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1.  Renal β-intercalated cells maintain body fluid and electrolyte balance 
The Journal of Clinical Investigation  2013;123(10):4219-4231.
Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1–/– mice) displayed renal loss of NaCl, K+, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na+-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E2 (PGE2) and ATP levels in Atp6v1b1–/– mice. Inhibition of PGE2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in β-ICs induced release of PGE2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE2 paracrine signaling originating from β-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.
doi:10.1172/JCI63492
PMCID: PMC3784520  PMID: 24051376
2.  SLC26A4 Targeted to the Endolymphatic Sac Rescues Hearing and Balance in Slc26a4 Mutant Mice 
PLoS Genetics  2013;9(7):e1003641.
Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
Author Summary
Mutations of SLC26A4 are the most common cause for hearing loss associated with a swelling of the inner ear. This human disease is largely recapitulated in a mutant mouse model. Mutant mice lack Slc26a4 expression and their inner ears swell during embryonic development, which leads to failure of the cochlea and the vestibular organs resulting in deafness and loss of balance. SLC26A4 is normally found in the cochlea and vestibular organs of the inner ear as well as in the endolymphatic sac, which is a non-sensory part of the inner ear. The multitude of sites where SLC26A4 is located made the goal to restore function through restoration look futile, unless some sites were more important than others. Here, we generated a new mutant mouse that expresses SLC26A4 in the endolymphatic sac but not in the cochlea or the vestibular organs of the inner ear. Fantastically, this mouse did not develop the detrimental swelling of the inner ear and even more exciting, the mouse developed normal hearing and balance. Our study provides the proof-of-concept that a therapy aimed at repairing the endolymphatic sac during embryonic development is sufficient to restore a life-time of normal hearing and balance.
doi:10.1371/journal.pgen.1003641
PMCID: PMC3708829  PMID: 23874234
3.  Plasma Fibroblast Growth Factor 23 Concentration Is Increased and Predicts Mortality in Patients on the Liver-Transplant Waiting List 
PLoS ONE  2013;8(6):e66182.
High plasma fibroblast growth factor-23 (FGF23) concentration predicts the risk of death and poor outcomes in patients with chronic kidney disease or chronic heart failure. We checked if FGF23 concentration could be modified in patients with end stage liver disease (ESLD) and predict mortality. We measured plasma FGF23 in 200 patients with ESLD registered on a liver transplant waiting list between January 2005 and October 2008. We found that median plasma FGF23 concentration was above normal values in 63% of the patients. Increased FGF23 concentration was not explained by its classical determinants: hyperphosphataemia, increased calcitriol concentration or decreased renal function. FGF23 concentration correlated with the MELD score, serum sodium concentration, and GFR. Forty-six patients died before being transplanted and 135 underwent liver transplantation. We analyzed the prognostic value of FGF23 levels. Mortality was significantly associated with FGF23 levels, the MELD score, serum sodium concentration and glomerular filtration rate. On multivariate analyses only FGF23 concentration was associated with mortality. FGF23 levels were independent of the cause of the liver disease. To determine if the damaged liver can produce FGF23 we measured plasma FGF23 concentration and liver FGF23 mRNA expression in control and diethyl-nitrosamine (DEN)-treated mice. FGF23 plasma levels increased with the apparition of liver lesions in DEN-treated mice and that FGF23 mRNA expression, which was undetectable in the liver of control mice, markedly increased with the development of liver lesions. The correlation between FGF23 plasma concentration and FGF23 mRNA expression in DEN-treated mice suggests that FGF23 production by the liver accounts for the increased plasma FGF23 concentration. In conclusion chronic liver lesions can induce expression of FGF23 mRNA leading to increased FGF23 concentration, which is associated with a higher mortality in patients on a liver-transplant waiting list. In these patients FGF23 concentration was the best predictor of mortality.
doi:10.1371/journal.pone.0066182
PMCID: PMC3692511  PMID: 23825530
4.  A mouse model for distal renal tubular acidosis reveals a previously unrecognized role of the V-ATPase a4 subunit in the proximal tubule 
EMBO Molecular Medicine  2012;4(10):1057-1071.
The V-ATPase is a multisubunit complex that transports protons across membranes. Mutations of its B1 or a4 subunit are associated with distal renal tubular acidosis and deafness. In the kidney, the a4 subunit is expressed in intercalated cells of the distal nephron, where the V-ATPase controls acid/base secretion, and in proximal tubule cells, where its role is less clear. Here, we report that a4 KO mice suffer not only from severe acidosis but also from proximal tubule dysfunction with defective endocytic trafficking, proteinuria, phosphaturia and accumulation of lysosomal material and we provide evidence that these findings may be also relevant in patients. In the inner ear, the a4 subunit co-localized with pendrin at the apical side of epithelial cells lining the endolymphatic sac. As a4 KO mice were profoundly deaf and displayed enlarged endolymphatic fluid compartments mirroring the alterations in pendrin KO mice, we propose that pendrin and the proton pump co-operate in endolymph homeostasis. Thus, our mouse model gives new insights into the divergent functions of the V-ATPase and the pathophysiology of a4-related symptoms.
doi:10.1002/emmm.201201527
PMCID: PMC3491836  PMID: 22933323
deafness; distal renal tubular acidosis; H+-ATPase; kidney; proteinuria
5.  PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor 
The Journal of Clinical Investigation  2012;122(9):3355-3367.
Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.
doi:10.1172/JCI57407
PMCID: PMC3428075  PMID: 22886306
6.  Determination of the best method to estimate glomerular filtration rate from serum creatinine in adult patients with sickle cell disease: a prospective observational cohort study 
BMC Nephrology  2012;13:83.
Background
Sickle cell disease (SCD) leads to tissue hypoxia resulting in chronic organ dysfunction including SCD associated nephropathy. The goal of our study was to determine the best equation to estimate glomerular filtration rate (GFR) in SCD adult patients.
Methods
We conducted a prospective observational cohort study. Since 2007, all adult SCD patients in steady state, followed in two medical departments, have had their GFR measured using iohexol plasma clearance (gold standard). The Cockcroft-Gault, MDRD-v4, CKP-EPI and finally, MDRD and CKD-EPI equations without adjustment for ethnicity were tested to estimate GFR from serum creatinine. Estimated GFRs were compared to measured GFRs according to the graphical Bland and Altman method.
Results
Sixty-four SCD patients (16 men, median age 27.5 years [range 18.0-67.5], 41 with SS-genotype were studied. They were Sub-Saharan Africa and French West Indies natives and predominantly lean (median body mass index: 22 kg/m2 [16-33]). Hyperfiltration (defined as measured GFR >110 mL/min/1.73 m2) was detected in 53.1% of patients. Urinary albumin/creatinine ratio was higher in patients with hyperfiltration than in patients with normal GFR (4.05 mg/mmol [0.14-60] versus 0.4 mg/mmol [0.7-81], p = 0.01). The CKD-EPI equation without adjustment for ethnicity had both the lowest bias and the greatest precision. Differences between estimated GFRs using the CKP-EPI equation and measured GFRs decreased with increasing GFR values, whereas it increased with the Cockcroft-Gault and MDRD-v4 equations.
Conclusions
We confirm that SCD patients have a high rate of glomerular hyperfiltration, which is frequently associated with microalbuminuria or macroalbuminuria. In non-Afro-American SCD patients, the best method for estimating GFR from serum creatinine is the CKD-EPI equation without adjustment for ethnicity. This equation is particularly accurate to estimate high GFR values, including glomerular hyperfiltration, and thus should be recommended to screen SCD adult patients at high risk for SCD nephropathy.
doi:10.1186/1471-2369-13-83
PMCID: PMC3465224  PMID: 22866669
Sickle cell disease; Glomerular hyperfiltration; Albuminuria; Glomerular filtration rate; CKD-EPI equation; Iohexol plasma clearance; Ethnicity
7.  Insulin receptor-related receptor as an extracellular alkali sensor 
Cell metabolism  2011;13(6):679-689.
SUMMARY
The insulin receptor-related receptor (IRR), an orphan receptor tyrosine kinase of the insulin receptor family, can be activated by alkaline media both in vitro and in vivo at pH>7.9. The alkali-sensing property of IRR is conserved in frog, mouse and human. IRR activation is specific, dose-dependent, quickly reversible and demonstrates positive cooperativity. It also triggers receptor conformational changes and elicits intracellular signaling. The pH sensitivity of IRR is primarily defined by its L1F extracellular domains. IRR is predominantly expressed in organs that come in contact with mildly alkaline media. In particular, IRR is expressed in the cell subsets of the kidney that secrete bicarbonate into urine. Disruption of IRR in mice impairs the renal response to alkali loading attested by development of metabolic alkalosis and decreased urinary bicarbonate excretion in response to this challenge. We therefore postulate that IRR is an alkali sensor that functions in the kidney to manage metabolic bicarbonate excess.
doi:10.1016/j.cmet.2011.03.022
PMCID: PMC3119365  PMID: 21641549
9.  Increased renal renin content in mice lacking the Na+/H+ exchanger NHE2 
Macula densa (MD) cells express the Na+/H+ exchanger (NHE) isoform NHE2 at the apical membrane, which may play an important role in tubular salt sensing through the regulation of cell volume and intracellular pH. These studies aimed to determine whether NHE2 participates in the MD control of renin synthesis. Renal renin content and activity and elements of the MD signaling pathway were analyzed using wild-type (NHE2+/+) and NHE2 knockout (NHE2−/−) mice. Immunofluorescence studies indicated that NHE2−/− mice lack NHE3 at the MD apical membrane, so the other apical NHE isoform has not compensated for the lack of NHE2. Importantly, the number of renin-expressing cells in the afferent arteriole in NHE2−/− mice was increased ∼2.5-fold using renin immunohistochemistry. Western blotting confirmed ∼20% higher renal cortical renin content in NHE2−/− mice compared with wild type. No-salt diet for 1 wk significantly increased renin content and activity in NHE2+/+ mice, but the response was blunted in NHE2−/− mice. Renal tissue renin activity and plasma renin concentration were elevated three- and twofold, respectively, in NHE2−/− mice compared with wild type. NHE2−/− mice also exhibited a significantly increased renal cortical cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase (mPGES) expression, indicating MD-specific mechanisms responsible for the increased renin content. Significant and chronic activation of ERK1/2 was observed in MD cells of NHE2−/− kidneys. Removal of salt or addition of NHE inhibitors to cultured mouse MD-derived (MMDD1) cells caused a time-dependent activation of ERK1/2. In conclusion, the NHE2 isoform appears to be important in the MD feedback control of renin secretion, and the signaling pathway likely involves MD cell shrinkage and activation of ERK1/2, COX-2, and mPGES, all well-established elements of the MD-PGE2-renin release pathway.
doi:10.1152/ajprenal.00591.2007
PMCID: PMC3018236  PMID: 18287398
macula densa; cell volume; renin release; mitogen-activated protein kinases; cyclooxygenase-2
10.  The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice  
The Journal of Clinical Investigation  2010;120(5):1627-1635.
Regulation of sodium balance is a critical factor in the maintenance of euvolemia, and dysregulation of renal sodium excretion results in disorders of altered intravascular volume, such as hypertension. The amiloride-sensitive epithelial sodium channel (ENaC) is thought to be the only mechanism for sodium transport in the cortical collecting duct (CCD) of the kidney. However, it has been found that much of the sodium absorption in the CCD is actually amiloride insensitive and sensitive to thiazide diuretics, which also block the Na-Cl cotransporter (NCC) located in the distal convoluted tubule. In this study, we have demonstrated the presence of electroneutral, amiloride-resistant, thiazide-sensitive, transepithelial NaCl absorption in mouse CCDs, which persists even with genetic disruption of ENaC. Furthermore, hydrochlorothiazide (HCTZ) increased excretion of Na+ and Cl– in mice devoid of the thiazide target NCC, suggesting that an additional mechanism might account for this effect. Studies on isolated CCDs suggested that the parallel action of the Na+-driven Cl–/HCO3– exchanger (NDCBE/SLC4A8) and the Na+-independent Cl–/HCO3– exchanger (pendrin/SLC26A4) accounted for the electroneutral thiazide-sensitive sodium transport. Furthermore, genetic ablation of SLC4A8 abolished thiazide-sensitive NaCl transport in the CCD. These studies establish what we believe to be a novel role for NDCBE in mediating substantial Na+ reabsorption in the CCD and suggest a role for this transporter in the regulation of fluid homeostasis in mice.
doi:10.1172/JCI40145
PMCID: PMC2860930  PMID: 20389022

Results 1-10 (10)