BASIC RESEARCH www.jasn.org The Role of Intercalated Cell Nedd4–2 in BP Regulation, Ion Transport, and Transporter Expression Masayoshi Nanami,1 Truyen D. Pham,1 Young Hee Kim,1 Baoli Yang,2 Roy L. Sutliff,3 Olivier Staub,4,5 Janet D. Klein,1 Karen I. Lopez-Cayuqueo,6,7 Regine Chambrey,8 Annie Y. Park,1 Xiaonan Wang,1 Vladimir Pech,1 Jill W. Verlander,9 and Susan M. Wall1,10 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Nedd4–2 is an E3 ubiquitin-protein ligase that associates with transport proteins, causing their ubiquitylation, and then internalization and degradation. Previous research has suggested a corre- lation between Nedd4–2 and BP. In this study, we explored the effect of intercalated cell (IC) Nedd4–2 gene ablation on IC transporter abundance and function and on BP. Methods We generated IC Nedd4–2 knockout mice using Cre-lox technology and produced global pen- 2/2 drin/Nedd4–2 null mice by breeding global Nedd4–2 null (Nedd4–2 ) mice with global pendrin null 2/2 (Slc26a4 ) mice. Mice ate a diet with 1%–4% NaCl; BP was measured by tail cuff and radiotelemetry. We 2 measured transepithelial transport of Cl and total CO2 and transepithelial voltage in cortical collecting ducts perfused in vitro. Transporter abundance was detected with immunoblots, immunohistochemistry, and immunogold cytochemistry. 2 2 Results IC Nedd4–2 gene ablation markedly increased electroneutral Cl /HCO3 exchange in the cortical col- lecting duct, although benzamil-, thiazide-, and bafilomycin-sensitive ion flux changed very little. IC Nedd4–2 gene ablation did not increase the abundance of type B IC transporters, such as AE4 (Slc4a9), H+-ATPase, barttin, + 2 2 or the Na -dependent Cl /HCO3 exchanger (Slc4a8). However, IC Nedd4–2 gene ablation increased CIC-5 total protein abundance, apical plasma membrane pendrin abundance, and the ratio of pendrin expression on the apical membrane to the cytoplasm. IC Nedd4–2 gene ablation increased BP by approximately 10 mm Hg. Moreover, pendrin gene ablation eliminated the increase in BP observed in global Nedd4–2 knockout mice. 2 2 Conclusions IC Nedd4–2 regulates Cl /HCO3 exchange in ICs., Nedd4–2 gene ablation increases BP in part through its action in these cells. J Am Soc Nephrol 29: 1706–1719, 2018. doi: https://doi.org/10.1681/ASN.2017080826 In people and rodent models of salt-sensitive hy- through a mechanism that involves the E3 ubiq- pertension, BP elevation requires increased intake uitin-protein ligase neuronal precursor cell 2 of Na+ and Cl .1,2 One commonly used rodent model of human salt-sensitive hypertension is Received August 2, 2017. Accepted March 29, 2018. achieved with the administration of aldosterone J.W.V. and S.M.W. contributed equally to this work. and a high-NaCl diet. This treatment model pro- duces salt-sensitive hypertension partly by stimu- M.N. and T.D.P. contributed equally to this work. + 2 lating renal Na and Cl transporters, such as the Published online ahead of print. Publication date available at epithelial Na+ channel (ENaC),3 the thiazide-sensitive www.jasn.org. 4 5 NaCl cotransporter, and pendrin. Aldosterone Correspondence: Dr. Susan M. Wall, Renal Division, Emory modulates NaCl absorption, at least in some renal University School of Medicine, WMB Room 338, 1639 Pierce cell types, by changing the number of func- Drive, NE, Atlanta, GA 30322. Email: [email protected] tional transporters in the cell membrane partly Copyright © 2018 by the American Society of Nephrology 1706 ISSN : 1046-6673/2906-1706 JAmSocNephrol29: 1706–1719, 2018 www.jasn.org BASIC RESEARCH Significance Statement Nedd4–2 associates with the epithelial Na+ channel (ENaC), resulting in its ubiquitylation and then, internalization and deg- radation. In the absence of Nedd4–2, increased renal Na+ ab- sorption and increased BP are observed. Despite high levels of Nedd4–2 expression in intercalated cells, the role that these cells play in the changes in BP observed with Nedd4–2 gene ablation is unknown. This study is the first to show that Nedd4–2-dependent changes in BP occur in part from Nedd4–2 expressed in in- tercalated cells. Moreover, this study shows that Nedd4–2 gene ablation within intercalated cells stimulates electroneutral apical 2 2 Cl /HCO3 exchange in mouse CCD, in part by upregulating pendrin. In the cortical collecting duct (CCD), Na+ is absorbed primarily 2 by principal cells, whereas Cl is absorbed primarily across 2 2 15 ICs, largely through electroneutral Cl /HCO3 exchange across type B ICs.16 Apical anion exchange occurs through apical + 2 2 Na -independent Cl /HCO3 exchange mediated principally by pendrin (Slc26a4),17,18 which acts in parallel with the + 2 2 Figure 1. Ion transporters in mouse cortical collecting duct. Na -dependent Cl /HCO3 exchanger, NDCBE, encoded by + Mouse cortical collecting duct is made up of principal cells, which Slc4a819. NaCl and net H equivalentsexitacrossthetypeBIC 2 mediate electrogenic Na+ absorption through the benzamil- basolateral plasma membrane through a Cl channel (ClC-K2/ + 20,21 22 sensitive epithelial Na channel (ENaC) on the apical plasma barttin or ClC-Kb), an NaHCO3 cotransporter (AE4), membrane. Na+ exits principal cells across the basolateral plasma and an H+ pump (H+-ATPase)22. This NaCl and H+ exit in- + membrane through the Na,K-ATPase. ENaC-mediated Na ab- creases the electrochemical gradient for apical anion sorption creates a lumen-negative voltage, which provides the 2 2 exchange, thereby increasing Cl absorption and HCO3 driving force for K+ secretion. Type B ICs mediate electroneutral 2 secretion. In contrast to type B ICs, type A ICs mediate net NaCl absorption and HCO3 secretion through an apical plasma 23–26 2 + 2 2 HCl secretion into the luminal fluid in series with Cl membrane Na -independent electroneutral Cl /HCO3 ex- 2 + uptake and HCO3 exit across the basolateral membrane changer (pendrin) that acts in tandem with an Na -dependent 2 2 2 2 2 + + through Cl /HCO3 exchange (AE1), Na -K -2Cl cotrans- Cl /HCO3 exchanger. NaCl exits the cell through a basolateral 2 2 20,21,26,27 plasma membrane Cl channel and an NaHCO3 cotransporter porter 1, and a Cl channel (Figure 1). (AE4). Net H+ equivalents exit across the basolateral plasma Nedd4–2 is expressed in the aldosterone-sensitive region membrane through the H+-ATPase. The type A IC mediates up- of the nephron,10 which includes the connecting tubule 2 take of H+ equivalents and Cl across the basolateral plasma (CNT), the CCD, and the OMCD. Mouse CNT is made 2 2 membrane through Na+-K+-2Cl cotransporter 1 (NKCC1), Cl / 2 2 up of CNT cells and ICs, whereas mouse CCD is composed HCO3 exchange (AE1), and possibly, a Cl channel. This cell 28 – + 2 of principal cells and ICs. Nedd4 2 is highly expressed in type secretes HCl through an apical H -ATPase and an apical Cl 10 2 2 the CCD and the CNT, particularly within type B and channel or Cl /HCO3 exchanger. non-A, non-B ICs; CNT cells; and principal cells, with much lower abundance in type A ICs.10 Although the role expressed developmentally downregulated 4–2 (Nedd4–2).6–9 of Nedd4–2 in principal cells has been well studied, little is When a transporter or a channel associates with Nedd4–2,it known about its function in ICs. Our ability to generate is ubiquitylated, and then, endocytosed and degraded in mice in which Nedd4–2 gene ablation has occurred specif- proteasomes or lysosomes.9,10,11 Conversely, in the absence ically within ICs of the CCD plus our ability to perfuse of Nedd4–2 (i.e.,inNedd4–2 knockout mice), channel in- CCDs in vitro from these mice29 provide a unique oppor- ternalization and degradation fall, which increases plasma tunity by which to explore the physiologic role of IC membrane abundance of channels such as ENaC, thereby Nedd4–2 in native tissue. contributing to the salt-sensitive hypertension observed in Aldosterone’s signal transduction mechanism in type B ICs global Nedd4–2 null mice.8 As such, increased BP is ob- is poorly understood. Because Nedd4–2 participates in aldo- served in mice with embryonic, global Nedd4–2 gene abla- sterone signaling in many cell types and because Nedd4–2 is tion8; mice with inducible, kidney-specific Nedd4–2 gene expressed in ICs, we sought to determine if Nedd4–2 changes ablation12; and people with certain polymorphisms of BP by altering IC function. The purpose of this study was to NEDD4-L, the human homolog of rodent Nedd4–2.13,14 determine if IC Nedd4–2 gene ablation changes CCD ion 2 The Na+ and Cl transporters expressed in principal cells and transport or BP and determine the transporter(s) regulated the various intercalated cell (IC) subtypes are displayed in Figure 1. by Nedd4–2 within ICs. J Am Soc Nephrol 29: 1706–1719, 2018 Intercalated Cell Nedd4–2 1707 BASIC RESEARCH www.jasn.org METHODS Animals IC Nedd4–2 null mice were generated by breeding floxed Nedd4–2 mice8 with transgenic mice expressing Cre recombi- nase driven by the ATP6V1B1 promoter (B1-H+-ATPase Cre),30 asubunitoftheH+-ATPase that is expressed in renal ICs.30 The Cre was carried through the female line. We com- pared IC Nedd4–2 null (Nedd4–2loxloxcre) with Cre(2)sex- matched, wild-type littermates (Nedd4–2loxlox). Unless otherwise stated, IC Nedd4–2 knockout and wild-type littermates will refer to Nedd4–2loxloxcre and Nedd4–2loxlox, respectively. Mice were genotyped by quantitative PCR (Transnetyx) and sometimes, standard PCR.8,30 Global Nedd4–2 null mice were generated as described previously8 by breeding floxed Nedd4–2 mice with mice ex- pressing Cre recombinase globally (EIIa-Cre, Stock 003724; 2 2 2 2 Jackson Labs).8 To generate Nedd4–2 / /Slc26a4 / ; Nedd4–2+/+ 2 2 2 2 /Slc26a4 / , Nedd4–2 / /Slc26a4+/+, and wild-type littermates on a C57Bl/6 background, we first bred global pendrin null 2 2 (Slc26a4 / ) on a 129 SvEv Tac background with wild-type mice on a C57Bl/6 background over ten generations.