Renal COP9 Signalosome Deficiency Alters CUL3-KLHL3-WNK Signaling

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Renal COP9 Signalosome Deﬁciency Alters CUL3-KLHL3-WNK Signaling Pathway

Ryan J. Cornelius,1 Jinge Si,1 Catherina A. Cuevas,1 Jonathan W. Nelson,1 Brittany D.K. Gratreak,1 Ruggero Pardi,2 Chao-Ling Yang,1 and David H. Ellison1,3

1Division of Nephrology and Hypertension, Department of Medicine, Oregon Health and Science University, Portland, Oregon; 2School of Medicine and Scientiﬁc Institute, San Raffaele University, Milan, Italy; and 3Renal Section, Veterans Affairs Portland Health Care System, Portland, Oregon

ABSTRACT Background The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3D9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin- RING ligases. CUL3D9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3D9phenotype.

Methods The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN BASIC RESEARCH 2 2 subunit, Jab1, was deleted only along the nephron, after full development (KS-Jab1 / ). Results Western blot analysis demonstrated that Jab1 deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was 2 2 almost completely absent in KS-Jab1 / mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunoﬂu- orescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na+ and K+ wasting. Additionally, long-term Jab1 deletion resulted in kidney damage. Conclusions Together, the results indicate that deﬁcient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3D9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.

J Am Soc Nephrol 29: 2627–2640, 2018. doi: https://doi.org/10.1681/ASN.2018030333

The thiazide-sensitive Na-Cl cotransporter (NCC) disease is caused by mutations in WNK1 and in the distal convoluted tubule (DCT) of the kidney WNK4,5 and also mutations in the cullin-RING is modulated through the WNK-SPAK/OSR1 kinase ligase (CRL) proteins kelch-like 3 (KLHL3)6 and pathway. WNKs (with-no-lysine kinases) phosphorylate intermediary kinases serine/threonine protein kinase 39 (SPAK; STK39) and oxidative stress- Received March 28, 2018. Accepted September 7, 2018. response 1 (OxSR1 or OSR1; OXSR1), which then C-L.Y. and D.H.E. contributed equally to this work. directly phosphorylate NCC, activating the cotrans- Published online ahead of print. Publication date available at 1,2 porter. Increased WNK activity causes the disease www.jasn.org. familial hyperkalemic hypertension (FHHt), also Correspondence: Dr. David H. Ellison, Division of Nephrology known as pseudohypoaldosteronism type 2 or and Hypertension, SON440, Oregon Health and Science Uni- Gordon syndrome.3 FHHt is characterized by hy- versity, 3181 SW Sam Jackson Park Road, Portland, OR 97239. pertension, hyperkalemia, and metabolic acidosis, Email: [email protected] which can be treated with thiazide diuretics.4 The Copyright © 2018 by the American Society of Nephrology

J Am Soc Nephrol 29: 2627–2640, 2018 ISSN : 1046-6673/2911-2627 2627 BASIC RESEARCH www.jasn.org cullin 3 (CUL3).7 Although the effects of CUL3 mutations Significance Statement have been studied extensively, a consensus understanding of the mechanisms by which they cause human disease has not The mechanism by which mutations in cullin 3 (CUL3) cause familial emerged. hyperkalemic hypertension (FHHt) is controversial. Previous work D All mutations causing FHHt lead to accumulation of WNK showed that the CUL3 9 mutant does not bind normally to the COP9 signalosome (CSN). Here, genetic deletion of the CSN cat- proteins. WNK4, KLHL3,andCUL3 mutations all increase alytic subunit Csn5/Jab1 in mouse kidney upregulated the WNK- – WNK abundance by impairing WNK protein degradation,8 12 SPAK pathway, probably resulting from the decreased abundance via CRLs. CRLs are modular protein complexes containing one of KLHL3, which links WNKs with CUL3 for ubiquitination and of eight cullin proteins (CUL1, 2, 3, 4A, 4B, 5, 7, and 9),13 degradation. NCC expression decreased despite the upregulation bound to a RING protein, which transfers ubiquitin to a sub- of WNK signaling, and the mice developed polyuria, salt wasting, hypokalemia, and kidney tubule damage. The findings present a 2 2 strate targeting it to the proteasome for degradation. The sub- mixed phenotype that resembles the kidney-specific Cul3 / strate does not typically interact directly with the cullin but phenotype but also suggests that the inability of the CUL3D9 mu- instead through an adaptor protein and substrate receptor. In tant to interact with the CSN contributes to FHHt by enhancing the case of CUL3, however, the substrate adaptor and receptor KLHL3 degradation. are merged into a single protein.14 For simplicity we will refer to CRL adaptors and receptors as substrate adaptors. In hu- features, with regard to specific substrates and substrate fi mans, hundreds of substrate adaptors have been identi ed, adaptors.36 As CSN’s catalytic activity is contained within the fi 15 each able to bind to one or a few speci c proteins. WNKs JAB1 subunit,21 and CUL3D9 is hyperneddylated in cultured 16 interact with CUL3 via the substrate adaptor KLHL3. cells,11,37 we postulated that hyperneddylation contributes to fi Before ubiquitylation can occur the CRL must rst be ac- substrate accumulation and substrate adaptor instability. tivated. Activation occurs via neddylation, a process in which Here, we generated mice in which JAB1 could be deleted from 2 2 a NEDD8 (neuronal precursor cell expressed developmentally kidney-tubule cells (KS-Jab1 / ) to test the importance of the downregulated protein 8) protein is covalently attached to the CSN-binding deficiency in disease pathogenesis, in vivo. cullin.17,18 Neddylation increases flexibility of the cullin-RING structure, which closes the gap between the ubiquitin-charged E2 (ubiquitin-conjugating enzyme) and the substrate.19 In vivo, METHODS neddylation activates the CRL, but normal function can only be maintained through subsequent NEDD8 removal, as hyperned- Antibodies dylation paradoxically impairs substrate degradation.20 NEDD8 Antibodies used are described in Supplemental Table 1. removal occurs via attachment of the multisubunit deneddylase COP9 signalosome (CSN).21 Deneddylation prevents overac- Animals tivation of the CRL, which can lead to aberrant ubiquitylation Animal studies were approved by the Oregon Health and Sci- targeting the substrate adaptor and/or the cullin protein itself.22 ence University Institutional Animal Care and Use Commit- The CSN is one of the three Proteasome-COP9 signalo- tee (protocol IP000286). The Pax8-rtTA/LC1 system38,39 was 2 2 some-Initiation factor 3 (PCI) domain-containing complexes used to generate inducible renal epithelia-specific Jab / fl fl and consists of eight subunits.23 The subunits CSN1–4, and mice. Jab1 x/ x mice were generated by R.P.40 and generously CSN7–8 make up a horseshoe-shaped ring structure and each provided by Dr. Guang Zhou. These mice developed normally, contain a PCI domain, whereas the core subunits CSN5 and were phenotypically similar to wild-type (WT) mice, and had fl fl CSN6 are Mpr1-Pad1-N-terminal (MPN) domain-containing normal JAB1 expression. Jab1 x/ x mice were crossed with Pax8/ fl fl proteins. Unlike CSN6, CSN5 contains a JAB1/MPN/Mov34 LC1 mice to generate Jab1 x/ x-Pax/LC1 mice. Doxycycline was motif conferring Zn2+-dependent metalloprotease activity. administered at 2 mg/ml with 5% sucrose in drinking water for 2 2 Although CSN5 (also known as JAB1 or jun activation do- 3 weeks to generate renal epithelia-specific Jab1 / mice 2 2 main-binding protein-1) is the sole isopeptidase, all subunits (referred to as KS-Jab1 / ). Heterozygous Jab1 mice were gen- fl 2 are required for enzymatic activity.24 In mice, genetic deletion erated by giving doxycycline to Jab1 x/ mice (referred to as 2 2 of CSN subunits 2, 3, 5, 6, or 8 is embryonic lethal.25–29 In- KS-Jab1+/ ). Some KS-Jab1+/ mice were fed a high sodium hibition of any subunit, either in vitro30–34 or in vivo,33,35 leads (6%), low potassium diet (0%). Control mice were littermates to decreased expression of substrate adaptor proteins. Two that received 5% sucrose in drinking water. Both males and groups have shown that the CUL3 FHHt mutant (CUL3D9) female were used for experiments except for BP measurement, has impaired binding to the CSN11,36; our group found that in which only male mice were used, and all mice were on a CUL3D9 also targets KLHL3 for degradation. The loss of in- C57Bl/6J background. teraction with the CSN could account for enhanced KLHL3 Themicewerereturnedtoregulardrinkingwaterfor degradation, perhaps explaining the disease phenotype, as we 3 weeks before they were placed in metabolic cages for postulated previously.37 24-hour urine collection and analysis of food and water con- We recently proposed, on the basis of work primarily in sumption. Toprevent urine contamination, mice were fed a gel cells, that CUL3D9 exhibits both gain- and loss-of-function diet. Mice were acclimated in the metabolic cages for 3 days

2628 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018 www.jasn.org BASIC RESEARCH before analysis. Urine K+ and Na+ were determined by flame Signal (TOYOBO, Osaka, Japan). Appropriate horseradish photometry. Plasma Mg2+ and urinary Ca2+ was analyzed peroxidase–conjugated secondary antibody in blocking buffer using colorimetric assay kits (Pointe Scientific, Canton, MI). was added to membranes for 1 hour at room temperature. Mem- To determine the effects of long-term deletion of Jab1, mice branes were developed using enhanced chemiluminescence, were returned to regular drinking water for 9 weeks before Western Lightning Plus–ECL (Perkin Elmer, Waltham, MA), kidneys were harvested. and proteins were visualized using PXi digital imaging system (Syngene, Frederick, MA). Immunohistochemistry For hematoxylin and eosin staining, mice were anesthetized, Quantitative PCR kidneys were removed and placed in a 10% formalin solution Kidneys were harvested and RNA was preserved by treating (Thermo Fischer Scientific, Waltham, MA) for 24 hours. The with RNAlater RNA Stabilization Reagent (Qiagen, Hilden, kidneys were then transferred to a 70% ethanol solution for Germany) overnight at 4°C. Kidney tissue was then homoge- 72 hours. The Oregon Health and Science University Histopa- nized in PBS and immediately transferred to QIAzol lysis thology Core Facility embedded the kidney in paraffinand reagent, where it was further homogenized by running the performed hematoxylin and eosin staining. sample through a 20 gauge blunt cannula. Total RNA was isolated using RNeasy Plus Universal Mini kit (Qiagen). The total Immunofluorescence RNA (1000 ng) was reverse transcribed using a High-Capacity Mice were anesthetized with a ketamine/xylazine/acepromazine RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). The cocktail and kidneys were perfusion-fixed with retrograde ab- resulting complementary DNA was diluted 1:10 and real-time dominal aortic perfusion of 3% paraformaldehyde in PBS quantitative PCR was performed on a QuantStudio 7 Flex (pH 7.4). Kidneys were removed, dissected, and cryopreserved Real-Time PCR System using TaqMan Universal Master Mix II in 800 mOsm sucrose in PBS overnight before embedding in (Applied Biosystems). The primers used were TaqMan Gene Ex- Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA). pression Assays ID Mm00490213_m1 for NCC (Slc12a3)and Slideswerepreparedbycutting5mmsections andwerestoredat Mm03928990_g1 for 18S (Rn18s) containing FAM dye. mRNA 280°C until use. Immunofluorescence staining was prepared expression levels were calculated with the comparative thresh- 2DD as follows. Slides were incubated with 0.5% Triton-X 100 old cycle method (2 Ct)and18SmRNAwasusedfor in PBS for 30 minutes. Sections were then blocked with 5% normalization. milk in PBS for 30 minutes, followed by incubation with primary antibody, diluted in blocking buffer, for 1 hour at room Blood Analysis temperature or overnight at 4°C (details are provided in Sup- Whole blood was collected via cardiacpuncturefrommiceunder plemental Table 1). Sections were incubated with fluorescent isoflurane anesthesia and transferred to heparin-containing dye–conjugated secondary antibody, diluted in blocking buffer, tubes. The blood was analyzed via an iSTAT analyzer (Abbott, for 1 hour at room temperature before being mounted with Princeton, NJ) using Chem8+ cartridges. Aldosterone was mea- Prolong Diamond Antifade Mountant with DAPI (Invitrogen, sured by ELISA (IBL-America, Minneapolis, MN) from the Carlsbad, CA). Images were captured using a Zeiss AXIO Im- plasma by centrifuging whole blood at 20003g for 10 minutes ager M2 microscope and AxioVision software. Image process- at 4°C. ing was completed using ImageJ software (National Institutes of Health). BP Measurement Radio telemetry was used to measure BP in male mice only, Western Blotting using PA-C10 transmitters (Data Sciences International, Kidneys were harvested and flash frozen in liquid nitrogen and St. Paul, MN). Mice were anesthetized with isoflurane and stored at 280°C. Whole kidney was homogenized in buffer transmitters were implanted in the left carotid artery. Measure- containing enzyme inhibitors with 1 mM dithiothreitol and ments were recorded for 10 seconds every 10 minutes. Mean of 1 mM PMSF. Protein samples were separated by electropho- the hourly averages were used to calculate mean systolic BP. resis on 4%–15% Criterion TGX stain-free gels (Bio-Rad Laboratories, Hercules, CA) and transferred to Immobilon- Cell Culture, Plasmids, and Transfections Ppolyvinylidenedifluoride membranes (EMD Millipore, HEK293 cells were maintained in DMEM supplemented with Billerica, MA) using the Trans-Blot Turbo transfer system 10% FBS, 25 mM HEPES, 100 U/ml penicillin, and 100 mg/ml (Bio-Rad Laboratories). Stain-free imaging was used as a total streptomycin. Cells were transiently transfected using lipofect- protein loading control (Supplemental Figure 1). Membranes amine 2000 (Ambion, Foster City, CA; Invitrogen). Constructs were blocked with 5% nonfat milk in PBS-Tween for 1 hour at were made by amplifying FLAG-CUL3 WT DNA using Phu- room temperature, before incubation with primary antibody in sion Hot Start II DNA Polymerase (Thermo Fisher Scientific, blocking buffer for 1 hour at room temperature or overnight at Boston, MA) with the appropriate primers, purified with 4°C (details are provided in Supplemental Table 1). KLHL3 anti- the PureLink PCR Purification Kit (Invitrogen) and prop- body (Proteintech, Rosemont, IL) was incubated in Can Get erly digested. The products were then extracted using the

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UltraClean GelSpin DNA Extraction Kit (MoBio Laboratories, Statistical Analyses Inc., Carlsbad, CA) and ligated into mammalian plasmids with Data are presented as individual values as well as mean6SEM. T4 DNA Ligase (New England BioLabs, Ipswich, MA). Ligated Differences between two groups were determined using two- constructs were transformed using DH5a competent cells tailed unpaired t test. Differences between multiple groups (Thermo Fisher Scientific) and plasmid DNA was purified were determined using one-way ANOVA with Tukey post with either the QIAprep Spin Miniprep Kit or HiSpeed Plasmid hoc analysis. A P-value of ,0.05 was considered significant. Midi Kit (Qiagen). Sanger sequencing was performed for all Statistical analyses were performed using GraphPad Prism 7 constructs. For small interfering RNA (siRNA) experiments, software (GraphPad Software, San Diego, CA). either 40 nM of COPS5 siRNA (Ambion) or control siRNAwas transfected along with DNA plasmids. Cells were harvested at 36 hours post-transfection. Transfected cells were harvested in RESULTS 0.5% Triton X-100 in PBS cell lysis buffer containing enzyme inhibitors. All experiments were performed in triplicate, with CSN Is Abundant in the Distal Nephron each well representing a unique transfection. Western blotting Immunofluorescence staining of mouse kidney sections was performed as explained above. showed that JAB1 is most highly expressed in segments that

Figure 1. JAB1 expression is abundant in the distal nephron. (A) Immunofluorescence staining of kidney cortex sections in control mice. Top, JAB1 localization was examined in the distal nephron by costaining with the distal nephron marker calbindin. The staining intensity of JAB1 was higher in calbindin-positive cells. Bottom, JAB1 localization was examined in the proximal tubule by costaining with the Na-H exchanger 3 (NHE3). JAB1 did not colocalize with NHE3-positive tubules. (B and C) Doxycycline-induced Jab1 deletion. Doxycycline or fl fl vehicle control was administered to Jab1 x/ x Pax8/LC1 mice for 3 weeks. After 3 weeks of recovery, JAB1 protein abundance and localization was measured by Western blot (B) and immunofluorescence (C). Using whole kidney lysates, immunoblotting for JAB1 showed 2 2 2 2 62% knockdown in KS-Jab1 / mice compared with controls. JAB1 staining was reduced in KS-Jab1 / mice. Data represent individual values as well as mean6SEM relative to controls. Statistical differences were examined using two-tailed unpaired t test.

2630 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018 www.jasn.org BASIC RESEARCH also express calbindin (Figure 1A), a marker for the distal nephron. Expression of JAB1 was most prominent in apical and subapical regions. In contrast, JAB1 was not abundant in proximal tubules, as indicated by costaining with Na-H exchanger 3.

Disruption of the CSN along the Nephron Increases Both Neddylation and Degradation of CUL3 To examine the role of the CSN in kidney in vivo, we crossed fl fl fl fl Jab1 x/ x mice with Pax8/LC1. Homozygotic Jab1 x/ x and Pax8/LC1-positive mice were then administered doxycycline for 3 weeks before a 3-week recovery period. Protein analysis by Western blot showed a 62% knockdown of JAB1 in whole kidney lysates (Figure 1B). Although the Pax8/LC1 system is not 100% efficient, the residual JAB1 protein probably reflects expression in nontubular cells. Examination of JAB1 by immunofluorescence staining demonstrated lower stain- Figure 2. Nephron-specific Jab1 deletion increases neddylation and degradation of CUL3. Western blot of whole kidney lysates ing intensity in the distal nephron of kidney sections, as de- 2/2 termined by costaining with calbindin (Figure 1C). We have from control and KS-Jab1 mice. Left, immunoblotting was performed with antibodies against NEDD8 and CUL3. Immuno- called these mice kidney-specific Jab1 knockout mice 2/2 blotting for NEDD8 showed increased protein abundance in 2 2 (KS-Jab1 mice). KS-Jab1 / mice compared with controls. Immunoblotting for Inhibition of the CSN should block deneddylation, there- CUL3 showed a neddylated (top) and unneddylated (bottom) fore increasing the amount of neddylated cullin. Totest this, we band. The neddylated CUL3 band showed a higher abundance in 2 2 immunoblotted for NEDD8. Western blot analysis showed a KS-Jab1 / mice compared with controls. Additionally, total CUL3 2 2 distinct band at approximately 85 kDa, which is the same as protein abundance was lower in KS-Jab1 / mice compared with the molecular mass of cullins (Figure 2). The NEDD8-cullin controls. Right, quantitative analysis of NEDD8 and CUL3 protein 2 2 band was strikingly more abundant in KS-Jab1 / mice com- abundance. Data represent individual values as well as mean6SEM pared with controls. To confirm enhanced CUL3 neddylation relative to controls. Statistical differences were examined using two- we examined CUL3 protein. Although immunoblotting for tailed unpaired t test. 2 2 CUL3 revealed two bands in the in KS-Jab1 / mice, the higher molecular mass band, representing the neddylated Keap1 (kelch-like ECH-associated protein 1). Keap1 protein 2 2 form of CUL3, was absent in control mice. Additionally, quan- abundance was increased in KS-Jab1 / mice (Figure 3C). titative analysis of total CUL3 showed a 37% reduction in 2 2 protein abundance in KS-Jab1 / mice compared with con- Disruption of CSN along the Nephron Activates the trols. The results indicate that most of the CUL3 protein under WNK-SPAK/OSR1 Pathway normal conditions is in the unneddylated state. Inhibition of We next studied the effects of CSN inhibition on WNK/SPAK 2 2 the CSN causes hyperneddylation of CUL3, which also desta- abundance. Here, KS-Jab1 / mice had an increase in the total bilizes the protein. protein abundance of WNK1 and WNK4 (Figure 4A). Phos- 2 2 phorylation of WNK4 at S1196 was also higher in KS-Jab1 / Disruption of CSN along the Nephron Reduces KLHL3 mice. Furthermore, the ratio of phosphorylated WNK4 to Protein Abundance total WNK4 was increased. Immunofluorescence staining of To examine the effect of CSN inhibition on KLHL3 protein WNK4 showed increased intensity and translocalization into 2 2 abundance in vivo,weusedKS-Jab1 / mice and studied puncta (Figure 4B) in the distal nephron, as identified by cos- them using a commercial KLHL3 antibody that has been val- taining with calbindin (Supplemental Figure 2). This punctate idated with KLHL3 knockout mice.41 Other KLHL3 antibodies formation has been observed in CUL3D9mice,11,43 and under validated in this way have proven nonspecific.42 KLHL3 protein conditions in which the protein is activated.44 abundance was substantially diminished, as shown by Western The intermediary kinases SPAK and OSR1 link WNKs to blot (Figure 3A) and immunofluorescence (Figure 3B). Costain- NCC, and were examined by Western blot and immunoflu- ing with the DCT1 marker parvalbumin in control mice dem- orescence. Western blotting for SPAK showed an increase in onstrated high levels of KLHL3 protein localized in the DCT1. the full-length SPAK, with lower abundance of the shorter 2 2 Additionally, KLHL3 colocalized with calbindin (data not forms, SPAK, SPAK2, and KS-SPAK in KS-Jab1 / mice shown) indicating expression in the distal nephron, consistent (Figure 4C). Additionally, immunoblotting for phosphory- with our previous work.37 Because CSN inhibition has been lated SPAK and OSR1 using an antibody that is specificfor shown to differentially affect substrate adaptors, we examined phospho-SPAK at serine 373 and phospho-OSR1 at serine 325 the effect of Jab1 deletion on another CUL3 substrate adaptor showed an increase in phosphorylated protein (Figure 4D).

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Figure 3. Nephron-speciﬁc Jab1 deletion reduces KLHL3 protein abundance. (A and C) Western blot of whole kidney lysates from control 2 2 and KS-Jab1 / mice. (A) Immunoblotting was performed with antibodies against KLHL3. KLHL3 protein abundance was almost completely 2 2 diminished in KS-Jab1 / mice compared with controls. (B) Immunoﬂuorescence staining of kidney cortex sections from control and 2 2 KS-Jab1 / mice. KLHL3 expression was examined in kidney cortex by costaining with the DCT1 marker parvalbumin. In control mice, 2 2 KLHL3 staining intensity was most prominent in tubules expressing parvalbumin. KS-Jab1 / mice showed lower levels of KLHL3 staining 2 2 compared with controls. (C) Immunoblotting was performed with antibodies against Keap1. KS-Jab1 / mice showed higher Keap1 protein levels compared with control mice. The graph shows the quantitative analysis of Keap1 protein abundance. Data represent individual values as well as mean6SEM relative to controls. Statistical differences were examined using two-tailed unpaired t test.

2 2 Immunoﬂuorescence staining of SPAK and OSR1 showed a that the WNK-SPAK/OSR1 pathway is activated in KS-Jab1 / pattern similar to WNK4, with control mice displaying low mice. 2 2 staining intensity and KS-Jab1 / mice showing staining The results here also demonstrate a distal nephron-speciﬁc localized into puncta (Figure 4E). Puncta formation was lo- phenotype. WNK4 is expressed in both the DCT and thick calized to distal nephron cells as shown by costaining with ascending limb (TAL).45 Although SPAK and OSR1 are also calbindin (Supplemental Figure 2). The results demonstrate expressed in these nephron segments, SPAK is the main

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Figure 4. Nephron-specific Jab1 deletion activates the WNK-SPAK/OSR1 pathway. (A, C, and D) Western blot of whole kidney lysates 2 2 from control and KS-Jab1 / mice. (A) Left, immunoblotting was performed with antibodies against WNK4, phosphorylated WNK4 at 2 2 serine 1196 (pWNK4S1196), and WNK1. WNK4, pWNK4S1196, and WNK1 protein abundance was higher in KS-Jab1 / mice compared with controls. Right, quantitative analysis of total WNK4 protein abundance and the ratio of pWNK4S1196 to total WNK4 (pWNK4/ 2 2 tWNK4). Total WNK4 abundance and pWNK4/tWNK4 was higher in KS-Jab1 / mice compared with controls. Data represent individual values as well as mean6SEM relative to controls. Statistical differences were examined using two-tailed unpaired t test. (B and E) 2 2 Immunofluorescence staining of kidney cortex sections from control and KS-Jab1 / mice. (B) Immunofluorescence staining was 2 2 performed with antibodies against WNK4. There was low abundance of WNK4 staining in control mice relative to KS-Jab1 / mice. 2 2 Staining of KS-Jab1 / mice kidney sections showed WNK4 protein translocated into puncta. (C) Immunoblotting was performed with antibodies against SPAK and OSR1. Protein abundance of full-length SPAK was higher, whereas SPAK2 and KS-SPAK were lower in 2 2 KS-Jab1 / mice compared with controls. (D) Immunoblotting was performed with antibodies against pSPAK/pOSR1 at serine 373 for 2 2 SPAK and serine 325 for OSR1. Phosphorylation levels were higher in KS-Jab1 / mice compared with controls. (E) Immunofluores- cence staining was performed with antibodies against SPAK and OSR1. There was low abundance of SPAK (top) and OSR1 (bottom) 2 2 2 2 staining in control mice relative to KS-Jab1 / mice. Staining of KS-Jab1 / mice kidney sections showed SPAK/OSR1 protein translocated into puncta.

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2 2 Figure 5. JAB1 dysfunction reduces NCC expression. (A) Top, Western blot of whole kidney lysates from control and KS-Jab1 / mice. Immunoblotting was performed with antibodies against NCC or pNCC at threonine 53 (pNCCT53). Both NCC and pNCCT53 were lower 2 2 in KS-Jab1 / mice compared with controls. Bottom, quantitative analysis of NCC and pNCCT53 protein abundance. Both NCC and 2 2 pNCCT53 protein abundance were lower in KS-Jab1 / mice compared with controls. The ratio of pNCCT53 to tNCC was approximately 2 2 three-fold higher in KS-Jab1 / mice compared with controls. (B) Quantitative RT-PCR for the expression of NCC (Slc12a3 gene)inthe 2 2 kidney. The results were normalized to 18S mRNA expression. NCC mRNA expression was decreased in KS-Jab1 / mice compared with controls. (C) NCC was cotransfected into HEK293 cells with either Jab1 siRNA or control siRNA. Top, Protein abundance was examined by immunoblotting using antibodies against NEDD8, CUL3, NCC, and JAB1. Bottom, quantitative analysis of NEDD8, NCC, and JAB1 protein abundance. Jab1 siRNA decreased JAB1 and NCC protein abundance and caused an increase in cullin neddylation as shown by increase in NEDD8 abundance at the molecular mass of cullins, and by an increase in the neddylated CUL3 band. (D) NCC was cotransfected into HEK293 cells together with increasing amounts of HA-tagged JAB1. Left, Immunoblotting for NCC showed an increase in protein abundance when cotransfected with 25 mg of JAB1, and trended higher when cotransfected with 50 mgofJAB1. Right, quantitative analysis of NCC protein. Data represent individual values as well as mean6SEM relative to controls. Statistical differences were examined using two-tailed unpaired t test (A–C) or by one-way ANOVA with Tukey post hoc analysis (D).

2634 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018 www.jasn.org BASIC RESEARCH activator of NCC in the DCT and OSR1 is the main activator of (Supplemental Figure 3). To test whether NCC synthesis was the Na-K-2Cl cotransporter (NKCC2) in the TAL.46–48 SPAK decreased, we performed quantitative RT-PCR. Although and OSR1 immunofluorescence showed punctate formation NCC proteins levels were lower by approximately 85%, NCC in the distal nephron, whereas there was no change in expression (Slc12a3 gene) mRNA levels were decreased by approximately 2 2 in the TAL (Supplemental Figure 2). Furthermore, total and 50% in KS-Jab1 / mice compared with controls (Figure 5B). phosphorylated NKCC2 protein levels were unchanged in This indicates that some, but not all, of the decreased NCC 2 2 KS-Jab1 / mice (Supplemental Figure 3), indicating that the abundance may be due to effects on transcription. 2 2 effects of KS-Jab1 / mice on the WNK-SPAK/OSR1 pathway are localized to the distal nephron. Changes in JAB1 Expression Affect NCC Abundance in HEK293 Cells Disruption of CSN along the Nephron Reduces To determine whether the decrease in NCC abundance in 2 2 NCC Abundance KS-Jab1 / mice is directly caused by Jab1 deletion and not 2 2 Because KS-Jab1 / mice demonstrated activation of the WNK- from systemic effects, we examined the effects of JAB1 expres- SPAK pathway, it might be suspected that NCC protein phos- sion on NCC abundance in vitro using HEK293 cells. First, 2 2 phorylation would also be higher in these mice. In contrast, both to mimic the KS-Jab1 / mice we inhibited endogenous total NCC (tNCC) and phosphorylated NCC (pNCC) at T53 JAB1 expression by using siRNA in cells overexpressing were dramatically lower than in control (Figure 5A). Although NCC (Figure 5C). As shown previously,36 areductionin both total and activated (phosphorylated) forms of NCC JAB1 protein caused an increased in cullin neddylation com- were low, there was a three-fold increase in the ratio of pNCC pared with control siRNA. NCC abundance was dramatically 2 2 to tNCC in KS-Jab1 / mice compared with controls. This sug- reduced in cells transfected with Jab1 siRNA compared with gests that, although the WNK-SPAK pathway may be function- cells containing control siRNA. Next, to see if the opposite ing to activate NCC, a second process, either increased NCC result could be achieved, we transfected NCC together with degradation or decreased NCC synthesis must be present. overexpressed JAB1 (Figure 5D). Cells overexpressing Evidence for destructive effects outside of the DCT is the low JAB1 caused NCC abundance to increase compared with cells aquaporin 2 abundance, as shown by Western blot without transfected JAB1. Doubling the amount of JAB1 transfected into cells caused NCC abundance to trend higher; however, the result Table 1. Plasma and urine electrolytes and renal function was not significant. Together the results in- 2 2 Control KS-Jab1 / Significance P-Value dicate that JAB1 affects NCC through a Plasma WNK-independent pathway. Na+, mM 144.860.75 144.360.76 NS 0.65 K+,mM 3.5560.06 3.2060.07 a 0.004 Disruption of CSN along the Nephron Mg2+ (mg/dl) 1.8460.10 1.7660.11 NS 0.58 Causes Hypokalemia and Electrolyte 2 Cl (mM) 112.761.26 117.561.67 b 0.04 Loss 2 2 iCa2+ (mM) 1.2460.02 1.2660.02 NS 0.55 KS-Jab1 / mice were hypokalemic, hy- 6 6 c , TCO2 (mM) 18.0 0.58 11.8 0.87 0.001 perchloremic, and acidemic (Table 1). 6 6 c , BUN (mg/dl) 28.7 1.15 94.5 13.41 0.001 Additionally, the knockout mice exhibited 6 6 Glucose (mg/dl) 325.5 14.3 288.5 17.8 NS 0.14 Na+ and K+ wasting with hematocrits that Hct (% PCV) 36.760.2 39.261.3 NS 0.09 6 6 tended to be higher, suggesting mild extra- Hb (g/dl) 12.5 0.08 13.3 0.44 NS 0.08 fl AnGap (mM) 18.860.7 19.360.5 NS 0.57 cellular uid volume contraction. The Aldosterone (pg/ml) 466645.4 10316117.1 a 0.001 plasma aldosterone concentration in KS- 2/2 Urine Jab1 mice was twice the value of control b UV (ml/d) 4.7160.50 9.4161.60 0.02 mice (Table 1). Furthermore, telemetric Na+ clearance 0.08860.006 0.13560.013 a 0.009 analysis of BP showed no difference between 2 2 (ml/d per gram body wt) KS-Jab1 / and control mice (Supplemental K+ clearance 3.6860.28 4.9060.40 b 0.03 Figure 4, Table 1). (ml/d per gram body wt) 2+ 6 6 Ca (mg/dl) 2.15 0.63 2.64 0.44 NS 0.53 Heterozygous Jab1 Mice on a High 6 6 Systolic BP (mm Hg) 128.7 1.1 126.3 7.5 NS 0.76 Sodium, Low Potassium Diet Have an Kidney weight/body wt (mg/g) 10.7160.39 15.6961.03 a 0.002 Exaggerated Effect on WNK-NCC Data are presented as mean6SEM. Systolic BP measure is the mean of 1 hour average values during three dark periods. For systolic blood pressure n55. For all other measurements n56. iCa2+,ionized Pathway calcium; TCO2, total carbon dioxide; Hct, hematocrit; PCV, packed cell volume; Hb, hemoglobin; Although the WNK-SPAK/OSR1 pathway 2 2 AnGap, anion gap; UV, urine volume. / a is activated in KS-Jab1 mice, the deple- P,0.01, two-tailed unpaired t test. fl bP,0.05, two-tailed unpaired t test. tion of the extracellular uid volume or hy- cP,0.001, two-tailed unpaired t test. pokalemia might have contributed to the

J Am Soc Nephrol 29: 2627–2640, 2018 Role of the CSN in the Kidney 2635 BASIC RESEARCH www.jasn.org phenotype. To determine whether WNKs are activated by a depletion, and high plasma aldosterone levels may cause an 2 2 compensatory physiologic response, we examined mice with increase in WNK4 activity in KS-Jab1 / mice, the results 2 heterozygous expression of Jab1 (KS-Jab1+/ ). Mice express- indicate that JAB1 deﬁciency does contribute to the activation ing one Jab1 allele showed no phenotypic differences on con- of the WNK-SPAK/OSR1 pathway, which is most likely caused trol diet (data not shown). Additionally, plasma and urine by the substantial decrease in KLHL3. 2 electrolyte levels were similar between KS-Jab1+/ mice and control mice when place on a high sodium, low potassium Chronic Disruption of the CSN in Kidney Leads to diet. However, the high sodium, low potassium diet caused a Tubule Damage more abundant increase in neddylated cullin, WNK4, and CUL3 also facilitates degradation of cyclin-dependent 2 pNCC protein in KS-Jab1+/ mice compared with control kinases, including cyclin E, a protein involved in cell cycle mice (Figure 6). Thus, although the hypokalemia, volume regulation.49 Our laboratory has previously shown that

Figure 6. A high sodium, low potassium diet exaggerates the activation of the WNK-NCC pathway in heterozygous Jab1 mice. Mice containing 2 2 2 only one ﬂoxed Jab1 allele were treated with doxycycline, similar to KS-Jab1 / mice, to generate heterozygous Jab1 mice (KS-Jab1+/ ). Top, protein was analyzed by Western blot with whole kidney lysate for NEDD8, WNK4, NCC, and pNCCT53 in mice on control and high sodium, low 2 potassium diet (HNa/LK). Bottom, quantitative analysis of protein abundance. KS-Jab1+/ mice on control diet showed no differences in protein levels compared with control mice. Placing control mice on a HNa/LK diet caused an increase in neddylated cullin, WNK4, NCC, and pNCCT53 2 compared with control diet. When KS-Jab1+/ mice were fed a HNa/LK diet, neddylated cullin, WNK4, and pNCCT53 protein levels were further increased compared with control mice and the ratio of pNCCT53 to tNCC was signiﬁcantly higher. Data represent individual values as well as mean6SEM relative to controls. Statistical differences were examined by one-way ANOVA with Tukey post hoc analysis.

2636 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018 www.jasn.org BASIC RESEARCH chronic kidney-tubule specific Cul3 deletion led to increased Although this hypothesis has proved controversial,11 genetic cyclin E, and histologic analysis showed tubulointerstitial in- deletion of KLHL3 does lead to a mouse phenotype that re- flammation and fibrosis.37 Similar to chronic Cul3 deletion, sembles FHHt,41 and recent results confirm that low KLHL3 2 2 cyclin E abundance was greater in kidneys from KS-Jab1 / abundance in mice carrying the pathogenic CUL3D9 muta- 2 2 mice than in controls (Figure 7A). Kidneys from KS-Jab1 / tion contributes to the phenotype.42 Here, we tested the hy- mice were heavier and appeared discolored (Table 1), although pothesis that defective CSN action contributes to the FHHt histologic analysis of kidney sections did not show tubule dam- phenotype mice by deleting a component of the key enzyme age (Figure 7B). However, when the period after Jab1 deletion involved in removing NEDD8 from the cullin proteins. The was longer (9 weeks after doxycycline treatment), kidney dam- results confirm a role for deficient deneddylation for the dis- age was present, with increased nuclear staining and dilated ease process, but also provide novel insight into factors regu- tubules. lating NCC abundance, in vivo. To disturb cullin deneddylation, we deleted CSN5, also known as JAB1, in kidney tubules; this deletion renders CSN DISCUSSION ineffective. As expected, CUL3, and likely other cullins, was 2 2 hyperneddylated in kidney tissue from KS-Jab1 / mice. As In cultured cells, the protein produced by the disease-causing predicted, the results also show low KLHL3 abundance, high mutant CUL3D9 does not associate normally with the WNK4 abundance, and a higher ratio of pNCC to tNCC, CSN,11,36 is hyperneddylated,11,36 and degrades KLHL3 compared with control mice. Surprisingly, however, tNCC anomalously.37 On the basis of these findings, we postulated abundance was drastically reduced by approximately 85%, that the resulting low KLHL3 abundance impairs WNK kinase and the mice had polyuria, salt wasting, and hypokalemia. degradation, contributing to the phenotype of FHHt.37 Although these features resemble Gitelman syndrome, the mice were also hyperchloremic and acidemic, indicating a mixed phenotype. The results suggest tubule dysfunction through- out the distal nephron, and more closely resemble another mouse model recently characterized by our laboratory, namely 2 2 KS-Cul3 / mice. 2 2 As shown previously,37 KS-Cul3 / mice also develop polyuria, salt and water wasting, and hypokalemia, with increased WNK abundance and an increased ratio of 2 2 pNCC to tNCC. Similar to KS-Jab1 / mice, chronic Cul3 deletion also led to increased hematocrit and aldosterone levels, a lower abundance of aquaporin 2, and later to renal inflammation and fibrosis. One factor that likely contributed to the kidney damage is the increased abundance of 2 2 2 2 cyclin E in both KS-Cul3 / and KS-Jab1 / mice; activation of this cyclin-dependent kinase favors cell proliferation and potential 2 2 damage.50 Unlike KS-Jab1 / mice, how- 2 2 ever, KS-Cul3 / mice are alkalemic and hypochloremic, and tNCC abundance is unchanged. Figure 7. Chronic nephron-specific Jab1 deletion induces kidney tubule damage. (A) 2/2 2 2 An interesting feature of the KS-Jab1 Immunoblotting was performed on whole kidney lysates from control and KS-Jab1 / 2/2 mice is that CUL3 abundance is low. This mice using antibodies against cyclin E. Cyclin E abundance was higher in KS-Jab1 51 fl fl likely reflects that, as reported previously, mice. (B) Doxycycline or vehicle control was administered to Jab1 / Pax8/LC1 mice neddylated cullins are unstable, and sug- for 3 weeks. Mice were either allowed to recover the normal 3 weeks postdoxycycline treatment, or were euthanized after 9 weeks of recovery (chronic). Kidneys were gests that hyperneddylation contributes harvested for immunohistochemistry and hematoxylin and eosin staining was per- substantially to the low abundance of formed with kidney sections after paraffin embedding. Mice with chronic deletion of CUL3D9 protein that has been described Jab1 developed kidney tubule damage as observed by increased nuclear staining and in models of CUL3D9-related FHHt.11,43 2 2 dilated tubules. Interestingly, the KS-Jab1 / mice also

J Am Soc Nephrol 29: 2627–2640, 2018 Role of the CSN in the Kidney 2637 BASIC RESEARCH www.jasn.org exhibited lower tNCC abundance despite the elevated WNK kinase abundance. This may reflect a previously unreported direct effect of JAB1 disruption on NCC (as suggested by our results in HEK293 cells), which would not be present in 2 2 KS-Cul3 / mice. CSNdisruptiondidnotresultinmonotoniceffectsonall CUL3 BTB-Kelch substrate adaptors, as the effects on KLHL3 and Keap1 were different. This selectivity of substrate adaptor degradation by CSN inhibition has been shown in multiple in vitro and in vivo studies.30–35 A current model for CSN function is that it competitively binds to the neddylated-CRL when substrates are low and prevents erroneous autoubiquitylation of substrate adaptors and the cullin scaffold protein.33,52 Thus, hyperneddylation can disrupt regulated degradation of substrates by abnormally targeting its substrate adaptor. The results here are consistent with previous work in cultured cells using CSN inhibitors; KLHL3 was degraded36 and Keap1 abundance increased.53 Furthermore, CUL3D9 showed a sim- Figure 8. Simplified schematic of the effects of nephron-specific 36,37 ilar effect on the substrate adaptors, both in vitro and in Jab1 deletion on the CUL3-KLHL3-WNK pathway. Cullin-RING 42 D vivo. Because CUL3 9 also has an impaired interaction ubiquitin ligases cycle between an “inactive” unneddylated and with the CSN, the results are consistent with decreased CSN an “active” neddylated state. Left, when in homeostasis, NEDD8 activity as a partial mechanism for the disease. Additionally, (N) is removed from CUL3 by CSN and the substrate, WNK4, is the selectiveness of CSN inactivation to decrease KLHL3 but ubiquitylated and degraded via interaction with CUL3 through not Keap1 abundance may explain why the disease phenotype the adaptor KLHL3, preventing NCC phosphorylation. Right, in 2 2 is only observed in the kidney and smooth muscle.54 Although KS-Jab1 / mice, disturbed interaction of CUL3 with the CSN CUL3 is expressed ubiquitously, KLHL3 is expressed most causes more neddylation of CUL3 and abnormal ubiquitylation abundantly in the kidney and brain.41 Thus, the effects of and degradation of KLHL3, leading to an increase in WNK4 KLHL3 degradation by CUL3D9 would only be observed in abundance. Phosphorylation of NCC is increased through downstream kinases SPAK/OSR1; however, deletion of Jab1 these tissues. “ ” causes a decrease in NCC abundance that is independent from Wepropose that decreased KLHL3 is a second hit required the WNK-SPAK pathway. for FHHt to occur. As noted above, CUL3D9 is unstable and its abundance in vivo is quite low.11,43 Yet, CUL3D9-FHHt is an autosomal dominant disease, meaning that the second WT the low KLHL3 abundance suggests that an important effect of CUL3 allele remains. Ferdaus et al.43 recently showed that the CUL3D9 mutation is to disrupt CUL3 binding to the CSN, CUL3 haploinsufficiency itself does not recapitulate the dis- thus enhancing cullin neddylation, activating KLHL3 degra- ease phenotype. The decreased abundance of CUL3 may, dation, and thereby contributing to the disease. The results therefore, represent a “first factor” in the disease pathogenesis. reveal unexpected complexity in the effects of CRL modula- When Ferdaus et al. introduced CUL3D9onahaploinsuffi- tion of kidney structure and function. The dissociation be- cient background, the disease phenotype was reproduced. tween WNK kinase abundance and NCC activity observed This indicates clearly that a dominant effect of CUL3D9is here suggests that small molecule strategies to recapitulate also essential, as a second hit. Previous results suggest that this effect in humans might be useful to treat hypertension the second consequence of CUL3D9involvesenhanced or edema. KLHL3 degradation, leading to KLHL3 deficiency. In a cell culture model that expressed both WT and CUL3D9, depletion of KLHL3, driven by CUL3D9-mediated degradation, prevented the remaining WT CUL3 from effectively degrading ACKNOWLEDGMENTS WNK4 (it exerted a dominant-negative effect).36 The results here show that the KLHL3 degradation from CUL3D9maybe We thank Dr. Guang Zhou from Case Western Reserve University for fl fl caused by its impaired interaction with the CSN. providing the Jab1 x/ x mice, and Dr. Jim McCormick and Dr. Jeffrey Thus, our results show that JAB1 dysfunction in vivo Singer for the helpful discussions, as well as Lauren Miller for breeding causes a mixed phenotype that in some respects resembles and genotyping the mice. We thank the Oregon Health and Science 2 2 KS-Cul3 / mice, with polyuria, salt wasting, hypokalemia, University Histopathology Core Facility for performing hematoxylin 2 2 and kidney tubule damage. The KS-Jab1 / mice also have and eosin staining. unique features, including activation of the WNK-SPAK path- C-L.Y., D.H.E., and R.J.C. conceived the study. R.J.C. performed way with decreased NCC abundance (Figure 8). Furthermore, most of the experiments. J.S., C.A.C., J.W.N., and B.D.K.G. helped

2638 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018 www.jasn.org BASIC RESEARCH

fl fl with some of the experiments. R.P. generated Jab1 x/ x mice. R.J.C., 14. Wimuttisuk W, West M, Davidge B, Yu K, Salomon A, Singer JD: Novel C-L.Y., and D.H.E. analyzed data. R.J.C. and D.H.E. wrote the paper. Cul3 binding proteins function to remodel E3 ligase complexes. BMC R.J.C., C-L.Y., and D.H.E. edited the paper. Cell Biol 15: 28, 2014 15. Petroski MD, Deshaies RJ: Function and regulation of cullin-RING This work was supported by National Institutes of Health ubiquitin ligases. Nat Rev Mol Cell Biol 6: 9–20, 2005 (NIH) grants R01 DK51496 (D.H.E. and C-L.Y.) and T32 DK067864 16. Wu G, Peng JB: Disease-causing mutations in KLHL3 impair its effect on (D.H.E.), as well as by the merit review grant 1I01BX002228-01A1 WNK4 degradation. FEBS Lett 587: 1717–1722, 2013 from the Department of Veteran Affairs (D.H.E.), and by American 17. Saha A, Deshaies RJ: Multimodal activation of the ubiquitin ligase SCF – Heart Association grant 16POST3064003 and NIH grant F32 by Nedd8 conjugation. Mol Cell 32: 21 31, 2008 18. Pan ZQ, Kentsis A, Dias DC, Yamoah K, Wu K: Nedd8 on cullin: Building DK112531 (R.J.C.). an expressway to protein destruction. Oncogene 23: 1985–1997, 2004 19. Boh BK, Smith PG, Hagen T: Neddylation-induced conformational control regulates cullin RING ligase activity in vivo. J Mol Biol 409: 136– 145, 2011 DISCLOSURES 20. Pintard L, Kurz T, Glaser S, Willis JH, Peter M, Bowerman B: Neddyla- None. tion and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans. Curr Biol 13: 911–921, 2003 21. Chung D, Dellaire G: The role of the COP9 signalosome and neddylation REFERENCES in DNA damage signaling and repair. Biomolecules 5: 2388–2416, 2015 22. Wolf DA, Zhou C, Wee S: The COP9 signalosome: An assembly and 1. 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2640 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 2627–2640, 2018