Why Retinoic Acid Receptor Responder1 can Promotes Development Of Glomerular Diseases?
Mar 11, 2022
Retinoic Acid Receptor Responder1 Promotes Development Of Glomerular Diseases Via The Nuclear Factor-kB Signaling Pathway
Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791
Katja Mo¨ller-Hackbarth1,2 , Dina Dabaghie1,2 , Emmanuelle Charrin1,2 , Sonia Zambrano1,2, Guillem Genove´ 1,3 , Xidan Li1,3 , Annika Wernerson4 , Mark Lal5 and Jaakko Patrakka1,2
1 KI/AZ Integrated Cardio Metabolic Centre, Karolinska Institutet at Karolinska University Hospital, Huddinge, Stockholm, Sweden; 2 Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet at Karolinska University Hospital, Huddinge, Stockholm, Sweden; 3 Department of Medicine Huddinge, Karolinska Institutet at Karolinska University Hospital, Huddinge, Stockholm, Sweden; 4 Department of Clinical Sciences, Intervention and Technology, Division of Renal Medicine, Karolinska Institutet, Stockholm, Sweden; and 5 Bioscience Renal, Research, and Early Development Cardiovascular, Renal and Metabolism (CVRM), R&D Biopharmaceuticals, AstraZeneca, Gothenburg, Sweden
Kidney International (2021) 100, 809–823; https://doi.org/10.1016/ j.kint.2021.05.036
Copyright ª 2021, International Society of Nephrology. Published by Elsevier Inc. This is an open-access article under the CC BY license
Correspondence: Jaakko Patrakka, Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Blickagången 6, SE-141 57 Huddinge, Sweden. E-mail: jaakko.patrakka@ki.se Received 27 July 2020; revised 11 May 2021; accepted 20 May 2021; published online 18 June 2021
KEYWORDS: glomerular endothelial cell; NF-kB; podocyte; Rarres1
Inflflammatory pathways are activated in most glomerular diseases but molecular mechanisms driving them in kidney tissue are poorly known. We identified retinoic acid receptor responder 1 (Rarres1) as a highly podocyte-enriched protein in healthy kidneys. Studies in podocyte-specific knockout animals indicated that Rarres1 was not needed for the normal development or maintenance of the glomerulus filtration barrier and did not modulate the outcome of kidney disease in a model of glomerulonephritis. Interestingly, we detected an induction of Rarres1 expression in glomerular and peritubular capillary endothelial cells in IgA and diabetic kidney disease, as well as in ANCA-associated vasculitis. Analysis of publicly available RNA data sets showed that the induction of Rarres1 expression was a common molecular mechanism in chronic kidney diseases. A conditional knock-in mouse line, overexpressing Rarres1 specifically in endothelial cells, did not show any obvious kidney phenotype. However, the overexpression promoted the progression of kidney damage in a model of glomerulonephritis. In line with this, conditional knock-out mice, lacking Rarres1 in endothelial cells, were partially protected in the disease model. Mechanistically, Rarres1 promoted inflflammation and fifibrosis via transcription factor Nuclear Factor-kB signaling pathway by activating receptor tyrosine kinase Axl. Thus, induction of Rarres1 expression in endothelial cells is a prevalent molecular mechanism in human glomerulopathies and this seems to have a pathogenic role in driving inflflammation and fifibrosis via the Nuclear Factor-kB signaling pathway.
Translational Statement
We identified the upregulation of retinoic acid receptor responder protein 1 (Rarres1) in endothelial cells (ECs) of common human glomerulopathies. The induction seems to have a pathogenic role in glomerular disease as the progression of glomerulonephritis was (i) aggravated in a mouse overexpressing Rarres1 specifically in ECs, and (ii) ameliorated in an EC-specific Rarres1 knockout line. Rarres1 can be a biomarker to detect microvascular damage in kidney diseases and potentially a novel therapeutical target.
Glomerular disease processes are a major cause of end-stage renal disease. Diabetic nephropathy (DN) is globally the most common cause of end-stage renal disease, whereas IgA nephropathy (IgAN) is the most prevailing primary glomerulonephritis (GN).1,2 Histologically, glomerular damage in these common disorders manifests in a very similar fashion. This includes activation of glomerular endothelial cells (GECs), expansion of extracellular matrix, the proliferation of mesangial cells, podocyte damage, and eventually podocyte loss.3
Global transcript profiling studies in human renal biopsies have shown a strong inflflammatory signature in glomerular disorders,4–7 and mouse studies have demonstrated that the activation of nuclear factor-kB (NF-kB) and transforming growth factor-b (TGF-b) signaling pathways have important roles in disease progression.8,9 Importantly, pharmaceutical targeting of the NF-kB pathway has renoprotective effects in animal models, indicating that the pathway is a potential target to treat glomerular diseases.10,11 In podocytes, we recently identified G protein-coupled receptor class C group 5 member B as a modulator of NF-kB–mediated inflflammatory response.12 However, the activation of inflflammatory pathways in glomerulopathies is probably driven by multiple cell types. In fact, the activation of the NF-kB signaling pathway is detected in GECs in many human kidney diseases.13,14 The molecular mechanisms involved in NF-kB activation in GECs are poorly understood.
Retinoic Acid Receptor Responder1 can Promotes Development Of Glomerular Diseases
In this study, we analyzed molecular signatures of human glomerular diseases and identified the induction of retinoic acid receptor responder 1 (Rarres1) expression in GECs as a common molecular signature in DN, IgAN, and anti-neutrophil cytoplasmic autoantibody (ANCA)–associated vasculitis. Studies in mouse lines with activation/inactivation of Rarres1 expression in endothelial cells (ECs) demonstrate that this induction has a pathogenic role in a mouse model of GN. Mechanistically, Rarres1 activates receptor tyrosine kinase Axl, leading to activation of the NF-kB signaling pathway. We propose Rarres1 as a new biomarker of microvascular damage that may be an attractive therapeutic target for glomerular disease processes by modulating inflflammatory responses.
Figure 1 | (Continued) (c) BackSPIN analysis showing Rarres1 expression specifically in podocytes (PD) in RNAseq data of murine kidney tissue (data from Woroniecka et al.6 ). (d) Relative mRNA levels of Rarres1 in isolated Tub and Glom from human samples. Data are expressed as a fold change of Rarres1 expression in the Glom compared with the Tub. *P < 0.05 versus Tub. (e) Representative confocal FL fluorescent microscopic image of Rarres1 expression (green) by podocytes in human glomeruli from normal subjects. (f) Representative confocal microscopic images showing the expression of Rarres1 in podocytes from human subjects; vimentin was used as a marker for major processes and nephrin as a marker for foot processes of podocytes. CD31 was used as a marker for endothelial cells, and ⍺-smooth muscle actin (⍺SMA) was used as a mesangial cell marker. Bars = 50/10 mm. Data are expressed as means ± SEM. The student’s t-test was employed for comparisons between 2 groups. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
METHODS
RNA extraction and real-time polymerase chain reaction
Glomeruli were isolated as described previously.15 RNA was extracted using TRIzol reagent (Invitrogen) and RNeasy Kit (Qiagen Inc.). The iScript cDNA Synthesis Kit was used to generate cDNA. The quantitative polymerase chain reaction was performed using the CFX96 Touch Real-Time PCR Detection System with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad). Primers used are listed in Supplementary Table S1. The relative expression levels were calculated using the 2-DDCT method.
Cell culture
Human podocytes were cultured as described.16 JetPEI transfection reagent (Polyplus-transfection SA) was used for stable transfection of pRP[Exp]-CMV>hRARRES1[ORF011242]:T2A: Bsd – plasmid (VectorBuilder Inc. Clones have expanded under puromycin (Merck KGaA) selection. Transfection of short interfering RNA (siRNA) for Axl/Rarres1 was performed using siRNA from Origene. Cells were transfected with 30 nM siRNA followed by complexation with JetPEI transfection reagent (Polyplus-transfection SA) with or without TGF-b1 stimuli (10 ng/ml in culture medium). The Axl-inhibitor R428 (Selleck Chemicals) was added 1 hour before treatment (3 mmol/l).
Why Retinoic Acid Receptor Responder1 can Promotes Development Of Glomerular Diseases?
Western blot
Cells were lysed with RIPA lysis buffer supplemented by protease/ phosphatase inhibition cocktails (Roche). Proteins from nuclear fractions were isolated using Nuclear Extract Kit (Active Motif Europe). Lysates were separated on 4%–12% Tris-glycine sodium dodecyl sulfate–polyacrylamide gel (Invitrogen) and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% bovine serum albumin (Merck). Incubation with primary antibodies was carried out overnight (antibodies in Supplementary Table S2). Horseradish peroxidase-conjugated secondary antibodies and Clarity Western ECL Substrate (Bio-Rad) were used to detect signals. Western blot analyses were performed at least twice.
RNA in situ hybridization
The RNAscope analyses were performed in paraffin-embedded human kidneys according to the manufacturer’s protocol (ACD). The probes used were NPHS1 - Cat No. 416071-C2, PECAM1 - Cat No. 487381-C3, and RARRES1 - customized probe targeting nucleotides 291-1515.
Assessment of kidney injury
Urinary albumin and creatinine ratio was evaluated using the Mouse Albumin ELISA Kit (Allbuwell M; Ethos Biosciences) and the QuantiChrom Creatinine Assay Kit (BioAssay Systems). Kidney tissues were fixed in 10% neutral buffered formalin solution and embedded in paraffin, followed by cutting and periodic acid-Schiff staining. Electron microscopy was performed in samples fixed in 2.5% glutaraldehyde.
Immunostainings
Paraffin-embedded tissues were fixed for 24 hours in formalin. Five-micrometer sections were deparaffinized and microwave-treated either in Tris-ethylenediamine tetraacetic acid buffer (10 mM Tris base, 1 mM ethylenediaminetetraacetic acid buffer solution, 0.05% Tween 20, pH 9.0) or sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0), followed by permeabilization with 0.3% Triton X-100 and blocked with 10% normal goat serum, 3% hydrogen peroxidase, and the Vector Blocking Kit (Vector Laboratories). Slides were developed using the DAB peroxidase (horseradish peroxidase) substrate kit, 3,30 -diaminobenzidine (Vector Laboratories).
For frozen sections, acetone-fixed sections were permeabilized with 0.1% Tween 20 and blocked with 10% normal goat serum. Primary antibodies used are listed in Supplementary Table S1. Alexa-Fluor secondary antibodies for immunofluorescence were obtained from Molecular Probes (Life Technologies). Images were obtained using the Leica SP8 Confocal Microscope (Leica Microsystems).
Human samples
Renal biopsies were obtained from Karolinska University Hospital (Stockholm, Sweden). Control samples were obtained from the healthy kidney poles of individuals who underwent tumor nephrectomies. The local ethical committee approved the study (approval no. 2010/579-31/1, Stockholm, Sweden).
versus control (Ctrl.) or ***P < 0.001 versus si-negative control (scramble) (Rarres1, n = 6; siRarres1, n = 4). (c) Representative Western blot documents and summarized data showing the relative protein levels of pP65 in isolated nuclear fractions of podocytes treated with 10 ng/ml TGF-b1. **P < 0.01, ***P < 0.001 versus control podocytes (Ctrl.) (n = 3). (d) Representative Western blot documents and summarized data showing the relative protein levels of pP65 in isolated nuclear fractions of podocytes treated with 10 ng/ml TGF-b1 ±3 mmol/l R428, n = 2. (e) Representative Western blot showing that overexpression of Rarres1 activates Axl constitutively. *P < 0.05 (n = 2). (f) Relative mRNA levels of EMT-related genes in podocytes overexpressing Rarres1 (Rarres1) or control podocytes (Ctrl.) transfected with siRNA for Axl (siAxl), si-negative control (scramble), ##P < 0.01, ###P < 0.001 versus scramble, n = 3, or treated with 3 mmol/l R428. ##P < 0.01, ###P < 0.001 versus ctrl., **P < 0.01, ***P < 0.001 versus Rarres1, *P < 0.05 versus Rarres1, n = 4. Data are expressed as means ± SD. Student’s t test was employed for comparisons between 2 groups. UT, untreated.
Figure 3 | Establishment of podocyte-specific Rarres1 knockout (CreD/Rarres1flfl/flfl) mice. (a) Generation of conditional knockout mice in which Rarres1 is specifically ablated in podocytes by using the Cre-LoxP recombination system. Exon 3 is deleted on NPHS2-Cre–mediated recombination. (b) Genotyping was confirmed by tail preparation and polymerase chain reaction at 4 weeks of age. (Continued)
Figure 3 | (Continued) (c) Representative Western blot showing the decreased expression of Rarres1 in isolated glomeruli from podocyte-specific Rarres1 knockout (Creþ/Rarres1flfl/flfl ) mice. (d) Podocyte-specific loss of Rarres1 does not affect albuminuria levels in mice treated with nephrotoxic serum (NTS) as analyzed by urine albumin-to-creatinine ratio in different groups of mice (NTS-ctrl., n = 5–7; NTS-Rarres1_cKO, n - 7). (e) Photomicrographs and quantifications showing typical glomerular structure changes in different groups of mice. Glomerular lesions were identified in periodic acid–Schiff–stained sections. #P < 0.0001 versus untreated control (Ctrl.) (Ctrl., n - 6; NTS-ctrl., n - 9; NTS-Rarres1_cKO, n = 9). (f) Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness and the number of slits per mm GBM in different groups of mice by transmission electron microscopy analyses. ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus untreated control (Ctrl., n = 3; NTS-ctrl., n = 4; NTS-Rarres1_cKO, n = 6). Data are expressed as means ± SEM. One-way analysis of variance followed by Tukey’s post-test for multiple comparisons was used for groups of 3 or more. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Mouse lines
Floxed Rarres1 mice with C57Bl/6J background (Rarres1flfl/flfl; Cyagen) were podocin (B6.Cg-Tg [NPHS2-cre] 295Lbh/J; Jackson Laboratory) and Tie2 cre mice (B6.Cg-Tg(Tek-cre)12Flv/J) to generate cell-specific knockout mice. The GT(ROSA)26Sortm1(CAG-Rarres1-T2AEGFP)/J mice were crossed with a Tie2-cre line to activate Rarres1 expression specifically in ECs.17 Breeding and genotyping were done according to standard procedures. All animal studies were carried out in Preclinical Laboratory (Karolinska Institutet) according to the relevant guidelines and regulations and approved by the Ethical Committee on Research Animal Care (Linköpings djurförsöksestiska nämd; DNR 1336).
Nephrotoxic serum (NTS)–induced nephropathy in mice
Seven- to 9-week-old mice received 50 ml Complete Freund’s Adjuvant (Merck) s.c., followed by NTS injection (i.v.; 6 ml/kg body weight; Probetex Inc.) on day 4.18 Urine was collected 3, 7, 10, and 14 days after the injection, and mice were killed at day 14.
Statistical analysis
Statistical analysis was performed using Prism 9.0 software (GraphPad).
The data were first examined for normality (Shapiro-Wilk test). When the distribution was Gaussian, the data were analyzed using a parametric test (Student’s t-test for 2 groups, and analysis of variance with Tukey’s post-test for more than 2 groups). When the distribution was not Gaussian, nonparametric tests (Friedman test with Dunn’s post-test for more than 2 groups and the Mann-Whitney U test for 2 groups) were used. The differences were considered significant when P < 0.05.
RESULTS
Rarres1 is expressed by podocytes
We first assessed the expression pattern of Rarres1 in murine and human kidney tissue. Polymerase chain reaction analyses showed that Rarres1 is expressed signifificantly higher in the glomerular than in the tubulointerstitial fraction of mouse renal cortex (Figure 1a and b6 ). Single-cell RNA sequencing analysis of murine kidney tissue revealed that Rarres1 is exclusively expressed by podocytes (Figure 1c). In humans, Rarres1 was also enriched in glomerular tissue (Figure 1d). In immunofluorescence of normal human kidney tissue, Rarres1 showed a high signal in glomeruli (Figure 1e). Double stainings showed that Rarres1 was located “outside” nephrin reactivity in the capillary loops, suggesting podocyte expression (Figure 1f). Colocalization with vimentin indicated localization to major processes. No overlapping reactivity was seen for EC marker CD31 or mesangial marker α-smooth muscle actin (Figure 1f).
Rarres1 promotes NF-kB and TGF-b1 signaling via receptor tyrosine kinase Axl
To elucidate the role of Rarres1 in podocytes, we manipulated its expression levels in immortalized human podocytes.16 Rarres1 levels were signifificantly upregulated in podocytes stably transfected with human Rarres1 cDNA and down-regulated after transfection with Rarres1-targeting siRNA (Figure 2a). Overexpression of Rarres1 upregulated the expression of epithelial-mesenchymal transition-related genes, whereas the downregulation of the endogenous Rarres1 had the opposite effect (Figure 2b). As NF-kB signaling has an important role in the epithelial-mesenchymal transition leading to fifibrosis and inflflammation in GN,19 we measured TGF-b1–induced NF-kB signaling activation in vitro. Rarres1 overexpression caused increased activation of the NF-kB pathway at baseline and after TGF-b1 stimulation (Figure 2c). As Rarres1 has been reported to activate receptor tyrosine kinase Axl, and in that way promote the NF-kB signaling pathway in inflflammatory breast cancer,20 we assessed whether Axl is a potential functional partner of Rarres1 in kidney tissue. TGF-b1–induced NF-kB activation could be inhibited by R428, a selective small-molecule inhibitor of Axl kinase21,22 (Figure 2d), and the overexpression of Rarres1 correlated positively with the constitutive activation of Axl kinase (Figure 2e). The depletion of Axl activity with R428 but not gene silencing of Axl by siRNA could diminish the Rarres1-induced expression of epithelial-mesenchymal transition-related genes, as evidenced by reduced mRNA levels (Figure 2f). Of note, no activation of noncanonical NF-kB pathways was detected (Supplementary Figure S1). Thus, Rarres1 promoted NF-kB signaling in cultured podocytes via activation of Axl kinase.
Why Retinoic Acid Receptor Responder1 can Promotes Development Of Glomerular Diseases?
Inactivation of Rarres1 in podocytes does not lead to glomerular abnormalities or modulate the outcome of nephropathy in an anti-glomerular basement membrane (GBM) GN model
To analyze the role of Rarres1 in podocytes in vivo, we generated a novel Rarres1flfl/flfl (floxed) mouse line and crossed it with podocin-Cre mice to produce Podocin-Cre Rarres1flfl/flfl mice (Creþ/Rarres1flfl/flfl ) (Figure 3a). Genotypes for floxed/Cre alleles were identified by ear genotyping (Figure 3b). Polymerase chain reaction analysis of tubulointerstitial and glomerular fractions, as well as of liver tissue, confirmed the deletion of exon 3 specifically in the glomerulus (Supplementary Figure S2A), and Rarres1 protein levels were reduced in glomeruli from Creþ/Rarres1flfl/flfl mice (Figure 3c).
Figure 4 | Upregulation of Rarres1 in glomerular and peritubular capillary endothelial cells in patients with chronic kidney disease. (a) Analyses of Rarres1, collagen1a2 (COL1A2), and nephrin (NPHS1) gene expression in microarray data of glomeruli isolated from patients with diabetic nephropathy (DN), vasculitis, IgA nephropathy (IgAN), and control (Ctrl.) subjects.4,6,24–26 *P < 0.05, **P < 0.01, ***P < 0.001 versus control subjects. (b) Quantitative polymerase chain reaction analysis for Rarres1, COL1A2, and NPHS1 genes in isolated glomeruli from patients with DN (n = 5) and controls (n = 5). **P < 0.01, ***P < 0.001 versus control subjects. (c) Analyses of Rarres1 and COL1A2 gene expression in microarray data of tubular fraction isolated from DN, vasculitis, IgAN, and control patients. *P < 0.05, **P < 0.01, ***P < 0.001 versus control subjects. (d) Representative photomicrographs of Rarres1 immunohistochemical staining in human renal cortical tissue from controls and patients with DN (n = 5), anti-neutrophil cytoplasmic autoantibody (ANCA) vasculitis (n =4), and IgAN (n = 5). The arrows indicate Rarres1-positive podocytes (control) and endothelial cells (ECs) (DN, IgAN, and vasculitis). (Continued)
Figure 4 | (Continued) (e) Representative confocal microscopic images showing the expression of Rarres1 in glomeruli of patients with DN. CD31 was used as a marker for ECs. Bars = 50/10 mm. (f) Representative confocal FL fluorescent pictures of RNAscope showing Rarres1 gene expression in PECAM1-coexpressing cells in the tubular fraction of patients with DN. Data are expressed as means±SEM. The student’s t-test was employed for comparisons between 2 groups. Bars = 50/10 mm. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Creþ/Rarres1flfl/flfl mice were born at an expected Mendelian inheritance ratio and they were viable and fertile (data not shown). Kidneys were indistinguishable from control littermates (Cre- /Rarres1flfl/flfl ) as analyzed by routine light and electron microscopic examination (Supplementary Figure S2B and C). Also, the distribution of podocyte proteins nephrin and synaptopodin was unaffected in Creþ/ Rarres1flfl/flfl mice (Supplementary Figure S2D)
To elucidate the role of Rarres1 in kidney injury in vivo, Creþ/Rarres1flfl/flfl were treated with NTS that leads to IgG deposits in the GBM, albuminuria, and progressive GN.23 Albuminuria levels were similar in both Creþ/Rarres1flfl/flfl and control mice (Figure 3d). Light microscopic examination revealed no significant differences in the proportion of affected glomeruli (Figure 3e). Electron microscopy showed no difference in the thickness of the GBM or foot process effacement, and GECs seemed to be similarly affected in both groups (Figure 3f). Podocyte markers nephrin, Wt1, and synaptopodin were affected in a similar fashion in both groups (Supplementary Figure S3A). Also, staining for ECs using CD31 showed no differences in glomeruli or peritubular capillaries between the groups (Supplementary Figure S3B and C). In addition, the expression of fifibrosis and inflflammation-related genes were similarly elevated in both groups treated with NTS (Supplementary Figure S4).
Rarres1 expression is induced in glomerular and peritubular capillary ECs in chronic kidney disease (CKD)
To explore the role of Rarres1 in CKD, we analyzed its expression in publicly available RNA data sets. We observed signifificantly elevated Rarres1 levels in glomeruli isolated from patients with DN, ANCA-associated vasculitis, and IgAN (Figure 4a),4,6,24–26 whereas other podocyte-associated genes, for example, nephrin, were signifificantly downregulated or unchanged. Our quantitative polymerase chain reaction analysis of glomeruli isolated from patients with DN (n = 5) validated the induction of Rarres1 expression (Figure 4b). Because of loss of podocytes, the expression of podocyte genes is usually decreased in progressive glomerular diseases,6 and the ratio of glomerular Rarres1 to nephrin expression was increased by approximately 20-fold in DN versus healthy glomeruli (Figure 4b). The expression of Rarres1 was also upregulated in tubulointerstitial tissue data sets in DN, ANCA-associated vasculitis, and IgAN (Figure 4c).8–11
Next, we analyzed Rarres1 expression immunohistochemically in renal biopsies from patients with DN (n ¼ 5), IgAN (n ¼ 5), and ANCA-associated vasculitis (n ¼ 4). In diseased glomeruli, Rarres1 staining seemed to be localized to GECs as shown by immunoreactivity on the inner face of capillary walls (Figure 4d, insets), whereas the signal from podocytes appeared weaker. In tubulointerstitial space, the staining was detected in peritubular capillaries (Figure 4d, insets), suggesting EC expression. Double immunolabeling with CD31 validated the localization to ECs in both glomeruli and peritubular capillaries (Figure 4e). Similarly, double RNAscope experiments showed the colocalization of Rarres1 mRNA with PECAM1 mRNA in DN (Figure 4f). Taken together, the induction of Rarres1 expression in glomerular and peritubular capillary ECs was a common phenomenon in CKD.
Generation and characterization of a mouse line overexpressing Rarres1 in ECs
As Rarres1 is induced in human CKD, we generated a novel transgenic mouse line in which Rarres1 cDNA was introduced into the Rosa26 locus under the CAG promoter,27 with a lox-STOP-lox cassette between the splice acceptor sites (Figure 5a). The line was crossed with a Tie2-cre line to generate Rarres1_Tie2_KI mice, and mice were identified by ear genotyping for knockin and Cre alleles (Figure 5a). Rarres1_Tie2_KI mice showed signifificantly increased levels of Rarres1 protein in kidney tissue (Figure 5b), and the successful knockin strategy was further validated by analysis of Rarres1 mRNA expression in FACS-sorted GFP-positive GECs (Figure 5c).
We phenotyped kidneys of Rarres1_Tie2_KI mice and control mice in age-matched groups. By light microscopic examination, the kidney morphology was indistinguishable in Rarres1_Tie2_KI mice from control littermates (Figure 5d). Similarly, electron microscopy showed an unchanged GBM thickness and podocyte foot process width (Figure 5e). Moreover, the expression of podocyte markers nephrin and synaptopodin seemed unaffected in Rarres1 overexpressing glomeruli (Figure 5f). Of note, other major organs did not show macroscopically or microscopically any obvious abnormalities (data not shown).
Figure 5 | Establishment of endothelial cell-specific Rarres1 knockin (Rarres1_Tie2_KI) mice. (a) A generation of conditional knockin mice in which Rarres1 expression is specifically induced in endothelial cells (ECs) by using the Cre-LoxP recombination system. Tie2-Cre mice were crossed with mice expressing the targeting vector (Rarres1_casetteþ), containing a lox-STOP-lox cassette between the splice (continued)
Figure 5 | (continued) acceptor sites and Rarres1 cDNA under the CAG promoter, which was introduced into the Rosa26 locus, were identified by ear genotyping. Genotyping was confirmed by ear preparation and polymerase chain reaction (PCR) at 4 weeks of age. (b) Representative Western blot showing the increased expression of Rarres1 in the renal cortex from EC-specific Rarres1 knockin (Rarres1_Tie2_KI) mice. **P < 0.01 versus control (Ctrl.; Cre- /Rarres1_Tie2_KI, n = 5, Mann-Whitney U test). (c) Quantitative PCR for Rarres1 in isolated ECs. **P < 0.01 versus control (Cre- /Rarres1_Tie2_KI, n = 3, Student’s t-test). (d) Representative photomicrographs of periodic acid–Schiff–stained sections showing typical glomerular structures in different groups of mice. (e) Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness, and the number of slits per mm GBM in different groups of mice by transmission electron microscopy analyses (Student’s t-test). (f) Representative confocal microscopic images showing the expression of the podocyte-specific marker synaptopodin and nephrin in EC-specific Rarres1 knock-in mice (Rarre1_Tie2_KI) and Ctrl. mice. Bars = 10 mm. Data are expressed as means ± SEM. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
Induction of Rarres1 in ECs modulates the NF-kB signaling pathway and the outcome of nephropathy by regulating Axl activation
To evaluate whether Rarres1 expression in ECs modulates disease progression, we induced GN in transgenic mice and their littermates with a single injection of NTS. Rarres1_- Tie2_KI mice developed signifificantly higher albuminuria levels than controls after 3 days, whereas no significant difference in albuminuria was seen at 7, 10, and 14 days (Figure 6a). Histologic analysis 14 days after the induction showed that Rarres1_Tie2_KI mice did not have more glomerular alterations, such as sclerotic and crescents lesions (Figure 6b). Electron microscopic evaluation showed, however, a signifificantly thicker GBM and more foot process effacement in Rarres1_Tie2_KI mice (Figure 6c). Furthermore, Rarres1_Tie2_KI mice showed increased levels of -smooth muscle actin in the kidney cortex (Figure 6d). The analysis of podocyte markers nephrin, synaptopodin, and WT1 showed a profound reduction suggesting dedifferentiation and a significant loss of podocytes (Figure 6d). Importantly, Rarres1_Tie2_KI glomeruli showed fewer WT1-positive podocytes in comparison with control animals, indicating that EC-expressed Rarres1 was promoting podocyte damage. Also, more fibrin thrombi were detected in Rarres1_Tie2_KI glomeruli (Figure 6e). In line with this, the expression of different inflflammation, fifibrosis, and renal tubular injury-related genes were signifificantly upregulated in Rarres1_- Tie2_KI mice as shown by real-time polymerase chain reaction analysis for Il-1beta, Col1a1, TGF-b1, and kidney injury molecule-128 in kidney cortex tissue (Figure 6f). However, no differences were detected in blood urea nitrogen and S-creatinine values between the groups (Figure 6g). Also, staining for ECs using CD31 showed no differences in glomeruli or peritubular capillaries between the groups (Supplementary Figure S5A and B). Taken together, EC-specific induction of Rarres1 expression aggravated glomerular pathology in NTS-induced nephropathy.
Mechanistically, we analyzed whether Rarres1 over-expression in ECs promoted the activation of Axl and NF-kB signaling in vivo, similar to our in vitro observations (Figure 2). Both Rarres1_Tie2_KI and control mice treated with NTS exhibited an activation of the Axl signaling pathway as shown by increased Axl phosphorylation (Figure 6h). However, this activation was more significant in Rarres1_- Tie2_KI mice (Figure 6h). Correspondingly, NF-kB pathway activation was promoted in these mice as shown by the number of pP65-positive nuclei in glomeruli (Figure 6i). When pP65 levels were measured by Western blotting, we detected a similar trend for NF-kB pathway activation although this was not significant, possibly due to one outlier (Supplementary Figure S5C).
Inactivation of Rarres1 in ECs decreases glomerular damage in the anti-GBM glomerulonephritis model
To validate the pathogenic role of EC-derived Rarres1 in CKD, we generated a mouse line in which Rarres1 was deleted in ECs using Tie2-Cre (Figure 7a). Genotypes for floxed and Cre alleles were identified by ear genotyping (Figure 7b). There was a trend for upregulation of Rarres1 in NTS-treated mice, which was abolished in Rarres1_Tie2_KO mice (Figure 7c). The mice did not show any obvious renal abnormalities (data not shown). The injection of NTS induced similar albuminuria in Rarres1_Tie2_KO and controls (Figure 7d). However, histologic analysis 14 days after the induction showed fewer glomerular alterations in Rarres1_- Tie2_KO mice (Figure 7e). In electron microscopy, Rarres1_Tie2_KO mice showed less foot process effacement and thickening of the GBM (Figure 7f). Immunostaining showed no significant differences for aSMA, nephrin, or synaptopodin (Figure 7g). However, the number of WT1-positive podocytes was signifificantly more in Rarres1_Tie2_KO mice (Figure 7g). Staining for pP65 showed a decreased number of positive nuclei in Rarres1_Tie2_KO glomeruli suggesting the involvement of the NF-kB signaling pathway (Figure 7h).
Figure 6 | (continued) quantification of mean glomerular basement membrane (GBM) thickness and the number of slits per mm GBM in different groups of mice by transmission electron microscopy analyses sections (Ctrl., n = 4; NTS-ctrl., n = 9; NTS-Rarres1_Tie2_KI, n = 12). (d) Representative confocal microscopic images showing the expressions of nephrin, synaptopodin, ⍺-smooth muscle actin (⍺Sma), and WT1 in the kidney from different groups of mice. Bars = 100/10 mm. The ⍺Sma–positive area was counted per cortical cross-section, and WT1 was counted per glomerulus (Ctrl., n = 2; NTS-ctrl., n = 3; NTS-Rarres1_Tie2_KI, n = 3). (e) Presence of fibrin thrombi in glomeruli as detected by trichome staining (counted per glomerular cross-section) (Ctrl., n = 3; NTS-ctrl., n = 4; NTS-Rarres1_Tie2_KI, n = 5). (f) Relative mRNA levels of different inflflammation, fifibrosis, and renal tubular injury-related genes in the renal cortex of NTS-treated mice (Ctrl., n = 4; NTS-ctrl., n = 6–10; NTS-Rarres1_Tie2_KI, n = 6–10). (g) Serum creatinine and blood urea nitrogen (BUN) levels in NTS-ctrl. (n = 3) and NTS-Rarres1_Tie2_KI (n = 3) mice. (h) Representative Western blot documents and summarized data showing the relative protein levels of Paxil in the renal cortex in different groups of NTS-treated mice (NTS-ctrl., n = 4; NTS-Rarres1_Tie2_KI, n = 5). (i) Representative photomicrographs of pP65 immunohistochemical staining in renal cortical tissue from different groups of NTS-treated mice. pP65-positive nuclei per glomeruli were quantified in the different groups of mice (Ctrl., n = 7; NTS-ctrl., n = 8; NTS-Rarres1_Tie2_KI, n = 14). EC-specific Rarres1 knock-in mice (Rarres1_Tie2_KI mice); mice with Rarres1 cassette and without Cre expression (Ctrl.) were used as control. #P < 0.05, ##P < 0.01, ###P < 0.001 versus untreated control (Ctrl.), *P < 0.05 versus NTS-treated control (NTS-ctrl.). Data are expressed as means ± SEM. The student’s t-test was employed for comparisons between 2 groups. One-way analysis of variance followed by Tukey’s post-test for multiple comparisons was used for groups of 3 or more. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
DISCUSSION
Rarres1 was first identified in skin raft cultures treated with tazarotene29 and has been shown to act as an invasion repressor in prostate cancer cell lines.30 In inflflammatory breast cancer, Rarres1 regulates in vitro the invasion of cancer cells through the mediation of the Axl signaling pathway. In detail, Rarres1 stabilizes Axl by inhibition of the proteasome-dependent degradation of Axl. Furthermore, the depletion of Rarres1 in SUM149 cells downregulates Axl expression and inactivates NF-kB, leading to reduced invasion of inflflammatory breast cancer cells.20 In addition, Rarres1 expression is induced in patients with advanced fifibrosis, and a profibrotic role of Rarres1 has been suggested in a rat lung fifibrosis model and during fibrogenic activation of hepatic stellate cells.31 Taken together, previous in vitro studies suggest a role for Rarres1 in fifibrosis, inflflammation, and cancer invasion.
Initially, Rarres1 raised our interest as it was highly enriched in podocyte cells. We could show that Rarres1 promoted inflflammatory and profibrotic signaling in podocytes via Axl-mediated NF-kB pathway activation. On the other hand, podocyte-specific knockout animals failed to show any obvious difference in comparison with controls under healthy conditions as well as in a mouse model of GN. This could be due to compensatory mechanisms as the NF-kB signaling pathway is regulated by a number of molecules and biological processes.
Molecular profiling of patients with diabetes indicated that Rarres1 was induced in glomerular and peritubular capillary ECs in DN. Importantly, Rarres1 expression was not detected in ECs in diabetic patients without nephropathy. Therefore, we speculate that Rarres1 could be a new marker for diabetic microvascular disease, and studies in plasma and urine samples are indicated to explore the feasibility of Rarres1 as a non-invasive biomarker. We identified the induction of Rarres1 also in patients with IgAN and vasculitis. It is tempting to speculate that Rarres1 has a pathogenic role in these glomerulopathies and contributes to disease progression. Functionally, we showed that the induction of Rarres1 in the ECs augmented renal injury in a murine model of GN, whereas the inactivation of Rarres1 in ECs ameliorated the injury. Phenotypical analysis showed that podocytes were affected in mice overexpressing Rarres1 in ECs. This may be a direct effect of Rarres1 to podocytes as a soluble form of Rares has been described.32 Alternatively, podocyte injury may be secondary to the damage of the glomerulus filtration barrier.
Previously, endothelial NF-kB activation has been shown to play a pathogenic role in the development of kidney disease in vasculitis.13 Notably, inhibition of the endothelial NF-kB pathway in a model of crescentic GN abrogated the progression. Axl inhibitors have been shown to be beneficial in experimental anti-GBM nephritis. Mice treated with the most selective small-molecule inhibitor, R428, showed significant preservation of kidney function and decreased inflflammatory cytokine production.33 This indicates that the pathway could be a therapeutic option for glomerular disease processes. However, as both Axl and NF-kB are modulating a number of different cellular processes throughout the body, it is unlikely to be a good candidate for pharmaceutical targeting in CKD. Rarres1 can provide a more kidney-associated target.
Tie2-Cre has been shown to drive expression in a subpopulation of hematopoietic cells.34 Some of these cells may modulate the inflflammatory response in the NTS-induced model, and therefore we cannot exclude that hematopoietic cell-derived Rarres1 contributes to disease progression in Tie2-Cre lines. However, as we did not detect any obvious Rarres1 expression in infiltrating immune cells, we think that this is less likely.
While this manuscript was in preparation, Chen et al.32 reported a soluble form of Rarres1 that promotes glomerular disease progression. They identified, similarly to us, the upregulation of Rarres1 in common human glomerular diseases. The study supports our finding that an increased Rarres1 expression correlates with renal function decline in human glomerular disease. However, in contrast to our results, they concluded that the upregulation of Rarres1 was due to overexpression in podocytes. We cannot exclude that possibility, but we detected in human diseases a distinct induction of Rarres1 expression in ECs. One mechanism explaining our findings could be that the soluble form of Rarres1 produced by podocytes is taken up by GECs. However, the results that we detected Rarres1 mRNA in ECs by in situ hybridization and that Rarres1 was also induced in peritubular capillary ECs strongly support the idea that the expression of Rarres1 in renal diseases is mainly of endothelial origin.
Figure 7 | Endothelial-specific ablation of Rarres1 attenuates renal injury in nephrotoxic serum (NTS)–treated mice. (a) Generation of conditional knockout mice in which Rarres1 is specififically ablated in endothelial cells using the Cre-LoxP recombination system. Exon 3 is deleted on Tie2-Cre–mediated recombination. (b) Genotyping was confirmed by tail preparation and polymerase chain reaction at (continued)
Figure 7 | (continued) 4 weeks of age. (c) Representative Western blot documents and summarized data showing the relative protein levels of Rarres1 in the renal cortex in different groups of NTS-treated mice (NTS-ctrl., n = 4; NTS-Rarres1_Tie2_KO, n = 4) and untreated mice (Control [Ctrl.], n = 3). **P < 0.01. (d) UACR (urine albumin-to-creatinine ratio) in different groups of mice on NTS treatment (Ctrl., n = 7–11; Rarres1_Tie2_KO, n = 6–8). (e) Photomicrographs and quantifications showing typical glomerular structure changes in different groups of mice. Glomerular lesions were identified in periodic acid-Schiff–stained sections (Ctrl., n = 3; NTS-treated control [NTS-ctrl.], n = 12; NTSRarres1_Tie2_KO, n = 8). (f) Representative photomicrographs and quantifications of mean glomerular basement membrane (GBM) thickness and the number of slits per mm GBM in different groups of mice by transmission electron microscopy analyses sections (Ctrl., n = 6; NTS-ctrl., n = 13; NTS-Rarres1_Tie2_KO, n = 16). (g) Representative confocal microscopic images showing the expressions of nephrin, synaptopodin, -smooth muscle actin (Sma), and WT1 in the kidney from different groups of mice. Bars = 50/10 mm. The Sma-positive area was counted per cortical cross-section (NTS-ctrl., n = 8; NTS-Rarres1_Tie2_KO, n = 4). (h) Representative photomicrographs of pP65 immunohistochemical staining in renal cortical tissue from different groups of NTS-treated mice. pP65-positive nuclei per glomeruli were quantified in the different groups of mice (NTS-ctrl., n = 7; NTS-Rarres1_Tie2_KO, n = 6). ####P < 0.001 versus untreated Ctrl., *P < 0.05 versus NTS-ctrl. Data are expressed as means± SEM. The student’s t-test was employed for comparisons between 2 groups. One-way analysis of variance followed by Tukey’s post-test for multiple comparisons was used for groups of 3 or more. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.
There are a few limitations in this study that should be acknowledged. First, in vitro studies have been performed in podocytes, whereas in vivo we manipulated the expression in ECs. Therefore, we cannot directly link Rarres1 to Axl activation in ECs. Second, whereas we could identify a Rarres1- induced (Axl-dependent) activation of NF-kB signaling in podocytes, we did not investigate the molecular mechanism behind this process. More studies are needed to explore the previous finding that Rarres1 promotes Axl activation by inhibiting its proteasome-dependent degradation. Finally, we challenged our knockout/-in animals only with the NTS-induced nephropathy model. Therefore, more studies in the animal models are indicated to see whether Rarres1 has a pathogenic role also in other disease processes.
To summarize, our study demonstrates that in common human glomerulopathies, the podocyte-enriched protein Rarres1 is induced in ECs, leading to inflflammation, podocyte injury, and fifibrosis. This is potentially mediated by enhancing the NF-kB signaling pathway via activation of receptor tyrosine kinase Axl (Figure 7). Rarres1 is a new biomarker of microvascular injury and could potentially be a novel therapeutic target to repress inflflammation and fifibrosis in CKD
DISCLOSURE
ML is an employee of AstraZeneca, Gothenburg, Sweden. JP’s research is supported by AstraZeneca. All the other authors declared no competing interests.
ACKNOWLEDGMENTS
This study was supported by KM-H from the Diabetes Wellness Sverige and to JP from KI/AZ Integrated Metabolic Center, Marianne och Marcus Wallenberg Foundation, Swedish Diabetes Foundation, Swedish Kidney Foundation and Center for Innovative Medicine.
AUTHOR CONTRIBUTIONS
KM-H, ML, and JP designed the research; KM-H, DD, EC, SZ, and GG performed the research; KM-H and XL analyzed the data; AW provided human samples; and KM-H and JP wrote the paper.
Why Retinoic Acid Receptor Responder1 can Promotes Development Of Glomerular Diseases?
SUPPLEMENTARY MATERIAL
Supplementary File (PDF)
Table S1. Primer pairs of target genes were used for real-time RT-PCR in this study.
Table S2. Antibodies were used in this study. Supplementary File (PowerPoint)
Figure S1. Rarres1 overexpression does not activate the noncanonical NF-kB signaling pathway. Representative Western blot document and summarized data showing that overexpression of Rarres1 does not activate p100/p52 in podocytes treated with 10 ng/ml TGF-b1 (n = 2). Data are expressed as means ± SD. The student’s t-test was employed for comparisons between two groups. UT, untreated.
Figure S2. Establishment of podocyte-specific Rarres1 knockout (Creþ/Rarres1flfl/flfl ) mice. (A) Conventional PCR analysis in tubular and glomerular fractions, as well as in liver tissue. (B) Histological examination (PAS-staining) of kidney pathology in Cre– /Rarres1flfl/flfl and Creþ/Rarres1flfl/flfl mice. (C) Representative transmission electron micrographs (TEM) and quantification of mean glomerular basement membrane (GBM) thickness and the number of slits per mm GBM in different groups of mice (n = 3). (D) Representative confocal microscopic images showing the expression of podocyte-specific markers, synaptopodin, and nephrin, in podocyte-specific Rarres1 knockout (Creþ/Rarres1flfl/flfl ) and control (Cre– /Rarres1flfl/flfl ) mice. Bar = 10 mm.
Figure S3. The expression levels of podocyte markers and CD31 in the kidneys of NTS-treated mice. (A) Representative confocal microscopic images showing the expression of nephrin, WT-1, and synaptopodin in podocytes from NTS-treated Rarres1 podocyte-specifific knockout mice (Creþ/Rarres1flfl/flfl ) and control littermates (Cre– /Rarres1flfl/flfl ). Bar = 10 mm (NTS Cre– /Rarres1flfl/flfl , n = 4; NTS-Creþ/Rarres1flfl/flfl , n = 5). (B) Proportion of CD31-positive capillaries in peritubular areas. Thirty peritubular areas were evaluated per mouse. (NTS Cre– /Rarres1flfl/flfl , n= 4; NTS-Creþ/Rarres1flfl/flfl , n = 4). Student’s t test. (C) Semiquantitative scoring of the CD31 signal in glomeruli: 0 = no signal, 1 = weak signal, 2 = moderate signal, 3 = strong signal. Ten glomeruli evaluated from each mouse (NTS Cre–/ Rarres1flfl/flfl , n = 4; NTS-Creþ/Rarres1flfl/flfl , n = 4). Student’s t test.
Figure S4. The expression levels for markers of fifibrosis, inflflammation, and tubular injury in the kidneys of NTS-treated mice. (A) Representative confocal microscopic images showing the expressions of -Sma in the kidney from NTS-treated Rarres1 podocyte-specific knockout mice (Creþ/Rarres1flfl/flfl ) and control littermates (Cre– /Rarres1flfl/flfl ). Bar = 100 mm. (B) Relative mRNA levels of different inflflammation-, fifibrosis-, and renal tubular injury-related–genes in the renal cortex of NTS treated mice. #P < 0.05, ##P < 0.01 versus control (ctrl.); data are expressed as means ± SEM. Student’s t-test was employed for comparisons between 2 groups (sham n = 2; NTS– Cre– /Rarres1flfl/flfl, n = 3; NTS-Creþ/Rarres1flfl/flfl , n = 3).
Figure S5. The expression of C31 and activated P65 in NTS-treated kidneys. (A) The proportion of CD31-positive capillaries in peritubular areas. Thirty peritubular areas were evaluated per mouse (NTS Cre–/ Rarres1flfl/flfl, n = 4; NTS Creþ/Rarres1flfl/flfl, n = 4). Student’s t-test. (B) Semiquantitative scoring of the CD31 signal in glomeruli: 0 = no signal, 1 = weak signal, 2 = moderate signal, 3 =strong signal. Ten glomeruli were evaluated from each mouse (NTS Cre –/Rarres1flfl/flfl, n =4; NTS-Creþ/Rarres1flfl/flfl, n= 4). Student’s t-test. (C) Representative Western blot documents and summarized data showing the relative protein levels of pP65 in the renal cortex in different groups of NTS treated mice and untreated controls (ctrl.) (ctrl., n = 3; NTS-ctrl., n = 9; NTS-Rarres1_Tie2_KI, n = 10). Data are expressed as means ± SEM. The student’s t-test was employed for comparisons between 2 groups.
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