COP9 signalosome deletion promotes renal injury and distal convoluted tubule remodeling

Abstract

Cullin-RING ligases are a family of E3 ubiquitin ligases that control cellular processes through regulated degradation. Cullin 3 targets with-no-lysine kinase 4 (WNK4), a kinase that activates the Na⁺-Cl⁻ cotransporter (NCC), the main pathway for Na⁺ reabsorption in the distal convoluted tubule (DCT). Mutations in the cullin 3 gene lead to familial hyperkalemic hypertension by increasing WNK4 abundance. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) regulates the activity of cullin-RING ligases by removing the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Genetic deletion of the catalytically active CSN subunit, Jab1, along the nephron in mice (KS-Jab1^−/−) led to increased WNK4 abundance; however, NCC abundance was substantially reduced. We hypothesized that the reduction in NCC resulted from a cortical injury that led to hypoplasia of the segment, which counteracted WNK4 activation of NCC. To test this, we studied KS-Jab1^−/− mice at weekly intervals over a period of 3 wk. The results showed that NCC abundance was unchanged until 3 wk after Jab1 deletion, at which time other DCT-specific proteins were also reduced. The kidney injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin demonstrated kidney injury immediately after Jab1 deletion; however, the damage was initially limited to the medulla. The injury progressed and expanded into the cortex 3 wk after Jab1 deletion coinciding with loss of the DCT. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury that leads to hypoplasia of the DCT.

NEW & NOTEWORTHY Cullin 3 (CUL3) targets with-no-lysine-kinase 4 (WNK4), which activates Na⁺-Cl⁻ cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Renal-specific genetic deletion of the constitutive photomorphogenesis 9 signalosome, an upstream regulator of CUL3, resulted in a reduction of NCC due to DCT hypoplasia, which coincided with cortical kidney injury. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury leading to hypoplasia of the DCT.

INTRODUCTION

Cullin-RING ligases (CRLs) attach a 76-amino acid ubiquitin protein to target substrates marking them for degradation (1). This is an important process in the maintenance of all cells to eliminate unwanted proteins. CRLs normally undergo cycling of neddylation and deneddylation (2, 3). Neddylation, a process in which a neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) ubiquitin-like protein is covalently attached to the cullin scaffold protein, initiates ubiquitin transfer to a target protein. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) is a deneddylase that removes NEDD8 from CRLs, stopping ubiquitin ligase activity. This cycling of neddylation/deneddylation is essential for both stability and activity of the CRL. Deneddylation is important for normal CRL function because it permits adaptor and substrate recycling and prevents anomalous self-ubiquitylation that can destabilize the CRL complex (4, 5). The CSN is a multisubunit protein containing eight subunits. All subunits, including the catalytically active subunit JAB1, are expected to be expressed in most, if not all, cells. Genetic deletion of these subunits causes embryonic lethality in mice (6, 7). Although disease-causing mutations in the CSN have not been reported, disruption of CRLs or their associated substrate adaptors/receptors have been implicated in renal diseases. Mutations in the cullin 2 substrate receptor von Hippel-Lindau can cause clear cell renal cell carcinoma (8); moreover, renal-specific deletion of cullin 3 (CUL3) in mice causes renal fibrosis and tubular injury (9, 10).

The Na⁺-Cl⁻ cotransporter (NCC) is the main pathway for NaCl reabsorption and blood pressure regulation in the distal convoluted tubule (DCT) of the kidney. Activation of NCC occurs via phosphorylation by with-no-lysine kinase 4 (WNK4), which phosphorylates the intermediary protein STE20/SPS1-related proline/alanine-rich kinase (SPAK; 11, 12). Thus, activation of WNKs leads to increased upregulation of NCC. CRLs regulate WNK protein abundance (13, 14). Our previous work, however, in which CRL function was disrupted throughout the nephron, resulted in a surprising disassociation between WNK and NCC expression patterns (15). In that study, we examined mice in which the main catalytic subunit of the CSN, JAB1, was deleted in kidney epithelial cells (KS-Jab1^−/−). The results showed that WNK4 abundance was significantly higher, but unexpectedly NCC abundance did not parallel the change in WNK4 and instead was substantially reduced. Alterations in WNK4 and NCC abundance are typically coupled together. Furthermore, phosphorylation of NCC inhibits its ubiquitylation and subsequent removal from the plasma membrane (16). Our previous results suggest that the decrease in NCC is not a regulatory effect but could be caused by morphological changes to the DCT. KS-Jab1^−/− mice had elevated blood urea nitrogen (BUN), indicating renal injury, and mice with chronic deletion of Jab1 developed chronic kidney disease with interstitial fibrosis and tubule atrophy. The results suggested that deletion of Jab1 causes kidney injury progression over time. Thus, the disassociation of WNK4 and NCC may result from progressive kidney injury. Therefore, in the present study, we examined the expression of NCC and other segment-specific proteins, as well as tubule injury markers, in KS-Jab1^−/− mice at multiple time points after Jab1 deletion. We hypothesized that the decrease in NCC is caused by hypoplasia of the DCT, which is associated with cortical tubular damage.

The results revealed that NCC is one of the multiple DCT-specific proteins that are reduced 3 wk after Jab1 deletion, indicating DCT hypoplasia. Moreover, mice developed a renal injury that progressed from the medulla to the cortex over time and paralleled the decrease in NCC protein abundance and reduction in DCT mass. The data revealed that nephron-specific Jab1 deletion causes a complex injury progression. Interestingly, sufficient damage to the cortex over time was associated with alterations in tubule morphology leading to hypoplasia of the DCT.

MATERIALS AND METHODS

Antibodies

Antibody sources, species, and dilutions are shown in Table 1.

Table 1.

Antibodies used for Western blot analysis and immunofluorescent staining

Target	Figures	Source	Id	Species	Application	Dilution
JAB1	Fig. 1	Santa Cruz Biotechnology	sc-9074	Rabbit	WB	1:2,000, o/n
NEDD8	Fig. 1	Cell Signaling	No. 2754	Rabbit	WB	1:1,000, o/n
CUL3	Fig. 1	Cell Signaling	No. 2759	Rabbit	WB	1:1,000, o/n
NCC	Figs. 2, 3, and 8	Ellison laboratory		Rabbit	WB; IFP	1:6,000, 1 h; 1:1,000, o/n
pNCCT53	Figs. 2, 3, and 4	Ellison laboratory		Rabbit	WB; IFC	1:2,000, o/n; 1:100, 4 days
WNK4	Fig. 2	Ellison laboratory		Rabbit	WB	1:1,000, o/n
pSPAK	Fig. 2	Millipore	07-2273	Rabbit	WB	1:1,000, o/n
KLHL3	Fig. 2	Proteintech	16951-1-AP	Rabbit	WB	1:500, o/n
Parvalbumin	Figs. 3, 7, and 9	Swant	GP72	Guinea pig	IFP	1:200, 2 h
Calbindin	Figs. 3 and 5	Swant	CB300	Mouse	WB; IFP	1:500, o/n; 1:200, o/n
NHE3	Fig. 5 and Supplemental Fig. S1	Millipore	AB3085	Rabbit	WB; IFF	1:1,000, o/n; 1:100, o/n
NKCC2	Fig. 5 and Supplemental Fig. S1	StressMarq	SPC-401	Rabbit	WB; IFP	1:1,000, o/n; 1:100, o/n
AQP3	Supplemental Fig. S1	StressMarq	SPC-504	Rabbit	IFF	1:100, o/n
AQP4	Fig. 5	StressMarq	SPC-505	Rabbit	WB	1:1,000, o/n
KIM-1	Figs. 7 and 8	R&D Systems	AF-1817	Goat	WB; IFP/IFF	1:500, o/n; 1:200, 2 h
NGAL	Fig. 7	Abcam	Ab63929	Rabbit	WB	1:1,000, o/n
Ki-67	Fig. 9	Vector Laboratories	VP-RM04	Rabbit	IFP/IFF	1:200, 2 h
LTL	Figs. 8 and 9	GlycoMatrix	21761116		IFP/IFF	1:250, 2 h

AQP, aquaporin; CUL3, cullin 3; IFC, immunofluorescence clearing; IFF, immunofluorescence frozen; IFP, immunofluorescence paraffin; KIM-1, kidney injury molecule-1; KLHL3, Kelch-like family member 3; LTL, Lotus tetragonolobus lectin; NCC, Na⁺-Cl⁻ cotransporter; NEDD8, neural precursor cell expressed developmentally downregulated protein 8; NGAL, neutrophil gelatinase-associated lipocalin; NHE3, Na⁺/H⁺ exchanger 3; NKCC2, Na⁺-K⁺-2Cl⁻ cotransporter; o/n, overnight; pNCC, phosphorylated NCC (at Thr⁵³); pSPAK, phosphorylated STE20/SPS1-related proline/alanine-rich kinase; WB, Western blot; WNK4, with-no-lysine kinase 4.

Animals

Animal experiments were approved by the Oregon Health and Science University Institutional Animal Care and Use Committee. All mice were housed in a temperature-controlled, 12:12-h light-dark cycle room with ad libitum access to food and water. Both male and female mice were used to examine sex as a biological variable. KS-Jab1^−/− mice were generated using the Pax8-rtTA/LC1 system (17, 18). Jab1^flx/flx mice were generated by Ruggero Pardi (19) and generously provided by Dr. Guang Zhou. Jab1^flx/flx mice were crossed with Pax8/LC1 mice to generate Jab1^flx/flx-Pax/LC1 mice. Cre recombinase expression was induced by administration of doxycycline (2 mg/mL with 5% sucrose) in the drinking water for 3 wk to generate renal epithelium-specific Jab1^−/− mice (referred to as KS-Jab1^−/− mice). Control mice were littermates that received 5% sucrose water for 3 wk. Mice were examined at weekly intervals after the end of doxycycline treatment through 3 wk (the groups were referred to as 0-wk, 1-wk, 2-wk, and 3-wk Jab1^−/−).

PCR Genotyping

Genomic DNA extracts were prepared from tail snips by heating overnight at 55°C in 300 μL digestion solution containing 5 mM EDTA (Thermo Fisher Scientific, Waltham, MA), 200 mM NaCl (Thermo Fisher Scientific), 100 mM Tris (pH 8.0, Millipore Sigma, Burlington, MA), 0.2% SDS (Millipore Sigma), and 0.4 mg/mL proteinase K (Viagen Biotech, Los Angeles, CA) followed by ethanol precipitation. The following primers were used: Pax8, forward 5′- CCATGTCTAGACTGGACAAGA-3′ and reverse 5′- CAGAAAGTCTTGCCATGACT-3′; CRE, forward 5′- TTTCCCGCAGAACCTGAACCTGAAGAT-3′ and reverse 5′- TCACCGGCATCAACGTTTTCTT-3′; and Jab1, forward 5′- GGTCAGAAAGCTAGGCCTAAGAAGG-3′ and reverse 5′- GGCATGCATCACCATTTTCAGTAG-3′.

Metabolic Cages for Urine Analysis

Mice were placed in metabolic cages for 24-h urine collection and analysis of food and water consumption. To prevent urine contamination, mice were given a gel diet. Mice were acclimated in the metabolic cages for 3 days before analysis. Urine was collected under mineral oil, and urine K⁺ and Na⁺ were determined by flame photometry.

Kidney Perfusion, Removal, and Blood Analysis

Mice were anesthetized with a ketamine-xylazine-acepromazine cocktail (50/5/0.5 mg/kg). The right kidney was tied off, removed, and flash frozen in liquid nitrogen for isolation of RNA or protein. Blood was collected via cardiac puncture and transferred into heparinized tubes; 80 μL were loaded into a Chem8+ cartridge for electrolyte measurement by an i-STAT analyzer (Abbot Point of Care, Princeton, NJ). The left kidney was perfusion fixed with retrograde abdominal aortic perfusion of 3% paraformaldehyde in PBS (pH 7.4), removed, and stored in 3% paraformaldehyde for clearing experiments.

Kidney Clearing via Ethyl Cinnamate

Paraformaldehyde perfusion-fixed kidneys were cut into 1-mm-thick kidney slices. The kidneys were postfixed for at least 3 days at room temperature. Slices were washed in 1× wash buffer (Envision Flex Wash Buffer, Cat. No. K800721-2, Agilent) overnight at room temperature on an orbital shaker. Antigen retrieval was performed by placing slices in antigen retrieval solution (Cat. No. H-3300, Vector Laboratories) for 1 h at 92°C–98°C. After being cooled to room temperature and washed overnight in wash buffer at room temperature, slices were immersed with NCC primary antibody in antibody diluent (Cat. No. S0809, Dako) at room temperature for 4 days. Next, slices were washed overnight at room temperature with wash buffer and immersed in a secondary antibody solution made of antibody diluent. The slices were washed again overnight and then dehydrated in 100% ethanol for 2 h at room temperature (with one change to fresh ethanol at 1 h). Afterward, slices were immersed in ethyl cinnamate (Cat. No. 112372, Sigma-Aldrich) and gently agitated overnight at room temperature under light protection. The now translucent slices were placed in a glass-bottom culture dish and bathed in ethyl cinnamate. Imaging was performed immediately on a confocal microscope (Zeiss LSM 880 with Airyscan, ×10 objective lens).

Immunofluorescence

Both male and female mice were analyzed for all immunofluorescent staining microscopy. Images shown are representative of both sexes.

Frozen Tissue

After paraformaldehyde perfusion fixation, the kidneys were cut in half and cryopreserved in 800 mOsm/kgH₂O sucrose in PBS overnight before being embedded in the Tissue-Tek OCT compound (Sakura Finetek, Torrance, CA). The tissue was sliced onto slides (5-µm sections) and stained as follows. Slides were incubated with 0.5% Triton X-100 in PBS for 30 min. Sections were then blocked with 5% milk in PBS for 30 min followed by an incubation with primary antibody and then diluted in blocking buffer for 1 h at room temperature or overnight at 4°C (antibody details are shown in Table 1). Sections were incubated with fluorescent dye-conjugated secondary antibody (diluted in blocking buffer) for 1 h at room temperature before being mounted with Prolong Diamond Antifade Mountant with DAPI (Invitrogen, Carlsbad, CA).

Paraffin-Embedded Tissue

Kidneys were removed from anesthetized mice, cut in half, and placed in 10% neutral buffer formalin overnight. They were then transferred to 70% ethanol before being embedded in paraffin by the Oregon Health and Science University Histopathology Core Facility and sectioned onto slides (5-µm sections) for staining. Paraffin was removed with kidney tissue was rehydrated with successive immersion for 5 min in 100% xylene and 100%, 90%, and 70% ethanol solutions. Antigen retrieval was performed by microwaving tissue in a citrate-based antigen unmasking solution (Cat. No. H-3300, Vector Laboratories). Immunostaining was performed with primary antibody diluted in 5% BSA-PBS for 2 h at room temperature (antibody details are shown in Table 1) followed by an incubation with fluorescent dye-conjugated secondary antibody diluted in 5% BSA-PBS for 1 h at room temperature before being mounted with Prolong Diamond Antifade Mountant with DAPI.

Imaging

For both frozen and paraffin-embedded tissue, images were captured using a Zeiss AXIO Imager M2 microscope and AxioVision software or a Keyance BZ-X810 fluorescence microscope. Image processing was completed using ImageJ software (National Institutes of Health). Ki-67 was quantified by manually counting the number of Ki-67-positive cells in the whole image of the cortex or medulla captured using a ×10 objective. All groups contained an n value of 3.

Analysis of Protein by Western Blot Analysis

Kidneys were harvested, flash frozen in liquid nitrogen, and stored at −80°C. The whole kidney was homogenized in a buffer containing enzyme inhibitors with 1 mM DTT and 1 mM PMSF. Protein samples were separated by electrophoresis on 4%–15% Criterion TGX stain-free gels (Bio-Rad Laboratories, Hercules, CA) and transferred to Immobilon-P PVDF membranes (EMD Millipore, Billerica, MA) using the Trans-Blot Turbo transfer system (Bio-Rad Laboratories). Stain-free imaging was used as a total protein loading control, as previously described (15). Membranes were blocked with 5% nonfat milk in PBS-Tween for 1 h at room temperature before incubation with primary antibody in blocking buffer for 1 h at room temperature or overnight at 4°C (details are shown in Table 1). The appropriate horseradish peroxidase-conjugated secondary antibody in blocking buffer was added to membranes for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence (Western Lightning Plus-ECL, Perkin-Elmer, Waltham, MA), and proteins were visualized using the PXi digital imaging system (Syngene, Frederick, MA).

Statistics

Data are presented as individual values as well as means ± SE. Differences between the experimental group and control were determined using one-way ANOVA with Dunnett’s multiple comparisons post hoc analysis. Differences between experimental groups were determined using one-way ANOVA with Tukey’s multiple comparisons post hoc analysis. P values of <0.05 were considered significant. Statistical analysis was performed using GraphPad Prism 8.3 software (GraphPad Software, San Diego, CA).

RESULTS

CUL3 Neddylation Is Enhanced After Jab1 Deletion

Kidney-specific Jab1 knockout (KS-Jab1^−/−) mice were generated and have been previously characterized by our laboratory (15). Jab1^flx/flx mice were crossed with Pax8/LC1 mice, and the resulting homozygous Jab1^flx/flx and positive Pax8/LC1 mice were given doxycycline. It was determined that 3 wk of doxycycline treatment was necessary for consistent and complete knockdown of JAB1. Immunofluorescent staining for JAB1 showed lower expression after doxycycline administration (15). Here, we examined the effects of Jab1 deletion at multiple time points after doxycycline treatment (Fig. 1A) starting at the end of the 3-wk doxycycline treatment (0-wk Jab1^−/−) and weekly through 3 wk (1-wk, 2-wk, and 3-wk Jab1^−/−). The protein abundance of JAB1, examined by Western blot analysis, was ∼75% lower in 0-wk Jab1^−/− mice and remained down through 3 wk (Fig. 1B). In addition, expression of Jab1 mRNA, as examined by quantitative PCR, was significantly lower at all time points after Jab1 deletion (Fig. 1C). To examine if Jab1 deletion affected CRL neddylation, we probed with antibodies against NEDD8 and CUL3. NEDD8 abundance at the molecular weight of cullin (indicative of neddylated cullin) was significantly higher and unneddylated CUL3 (lower band) was significantly lower in all groups of KS-Jab1^−/− mice (Fig. 1B). Neddylated CUL3 (top band) trended higher in 0-wk Jab1^−/− mice and was significantly higher in 1-wk, 2-wk, and 3-wk Jab1^−/− mice.

Figure 1.

Cullin 3 (CUL3) neddylation is enhanced after Jab1 deletion. A: diagram of the methods used to induce deletion of Jab1 in the kidney and examine mice at multiple time points after knockdown. Mice were given doxycycline in drinking water for 3 wk. The mice were either immediately analyzed (0-wk Jab1^−/− group) or given normal drinking water for a period of 1, 2, or 3 wk before analysis and kidney collection. Control mice received sucrose in drinking water for 3 wk. B: Western blot of whole kidney lysates from control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion. Immunoblot analysis for antibodies against JAB1 and neural precursor cell expressed developmentally downregulated protein 8 (NEDD8) showed an immediate decrease in JAB1 and increase in neddylated cullin protein (NEDD8 at the molecular weight of cullin) abundance at the 0-wk time point that was sustained through the 3-wk period. Immunoblot analysis for antibodies against CUL3 showed a neddylated (top) and unneddylated (bottom) band. Unneddylated CUL3 was decreased at all time points after Jab1 deletion, and neddylated CUL3 was increased in 1-wk, 2-wk, and 3-wk Jab1^−/− mice. n = 4 mice for control, 5 for 0-wk Jab1^−/−, 4 for 1-wk Jab1^−/−, 4 for 2-wk Jab1^−/−, and 5 for 3-wk Jab1^−/−. C: quantitative PCR showed that Jab1 mRNA expression was reduced after doxycycline treatment. n = 5 mice for all groups. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Dunnett’s post hoc analysis. **P < 0.01 vs. control and ***P < 0.001 vs. control. F, female; M, male.

NCC Abundance Is Reduced 3 wk After Jab1 Deletion

We next examined the effect of Jab1 deletion over time on the WNK4-SPAK-NCC pathway. WNK4 abundance was progressively increased after doxycycline treatment (Fig. 2). The abundance of phosphorylated (p)SPAK trended higher at all time points after Jab1 deletion and was significantly higher in 1-wk and 2-wk Jab1^−/− mice. pNCC trended higher in 0-wk Jab1^−/− mice and peaked in 1-wk Jab1^−/− mice, which was significantly higher than controls (Fig. 2). The abundance of pNCC began trending lower in 2-wk Jab1^−/− mice compared with 1-wk Jab1^−/− mice and was similar to control levels 3 wk after Jab1 deletion (P < 0.05 vs. 1-wk Jab1^−/− mice). The drop in pNCC abundance paralleled the reduction in total NCC. There was no difference in NCC abundance in 0-wk, 1-wk, and 2-wk Jab1^−/− mice compared with controls (Fig. 2). Similar to previous work (15), 3-wk Jab1^−/− mice had a significant reduction in NCC abundance. Furthermore, the ratio of pNCC to NCC increased 1 wk after the end of doxycycline administration and was still significantly higher even when total NCC abundance was reduced in 3-wk Jab1^−/− mice. The data demonstrate that although the WNK4-SPAK-NCC pathway was activated, there was a substantial decrease in NCC abundance 3 wk after Jab1 deletion.

Figure 2.

Na⁺-Cl⁻ cotransporter (NCC) abundance is reduced 3 wk after Jab1 deletion. Western blot analysis of whole kidney lysates from control and KS-Jab1^−/− mice was performed at weekly intervals after Jab1 deletion. With-no-lysine kinase 4 (WNK4) abundance was elevated at all time points after Jab1 deletion. Immunoblot analysis using an antibody against phosphorylated STE20/SPS1-related proline/alanine-rich kinase (pSPAK) and phosphorylated oxidative stress response 1 (pOSR1) showed that pSPAK (top band) was significantly higher in the 1-wk and 2-wk Jab1^−/− mice. Phosphorylated NCC (pNCC) abundance trended higher at 0 wk after Jab1 deletion and was significantly higher at 1 wk. In 3-wk Jab1^−/− mice, pNCC was significantly lower than the 1-wk time point. Total NCC abundance was not significantly different compared with controls at the 0-wk, 1-wk, or 2-wk time points. At the 3-wk time point, NCC abundance was lower than controls. The ratio of pNCC to NCC was calculated and showed higher levels at 1-wk, 2-wk, and 3-wk time points compared with controls. n = 4 mice for control, 5 for 0-wk Jab1^−/−, 4 for 1-wk Jab1^−/−, 4 for 2-wk Jab1^−/−, and 5 for 3-wk Jab1^−/−. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Tukey’s or Dunnett’s post hoc analysis. *P < 0.05 vs. control, **P < 0.01 vs. control. ***P < 0.001 vs. control, and #P < 0.05 vs. 1-wk Jab1^−/−. F, female; M, male.

Loss of NCC In 3-wk Jab1^−/− Mice Is Caused by DCT Remodeling

Normally, the expression of NCC parallels its phosphorylation status; however, 3 wk after Jab1 deletion, NCC abundance was reduced, even though the pNCC-to-NCC ratio was elevated (Fig. 2). This disassociation between pNCC and NCC suggests that NCC is not decreased via normal regulation but is being reduced via an unknown mechanism. Therefore, we attempted to identify other factors associated with the reduction in NCC and whether this was specific to NCC. To further examine the changes in NCC, we used immunofluorescence. Staining for NCC showed changes in expression that paralleled the Western blot data; NCC was not drastically reduced until the 3-wk time point (Fig. 3A). Interestingly, although the number of NCC-positive tubules was lower in 3-wk Jab1^−/− mice, the staining intensity did not appear to diminish over time. NCC is commonly used as a marker for the DCT; therefore, the results suggest that NCC may be downregulated due to a loss of DCT mass. To test this, we stained for an early DCT (DCT1)-specific protein, parvalbumin, and a late DCT (DCT2) marker, calbindin [calbindin is also expressed in the connecting tubule (CNT) and cortical collecting duct (CCD)]. Staining for parvalbumin showed similar levels in 0-wk and 1-wk Jab1^−/− mice compared with controls (Fig. 3B). In 2-wk Jab1^−/− mice, parvalbumin staining started to decline in both intensity and number of positively stained tubules, and in 3-wk Jab1^−/− mice, parvalbumin expression was almost completely absent. Calbindin showed less staining in 2-wk Jab1^−/− mice throughout the cortex of the kidney, but there was no reduction in staining intensity (Fig. 3C). In 3-wk Jab1^−/− mice, there was a major reduction in calbindin expression. Next, we costained pNCC together with parvalbumin and calbindin (Fig. 3D). The results showed a uniform pattern of all three proteins throughout the cortex in control mice. In 3-wk Jab1^−/− mice, there were areas throughout the cortex where pNCC, parvalbumin, and calbindin expression was either absent or substantially diminished. All pNCC-positive tubules in control mice were either parvalbumin or calbindin positive, whereas, in 3-wk Jab1^−/− mice, there were pNCC-positive tubules that were both parvalbumin and calbindin negative. Upon closer examination, some of these tubules contained cells with low parvalbumin expression, indicating that these are DCT1 segments.

Figure 3.

Jab1 deletion causes a time-dependent decrease in distal convoluted tubule (DCT)-specific proteins. Immunofluorescent staining of kidney sections for the DCT-specific proteins Na⁺-Cl⁻ cotransporter [NCC; early DCT (DCT1) and late DCT (DCT2); A] and parvalbumin (Parv; DCT1; B) as well as the DCT2/connecting tubule/cortical collecting duct protein calbindin (Calb; C) in control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion. In 2-wk Jab1^−/− mice, the number of tubules with NCC and calbindin staining and the intensity of parvalbumin staining were diminished. In 3-wk Jab1^−/− mice, the number of NCC- and calbindin-positive tubules were prominently reduced and parvalbumin staining was nearly absent. D: high-magnification images of phosphorylated NCC (pNCC), parvalbumin, and calbindin in control and 3-wk Jab1^−/− mice. In 3-wk Jab1^−/− mice, there were cortical sections with a reduction in the expression of pNCC, parvalbumin, and calbindin. n = 3 mice for all groups.

To better examine and quantify the changes to DCT structure over time, we used immunofluorescent staining coupled with tissue clearing to examine changes to whole DCT segments. Kidney slices from control, 0-wk and 3-wk Jab1^−/− mice were incubated with pNCC antibodies, the tissue was “cleared” using ethyl cinnamate, and a three-dimensional image was attained by capturing multiple z-stacks (Fig. 4). Quantitative analysis of DCT length showed an ∼33% decrease in 3-wk Jab1^−/− mice compared with control and 0-wk Jab1^−/− mice. In addition, the number of pNCC-positive tubules was decreased by ∼33% in 3-wk Jab1^−/− compared with control and 0-wk Jab1^−/− mice. Together, the data suggest remodeling of the distal nephron in which the DCT is reduced in both length and tubule segment quantity.

Figure 4.

Jab1 deletion causes remodeling of the distal convoluted tubule (DCT). Top: visualization of phosphorylated Na⁺-Cl⁻ cotransporter (pNCC)-positive DCT tubules in cleared kidney sections of control, 0-wk, and 3-wk Jab1^−/− mice. Kidney sections were cleared and labeled with pNCC antibodies to distinguish the DCT. Two-dimensional snapshot of a three-dimensional rendering of multiple z-stack images at low-power (top images) and high-power (bottom images) magnification are shown. Images were quantified by counting the number of pNCC positive tubules per field and by measuring tubule length. Bottom: quantification of DCT length and number of tubules showed no difference between 0-wk KS-Jab1^−/− and control mice; however, 3-wk KS-Jab1^−/− mice had a significantly lower number of tubules, and the tubules were significantly shorter than control and 0-wk KS-Jab1^−/− mice. n = 3 mice for all groups. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Tukey’s or Dunnett’s post hoc analysis.

Nephron Remodeling in 3-wk Jab1^−/− Mice Occurs Only in the Distal Nephron

To determine if tubule remodeling in KS-Jab1^−/− mice was specific to the DCT, we examined other segment-specific proteins. Western blot analysis of calbindin showed a significantly lower protein abundance in 3-wk Jab1^−/− mice, similar to NCC (Fig. 5). The abundance of proximal tubule-specific Na⁺/H⁺ exchanger 3 (NHE3) was unchanged at all time points after Jab1 deletion (Fig. 5). Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2), which is expressed in the thick ascending limb (TAL), trended higher in 0-wk Jab1^−/− mice and peaked in 1-wk Jab1^−/− mice, which was significantly higher than controls (Fig. 5). After the 1-wk time point, NKCC2 abundance declined and was similar to controls in 3-wk Jab1^−/− mice. Aquaporin (AQP)4 abundance, expressed in principal cells of the collecting duct (CD), showed a decrease in protein abundance in 1-wk Jab1^−/− mice that was sustained through the 3-wk time point (Fig. 5). In addition, immunofluorescent staining for NHE3 (Supplemental Fig. S1A; see https://doi.org/10.6084/m9.figshare.19491800.v1), NKCC2 (Supplemental Fig. S1B), and AQP3 (Supplemental Fig. S1C) showed similar expression patterns in all groups of mice. The results demonstrate that the changes in nephron structure found in KS-Jab1^−/− mice were most apparent in the DCT. The reduction in calbindin (via Western blot analysis and immunofluorescence) and AQP4 (via Western blot analysis) protein abundance suggest that the CNT and CCD segments may also atrophy due to Jab1 deletion; however, the lack of a significant reduction in NHE3 and NKCC2 (via Western blot analysis and immunofluorescence) and AQP3 (via immunofluorescence) suggest that this remodeling is specific to the cortical distal nephron.

Figure 5.

Nephron remodeling is limited to the distal nephron. Western blot analysis of whole kidney lysates for calbindin, Na⁺/H⁺ exchanger 3 (NHE3), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2), and aquaporin 4 (AQP4) from control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion is shown. Immunoblot analysis with antibodies against calbindin showed a significant decrease in band intensity in 2-wk and 3-wk Jab1^−/− mice compared with controls. Immunoblot analysis for NHE3 showed no difference in abundance in KS-Jab1^−/− mice compared with control mice. Immunoblot analysis for NKCC2 showed an increase in abundance in 1-wk Jab1^−/− mice compared with control mice. In 3-wk Jab1^−/− mice, NKCC2 abundance was significantly lower than 1-wk Jab1^−/− mice and similar to control mice. Immunoblot analysis for AQP4 showed a decrease in abundance in 1-wk, 2-wk, and 3-wk Jab1^−/− mice compared with control mice. n = 4 mice for control, 5 for 0-wk Jab1^−/−, 4 for 1-wk Jab1^−/−, 4 for 2-wk Jab1^−/−, and 5 for 3-wk Jab1^−/−. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Tukey’s or Dunnett’s post hoc analysis. *P < 0.05 vs. control, **P < 0.01 vs. control, ***P < 0.001 vs. control, and #P < 0.05 vs. 1-wk Jab1^−/− mice. F, female; M, male.

Jab1 Deletion Induces Renal Injury That Initiates in the Medulla and Progresses Into the Cortex

We postulated that the reduction in NCC and distal nephron remodeling in 3-wk Jab1^−/− mice was caused by renal injury. Previous work showed that in mice with short-term Jab1 deletion (3 wk), BUN was threefold higher than controls, and long-term deletion of Jab1 (9 wk) caused interstitial fibrosis and tubule atrophy (15). Here, analysis of 24-h urine volume in KS-Jab1^−/− mice showed that all groups had a similar urine output that was approximately, about twofold higher than controls, although only the 2-wk group was significantly higher (Fig. 6A). Analysis of the effects of Jab1 deletion on solute excretion showed an increase in osmolar excretion in 1-wk and 2-wk Jab1^−/− mice (Fig. 6C). This correlated with an increase in Na⁺ excretion at the same time points (Fig. 6D). K⁺ excretion was also significantly elevated in 2-wk Jab1^−/− mice (Fig. 6E). These data indicate a solute diuresis that is most apparent in 2-wk Jab1^−/− mice. BUN trended higher in 1-wk Jab1^−/− mice and was significantly higher than controls at the 2-wk and 3-wk time points (Fig. 6F). Kidney weight was significantly higher in 1-wk and 2-wk Jab1^−/− mice (Fig. 6G). Furthermore, analysis of hematocrit (Fig. 6H) and hemoglobin (Fig. 6I) showed a significant increase in 1-wk, 2-wk, and 3-wk Jab1^−/− mice compared with controls. Together, the data suggest that renal injury in KS-Jab1^−/− mice occurs at early time points after Jab1 deletion and is exacerbated over time.

Figure 6.

Jab1 deletion alters kidney weight and urine and blood parameters over time. Analysis of urine volume (A), urine osmolarity (B), osmolarity excretion (C), Na⁺ excretion (D), K⁺ excretion (E), blood urea nitrogen (BUN; F), kidney weight (G), hematocrit (H), and hemoglobin (I) in control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion. n = 10 mice for control, 4-5 for 0-wk Jab1^−/−, 6 for 1-wk Jab1^−/−, 4-5 for 2-wk Jab1^−/−, and 5 for 3-wk Jab1^−/−. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Dunnett’s post hoc analysis. *P < 0.05 vs. control, **P < 0.01 vs. control, and ***P < 0.001 vs. control. BW, body weight.

To determine the extent and localization of the kidney injury over time, we examined protein expression of the well-known injury markers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Western blot analysis of KIM-1, which identifies proximal tubule injury, showed no expression in control mice; however, there was a dense band observed in 0-wk Jab1^−/− mice and was approximately, about fourfold higher than controls (Fig. 7). The amount of KIM-1 protein progressively increased over time after Jab1 deletion and was significantly higher in 2-wk and 3-wk Jab1^−/− mice compared with controls. An increase in NGAL expression is considered a marker of distal nephron injury (20, 21). Recent RNA-sequencing analysis of adult mouse kidneys showed that NGAL (Lcn2 gene) is highly expressed in principal cells of the CD (22). NGAL abundance progressively increased over time in KS-Jab1^−/− mice, although it was not significantly higher than in controls until the 3-wk time point, which was approximately double the amount of 2-wk Jab1^−/− mice (Fig. 7). This suggests that distal nephron damage is substantially worse 3 wk after Jab1 deletion compared with earlier time points.

Figure 7.

Kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) abundance is progressively higher after Jab1 deletion. Western blot analysis of whole kidney lysates for proximal tubule injury marker KIM-1 and the distal nephron injury marker NGAL abundance in control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion is shown. Long exposure KIM-1 blot is the same image with a longer exposure time. There was no KIM-1 band observed in control mice. The abundance of KIM-1 progressively increased over time after Jab1 deletion and was significantly higher than controls in 2-wk and 3-wk Jab1^−/− mice. NGAL abundance was progressively increased in 0-wk, 1-wk, and 2-wk Jab1^−/− mice, although not significant compared with control mice. Three-week Jab1^−/− mice had NGAL abundance that was significantly higher than controls. n = 4 mice for control, 5 for 0-wk Jab1^−/−, 4 for 1-wk Jab1^−/−, 4 for 2-wk Jab1^−/−, and 5 for 3-wk Jab1^−/−. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Dunnett’s post hoc analysis. *P < 0.05 vs. control and ***P < 0.001 vs. control. F, female; M, male.

The localization of kidney damage in KS-Jab1^−/− mice over time was examined via immunofluorescence of KIM-1 and the cell proliferation marker Ki-67. KIM-1 expression was determined by costaining with the proximal tubule marker Lotus tetragonolobus lectin and NCC. The results showed that KIM-1 was limited to the late proximal tubule in the outer medulla in 0-wk, 1-wk, and 2-wk Jab1^−/− mice (Fig. 8). In 3-wk Jab1^−/− mice, KIM-1 expression extended throughout the outer medulla and cortex. Immunofluorescent staining for Ki-67 was completed together with Lotus tetragonolobus lectin and parvalbumin (Fig. 9). The results showed that the number of Ki-67-positive cells trended higher in the medulla at all time points after Jab1 deletion. Alternatively, in the cortex, the number of Ki-67 positive cells remained unchanged in 0-wk, 1-wk, and 2-wk Jab1^−/− mice. The 3-wk Jab1^−/− mice, however, had a higher number of Ki-67-positive cells compared with control mice. Together, the data demonstrate that tubule damage occurred almost immediately upon Jab1 deletion, beginning in the renal medulla, and progressed gradually to the cortex.

Figure 8.

Jab1 deletion causes tubule injury that progresses from the medulla to the cortex over time. Immunofluorescent staining of kidney sections for kidney injury molecule-1 (KIM-1) in control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion is shown. Left: costaining of KIM-1 (red) with the proximal tubule brush-border marker Lotus tetragonolobus lectin (LTL; gray) and the distal convoluted tubule marker Na⁺-Cl⁻ cotransporter (NCC; green) at low-power magnification. Right: higher-magnification images of KIM-1 focused on the cortex or medulla. The results showed no KIM-1 expression in control mice. In 0-wk, 1-wk, and 2-wk Jab1^−/− mice, KIM-1 was expressed in the outer medulla. In 3-wk Jab1^−/− mice, KIM-1 expression expanded into the cortex. n = 3 mice for all groups.

Figure 9.

Jab1 deletion causes cell proliferation that progresses from the medulla to the cortex over time. Immunofluorescent staining of kidney sections for the proliferation marker Ki-67 in control and KS-Jab1^−/− mice at weekly intervals after Jab1 deletion is shown. Left: costaining of Ki-67 (red) with the proximal tubule brush-border marker Lotus tetragonolobus lectin (LTL; gray) and the early distal convoluted tubule marker parvalbumin (Parv; green) in low-power images. Right: higher-magnification images of Ki-67 focused on the cortex or medulla. The results showed that the number of proliferating cells trended higher in the medulla immediately at the 0-wk time point and remained higher than controls through 3 wk. The number of proliferating cells did not increase in the cortex until 3 wk after Jab1 deletion. n = 3 mice for all groups. Data represent individual values as well as means ± SE relative to controls. Statistical differences were examined by one-way ANOVA with Dunnett’s post hoc analysis. ***P < 0.001 vs. control.

Jab1 Deletion Did Not Result in Sex-Specific Changes

It is well known that sex-specific differences are observed in rodent kidney injury models and that females are more protected (23, 24). Thus, we analyzed some of the markers of renal injury based on sex (Supplemental Fig. S2; see https://doi.org/10.6084/m9.figshare.19491665.v1). Linear regression analysis of urine volume (Supplemental Fig. S2A), BUN (Supplemental Fig. S2B), kidney weight (Supplemental Fig. S2C), hematocrit (Supplemental Fig. S2D), hemoglobin (Supplemental Fig. S2E), KIM-1 abundance (Supplemental Fig. S2F), and NGAL abundance (Supplemental Fig. S2G) in control, 0-wk, 1-wk, 2-wk, and 3-wk Jab1^−/− mice did not show any significant differences between male and female mice. Thus, the effects do not appear to be sexually dimorphic.

DISCUSSION

Nephron-specific deletion of the catalytic CSN subunit Jab1 caused activation of the WNK-SPAK pathway; however, it lowered NCC abundance (15). Here, we studied why NCC was reduced and if other proteins were similarly decreased. We examined the changes in NCC over time in KS-Jab1^−/− mice at 0, 1, 2, and 3 wk after Jab1 deletion. The results showed that total NCC abundance is only decreased 3 wk after Jab1 deletion. The decrease in NCC caused by Jab1 deletion paralleled changes in other DCT-expressed proteins, indicating that the loss of NCC was caused by tubule atrophy in the DCT segment. Moreover, loss of the DCT coincided with substantial kidney injury in the cortex 3 wk after Jab1 deletion. Kidney damage was present at all time points after Jab1 deletion, however, the localization and severity changed over time. In 0-wk Jab1^−/− mice, the damage was limited to the medulla. The tubule injury was exacerbated over time and in 3-wk Jab1^−/− mice was also observed in the cortex. The results suggest that CSN dysregulation and subsequently enhanced cullin neddylation causes kidney injury that is amplified over time and progresses from the medulla to the cortex. The injury correlates with remodeling of the nephron in which DCT mass is reduced.

WNK4 regulates NCC activity via indirect phosphorylation through the WNK-SPAK/oxidative stress response 1 (OSR1) phosphorylation cascade (11, 12). Thus, changes in WNK4 and NCC are normally positively correlated. Our previous work showed that although WNK4 abundance was elevated in KS-Jab1^−/− mice, NCC was reduced (15). Here, we show that the loss of NCC abundance occurs even though the pNCC-to-NCC ratio is still elevated (Fig. 2). This is unusual because normal downregulation of NCC occurs through ubiquitylation (25, 26), which is prevented by NCC phosphorylation (16). Therefore, the results demonstrate that the disassociation of WNK4 and NCC is not due to a compensatory mechanism but is rather due to an unusual reduction of NCC abundance that is not attributed to normal downregulation.

Immunofluorescent analysis showed that the number of NCC-positive tubules and not the loss of staining intensity was likely attributed to the decrease in protein abundance (Fig. 3A). We speculated that this was due to DCT remodeling and found other DCT-expressed proteins that underwent the same reduction in protein abundance. Therefore, we examined changes in abundance of multiple segment-specific proteins throughout the nephron. The DCT1-specific protein parvalbumin also showed a reduction in protein abundance over time after Jab1 deletion that was similar to NCC (Fig. 3B). Calbindin, which is expressed in the late DCT (DCT2) and CNT/CCD, protein abundance was decreased in both 2-wk and 3-wk Jab1^−/− mice (Fig. 3C). Immunofluorescent staining of pNCC costained with parvalbumin and calbindin in 3-wk Jab1^−/− mice showed large areas where expression of these proteins was largely reduced or absent (Fig. 3D). Although parvalbumin expression was almost completely diminished in 3-wk Jab1^−/− mice, there was an expression of pNCC in tubules that were calbindin negative that contained minimal parvalbumin expression, suggesting that these tubules are DCT1 segments. In 3-wk Jab1^−/− mice, the staining intensity of calbindin in pNCC-positive/calbindin-positive tubules was similar to control mice. The results suggest that the DCT1 segment is affected more than the DCT2 segment due to Jab1 deletion, although more experiments are needed, such as fate-mapping/lineage tracing analysis, to better understand the extent of the remodeling.

We also examined proteins expressed in segments outside the DCT. AQP3 and AQP4 are both expressed in the basolateral membrane of principles cells of the CNT/CD. Antibodies against AQP3 worked best with immunofluorescent staining, and antibodies against AQP4 worked best with Western blot analysis. Although there was a reduction in AQP4 (via Western blot analysis; Fig. 5) in Jab1^−/− mice, AQP3 immunofluorescence (Supplemental Fig. S1C) showed only minimal changes in the cortex and no significant changes in the medulla. NHE3 and NKCC2, which are expressed in the proximal tubule and TAL, respectively, did not show any major changes in protein abundance (Fig. 5 and Supplemental Fig. S1, A and B). We conclude from these results that Jab1 deletion causes a major loss of DCT mass that occurs 3 wk after genetic disruption. In addition, our results indicate that there is at least some reduction in the CNT/CCD, although more work is needed to determine if the changes are substantial. Based on these data, the tubule atrophy caused by Jab1 deletion is likely specific to the cortical distal nephron.

Although the DCT was atrophied in 3-wk Jab1^−/− mice, WNK4 abundance was still significantly elevated (Fig. 2). WNK4 is thought to mainly activate NCC in the DCT, but it is expressed in other nephron segments, including the TAL and CNT/CD (27). Since Jab1 is deleted along the entire nephron, WNK4 abundance would be increased in all these segments. Previous work showed increased immunofluorescent staining and puncta formation both within and outside of calbindin-positive tubules (15). Thus, the change in WNK4 abundance in the DCT, a relatively small nephron segment, may be overshadowed by changes in the longer TAL and CNT/CD segments.

Loss of the DCT coincided with substantial kidney injury in the cortex 3 wk after Jab1 deletion (Fig. 8). Although only correlative, the data suggest that the DCT remodeling is associated with the amplification of renal injury in the cortex. The plasticity of the DCT is well known and can be caused by many factors, including genetic modification (28–30), pharmacological treatment (31–33), and changes in Na⁺ and K⁺ handling (30, 32, 34–36). This is, however, the first known instance of isolated DCT loss associated with renal injury. The model used here deletes Jab1, which is likely expressed in all cells, simultaneously throughout the nephron. Although damage is observed throughout the kidney, the remodeling is most significant in the DCT and to a lesser extent in the CNT and CCD. The results suggest that Jab1 deletion directly or indirectly affects the mechanism for DCT remodeling, although another possible explanation is that the DCT, compared with other nephron segments, is just more susceptible to remodeling. More work in this area is needed to address this question.

Urine output (Fig. 6A) and kidney injury (KIM-1 and NGAL abundance; Fig. 7) were variable in Jab1^−/− mice. We further analyzed this to examine whether low urine output correlated with lower kidney injury. Although there was a low correlation between urine output and KIM-1 abundance, there was no correlation between urine output and BUN or urine output and NGAL abundance. Urine solute analysis suggested a solute diuresis that is most apparent in 2-wk Jab1^−/− mice (Fig. 6). At this time point, Na⁺ and K⁺ excretion is increased; however, pNCC abundance is enhanced. This discrepancy is likely due to the complicated effects of Jab1 being deleted throughout the nephron, including damage in the medulla. A DCT-specific JAB1 knockout model could be used to further study these issues. The injury caused by Jab1 deletion developed first in the medulla but eventually, over time, expanded into the cortex (Fig. 8). Interestingly, the injury pattern here in a genetically altered mouse model is similar to a mechanical model of renal injury, ischemia-reperfusion injury. The ischemia-reperfusion injury model allows for examination of the acute kidney injury to chronic kidney disease transition. Reperfusion after ischemia causes acute kidney injury, with damage in the outer medulla 1 day after reperfusion. The transition to chronic kidney disease starts with an expansion of the damage into the cortex 5 days after insult (37, 38). Thus, both models cause an injury pattern over time that is almost identical. DCT loss has not been reported in ischemia-reperfusion injury, but it is not clear if this has ever been examined.

We targeted the CSN because CUL3 mutations that cause the disease familial hyperkalemic hypertension were shown to have impaired binding to multiple CSN subunits, including Jab1 (39, 40). Thus, we postulated that CSN dysfunction in the kidney would phenocopy the disease. We used a tissue-specific and inducible system because global and constitutive deletion of any CSN subunit causes lethality in mice (6, 7). There are eight known cullins present in the human kidney (cullin 1, 2, 3, 4A, 4B, 5, 7, and 9), with varying expression throughout the nephron (41). Disruption of CRLs has been implicated in many kidney diseases. CUL3 is known to interact with cyclin E (42), an important protein in the regulation of the cell cycle, which when disrupted can impair cell proliferation (43). Cullin 2 regulates hypoxia-inducible factors through interactions with the substrate receptor von Hippel-Lindau. Mutations in this pathway cause von Hippel-Lindau disease characterized by cyst formation in the kidney and can lead to clear cell renal cell carcinoma (8). In addition, cullin 4 is known to be important in the DNA damage response pathway (44). Jab1, also known as Cops5, is expressed in all cells of the kidney, as shown by RNA-sequencing analysis (22). In addition, Jab1 is expressed in all kidney segments, with the highest expression found in intercalated cells. Inhibition of JAB1 and the CSN should affect all cullins expressed in the kidney and consequently disrupt multiple cellular processes. Kidney-specific Jab1 deletion did upregulate the WNK4-NCC pathway as expected; however, dysregulation of the numerous other proteins maintained by CRLs likely contributed to the tubular injury.

Although this is the first known study of CSN disruption in the kidney, renal disruption of the cullin-RING ubiquitin ligase system has been previously shown to cause injury. Work done by our group using a kidney-specific CUL3 knockout mouse (KS-Cul3^−/−) showed that chronic deletion caused tubulointerstitial fibrosis and inflammation (9). Kidneys from KS-Cul3^−/− mice were smaller and oddly shaped but denser than in wild-type controls. Similar to KS-Jab1^−/− mice, thorough examination of the injury showed higher KIM-1, NGAL, and Ki-67 as well as increases in other kidney injury markers (10). A difference between the two models, however, was that cortical injury progressed more rapidly in KS-Cul3^−/− mice compared with Jab1^−/− mice. KIM-1 and Ki-67 were expressed throughout the cortex in as little as 9 days after Cul3 deletion; in Jab1^−/− mice, KIM-1 and Ki-67 expression were limited to the outer medulla until 3 wk after Jab1 deletion (Figs. 8 and 9). One other major difference between the two models was that in contrast to Jab1^−/− mice, NCC was not decreased in KS-Cul3^−/− mice, further indicating that DCT remodeling can be attributed to the loss of Jab1.

In summary, we show that short-term, nephron-specific Jab1 deletion results in medullary injury and activation of the WNK4-NCC pathway. Long-term Jab1 deletion results in injury throughout the kidney and a decrease in NCC abundance caused by DCT hypoplasia. Regulation of ubiquitin ligase activity by the CSN is an essential process found in all cell types. Since CUL3 targets WNK4, the regulation of CLR activity in the kidney is important for electrolyte balance and blood pressure homeostasis. The data here show that by disrupting this system in kidney epithelial cells, we can get a better understanding of the relationship between the CSN, renal damage, and how the DCT remodels.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.19491800.v1.

Supplemental Fig. S2: https://doi.org/10.6084/m9.figshare.19491665.v1.

GRANTS

This work was supported by funding from National Institute of Diabetes and Digestive and Kidney Diseases Grants R01DK051496 (to D.H.E. and C.-L.Y) and K01DK120790 (to R.J.C.) and Fondation LeDucq: Transatlantic Network of Excellence 17CVD05 (to D.H.E.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

R.J.C., C.-L.Y., and D.H.E. conceived and designed research; R.J.C., J.W.N., and X.-T.S. performed experiments; R.J.C., C.-L.Y., and D.H.E. analyzed data; R.J.C., C.-L.Y., and D.H.E. interpreted results of experiments; R.J.C. prepared figures; R.J.C. drafted manuscript; R.J.C., J.W.N., X.-T.S., C.-L.Y., and D.H.E. edited and revised manuscript; R.J.C., J.W.N., X.-T.S., C.-L.Y., and D.H.E. approved final version of manuscript.