Rab35 Is Required for Embryonic Development and Kidney and Ureter Homeostasis through Regulation of Epithelial Cell Junctions

Visual Abstract

Abstract

Key Points

• Loss of Rab35 leads to nonobstructive hydronephrosis because of loss of ureter epithelium.

• Rab35 regulates kidney and ureter epithelial cell adhesion and polarity.

• Rab35 is required for embryonic development.

Background

Rab35 is a member of a GTPase family of endocytic trafficking proteins. Studies in cell lines have indicated that Rab35 participates in cell adhesion, polarity, cytokinesis, and primary cilia length and composition. In addition, sea urchin Rab35 regulates actin organization and is required for gastrulation. In mice, loss of Rab35 in the central nervous system disrupts hippocampal development and neuronal organization. Outside of the central nervous system, the functions of mammalian Rab35 in vivo are unknown.

Methods

We generated and analyzed the consequences of both congenital and conditional null Rab35 mutations in mice. Using a LacZ reporter allele, we assessed Rab35 expression during development and postnatally. We assessed Rab35 loss in the kidney and ureter using histology, immunofluorescence microscopy, and western blotting.

Results

Congenital Rab35 loss of function caused embryonic lethality: homozygous mutants arrested at E7.5 with cardiac edema. Conditional loss of Rab35, either during gestation or postnatally, caused hydronephrosis. The kidney and ureter phenotype were associated with disrupted actin cytoskeletal architecture, altered Arf6 epithelial polarity, reduced adherens junctions, loss of tight junction formation, defects in epithelial growth factor receptor expression and localization, disrupted cell differentiation, and shortened primary cilia.

Conclusions

Rab35 may be essential for mammalian development and the maintenance of kidney and ureter architecture. Loss of Rab35 leads to nonobstructive hydronephrosis, making the Rab35 mutant mouse a novel mammalian model to study mechanisms underlying this disease.

Introduction

Rab35 is a member of a family of small GTPases that regulate intracellular vesicular trafficking and other cellular processes. In vitro studies have shown that Rab35 promotes cytokinesis through recruitment of cleavage furrow proteins.¹ Rab35 also regulates cell adhesion through recycling of E-cadherin and other adhesion proteins.^2,3 In addition, Rab35 antagonizes Arf6, another small GTPase that functions to internalize surface proteins, such as epithelial growth factor receptor (EGFR), which inhibit cell adhesion and promote cell migration.² Depletion of Rab35 in cultured kidney cells led to an inversion of cell polarity in 3D cultures, suggesting that Rab35 not only plays a role in maintaining proteins at the membrane but also their trafficking or retention in specific cellular locations.⁴ In mammalian cells, Rab35 has been reported to mediate exosome secretion,⁵ autophagy,^6–8 retrograde transport between the endosomes and Golgi,^1,2,9 and have roles in phosphoinositide homeostasis.^10–12 Recently, Rab35 was identified as a novel regulator of primary cilia length and composition in mammalian cells and in zebrafish using siRNA-mediated and morpholino-mediated inhibition approaches, respectively.¹² In sea urchin, Rab35 has been implicated in the regulation of actin involved in gastrulation.¹³ Thus, as a small GTPase that can recruit different effectors to membranes, Rab35 may function in diverse cellular processes that are likely to be important for mammalian health and disease.

Little is known about the in vivo function of mammalian Rab35. Two studies demonstrated that loss of Rab35 in the central nervous system of mice disrupts axon elongation and distribution of hippocampal neurons, likely through altered cell adhesion.^14,15 Here, our goal was to evaluate the role of Rab35 in mouse embryonic development and in adult tissue homeostasis. To accomplish this, we used congenital and conditional mouse mutants to delete Rab35 in the germline, late in gestation, or in the postnatal mouse. On the basis of previous reports, we hypothesized that congenital loss of Rab35 would be embryonic lethal because of its role in cytoskeletal organization, and cytokinesis required for cleavage and gastrulation and that conditional deletion after these developmental stages would lead to shortened malfunctional cilia causing ciliopathic phenotypes.

Methods

Generation of Rab35 Mutant Alleles

All animal studies were conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animals Care and Use Committee at the University of Alabama at Birmingham. Mice were maintained on LabDiet JL Rat and Mouse/Irr 10F 5LG5 chow. The Rab35^KO allele (tm1a) was rederived from sperm obtained from the Knockout Mouse Project Repository into C57BL/6J strain mice.¹⁶ Mice were maintained on C57BL/6J background. Rab35 conditional allele (tm1c) mice were generated by mating Rab35^KO to FlpO recombinase mice (C57BL/6J) to remove the LacZ and Neo cassettes generating a conditional allele (tm1c or fl). Progeny that contained the recombined allele were crossed off the FlpO line and bred to males carrying CAGGCre-ER (Jax Stain 004682) ¹⁷ recombinase to generate the deletion allele (tm1d or delta). We refer to these alleles as tma1 (Rab35^KO), tm1c (Rab35^fl), and tm1d (Rab35^delta) alleles (Figure 1A and Supplemental Figure 1A). Primers used for genotyping are included in Supplemental Table 1.

Figure 1.

Rab35 expression in the mouse. (A) The tma1 allele that was used to generate Rab35^KO mice contains a LacZ reporter. $\beta$-galactosidase staining revealed Rab35 expression in embryos and postnatal tissues. (B) Staining for $\beta$-galactosidase activity in E8.5 heterozygous embryos indicated that Rab35 is ubiquitously expressed. $\beta$-galactosidase activity in P7 tissues was more restricted to (C) kidney (M, medulla; C, cortex; and RP, renal papilla) and (D) ureters (U, urothelium; SM, smooth muscle). $\beta$-galactosidase activity was also present in (E) testes, (F) lung, and (G) liver, but not in (H) heart and (I) pancreas.

Tamoxifen Cre Induction

Recombination of the conditional tm1c allele was induced in utero by injecting time pregnant females with a single intraperitoneal (IP) injection of 6 mg tamoxifen (Millipore Sigma, T5648)/40 g body weight dissolved in corn oil. Juvenile Rab35^fl/fl Cre⁺ and Rab35^fl/fl mice at postnatal day 7 (P7) were induced by a single IP injection of 3 mg tamoxifen/40 g body weight and adult animals at age 8 weeks by a single IP injection of 9 mg tamoxifen/40 g body weight. Cell culture media was supplemented with 1 mM 4-hydroxytamoxifen for 24 hours to achieve deletion of Rab35 in mouse embryonic fibroblasts (MEFs) and primary kidney epithelial cells. For simplicity throughout manuscript, all animals were induced, and controls are listed as Cre⁻ and mutants Cre⁺.

Embryo Isolation

Timed pregnancies were established with embryonic timepoint of E0.5 being noon on the morning of observing the copulatory plug. To isolate embryos, pregnant females were anesthetized using isoflurane followed by cervical dislocation. Isolated embryos were transferred to PBS, and yolk sac was taken for genotyping. Embryos or isolated embryonic tissues were then fixed in 4% paraformaldehyde (PFA; Sigma, 158127) in PBS.

β-Galactosidase Staining

For whole mount or section $\beta$-galactosidase staining, samples were fixed (0.2% glutaraldehyde (Sigma), 5 mM of EGTA, and 2 mM of MgCl₂ in 1× PBS) at 4°C for 30 minutes, rinsed three times for 15 minutes at 4°C (0.02% Igepal, 0.01% sodium deoxycholate, and 2 mM MgCl₂ in 1× PBS), and immersed in staining solution overnight in the dark at 37°C (1 mg/ml X-gal, 0.02% Igepal, 0.01% sodium deoxycholate, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl₂ in 1× PBS). Samples were postfixed in 4% PFA and stored at 4°C, and sections were counterstained with Nuclear Fast Red (Sigma). Embryos and sections were imaged using a Nikon SMZ800 stereo microscope.

MEF Isolation

Timed pregnancies between Rab35^fl/fl and Rab35^fl/fl Cre⁺ animals were set up to isolate embryos at E13.5 to culture MEFs. After the removal of the liver and head, embryos were mechanically dissociated and cultured in DMEM (Gibco, 11039-021) supplemented with 10% FBS, 1× penicillin and streptomycin, 0.05% Primocin, 3.6 μl/0.5 L β-mercaptoethanol. MEFs were cultured on glass cover slips treated with 0.1% gelatin 24–48 hours before tamoxifen induction. To induce cilia formation, cells were grown to confluency and then switched to DMEM medium containing 0.5% FBS.¹⁸

Primary Kidney Epithelial Cell Isolation

One-month-old Rab35^fl/fl Cre⁺ animals were anesthetized with isoflurane followed by cervical dislocation, and kidneys were removed and mechanically dissociated in a sterile environment. Minced tissue was filtered through a 70-µm cell strainer and resuspended in DMEM (Gibco, 11039-021) supplemented with 5% FBS, epidermal growth factor (recombinant human, 10 ng/ml), insulin (recombinant human, 5 µg/ml), hydrocortisone (36 ng/ml), epinephrine (0.5 µg/ml), triiodo-L-thyronine (4 pg/ml), and transferrin (recombinant human, 5 µg/ml) (Growth Medium 2 Supplement Pack, PromoCell, C-39605). Cells were allowed to grow until 80%–90% confluent, and culture medium promotes epithelial cell survival. Primary epithelial cells were cultured 48–72 hours before tamoxifen induction. Primary kidney epithelial cells were cultured for a maximum of four passages.

Tissue Isolation and Histology

Mice were anesthetized with 0.1 ml/10 g of body weight dose of 2.0% tribromoethanol (Sigma Aldrich, St. Louis, MO) and transcardially perfused with PBS followed by 4% PFA. Tissues were postfixed in 4% PFA overnight at 4°C and then cryoprotected by submersion in 30% sucrose in PBS for 24 hours, then frozen in O.C.T. (Fisher Scientific, 23-730-571) and cryosectioned at 10 or 20 µm thickness for immunofluorescence, or hematoxylin (Fisher Chemical, SH26-500D) and eosin (Sigma-Aldrich, HT110132-1L) staining was performed.

Necropsy Analysis

Animals were sent to the Comparative Pathology Laboratory (UAB) for necropsy where all tissues were fixed in 10% neutral buffered formalin overnight, processed into 5 µm sections, and stained with hematoxylin and eosin. Slides were evaluated for tissue histopathology by a board-certified veterinary pathologist. For blood chemistry analysis, blood was collected by axial bleed and provided to the Comparative Pathology Laboratory to measure serum calcium, BUN, creatinine, albumin, sodium, phosphate, and total protein. All analyses were performed blinded.

Immunofluorescence Microscopy

Tissue sections (10 µm, except kidney sections used for cilia length measurements, which were 20 µm) were fixed in 4% PFA for 10 minutes, permeabilized with 0.1% TritonX-100 in PBS for 8 minutes, and then blocked in a PBS or Tris-Buffered Saline (depending on antibodies) containing 1% BSA, 0.3% TritonX-100, 2% normal donkey serum, and 0.02% sodium azide for 1 hour at room temperature. Primary antibodies were incubated in blocking solution overnight at 4°C. Detailed information about primary antibody usage is in Supplemental Table 2. Cryosections were then washed with PBS/Tris-Buffered Saline three times for 5 minutes at room temperature. Secondary antibodies diluted in blocking solution were added for 1 hour at room temperature. Secondary antibodies were donkey-conjugated Alexa Fluor 647, 594, or 488 (Invitrogen, 1:1000). Samples were then washed in PBS and stained with Hoechst nuclear stain 33,258 (Sigma-Aldrich) for 5 minutes at room temperature. Cover slips were mounted using Immu-Mount (Thermo Scientific). All fluorescence images were captured on Nikon Spinning disk confocal microscope with Yokogawa X1 disk, using Hamamatsu flash4 sCMOS camera. We used 60× apo-TIRF (NA=1.49), 40× plan fluor (NA=1.3), or 20× Plan Flour multi-immersion (NA=0.8) objectives. Images were processed using Nikon Elements or Fiji software.

Intrapelvic Dye Injections

Timed pregnant females were injected with tamoxifen at E14.5 and killed at E16.5 for embryo isolations. Yolk sacs were taken for genotyping, and dye injection was performed blinded before genotyping. Whole kidneys with ureters and bladder attached were removed from embryos, and a 0.1% solution of Bromo-Phenol Blue in PBS was injected into the kidney pelvis through a pulled glass pipette. Injections were recorded by timelapse imaging and then converted into movie files. Ureters were recorded after dye injection for 5 minutes by timelapse imaging.

Statistical Analysis

Analyses were performed using GraphPad Prism and Microsoft Excel. Specific tests used are indicated in figure legends with significance indicated as follows: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. All error bars represent standard error of the mean (SEM).

Results

Rab35 Expression in the Mouse

The Rab35 tm1a allele (Rab35^KO) contains a LacZ reporter encoding $\beta$-galactosidase that allows for visual analysis of gene expression (Figure 1A).¹⁶ $\beta$-galactosidase staining of E8.5 Rab35 tm1a heterozygous embryos revealed that Rab35 was ubiquitously expressed (Figure 1B). At P7, $\beta$-galactosidase activity was more restricted to the kidney, ureter, and testes (Figure 1, C–E). Modest $\beta$-galactosidase activity was present in the lung and liver, and no β-galactosidase activity was evident in the heart and pancreas (Figure 1, F–I). In tissues where $\beta$-galactosidase activity was detected, it was enriched in epithelial cells and ciliated cells (e.g., biliary epithelial cells and spermatozoa, Figure 1G). In the kidney, β-galactosidase activity was not uniform, with greater activity in the tubule epithelium of the cortex and renal papilla than in the medulla (Figure 1, C and C′). β-galactosidase activity was also not uniform in the ureter, with greater activity in the urothelium than in the underlying smooth muscle (Figure 1, D and D′).

Rab35 Regulates Primary Cilia Length, but Is Not Required for Ciliogenesis in Mice

Previous studies have shown that Rab35 depletion using siRNA in mammalian cells or morpholinos in zebrafish reduces primary cilia length.¹² To confirm whether Rab35 regulates cilia length in mammalian cells, we generated primary MEFs and renal epithelium from conditional Rab35^tm1c/tm1c CAGG-CreER mice (Supplemental Figure 1A).¹⁶ Rab35 deletion was induced by addition of 4-hydroxytamoxifen to the culture medium to generate Rab35^tm1d/tm1d CAGG-CreER cells (hereafter referred to as Cre⁺ cells). The percentage of cells with cilia and cilia length was quantified after staining for acetylated α-tubulin, a ciliary component. The percentage of Cre⁺ cells with a cilium was not significantly different than the percentage of control cells with a cilium. However, MEFs and epithelial cells lacking Rab35 had shorter cilia (Supplemental Figure 1, B–G). Similarly, deletion of Rab35 in vivo reduced primary cilia length in the kidney or ureter (Supplemental Figure 1, H–K). Therefore, mammalian Rab35 maintains cilia length and elongation, but is not essential for ciliogenesis.

Rab35 Is Essential for Embryonic Development

No homozygous Rab35^KO embryos were isolated after E10.0, indicating that Rab35 is required for embryonic development, and genotyping of the embryos suggest lethality is occurring before or around E8.5 (Figure 2, A and B). This is in agreement with a possible role in gastrulation and shown in sea urchins.¹³ Rab35^KO embryos isolated at E8.5 were developmentally delayed (Figure 2C). Rab35^KO embryos had disorganized filamentous actin (F-actin, Figure 2D). Because Rab35 is required for actin organization in sea urchins,¹³ Rab35 function is evolutionarily conserved.

Figure 2.

Rab35 is required for embryonic development in the mouse. (A) Embryos were isolated between E8.5 and E11.5 and genotyped. Until E10.0, Rab35^KO mutant embryos were observed at sub-Mendelian ratios. Rab35^KO mutant embryos were not identified at E11.5. (B) Quantitation of embryo genotypes between E8.5 and E11.5 showed increasing resorptions, which explains the lack of mutants at timepoints after E8.5. (C) E7.5 Rab35^KO embryos were morphologically comparable with wild-type sibling controls. At E8.5, Rab35^KO embryos were developmentally delayed. (D) Phalloidin staining of wild-type sibling controls and Rab35^KO embryos. (E) Conditional deletion of Rab35 at E7.5 and analysis at E12.5 and (F) deletion at E8.5 with analysis at E10.5 reveals cardiac edema and developmental delay. Het, heterozygous; KO, knockout; WT, wild type.

Conditional Deletion of Rab35 Causes Bilateral Hydronephrosis and Decreased Kidney Function

To circumvent the early lethality in Rab35 germline null mutants and uncover other functions of Rab35, we deleted Rab35 during gestation (Figures 2, E and F and 3, A and B) using a ubiquitously expressed tamoxifen-inducible Cre transgene (CAGG-CreER, Cre⁺) (Supplemental Figure 1A). Deletion of Rab35 at E7.5, before organogenesis, resulted in developmental arrest and lethality (Figure 2E). We also induced Rab35 deletion at E8.5 and analyzed embryos at E10.5. Although Kuhns et al. implicated Rab35 in regulating ciliary Shh signaling, we did not detect phenotypes typically associated with altered Hedgehog signaling (e.g., polydactyly).¹² Deletion of Rab35 at E8.5 resulted in developmental arrest, cardiac edema, and lethality (Figure 2F).

Figure 3.

Conditional deletion of Rab35 leads to bilateral hydronephrosis and decline in kidney function. (A) Timeline for Rab35 embryonic inductions. Rab35^delta Cre⁺ embryos isolated at E18.5 had bilateral hydronephrosis observed by gross morphology and histological staining of 10 µm sections (n=16). (B) Timeline for Rab35 juvenile (P7) and adult (8 weeks) inductions. Independent of induction time, Rab35^delta cre⁺ mice have bilateral hydronephrosis 8 wkpi (n=16 and n=14, respectively). (C) Blood serum analysis of kidney function markers analyzed by unpaired t tests. BUN P value = 0.009 (**), calcium P value = 0.01 (*), sodium P value = 0.02 (*), phosphate P value = 0.01 (*), total protein P value = 0.01. wkpi, weeks post-tamoxifen induction.

Deletion of Rab35 at E14.5, midorganogenesis, and analysis of embryos at E18.5 revealed severe bilateral hydronephrosis in all Cre⁺ embryos (Figure 3A). To determine whether the hydronephrosis was necessarily of developmental origin, we deleted Rab35 postnatally in juvenile (P7) and adult (8 weeks) conditional mice (Figure 3B). All Cre⁺ animals induced at either P7 or at P56 and analyzed 8 weeks post-tamoxifen induction (wkpi) exhibited hydronephrosis, indicating that Rab35 is required postnatally to maintain kidney architecture. Blood serum chemistry analysis of the Cre⁺ animals induced at P7 revealed increased BUN, calcium, sodium, phosphate, and total protein, indicative of decreased kidney function (Figure 3C). There was no significant difference in serum creatinine and albumin in Cre⁺ animals compared with littermate controls at 8 wkpi (Figure 3C).

To characterize the onset of hydronephrosis in Cre⁺ embryos and in postnatal mice, we assessed kidneys at a series of timepoints after Rab35 deletion at E14.5 or P7 (Supplemental Figure 2). After deletion at E14.5, E16.5 Cre⁺ embryos did not display hydronephrosis (Supplemental Figure 2A). Similarly, after deletion at P7, 4 wpki Cre⁺ mice did not display hydronephrosis (Supplemental Figure 2B), but did at 6 wpki. We also confirmed deletion efficiency at 6 wkpi in kidneys and ureters by quantitative reverse transcription-PCR (Supplemental Figure 2, C and D). To assess the pathogenesis of the hydronephrosis caused by Rab35 deletion, we therefore examined E16.5 Cre⁺ embryos and 4 wpki Cre⁺ mice.

Rab35 Participates in Renal Epithelial Cell Adhesion and Polarity

In vitro studies indicate that Rab35 is important for maintaining epithelial cell junctions through opposing the activity of Arf6 and through E-cadherin membrane recycling.^2,3 Analysis of Arf6 localization in adult and embryonic Cre⁺ kidney tubule epithelium showed an expansion of Arf6 distribution across the cell membrane, instead of its normal restriction to the apical epithelium (Figures 4A and 5A). This expanded distribution was observed before the onset of hydronephrosis. Western blot analysis revealed that the expanded distribution could not be explained by increased Arf6 protein (Figure 4B).

Figure 4.

Rab35 regulates epithelial cell adhesion and polarity within the adult kidney. (A) IF staining for Arf6 in control, prehydronephrotic (4 wkpi), and hydronephrotic (8 wkpi) kidneys. (B) Representative western blots of Arf6 in kidney lysates from control, prehydronephrotic, and hydronephrotic and quantification normalized to GAPDH. (C) IF staining for E-cadherin and (D) representative western blot for E-cadherin with quantification in whole kidney lysates normalized to GAPDH. (E) α-acetylated tubulin staining indicated no change in primary cilia polarity in kidneys. (F) Phalloidin shows a decrease in actin organization and brush border. (G) Tight junction marker, ZO-1 is reduced in Rab35^delta Cre⁺ cultured primary epithelial cells independent of confluency and (H and I) significantly reduced in prehydronephrotic and posthydronephrotic kidneys. Statistical analysis was performed using one-way ANOVA and post hoc analysis for multiple comparisons between each timepoint from one representative blot out of at least two experimental repeats. Prehydronephrosis analysis was not compared with hydronephrosis analysis. Unpaired t test P value for 4 wkpi (prehydronephrosis) quantification of E-cad is 0.001 (**), and for 8 wkpi (hydronephrosis) analysis, unpaired t test P value < 0.001 (***). Unpaired t test P values for ZO-1 < 0.001 (****). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IF, immunofluorescence.

Figure 5.

Rab35 regulates epithelial cell adhesion and polarity within the embryonic kidney. (A) IF staining for Arf6 in control, prehydronephrotic (E16.5), and hydronephrotic (E18.5) kidneys shows similar localization changes as in adult kidneys. (B) E-cadherin is reduced and has changes in localization from basolateral to apical membranes (white arrow). (C) Primary cilia are present and apically oriented. (D) Reduction in brush border actin.

Along with expanded Arf6 localization, Cre⁺ kidney tubule epithelium had reduced E-cadherin, and Cre+ embryonic kidney tubule epithelium had apical E-cadherin expression (Figures 4C and 5B). Quantification of E-cadherin by western blot analysis showed reduced E-cadherin levels before the onset of hydronephrosis (Figure 4D). In addition to E-cadherin, Cre⁺ kidneys displayed a reduction in N-cadherin (Supplemental Figure 3), although to a lesser degree, and its localization was not altered. Following this same trend, primary cilia, although shorter, remained apically oriented in Cre⁺ kidneys (Figures 4E and 5C).

With the increased membrane localization of Arf6 and reduced E-cadherin maintaining adherens junctions, we suspected an overall reduction in epithelial cell junction integrity and adhesion. To assess this, we analyzed the tight junction marker, ZO-1, by immunofluorescence and western blot using Cre⁺ primary epithelial cells and in vivo in the kidney (Figure 4, G–I). In both, there is a significant reduction of ZO-1 evident prehydronephrosis and posthydronephrosis (Figure 4I). These data support a model in which Rab35 is required to maintain E-cadherin–mediated epithelial cell adherens and ZO-1 at tight junctions, within the kidney. It is likely that the effect of Rab35's loss on tight junctions is indirect because adherens junctions are required for tight junction formation and maintenance.¹⁹

Along with defects in maintaining junctional complexes in Cre⁺ kidneys, there is a loss of actin organization and, more specifically, a loss of brush border actin (Figures 4F and 5D) that may contribute to a loss of polarity and epithelial junction integrity. To assess polarity defects, we analyzed EGFR localization, which is normally restricted to the basal-lateral membrane of kidney epithelia. Furthermore, data from other groups indicate that EGFR localization is regulated by E-cadherin and Rab35/Arf6 activity in vitro.^20,21 In Cre⁺ kidneys, EGFR is no longer restricted to the basal-lateral membrane prehydronephrosis or posthydronephrosis, and its expression is significantly increased in hydronephrotic kidneys (Figure 6, A–C). This suggests that Rab35 regulates EGFR internalization and degradation in vivo in the kidney in addition to regulation of E-cadherin and Arf6 membrane localization and polarity. However, our analysis in vivo indicates there is not a complete loss of epithelial cell polarity or organization in Rab35 mutant cells as CD13 remains apically localized (Figure 6A) as does the position of the primary cilium (Figures 4E and 5C).

Figure 6.

Rab35 regulates EGFR expression and localization within the kidney. (A) IF of EGFR expression in control, prehydronephrotic, and hydronephrotic kidneys. CD13 is used as a marker for apical membrane in kidney epithelia to show changes in EGFR localization in Rab35^delta Cre⁺ kidneys. (B) Western blot of whole kidney lysates and (C) quantification of EGFR normalized to GADPH. Only hydronephrotic kidneys had significant increases in EGFR with unpaired t test P value < 0.001 (****). (D) A ratio was taken of phosphorylated EGFR out of total EGFR to determine whether there were changes in EGFR activity. There was no significant difference in the pEGFR/EGFR ratio across kidney samples. EGFR, epithelial growth factor receptor.

Rab35 Regulates Ureter Integrity through Maintenance of the Urothelium

Bilateral hydronephrosis is most commonly caused by an obstruction along the kidney-ureter-bladder tract. However, in Cre⁺ animals, there is no evidence of obstruction because adult animals urinate normally, and dye injections in Cre⁺ embryos show that the lumen of the ureter remains patent (Figure 7, A and B, Supplemental Video 1, and Supplemental Video 2). Thus, Rab35 mutants are a new model of nonobstructive hydronephrosis. Interestingly, the lumens of Cre⁺ mutants were dilated compared with the Cre-control ureters (Figure 7, A and B, and Supplemental Video 2). Histological analysis of cross sections through the ureters revealed the Cre⁺ ureters were thin even at the prehydronephrotic stage (Figure 7C).

Figure 7.

Rab35 regulates ureter epithelium and smooth muscle maintenance. Embryonic intrapelvic dye injections suggest defects in ureter lumen structure. (A) Snapshot of dye injection in two Rab35^fl kidneys and two Rab35^delta Cre⁺ kidneys (K, kidney; B, bladder). (B) Three-minute snapshot after dye injection suggests widening of lumens. (C) Histological analysis of prehydronephrotic embryonic ureters by H&E staining confirmed widening of ureter lumens in Rab35^delta Cre⁺ embryos. IF staining of E-cadherin and smooth muscle actin showed Rab35^delta Cre⁺ proximal ureters had cells expressing both epithelial and smooth muscle markers where the distal ureters no longer had any E-cadherin–expressing cells. This was similar to (D) ureters isolated after hydronephrosis (E18.5).

Because loss of Rab35 led to a reduction in E-cadherin in the kidney, we analyzed whether there is a similar reduction in the urothelium associated with the widening of the ureter lumen. The data confirm a marked reduction in E-cadherin expression in the proximal regions of the ureter and that the distal regions of the ureter lack E-cadherin positive urothelium (Figure 7, C and D). Normally the urothelium (E-Cad+) and smooth muscle (aSMA+) cells are localized in well-defined regions of the ureter. However, cells in the Cre⁺ proximal ureters costained for both smooth muscle actin and E-cadherin, suggesting these cells had lost their normal identity (Figure 7D). No overt defects in smooth muscle are evident in the kidney, and there were no other indications that smooth muscle in general was impaired in Cre⁺ as indicated by necropsy and immunofluorescence staining in other tissues (Supplemental Figure 4).

Because deletion of Rab35 was induced after the ureter epithelium and smooth muscle layers had been established, we hypothesized that the thinning of the ureter was due to loss of these cells through apoptosis. In the prehydronephrotic Rab3^delta cre⁺ kidney and ureter, there is an increase in expression of cleaved caspase-3 supporting this hypothesis. This is specific to the epithelial cells that no longer expressed E-cadherin and was observed before the onset of hydronephrosis (Figure 8, A and B).

Figure 8.

Loss of Rab35 leads to apoptosis in the kidney and ureter. (A) IF staining of kidneys with E-cadherin and a marker for apoptosis, CC3, indicated cell death was occurring in cells no longer expressing E-cadherin. This was also observed in (B) ureters and (C) working model: Rab35 maintains actin organization and E-cadherin membrane recycling in addition to inhibiting Arf6 activity. This creates a spatial boundary for Arf6 that is lost when Rab35 is removed. EGFR membrane expression is finely tuned by the presence of active Rab35 and E-cadherin. Arf6 activation leads to the internalization and recycling of EGFR to the membrane. (D) Representative images suggest loss of these boundaries set by Rab35 leads to increased recycling of EGFR, reduced adherens junctions, and loss of tight junction protein ZO-1. These changes in epithelial cells within the ureter and kidney lead to loss of epithelial integrity, cell death, and hydronephrosis. CC3, cleaved caspase-3.

Collectively, these data suggest that Rab35 is required for the maintenance of the urothelium through regulation of E-cadherin, and loss of this regulation subsequently affects the surrounding smooth muscle cells. Interestingly, this regulation is specific to the ureter and kidney because other tissues in Cre⁺ embryos and adults that we analyzed have E-cadherin and smooth muscle actin expression comparable with Cre⁻ at these timepoints (Supplemental Figure 4). We suspect that this may be due to the timepoint analyzed, with the kidney being more sensitive to Rab35 loss than other tissues and thus maybe the first tissue in which a phenotype manifests.

Discussion

Rab35 is a critical regulator of many cellular processes, including cilia length, vesicle trafficking, cytoskeletal organization, cell adhesion and migration, cytokinesis, and axonal elongation. While loss of Rab35 is essential for viability, very little is known in mammals regarding the effect of Rab35 functional loss in tissues other than in the central nervous system. Using a congenital Rab35^KO reporter allele, we showed that Rab35 is ubiquitously expressed in early gestational embryos but has more restricted expression in the postnatal mouse, with highest expression in the renal cortex and papilla and ureter epithelium. This expression, however, fits with single-cell RNA expression data showing Rab35 expressed throughout much of the tubule epithelium of the human and mouse kidney as well as the epithelium of the human ureter.^22,23 We have shown that Rab35 congenital null embryos die before E8.5, while others have reported viable embryos at E10.5.¹⁵

We then assessed whether mutant embryos are viable when loss is induced at later developmental timepoints. To address this, we used conditional null allele of Rab35 to bypass the early gestational lethality and evaluated the importance of Rab35 in late gestation and in postnatal tissues. Although we noted a decrease in cilia length in the absence of Rab35, our analysis of early embryos did not show any overt signs of left-right axis or neural tube closure defects, both classic ciliopathy phenotypes. This indicated it is not sufficient to cause cilia functional loss, although this may be confounded by the development arrest in the mutants. A consistent phenotype that we do observe in Rab35 mutants, and a likely contributor to their lethality, is a marked alteration in the actin cytoskeletal organization. This phenotype was also reported in sea urchins lacking Rab35, indicating conserved function.¹³ Rab35 is known to recruit effectors that modulate localized actin assembly and organization,²⁴ including Arf6,^2,10,11 RhoA, and Rac1,^25–27 in multiple cell types. This regulation is critical for many cell processes that are important for normal development.

To evaluate Rab35 mutant perinatal and postnatal phenotypes, we induced Rab35 deletion at E14.5 and P7 after organogenesis has occurred. In both cases, Rab35 mutants have severe and fully penetrant bilateral hydronephrosis, a novel finding because Rab35 has not previously been associated in congenital anomalies of the kidneys and urinary tracts. Hydronephrosis is not considered widely as a ciliopathy, although defects in the hedgehog pathway in the smooth muscle surrounding the ureter can cause this phenotype.^28,29 However, we did not observe typical ciliopathy kidney phenotypes such as cyst formation.

As we observed in the Rab35 mutant embryos, there are changes in actin cytoskeletal organization from its apical enrichment in kidney epithelial cells to a diffuse pattern lacking organization in Cre⁺ kidneys. F-actin can regulate epithelial adherens junctions, polarity, and organization, all of which Rab35 has been shown to regulate in vitro. To determine the molecular/cellular mechanism involved in hydronephrosis, we investigated Arf6 and E-cadherin expression and localization, which have been shown to be affected in previous studies. Interestingly, we observed a redistribution of Arf6 from an apical domain to both apical and basal lateral membranes in the Rab35 mutants. This is indicative of an overactivated Arf6.^30,31 We also found a lack of E-cadherin at the cell junctions and a loss of E-cadherin protein expression that would disrupt the adherens junctions. As Arf6 and Rab35 are counter pathways with Arf6 promoting membrane protein internalization, this could explain the large decrease in E-cadherin. The loss of Rab35 does not only affect the adherens junctions because we observe a similar effect on ZO-1. Tight junction proteins have not been previously shown to be regulated by Rab35, and this may be a consequence of changes within the actin cytoskeleton and loss of E-cadherin at adherens junctions.³² The effects on the adherens and tight junctions occur before the onset of hydronephrosis and are present both in vivo and in primary renal cells cultured from the Rab35 mutants. Thus, disruption of the adherens and tight junctions is not a secondary consequence of hydronephrosis. Interestingly, the changes in polarity and expression of these adherens and tight junction proteins were specific to Arf6, E-cadherin, and ZO-1 because N-cadherin remained localized to basal-lateral membranes despite there being data indicating its regulation by Rab35 in cell culture.³ In addition, apical/basal polarity is not fully disrupted because primary cilia and CD13 remain in apical orientation, although cilia are shorter than normal.

The extracellular domain of E-cadherin binds to EGFR, and this interaction helps create the boundary for EGFR localization, while in turn, EGFR activity and signaling can regulate E-cadherin internalization and expression.^20,32–35 Arf6 has also been shown to internalize E-cadherin in response to EGFR activity, and this internalization requires Arf6 activity.³⁶ While at later stages, we observe an increase in EGFR expression and changes in localization, it is only seen after significant changes in Arf6 and E-cadherin are present, suggesting the changes in EGFR are a consequence of the loss of E-cadherin and ZO-1 and increased Arf6 localization. These data further suggest that Rab35 may be involved in EGFR internalization and degradation in the kidney through regulation of these pathways. Previous studies have identified Rab35 to be a regulator of EGFR expression and activity is various human cancer cell lines, and this regulation is dependent on Rab35 activity, although neither have been associated with congenital anomalies of the kidneys and urinary tracts previously.^2,21,37,38 On the basis of our data in conjunction with single-cell RNA sequencing data, there is likely to be functional deficiencies within the tubule compartment because we observed alterations within cell polarity and cytoskeletal changes in the tubule epithelium at an early stage of the disease.

Because we found no obstruction in our model and Rab35 is highly expressed in the urothelium and underlying smooth muscle, we hypothesized that there would be similar epithelial changes in the ureter as in the kidney. Like in Rab3^delta cre⁺ kidneys, there was a dramatic reduction in E-cadherin–positive cells, and those that remained E-cadherin–positive also expressed smooth muscle actin. This suggests that Rab35 is required to maintain the epithelial cell integrity within the urothelium through regulation of E-cadherin, which also affects the adjacent smooth muscle. Loss of E-cadherin led to apoptosis within Rab35 mutant kidney ureters, resulting in widening of the ureter lumen. Other models of nonobstructive hydronephrosis, such as uroplakin mutation, that impair urothelium barrier lead to similar pathology and mutations in aquaporin 2 that affect its apical localization and fluid homeostasis.^39–41 Our model is that Rab35 creates an Arf6 apical boundary through regulation of Arf6 activity, actin organization, through E-cadherin localization on the basolateral membrane. This regulation is necessary to maintain kidney and ureter epithelial E-cadherin cell adherens and, subsequently, the tight junctions as well as membrane receptors that are associated with this response such as EGFR.

In summary, Rab35 mutant mice are a novel mouse model of nonobstructive hydronephrosis. We found that the role of Rab35 in regulating processes such as actin organization and primary cilia length is conserved across species and required for early embryonic development and tissue maintenance. Although we did not observe classic ciliopathic phenotypes at the timepoints assessed, this may be a result of the timing of developmental and juvenile inductions and allows for future investigation using tissue-specific deletions. We showed that many of the mechanisms observed in vitro are recapitulated in vivo, such as Rab35's regulation of actin, E-cadherin, Arf6, and EGFR expression and localization, linking these processes to kidney and ureter maintenance and disease. Finally, we showed that loss of Rab35 in kidneys and ureters leads to cell death. Our data provide valuable insight into not only the role of Rab35 in vivo but provides a novel avenue of study when assessing kidney and urinary system pathologies associated with hydronephrosis.

Supplementary Material

jasn-35-719-s001.pdf ^{(1.4MB, pdf)}

jasn-35-719-s002.pdf ^{(1.3MB, pdf)}

Download video file ^{(16.1MB, mp4)}

Download video file ^{(26.1MB, mp4)}

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/JSN/E610.

Funding

J.F. Reiter: Eunice Kennedy Shriver National Institute of Child Health and Human Development (5R01HD089918-05). B.K. Yoder: Eunice Kennedy Shriver National Institute of Child Health and Human Development (5R01HD089918-05) and National Institute of Diabetes and Digestive and Kidney Diseases (2R01DK115751). K.R. Clearman: National Institutes of Health (5T32GM008111-34 and 5T32DK116672-05).

Author Contributions

Conceptualization: Kelsey R. Clearman.

Data curation: Kelsey R. Clearman, Courtney J. Haycraft.

Formal analysis: Kelsey R. Clearman.

Funding acquisition: Jeremy F. Reiter, Bradley K. Yoder.

Investigation: Kelsey R. Clearman, Dharti Patel, Addison B. Rains, Napassawon Timpratoom.

Methodology: Kelsey R. Clearman.

Supervision: Courtney J. Haycraft, Bradley K. Yoder.

Writing – original draft: Kelsey R. Clearman.

Writing – review & editing: Kelsey R. Clearman, Mandy J. Croyle, Courtney J. Haycraft, Jeremy F. Reiter, Bradley K. Yoder.

Data Sharing Statement

All data are included in the manuscript and/or supporting information.

Supplemental Material

This article contains the following supplemental material online at http://links.lww.com/JSN/E611, http://links.lww.com/JSN/E612, http://links.lww.com/JSN/E613.

Supplemental Figure 1. Conditional deletion of Rab35 shortens cilia.

Supplemental Figure 2. Progression of hydronephrosis during development and in the juvenile mouse.

Supplemental Figure 3. Expression of N-cadherin in kidneys.

Supplemental Figure 4. Loss of Rab35 does not affect other tissues at these timepoints.

Supplemental Table 1. Primers for genotyping.

Supplemental Table 2. List of antibodies.

Supplemental Video 1. Intrapelvic dye injections into control embryonic kidneys.

Supplemental Video 2. Intrapelvic dye injections into prehydronephrotic embryonic kidneys.