Background
Chronic kidney diseases (CKD) affect more than 10% of world population causing a heavy social and economic burden [
1]. Renal tubulointerstitial fibrosis is the most important pathological feature of various CKD and is the best predictor for renal function outcomes [
2,
3]. Hallmarks of renal fibrosis include overexpression of extracellular matrix (ECM) fibrils (collagen-I, fibronectin, etc.), up-regulation of epithelial to mesenchymal transition (EMT) markers (α-SMA, N-cadherin, Snail, etc.), and activation of TGFβ/Smad3 signaling pathway [
4‐
6].
Besides the classic TGFβ/Smad3 signaling pathway, multiple signaling pathways are dysregulated in fibrotic kidneys. Impaired peroxisome proliferative activated receptor alpha (PPARA), hepatocyte nuclear factor 4 alpha (HNF4A) and activation of nuclear factor kappa-B (NFκB, such as c-Rel), Sex determining region Y-Box 6 (SOX6), early growth response protein 2 (EGR2), c-Myb signaling pathways are associated with progression of renal fibrosis [
7‐
13]. Krüppel-like factors (KLFs) belong to a family of zinc-finger transcription factors, which is a research hotspot in the field of kidney disease and several KLF family members have been identified as important players in renal tubulointerstitial fibrosis [
14]. STAT4 is a well-known regulator involved in autoimmunity and inflammation [
15,
16]. Since renal fibrosis is characterized by activation of inflammatory responses, STAT4 signaling pathway may also be involved in renal fibrosis [
17].
Asymmetrical dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) are both byproducts of protein methylation, and both of them are increased in the circulation of patients with end stage renal disease (ESRD) [
18‐
20]. Not like ADMA, SDMA is not a death predictor in ESRD [
20]. However, SDMA is still regarded as a risk factor for cardiovascular events [
18]. This is probably due to that SDMA is a well-known inducer of endothelial damages and thus endothelial-related disease is the focus of SDMA study so far [
21,
22]. The majority of SDMA is excreted through the kidney [
19]. Interestingly, chronic infusion of SDMA has no detrimental effect on normal kidneys [
23]. However, the effect of SDMA on kidney in a pathological condition has not been studied. The proximal tubule is the most vulnerable target of kidney injuries [
24]. The role of SDMA in tubular associated kidney disease is currently unknown.
In this study, we aimed to study the role of SDMA in chronic kidney disease with a focus on renal tubulointerstitial fibrosis and explored its underlying mechanisms.
Methods
Animal studies
Wild type male C57BL/6 mice with body weight between 20 and 25 g were purchased from Shanghai SLAC Laboratory Animal Co., Ltd and housed in a SPF grade animal facility in the Shanghai University of Traditional Chinese Medicine under the local regulations.
UUO surgery was performed through twice ligation of the left ureter with 4–0 nylon sutures as described in a previous study [
25]. Mice were randomly divided into five groups: (I) UUO + normal saline (NS, n = 10), (II) UUO + 2.5 µmol/kg symmetric dimethylarginine (SDMA, n = 10), (III)UUO + 25 µmol/kg SDMA (n = 10) group. In another experiment, sham operated mice were divided into three groups: (I) sham + no renal injection (n = 10), (II) sham + normal saline (n = 10), (III)sham + 25 µmol/kg SDMA (n = 10) group. Mice were sacrificed at day7 to collect renal tissue.
To measure the content of renal SDMA after intrarenal injection, UUO mice were injected with normal saline (n = 5) or 25 µmol/kg SDMA (n = 5). Mice were sacrificed at 1 h after the injection.
For the unilateral ischemia–reperfusion injury (UIRI) model, left renal pedicles were clamped for 35 min by using microaneurysm clamps in male mice. Mice were randomly divided into three groups: (1) UIRI + normal saline (n = 10), (2) UIRI + 25 µmol/kg SDMA (n = 10). Mice were sacrificed at day 11 for tissue collection.
Intrarenal SDMA administration
SDMA (T7344) was purchased from TargetMol Chemicals Inc. (Boston, MA, USA) and dissolved in normal saline as a 100 mM stock, which was further diluted with normal saline to working solution at different concentrations. 0.04% typan blue dye (A601140, Sangon, Shanghai, China) was added into the working solution to monitor the injection process. 50 μL of normal saline, 1 mM or 10 mM SDMA (2.5 µmol/kg or 25 µmol/kg) was injected retrogradely once into the left kidney via the ureter. Unilateral ureteral obstruction was performed after the injection. In UIRI mouse model, 50 μL of normal saline or 10 mM SDMA (25 µmol/kg) was injected into the left kidney via the ureter, which was clamped by using a microaneurysm clamps just before the ischemia surgery. After 35-min ischemia, ureter and renal artery clamps were released. The dosage of SDMA that we used in this study was calculated by considering a previous publication where SDMA (250 µmol/kg/day) was systemically infused into mice by using osmotic minipumps for a long-term [
23]. In sham operated mice, the left ureter was clamped for 30 min after normal saline or SDMA injection.
Quantitation of renal SDMA and asymmetrical dimethylarginine (ADMA)
Renal concentrations of SDMA and ADMA were measured by using a validated LC–MS/MS method with minor modification [
26]. In brief, kidney tissue samples were added 10 volumes of ice-cold normal saline (10 ml/g wet weight of tissue) and homogenized immediately. The kidney homogenates (50 μl) were mixed with acetonitrile-methanol mixture (1: 1, v/v) to which stable isotope-labelled internal standard (2,3,3,4,4,5,5-d
7-ADMA) had been added. Then, those compounds were converted into their butyl ester derivatives and measured by a Waters LC–MS/MS system, which contained an ACQUITY UPLC and an Xevo TQ-S tandem quadrupole mass spectrometry (Waters, Milford, MA, USA). Compounds were separated on an Acquity UPLC BEH C
18 column (2.1 × 100 mm, 1.7 µm, Waters). The coefficients of variation for both ADMA and SDMA were below 15%.
Cell culture
HK2 renal proximal tubular epithelial cells were purchased from the Cell Bank of Shanghai Institute of Biological Sciences (Chinese Academy of Science). Cells were seeded in 6-well plate to 40–50% confluence, which were starved overnight with DMEM/F12 medium containing 0.5% fetal bovine serum (FBS). On the next day, cells were changed with fresh 0.5% medium and exposed to 2.5 ng/ml TGF-β (Peprotech, Rocky Hill, NJ, USA) for 48 h in the presence of various concentration of SDMA or STAT4 inhibitor berbamine dihydrochloride (T2920, TargetMol).
Nonsense control (NC) or human STAT4 siRNA were transfected by Lipofectamine 2000 (11668–027; Invitrogen) in HK2 cells by using DMEM/F12 medium containing 10% fetal bovine serum according to the manufacturer’s instruction. After 48 h transfection, protein was extracted from cells. In another experiment, fresh medium containing 0.5% fetal bovine serum was changed in the second day after transfection, and cells were exposed to 2.5 ng/ml TGF-β for 48 h with or without SDMA. The NC siRNA sequences were as follows: forward, 5′-UUCUCCGAACGUGUCACGUTT -3′; and reverse, 5′-ACG UGACACGUUCGGAGAATT -3′. The human STAT4 siRNA sequences were as follows: forward, 5′-GCUGUUGCUAAAGGAUAAATT -3′; and reverse, 5′-UUUAUCCUUUAGCAACAGCTT -3′.
Empty vector (pENTER) and human STAT4 plasmid were purchased from WZ biosciences (Jinan, China) and transfected by Lipofectamine 2000 (11668–027; Invitrogen) in HK2 cells by using DMEM/F12 medium containing 10% fetal bovine serum according to the manufacturer’s instruction. On the next day cells were stimulated with TGF-β and treated with 10 µM SDMA. After another 24 h, protein was extracted from cells.
Masson’s trichrome
Mouse kidneys were sliced, fixed, and embedded in paraffin, and cut into 4 μm-thick sections. The paraffin-embedded kidney section was stained with hematoxylin, and then with ponceau red liquid dye acid complex, which was followed by incubation with phosphomolybdic acid solution. Finally, the tissue was stained with aniline blue liquid and acetic acid. Images were captured by using a microscope (Nikon 80i, Tokyo, Japan).
RNA-seq analysis
Total RNA was isolated from mouse kidneys by using TRIzol® Reagent (Invitrogen) and genomic DNA was removed by using DNase I (TaKara). RNA quality was determined by 2100 Bioanalyser (Agilent) and quantified by ND-2000 (NanoDrop Technologies). RNA sample with high-quality (OD260/280 = 1.8 ~ 2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28S:18S ≥ 1.0, > 1 µg) was used to construct sequencing library.
Differentially expressed genes (DEGs) were calculated by DESeq2/DEGseq/EdgeR with Q ≤ 0.05. DEGs with |log2FC|> 1 and Q ≤ 0.05(DESeq2 or EdgeR) /Q ≤ 0.001(DEGseq) were significantly different expressed genes. Functional-enrichment analysis including gene ontology (GO) were performed to identify which DEGs were significantly enriched in GO terms with Bonferroni-corrected P ≤ 0.05, when compared with the whole-transcriptome background. GO functional enrichment analysis were carried out by Goatools (
https://github.com/tanghaibao/Goatools). All raw data have been deposited under the Gene Expression Omnibus accession number PRJNA783810.
Quantitative PCR (qPCR)
Total RNA was extracted by using Trizol (R401-01, Vazyme, Nanjing, China) from kidney samples according to the manufacture’s instruction, which was reverse transcribed to cDNA by Takara PrimeScript RT reagent kit (RR0036A, Kyoto, Japan). The primer sequences for qPCR were listed in Additional file
2: Table S1. Hieff Unicon
® qPCR TaqMan Probe Master Mix (11205E, Yeasen Biotechnology, Shanghai, China) was used. qPCR was performed by using a StepOne Plus Sequence Detection System (Applied Biosystems). All gene expression levels were normalized to Gapdh, and results are expressed as fold change in mRNA expression.
Western blotting analysis
Cell or kidney protein was extracted by using lysis buffer bought from Beyotime Biotech (Nantong, China). BCA Protein Assay Kit (P0012S, Beyotime Biotech, Nantong, China) was used to determine the protein concentration. Protein samples were dissolved in 5 × SDS-PAGE loading buffer (P0015L, Beyotime Biotech, Nantong, China), which were further subjected to SDS-PAGE gel electrophoresis. After electrophoresis, proteins were electro-transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). Unspecific binding on the PVDF membrane was blocked by incubation with the blocking buffer (5% non-fat milk, 20 mM Tris–HCl, 150mMNaCl, PH = 8.0, 0.01%Tween 20) for 1 h at room temperature, which was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam), anti-pSmad3 (1:1000, ET1609-41, HUABIO), anti-Collagen I (1:500, sc-293182, Santa Cruz), anti-α-SMA (1:1000, ET1607-53, HUABIO), anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-Snail (1:1000, A11794, Abclonal), anti-STAT4 (1:1000, ET1701-42, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech) antibodies overnight at 4 ℃. Binding of the primary antibody was detected by an enhanced chemiluminescence method (SuperSignal™ West Femto, 34,094, Thermo Fisher Scientific) by using horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit IgG, 1:1000, A0208, Beyotime or goat anti-mouse IgG, 1:1000, A0216, Beyotime).
Statistical analysis
Results were presented as mean ± SD. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) and comparison between two groups was performed by unpaired student t-test by using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California USA). A P value of lower than 0.05 was considered statistically significant.
Discussion
SDMA is mostly eliminated through the kidney, and it has no effect on healthy kidneys [
23]. Although circulating SDMA is regarded as biomarker of renal function decline in aging animals, the effect of SDMA on diseased kidneys is still not known [
30]. In the current study, we used in vitro and in vivo models to study the effect of SDMA on renal tubulointerstitial fibrosis. We first showed that SDMA dose-dependently inhibited the expression of ECM protein, EMT marker and pSmad3 in TGF-β stimulated renal epithelial cells. Second, intrarenal injection of SDMA dose-dependently reduced ECM deposition and pro-fibrotic marker expression in UUO kidneys, which was further confirmed in UIRI kidneys. We concluded that SDMA is renal protective in CKD in terms of inhibition of renal tubulointerstitial fibrosis.
SDMA is a well-known inducer of endothelial dysfunction and cause endothelial-related diseases through a mechanism by which SDMA competes with arginine for cell transport and thus reducing the bioavailability of nitric oxide [
18,
31]. SDMA is associated with diastolic dysfunction in patients with heart failure and is a risk factor of CVD in CKD patients [
18,
32]. In glomerular endothelial cells, SDMA enhances oxidative stress and may lead to glomerular damages in kidney diseases [
21]. It is known that SDMA is mostly excreted through renal tubules, however the effect of SDMA on renal epithelial cells was not studied. To our knowledge, this is the first study showing that SDMA is beneficial to renal epithelial cells in a fibrotic condition.
The mechanisms downstream of SDMA is still largely unknown. Through RNA-seq analysis, we found that SDMA inhibited the expression of STAT4 in UUO kidneys, which was further shown in another fibrotic mouse model. The direct inhibitory effect of SDMA on STAT4 expression in renal epithelial cells was further confirmed. Finally, we also found that STAT4 signaling pathway mediates the inhibitory effect of SDMA on EMT and ECM protein production in renal epithelial cells. Thus, STAT4 is a signaling pathway downstream of SDMA.
We can not exclude the direct effect of SDMA on other renal cell types. STAT4 is a risk factor for inflammatory diseases such as rheumatoid arthritis and lupus erythematosus, and is initially identified as an important immunoregulator, which regulates various cytokine or chemokine production by immune cells [
15,
33,
34]. Thus, SDMA may inhibit renal tubulointerstitial fibrosis partly through inhibition of STAT4 mediated cytokine production or immune cell infiltration.
Partial EMT is a hallmark of tubulointerstitial fibrosis in chronic kidney disease [
35]. The involvement of STAT4 in EMT has been reported recently. Several studies revealed that STAT4 is not only involved in tumor metastasis, but also promotes EMT and fibroblast proliferation [
36,
37]. In this work, we showed that the expression of STAT4 is positively correlated with the expression of EMT markers and ECM proteins in fibrotic kidneys. Moreover, inhibition of STAT4 through pharmacological or genetic approaches in renal epithelial cells reduced the expression of EMT markers and ECM proteins. Thus, STAT4 signaling is involved in EMT and promotes renal tubulointerstitial fibrosis.
Clinical studies revealed that polymorphisms of STAT4 is associated with the severity of kidney disease such as IgA nephropathy, lupus nephritis and primary membranous glomerulonephritis, suggesting that the activation of STAT4 signaling pathway is involved in immune-mediated kidney diseases [
38‐
40]. Renal tubulointerstitial fibrosis is characterized by infiltration of immune cells and activation of inflammatory responses [
2]. Thus, it is interesting to study the association of STAT4 polymorphisms or activation of STAT4 with the degree of renal tubulointerstitial fibrosis in CKD patients.
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