimed to identify the biological pathways most important for ROMK regulation. Therefore, we used a synthetic genetic array to identify non-essential genes that reduce the plasma membrane pool of ROMK in potassium-sensitive yeast cells. Genes identified in this screen included several members of the endosomal complexes required for transport (ESCRT) and the class-C core vacuole/endosome tethering (CORVET) complexes. Mass spectroscopy analysis confirmed that yeast cells lacking an ESCRT component accumulate higher potassium concentrations. Moreover, silencing of ESCRT and CORVET components increased ROMK levels at the plasma membrane in HEK293 cells. Our results indicate that components of the post-endocytic pathway influence the cell-surface density of ROMK and establish that components in this pathway modulate channel activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.819086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836112PMC
March 2018

Renal and colonic potassium transporters in the pregnant rat.

Am J Physiol Renal Physiol 2018 02 18;314(2):F251-F259. Epub 2017 Oct 18.

Department of Medicine, University of Florida , Gainesville, Florida.

Gestational potassium retention, most of which occurs during late pregnancy, is essential for fetal development. The purpose of this study was to examine mechanisms underlying changes in potassium handling by the kidney and colon in pregnancy. We found that potassium intake and renal excretion increased in late pregnancy while fecal potassium excretion remained unchanged and that pregnant rats exhibited net potassium retention. By quantitative PCR we found markedly increased H-K-ATPase type 2 (HKA2) mRNA expression in the cortex and outer medullary of late pregnant vs. virgin. Renal outer medullary potassium channel (ROMK) mRNA was unchanged in the cortex, but apical ROMK abundance (by immunofluorescence) was decreased in pregnant vs. virgin in the distal convoluted tubule (DCT) and connecting tubule (CNT). Big potassium-α (BKα) channel-α protein abundance in intercalated cells in the cortex and outer medullary collecting ducts (by immunohistochemistry) fell in late pregnancy. In the distal colon we found increased HKA2 mRNA and protein abundance (Western blot) and decreased BKα protein with no observed changes in mRNA. Therefore, the potassium retention of pregnancy is likely to be due to increased collecting duct potassium reabsorption (via increased HKA2), decreased potassium secretion (via decreased ROMK and BK), as well as increased colonic reabsorption via HKA2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00288.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5866449PMC
February 2018

α-Ketoglutarate drives electroneutral NaCl reabsorption in intercalated cells by activating a G-protein coupled receptor, Oxgr1.

Curr Opin Nephrol Hypertens 2017 09;26(5):426-433

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland, USA.

Purpose Of Review: This review describes the recent discoveries about a powerful electroneutral NaCl reabsorption mechanism in intercalated cells, and its regulation by an intrarenal metabolite paracrine, α-ketoglutartate, and the G-protein coupled receptor, Oxgr1.

Recent Findings: The distal nephron fine-tunes sodium, chloride, potassium, hydrogen, bicarbonate and water transport to maintain electrolyte homeostasis and blood pressure. Intercalated cells have been traditionally viewed as the professional regulators of acid-base balance, but recent studies reveal that a specific population of intercalated cells, identified by the pendrin-transporter, have a surprising role in the regulation of salt balance. The pendrin-positive intercalated cells (PP-ICs) facilitate electroneutral NaCl reabsorption through the cooperative activation of multitransport protein network. α-Ketoglutartate is synthesized and secreted into the proximal tubule lumen in the combined state of metabolic alkalosis and intravascular volume contraction to activate Oxgr1 in PP-IC, which in turn activates the multitransport protein network to drive salt reabsorption and bicarbonate secretion by these cells.

Summary: Recent studies identify a novel salt transport pathway in intercalated cells that is activated by an intrarenal paracrine system, α-ketoglutartate/Oxgr1. Activation of the paracrine system and transport pathway, particularly during alkalosis and volume contraction, mitigates deleterious salt wasting while restoring acid-base balance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/MNH.0000000000000353DOI Listing
September 2017

Constitutively Active SPAK Causes Hyperkalemia by Activating NCC and Remodeling Distal Tubules.

J Am Soc Nephrol 2017 Sep 25;28(9):2597-2606. Epub 2017 Apr 25.

Department of Physiology, Maryland Kidney Discovery Center, University of Maryland Medical School, Baltimore, Maryland; and

Aberrant activation of with no lysine (WNK) kinases causes familial hyperkalemic hypertension (FHHt). Thiazide diuretics treat the disease, fostering the view that hyperactivation of the thiazide-sensitive sodium-chloride cotransporter (NCC) in the distal convoluted tubule (DCT) is solely responsible. However, aberrant signaling in the aldosterone-sensitive distal nephron (ASDN) and inhibition of the potassium-excretory renal outer medullary potassium (ROMK) channel have also been implicated. To test these ideas, we introduced kinase-activating mutations after Lox-P sites in the mouse gene, which encodes the terminal kinase in the WNK signaling pathway, Ste20-related proline-alanine-rich kinase (SPAK). Renal expression of the constitutively active (CA)-SPAK mutant was specifically targeted to the early DCT using a DCT-driven Cre recombinase. CA-SPAK mice displayed thiazide-treatable hypertension and hyperkalemia, concurrent with NCC hyperphosphorylation. However, thiazide-mediated inhibition of NCC and consequent restoration of sodium excretion did not immediately restore urinary potassium excretion in CA-SPAK mice. Notably, CA-SPAK mice exhibited ASDN remodeling, involving a reduction in connecting tubule mass and attenuation of epithelial sodium channel (ENaC) and ROMK expression and apical localization. Blocking hyperactive NCC in the DCT gradually restored ASDN structure and ENaC and ROMK expression, concurrent with the restoration of urinary potassium excretion. These findings verify that NCC hyperactivity underlies FHHt but also reveal that NCC-dependent changes in the driving force for potassium secretion are not sufficient to explain hyperkalemia. Instead, a DCT-ASDN coupling process controls potassium balance in health and becomes aberrantly activated in FHHt.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1681/ASN.2016090948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5576927PMC
September 2017

A Common Signal Patch Drives AP-1 Protein-dependent Golgi Export of Inwardly Rectifying Potassium Channels.

J Biol Chem 2016 07 23;291(29):14963-72. Epub 2016 May 23.

From the Department of Physiology, School of Medicine, University of Maryland, Baltimore, Maryland 21201 and

Nearly all members of the inwardly rectifying potassium (Kir) channel family share a cytoplasmic domain structure that serves as an unusual AP-1 clathrin adaptor-dependent Golgi export signal in one Kir channel, Kir2.1 (KCNJ2), raising the question whether Kir channels share a common Golgi export mechanism. Here we explore this idea, focusing on two structurally and functionally divergent Kir family members, Kir2.3 (KCNJ4) and Kir4.1/5.1 (KCNJ10/16), which have ∼50% amino identity. We found that Golgi export of both channels is blocked upon siRNA-mediated knockdown of the AP-1 γ subunit, as predicted for the common AP-1-dependent trafficking process. A comprehensive mutagenic analysis, guided by homology mapping in atomic resolution models of Kir2.1, Kir2.3, and Kir4.1/5.1, identified a common structure that serves as a recognition site for AP-1 binding and governs Golgi export. Larger than realized from previous studies with Kir2.1, the signal is created by a patch of residues distributed at the confluence of cytoplasmic N and C termini. The signal involves a stretch of hydrophobic residues from the C-terminal region that form a hydrophobic cleft, an adjacent cluster of basic residues within the N terminus, and a potential network of salt bridges that join the N- and C-terminal poles together. Because patch formation and AP-1 binding are dependent on proper folding of the cytoplasmic domains, the signal provides a common quality control mechanism at the Golgi for Kir channels. These findings identify a new proteostatic mechanism that couples protein folding of channels to forward trafficking in the secretory pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.729822DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946915PMC
July 2016

Roles and Regulation of Renal K Channels.

Authors:
Paul A Welling

Annu Rev Physiol 2016 11;78:415-35. Epub 2015 Dec 11.

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201; email:

More than two dozen types of potassium channels, with different biophysical and regulatory properties, are expressed in the kidney, influencing renal function in many important ways. Recently, a confluence of discoveries in areas from human genetics to physiology, cell biology, and biophysics has cast light on the special function of five different potassium channels in the distal nephron, encoded by the genes KCNJ1, KCNJ10, KCNJ16, KCNMA1, and KCNN3. Research aimed at understanding how these channels work in health and go awry in disease has transformed our understanding of potassium balance and provided new insights into mechanisms of renal sodium handling and the maintenance of blood pressure. This review focuses on recent advances in this rapidly evolving field.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1146/annurev-physiol-021115-105423DOI Listing
December 2016

Integrated compensatory network is activated in the absence of NCC phosphorylation.

J Clin Invest 2015 May 20;125(5):2136-50. Epub 2015 Apr 20.

Thiazide diuretics are used to treat hypertension; however, compensatory processes in the kidney can limit antihypertensive responses to this class of drugs. Here, we evaluated compensatory pathways in SPAK kinase-deficient mice, which are unable to activate the thiazide-sensitive sodium chloride cotransporter NCC (encoded by Slc12a3). Global transcriptional profiling, combined with biochemical, cell biological, and physiological phenotyping, identified the gene expression signature of the response and revealed how it establishes an adaptive physiology. Salt reabsorption pathways were created by the coordinate induction of a multigene transport system, involving solute carriers (encoded by Slc26a4, Slc4a8, and Slc4a9), carbonic anhydrase isoforms, and V-type H⁺-ATPase subunits in pendrin-positive intercalated cells (PP-ICs) and ENaC subunits in principal cells (PCs). A distal nephron remodeling process and induction of jagged 1/NOTCH signaling, which expands the cortical connecting tubule with PCs and replaces acid-secreting α-ICs with PP-ICs, were partly responsible for the compensation. Salt reabsorption was also activated by induction of an α-ketoglutarate (α-KG) paracrine signaling system. Coordinate regulation of a multigene α-KG synthesis and transport pathway resulted in α-KG secretion into pro-urine, as the α-KG-activated GPCR (Oxgr1) increased on the PP-IC apical surface, allowing paracrine delivery of α-KG to stimulate salt transport. Identification of the integrated compensatory NaCl reabsorption mechanisms provides insight into thiazide diuretic efficacy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/JCI78558DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463200PMC
May 2015

SPAK-mediated NCC regulation in response to low-K+ diet.

Am J Physiol Renal Physiol 2015 Apr 28;308(8):F923-31. Epub 2015 Jan 28.

Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland; and.

The NaCl cotransporter (NCC) of the renal distal convoluted tubule is stimulated by low-K(+) diet by an unknown mechanism. Since recent work has shown that the STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) can function to stimulate NCC by phosphorylation of specific N-terminal sites, we investigated whether the NCC response to low-K(+) diet is mediated by SPAK. Using phospho-specific antibodies in Western blot and immunolocalization studies of wild-type and SPAK knockout (SPAK(-/-)) mice fed a low-K(+) or control diet for 4 days, we found that low-K(+) diet strongly increased total NCC expression and phosphorylation of NCC. This was associated with an increase in total SPAK expression in cortical homogenates and an increase in phosphorylation of SPAK at the S383 activation site. The increased pNCC in response to low-K(+) diet was blunted but not completely inhibited in SPAK(-/-) mice. These findings reveal that SPAK is an important mediator of the increased NCC activation by phosphorylation that occurs in the distal convoluted tubule in response to a low-K(+) diet, but other low-potassium-activated kinases are likely to be involved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajprenal.00388.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4398835PMC
April 2015

Short forms of Ste20-related proline/alanine-rich kinase (SPAK) in the kidney are created by aspartyl aminopeptidase (Dnpep)-mediated proteolytic cleavage.

J Biol Chem 2014 Oct 27;289(42):29273-84. Epub 2014 Aug 27.

From the Departments of Anesthesiology,

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na(+) transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na(+)-K(+)-2Cl(-) cotransporter, NKCC2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.604009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4200278PMC
October 2014

A novel Ste20-related proline/alanine-rich kinase (SPAK)-independent pathway involving calcium-binding protein 39 (Cab39) and serine threonine kinase with no lysine member 4 (WNK4) in the activation of Na-K-Cl cotransporters.

J Biol Chem 2014 Jun 8;289(25):17680-8. Epub 2014 May 8.

From the Department of Anesthesiology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 and

Na(+)-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na(+)-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M113.540518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4067202PMC
June 2014

NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia.

J Biol Chem 2014 Mar 18;289(13):9121-35. Epub 2014 Feb 18.

From the Departments of Medicine.

The highly sialylated vascular endothelial surface undergoes changes in sialylation upon adopting the migratory/angiogenic phenotype. We recently established endothelial cell (EC) expression of NEU1 sialidase (Cross, A. S., Hyun, S. W., Miranda-Ribera, A., Feng, C., Liu, A., Nguyen, C., Zhang, L., Luzina, I. G., Atamas, S. P., Twaddell, W. S., Guang, W., Lillehoj, E. P., Puché, A. C., Huang, W., Wang, L. X., Passaniti, A., and Goldblum, S. E. (2012) NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). We asked whether NEU1 might regulate EC capillary-like tube formation on a Matrigel substrate. In human pulmonary microvascular ECs (HPMECs), prior silencing of NEU1 did not alter tube formation. Infection of HPMECs with increasing multiplicities of infection of an adenovirus encoding for catalytically active WT NEU1 dose-dependently impaired tube formation, whereas overexpression of either a catalytically dead NEU1 mutant, NEU1-G68V, or another human sialidase, NEU3, did not. NEU1 overexpression also diminished EC adhesion to the Matrigel substrate and restrained EC migration in a wounding assay. In HPMECs, the adhesion molecule, CD31, also known as platelet endothelial cell adhesion molecule-1, was sialylated via α2,6-linkages, as shown by Sambucus nigra agglutinin lectin blotting. NEU1 overexpression increased CD31 binding to Arachis hypogaea or peanut agglutinin lectin, indicating CD31 desialylation. In the postconfluent state, when CD31 ectodomains are homophilically engaged, NEU1 was recruited to and desialylated CD31. In postconfluent ECs, CD31 was desialylated compared with subconfluent cells, and prior NEU1 silencing completely protected against CD31 desialylation. Prior CD31 silencing and the use of CD31-null ECs each abrogated the NEU1 inhibitory effect on EC tube formation. Sialyltransferase 6 GAL-I overexpression increased α2,6-linked CD31 sialylation and dose-dependently counteracted NEU1-mediated inhibition of EC tube formation. These combined data indicate that catalytically active NEU1 inhibits in vitro angiogenesis through desialylation of its substrate, CD31.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.555888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979388PMC
March 2014
-->