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Chinese Medical Journal logoLink to Chinese Medical Journal
. 2024 Mar 6;138(2):193–204. doi: 10.1097/CM9.0000000000002978

Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway

Weijie Ni 1, Yajie Zhao 2, Jinxin Shen 3, Qing Yin 1, Yao Wang 4, Zuolin Li 1, Taotao Tang 1, Yi Wen 1, Yilin Zhang 1, Wei Jiang 1, Liangyunzi Jiang 1, Jinxuan Wei 1, Weihua Gan 2, Aiqing Zhang 2, Xiaoyu Zhou 3,✉, Bin Wang 1,✉, Bi-Cheng Liu 1,✉
Editor: Yuanyuan Ji
PMCID: PMC11745862  PMID: 38445356

Abstract

Background:

Chronic kidney disease (CKD) is associated with common pathophysiological processes, such as inflammation and fibrosis, in both the heart and the kidney. However, the underlying molecular mechanisms that drive these processes are not yet fully understood. Therefore, this study focused on the molecular mechanism of heart and kidney injury in CKD.

Methods:

We generated an microRNA (miR)-26a knockout (KO) mouse model to investigate the role of miR-26a in angiotensin (Ang)-II-induced cardiac and renal injury. We performed Ang-II modeling in wild type (WT) mice and miR-26a KO mice, with six mice in each group. In addition, Ang-II-treated AC16 cells and HK2 cells were used as in vitro models of cardiac and renal injury in the context of CKD. Histological staining, immunohistochemistry, quantitative real-time polymerase chain reaction (PCR), and Western blotting were applied to study the regulation of miR-26a on Ang-II-induced cardiac and renal injury. Immunofluorescence reporter assays were used to detect downstream genes of miR-26a, and immunoprecipitation was employed to identify the interacting protein of LIM and senescent cell antigen-like domain 1 (LIMS1). We also used an adeno-associated virus (AAV) to supplement LIMS1 and explored the specific regulatory mechanism of miR-26a on Ang-II-induced cardiac and renal injury. Dunnett’s multiple comparison and t-test were used to analyze the data.

Results:

Compared with the control mice, miR-26a expression was significantly downregulated in both the kidney and the heart after Ang-II infusion. Our study identified LIMS1 as a novel target gene of miR-26a in both heart and kidney tissues. Downregulation of miR-26a activated the LIMS1/integrin-linked kinase (ILK) signaling pathway in the heart and kidney, which represents a common molecular mechanism underlying inflammation and fibrosis in heart and kidney tissues during CKD. Furthermore, knockout of miR-26a worsened inflammation and fibrosis in the heart and kidney by inhibiting the LIMS1/ILK signaling pathway; on the contrary, supplementation with exogenous miR-26a reversed all these changes.

Conclusions:

Our findings suggest that miR-26a could be a promising therapeutic target for the treatment of cardiorenal injury in CKD. This is attributed to its ability to regulate the LIMS1/ILK signaling pathway, which represents a common molecular mechanism in both heart and kidney tissues.

Keywords: microRNA-26a, Chronic kidney disease, LIMS1 protein, Cardiorenal injury, Inflammation, Fibrosis

Introduction

Chronic kidney disease (CKD) affects approximately 10% of adults worldwide, and its global burden is substantial and growing.[1] CKD progression is highly associated with cardiovascular disease, with cardiovascular death accounting for approximately 50% of all deaths in patients with stages 4 and 5 CKD. Since inflammation and fibrosis are the most prominent features of cardiorenal injury during CKD,[2] understanding the common mechanism underlying cardiorenal inflammation and fibrosis is critical for developing a strategy that “kills two birds with one stone” in the treatment of CKD.

MicroRNAs (miRNA/miRs) are short, noncoding RNAs that downregulate protein expression by binding to complementary sequences in the 3′-untranslated regions (UTRs) of target mRNAs.[3] miR-26 is highly conserved between humans and mice, and comprehensive studies have reported that miR-26a plays a protective role in a variety of diseases. miR-26a inhibits cell inflammatory responses in lipopolysaccharide-induced acute lung injury and can improve inflammation in cerebral ischemia–reperfusion injury.[4,5] Additionally, miR-26a has been found to limit renal fibrosis by suppressing connective tissue growth factor (CTGF) directly.[6] Our previous studies of CKD mice showed that miR-26a was decreased in the heart and kidneys, and that exogenous miR-26a could improve both renal fibrosis and cardiomyopathy.[7,8] However, how miR-26a markedly improves cardiorenal injury remains unclear.

Integrin-linked kinase (ILK) is an intracellular serine/threonine protein kinase that interacts with the cytoplasmic domains of integrins and mediates integrin signaling in diverse types of cells.[9] ILK forms a tripartite complex with LIM and senescent cell antigen-like domain 1 (LIMS1) and α-parvin (IPAP1 complex), which serves as a signaling mediator that transduces mechanical signals to downstream effectors.[10] ILK signaling activation has been reported to be essential for cardiac hypertrophy under various stress conditions.[11,12] A previous study demonstrated that the Ang-II-stimulated profibrotic process is regulated by a complex mechanism involving crosstalk between ILK and NF-κB signaling activation.[13] In addition to the heart, upregulation of ILK expression and/or activity has also been implicated in the pathogenesis of a wide variety of CKDs as a mediator during proteinuria and diabetic nephropathy.[14,15,16,17] Therefore, targeting ILK signaling could be a potential strategy to ameliorate both cardiac and renal diseases.

Studies from our laboratory and others have confirmed the potential role of miR-26a in CKD injury, and also demonstrated the critical role of LIMS1 in renal fibrosis. Our research aims to explore the role and mechanism of miR-26a in CKD cardiac and renal injury through miR-26a knockout (KO) mice and other methods.

Methods

Animals

miR-26a−/− (miR-26a KO, C57BL/6J) mice were generated as described in our previous study.[18] Both miR-26a-KO mice and wild type (WT) mice were produced by hybridization of miR-26a+/− mice, and the mice were age- and weight-matched. We divided the mice into the following groups: control (Ctrl) group, miR-26a KO group, Ang-II group, Ang-II + miR-26a KO group, Ang-II + shLIMS1 group, Ang-II + miR-26a KO + shLIMS1 group, Ang-II + miR-26a overexpression group, and Ang-II + miR-26a overexpression + LIMS1 over-expression group, with six mice in each group. WT and miR-26a-KO mice that were 6–7 weeks old (18–22 g) were divided into groups and subjected to unilateral nephrectomy. One week later, Ang-II (HY-P7503; MCE, New Jersey, USA) or vehicle (double distilled H2O) was administered continuously with a micro-osmotic pump (1004; Alzet, California, USA) for four weeks (0.11 µL/h [980 ng·kg–1·min–1]). During Ang-II infusion, normal saline was provided as drinking water to aggravate hypertension. After Ang-II infusion, blood pressure and heart ultrasound tests were performed, and urine was collected to measure 24 h-urinary protein (C035-2-1; Jiancheng, Nanjing, China). Mice were sacrificed after Ang-II infusion, and heart and kidney tissues were collected for various studies. Blood samples were taken before sacrifice and transferred into tubes. The plasma was separated by centrifugation at 3000 r/min at 4°C for 30 min and collected for further analysis. Serum creatinine (SCr) levels were measured by a creatinine assay kit (C011-2-1; Jiancheng). The experimental procedures were approved by the Ethics Committees for Animal Experimentation of Southeast University (No. 20211025052).

Morphological studies and immunohistochemistry (IHC)

Hearts or kidneys were harvested from mice; tissue fragments were fixed in Bouin’s solution (HT1013; Sigma–Aldrich, St. Louis, USA) overnight and embedded in paraffin. Masson’s trichrome staining was used to assess collagen levels. For IHC, the sections were incubated with anti-ILK (1:200) (GTX01046; Genetex, SAN Antonio, USA) and anti-LIMS1 (1:200) (ab154331; Abcam, Cambridge, United Kingdom) overnight at 4°C, and the staining was visualized using horseradish peroxidase-coupled secondary antibodies (KIT-9707; MXB, Wuhan, China).

Fluorescence in situ hybridization (FISH) and immunofluorescence (IF)

Proteinase K (20 μg/mL) was used to digest 3 µm paraffin sections of heart or kidney tissue sections (deparaffinized and hydrated). The samples were then prehybridized in prehybridization buffer for 8 min (78°C), followed by hybridization using Cy3-labeled miR-26a probes (devised by Genepharma, Shanghai, China) for 5 min (at 73°C) and overnight at 37°C. The sections were then washed with saline-sodium citrate buffer at 43°C to remove the unhybridized probes. FISH images were taken under a confocal microscope.

For IF, the sections were incubated with anti-CD68 antibody (1:200) (GB113109; Servicebio, Wuhan, China) and anti-wheat germ agglutinin (WGA) antibody (1:500) (Q-0087712; Xi’an Qiyue Biotechnology, Xi’an, China) overnight at 4°C, and incubated with the second antibody (1:500) (ab300671; Abcam) at room temperature for 1 h the next day.

RNA extraction and real-time polymerase chain reaction (PCR) analysis of miRNA

RNA extracts were quantified using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Massachusetts, USA). Total RNA was purified from tissues or cells using TRIzol reagent (15596026; Takara, Dalian, China) according to the manufacturer’s protocol. Then, we used the SYBR Green Master Mix Kit (Q311-02; Vazyme, Nanjing, China) to measure the expression levels of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC). Mature miRNA expression was quantified using a miRNA-specific primer (GeneCopoeia, Maryland, USA) real-time PCR assay kit. Relative gene expression was calculated using the 2–ΔΔCT formula.

Luciferase reporter assay

Effectene transfection reagent (Qiagen, Dusseldorf, Germany) was used for transfection of 293T cells. Firefly and Renilla luciferase activities were measured using a dual luciferase assay (Promega, Madison, USA) and a luminometer (Turner Designs, California, USA).

Western blotting

Frozen heart or kidney tissues from each group were lysed in radio immunoprecipitation assay (RIPA) lysis buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) method (P0013D; Beyotime Institute of Biotechnology, Beijing, China). A total of 50 mg of total protein was separated by 10–16% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% nonfat milk in tris buffered saline (TBS) with 0.5% Tween-20 (TBS-T) overnight, the membranes were incubated with primary antibodies against ILK (1:1000) (GTX01046; Genetex), LIMS1 (1:1000) (ab154331; Abcam), interleukin-1β (IL-1β) (1:1000) (16806-1-AP; Proteintech, Wuhan, China), interleukin-18 (IL-18) (1:2000) (10663-1-AP; Proteintech), α-smooth muscle actin (α-SMA) (1:200) (ab5694; Abcam), fibronectin (FN) (1:2000) (F3648; Merck, California, USA), β-actin (1:10000) (ZF2001; ZFdows Bio, Nanjing, China) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000) (ZF2000; ZFdows Bio). After incubation with the corresponding secondary antibody, immune complexes were detected using enhanced chemiluminescence (ECL) Western blotting reagents. The levels of the detected proteins were normalized to GAPDH or β-actin.

Cell culture, transfection, and cell treatment

HK2 and AC16 cells were purchased from American Type Culture Collection (ATCC). HK2 and AC16 cells were cultured in dulbecco’s modified eagle medium (DMEM)/F12 supplemented with 10% fetal calf serum (FBS) in a 37°C incubator with 5% CO2. miR-26a mimics and miR-26a inhibitors were designed and synthesized by Hanbio (Nanjing, China). HK2 and AC16 cells were transfected with Lipofectamine 3000 (L3000150; Invitrogen, CA, USA) according to the manufacturer’s protocol. Then, 1×10–6 mol Ang-II (HY-P7503; MCE) was added after transfection.

Statistical analysis

All data were tested for normality and homogeneity of variance before statistics. The data are presented in a histogram. Statistically significant differences between the Ctrl and treatment groups were determined using a simple analysis of variance (ANOVA), followed by Dunnett’s multiple comparison tests. When two independent sets of data were compared, the unpaired t-test was used. SPSS 19.0 (Chicago, IL, USA) was used for statistical analysis. P <0.05 was considered to indicate a statistically significant difference.

Results

Effect of Ang-II infusion on systolic blood pressure, body weight, and mortality in WT and miR-26a-KO mice

Kidney and heart tissues were excised from miR-26a-KO and WT mice, and miR-26a expression levels were measured by FISH and real-time PCR. As shown in Supplementary Figure 1A–C, http://links.lww.com/CM9/B854, compared with the Ctrl mice, miR-26a expression was significantly downregulated in both the kidney and heart after Ang-II infusion. As expected, miR-26a expression was absent in both the kidney and heart of miR-26a-KO mice, suggesting the successful construction of the miR-26a KO model [Supplementary Figure 1A–C, http://links.lww.com/CM9/B854]. Body weights were similar in the Ctrl and miR-26a-KO groups [Supplementary Figure 1D, http://links.lww.com/CM9/B854]. As expected, under continuous Ang-II infusion for four weeks, the body weight of Ctrl mice had decreased moderately, whereas a significant reduction in body weight was observed in miR-26a-KO mice compared with that of the Ctrl mice [Supplementary Figure 1D, http://links.lww.com/CM9/B854]. At baseline, systolic blood pressure was similar in Ctrl and miR-26a-KO mice, while knockout of miR-26a aggravated the Ang-II-induced increase in blood pressure [Supplementary Figure 1E, http://links.lww.com/CM9/B854]. Similarly, analysis of mortality showed that loss of miR-26a resulted in more severe outcomes after Ang-II infusion [Supplementary Figure 1F, http://links.lww.com/CM9/B854]. Together, our data suggested that miR-26a deficiency could lead to further damage in the Ang-II-induced CKD mouse model.

Effect of Ang-II infusion on cardiac injury in WT and miR-26a-KO mice

Ang-II infusion markedly induced distention of the left ventricular (LV) chamber, posterior wall thickness and cardiac dysfunction compared with those of the Ctrl group [Figure 1A]. Notably, knockout of miR-26a significantly aggravated these injuries [Figure 1A, Supplementary Table 1, http://links.lww.com/CM9/B854]. Additionally, compared with Ctrl mice, with Ang-II infusion, the expression of cardiac stress genes (ANP, BNP) increased markedly in miR-26a-KO mice [Figure 1B]. In a similar manner, knockout of miR-26a significantly aggravated Ang-II-induced cardiac hypertrophy, which was reflected by the expression of β-MHC and the cardiomyocyte diameter [Figure 1B and C]. Strikingly, a significant induction of proinflammatory factors and CD68-positive macrophages was observed after Ang-II infusion, and knockout of miR-26a aggravated inflammation in the heart [Figure 1D–F]. Similarly, compared with the Ctrl mice, miR-26a-KO mice exhibited worsened cardiac fibrosis, as demonstrated by the further increased expression of fibronectin and α-SMA, as well as extracellular matrix (ECM) accumulation after Ang-II infusion [Figure 1E–G].

Figure 1.

Figure 1

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miR-26a KO aggravates Ang-II infusion-induced cardiac damage. (A) M-mode echocardiograms showing left ventricular dimensions. (B) Real-time PCR analysis of ANP, BNP, and β-MHC mRNA levels (n = 6). (C) Representative immunofluorescence staining of WGA (green) in heart. Scale bars: 40 μm. (D) Representative immunofluorescence staining of CD68 (green) in heart. Scale bars: 40 μm. (E) Representative Western blot figure showing the levels of IL-1β, IL-18, α-SMA, and FN in the heart of mice. (F) Semi-quantitative statistical analysis of IL-1β, IL-18, α-SMA, and FN protein levels for Western blotting (n = 6). (G) Representative Masson’s staining of heart. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; ANP: Atrial natriuretic peptide; BNP: Brain natriuretic peptide; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IL: Interleukin; KO: Knockout; miR: MicroRNA; PCR: Polymerase chain reaction; SD: Standard deviation; WGA: Wheat germ agglutinin; WT: Wild type; α-SMA: α-smooth muscle actin; β-MHC: β-myosin heavy chain.

Effect of Ang-II infusion on renal injury in Ang-II-infused WT and miR-26a-KO mice

We then measured renal injury in WT and miR-26a-KO mice. Ang-II infusion moderately increased albuminuria and SCr in WT mice, and this damage was more serious in miR-26a-KO mice [Figure 2A and B]. Increased renal inflammation was further confirmed by the exacerbated expression of IL-1β and IL-18 in Ang-II-infused miR-26a-KO mice [Figure 2C and D]. Similar to the heart, no significant difference was observed in renal infiltration of CD68-positive macrophages between Ctrl and miR-26a-KO mice at baseline [Figure 2E]. With Ang-II infusion, the renal infiltration of CD68-positive macrophages increased significantly in miR-26a-KO mice compared with WT mice [Figure 2E]. To further clarify the effect of miR-26a knockout on Ang-II-induced renal injury, renal fibrosis was analyzed. miR-26a-KO mice exhibited worsened kidney fibrosis, as indicated by the increased ECM deposition and expression of fibronectin and α-SMA [Figure 2C, D and F].

Figure 2.

Figure 2

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miR-26a KO aggravates Ang-II infusion-induced kidney damage. (A) 24-h urinary protein quantity at the 4th week post Ang-II infusion in WT and miR-26a KO mice (n = 6). (B) SCr levels at the 4th week of Ang-II infusion in WT and miR-26a-KO mice (n = 6). (C) Representative Western blot figure showing the levels of IL-1β, IL-18, α-SMA, and FN in the kidney of mice. (D) Semi-quantitative statistical analysis of IL-1β, IL-18, α-SMA, and FN protein levels by Western blotting test (n = 6). (E) Representative immunofluorescence staining of CD68 (green) in kidney. Scale bars: 40 μm. (F) Representative Masson’s staining of kidney. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IL: Interleukin; KO: Knockout; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WT: Wild type; α-SMA: α-smooth muscle actin.

miR-26a knockout exacerbates ILK signaling activation in heart and kidneys of Ang-II-infused mice

To answer the question of whether ILK signaling is involved in cardiorenal injury and whether miR-26a deficiency exacerbates ILK signaling activation in our mouse model, Western blotting analysis and IHC measurements of the ILK and LIMS1 expression levels in the heart and kidney tissues were performed. Compared with the Ctrl group, Ang-II infusion caused a significant increase in the levels of LIMS1 and ILK in both the kidney and the heart. As expected, LIMS1 and ILK expression was further enhanced in miR-26a-KO mice [Figure 3A–E]. LIMS1 and ILK form a complex that serves as a signaling mediator that transduces mechanical signals to downstream effectors. We then detected the interaction via co-immunoprecipitation in our model. Consistent with previous studies,[10] our results suggested that LIMS1 and ILK interacted in both the heart and the kidney [Figure 3F–G]. These results suggested that ILK signaling in the kidney and heart was activated after Ang-II infusion, which was enhanced by miR-26a loss.

Figure 3.

Figure 3

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ILK signaling was activated after Ang-II infusion in WT and miR-26a-KO mice. (A) Representative Western blotting figure showing the levels of ILK and LIMS1 in the heart of mice. (B) Semi-quantitative statistical analysis of ILK and LIMS1 protein levels in the heart for Western blotting results (n = 6). (C) Representative Western blotting figure showing the levels of ILK and LIMS1 in the kidney of mice. (D) Semi-quantitative statistical analysis of ILK and LIMS1 protein levels in the kidney for Western blotting results (n = 6). (E) Representative IHC staining of ILK and LIMS1 in the heart and kidney. Scale bars: 40 μm. (F & G) Co-immunoprecipitation demonstrated an interaction between LIMS1 and ILK. Heart or kidney tissue lysates were immunoprecipitated with specific antibody against LIMS1, followed by immunoblotting with antibodies against ILK. Data are presented as mean ± SD. Ang: Angiotensin; Ctrl: Control; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IB: Immunoblotting; IHC: Immunohistochemistry; ILK: Integrin-linked kinase; IP: Immunoprecipitation; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SD: Standard deviation; WT: Wild type.

LIMS1 is a target of miR-26a in tubular cells and cardiomyocytes

A single miR-26a binding site was predicted in the LIMS1 3′-UTR (site: 549–555; Supplementary Figure 2A, http://links.lww.com/CM9/B854). In HEK293T cells transfected with pMIR-LIMS1/1477–1535, the miR-26a mimic decreased luciferase activity by 55% (P <0.05), and mutation of the binding site prevented this effect [Supplementary Figure 2B, http://links.lww.com/CM9/B854]. The hairpin inhibitor of miR-26a increased luciferase activity 1.6-fold (P <0.05) in cells transfected with pMIR-LIMS1/1477–1535 [Supplementary Figure 2B, http://links.lww.com/CM9/B854]. We further measured LIMS1 expression by Western blotting after transfection of cultured AC16 and HK2 cells with miR-26a mimic or inhibitor, and we found that the miR-26a mimic repressed LIMS1 expression. In contrast, the miR-26a inhibitor increased LIMS1 expression in the presence of Ang-II in both cultured cell lines [Supplemental Figure 2C and D, http://links.lww.com/CM9/B854]. These results indicated that miR-26a negatively regulates LIMS1 expression in both AC16 and HK2 cells.

Inhibiting LIMS1 expression attenuates Ang-II-induced cardiac and renal injury in WT and miR-26a-KO mice

To clarify the role of LIMS1 in cardiorenal injury after Ang-II infusion, an adeno-associated virus (AAV) that expresses LIMS1 short hairpin RNA (shRNA) was generated to knockdown LIMS1 in both the heart and the kidney. AAV-LIMS1 shRNA attenuated the expression of LIMS1 and ILK in the heart and kidney [Figure 4A and Figure 5A]. Interestingly, downregulation of LIMS1 in the heart ameliorated Ang-II-induced cardiac remodeling, as evidenced by the significantly reduced expression of ANP, BNP, β-MHC, proinflammatory factors and fibrosis indicators [Figure 4A and B]. Similarly, cardiomyocyte diameter, macrophage infiltration and cardiac fibrosis showed a consistent trend [Figure 4C–E]. In the kidney, inhibiting LIMS1 also improved all renal injuries, as demonstrated by reduced proteinuria and protein cast formation and alleviated renal fibrosis and inflammation [Figure 5A–E]. These results indicated that LIMS1 was responsible for cardiac remodeling and renal tubular injury in Ang-II-induced cardiorenal injury.

Figure 4.

Figure 4

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Inhibition of LIMS1 significantly improved Ang-II-induced heart injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in the heart for Western blotting results (n = 6). (B) Real-time PCR analysis of ANP, BNP and β-MHC mRNA levels (n = 6). (C) Representative immunofluorescence staining of WGA (green) in heart. Scale bars: 40 μm. (D) Representative immunofluorescence staining of CD68 (green) in heart. Scale bars: 40 μm. (E) Representative Masson’s staining of heart. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; ANP: Atrial natriuretic peptide; BNP: Brain natriuretic peptide; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WGA: Wheat germ agglutinin; WT: Wild type; α-SMA: α-smooth muscle actin; β-MHC: β-myosin heavy chain.

Figure 5.

Figure 5

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Inhibition of LIMS1 significantly improved Ang-II-induced kidney injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results (n = 6). (B) 24-h urinary protein quantity at the 4th week of Ang-II infusion in WT and miR-26a-KO mice (n = 6). (C) SCr levels at the 4th week of Ang-II infusion in WT and miR-26a KO mice (n = 6). (D) Representative immunofluorescence staining of CD68 (green) in kidney. Scale bars: 40 μm. (E) Representative Masson’s staining of kidney. Scale bars: 20 μm. Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of Differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS1: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; WT: Wildtype; α-SMA: α-smooth muscle actin.

Overexpression of miR-26a improves Ang-II-induced cardiac and renal injury by negatively targeting LIMS1 in WT and miR-26a-KO mice

To further investigate the function of miR-26a in our model, we injected miR-26a AAV into mice. We found that miR-26a AAV treatment significantly reduced the expression of both LIMS1 and ILK [Figure 6A] in the heart. Moreover, indicators of cardiac function, such as ANP, BNP and β-MHC, were significantly decreased after miR-26a AAV treatment, as were proinflammatory factors and fibrosis indicators [Figure 6A, Supplementary Figure 3, http://links.lww.com/CM9/B854]. Furthermore, cardiomyocyte diameter, macrophage infiltration and cardiac fibrosis were also significantly improved after miR-26a AAV treatment [Figure 6B and C, Supplementary Figure 3, http://links.lww.com/CM9/B854]. Similar to the heart, miR-26a AAV treatment abolished LIMS1 and ILK expression in the kidney [Figure 6D], which was accompanied by attenuated proteinuria, SCr, proinflammatory factors and renal fibrosis in the kidneys [Figure 6B–D, Supplementary Figure 3, http://links.lww.com/CM9/B854].

Figure 6.

Figure 6

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Overexpression of miR-26a significantly improved Ang-II-induced cardiorenal injury. (A) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in heart for Western blotting results (n = 6). (B) Representative Masson’s staining of heart and kidney. Scale bars: 20 μm. (C) Representative immunofluorescence staining of CD68 (green) in heart and kidney. Scale bars: 40 μm. (D) Semi-quantitative statistical analysis of LIMS1, ILK, IL-1β, IL-18, α-SMA and FN protein levels in kidney for Western blotting results (n = 6). Data are presented as mean ± SD. Ang: Angiotensin; CD: Cluster of differentiation; Ctrl: Control; DAPI: 4′,6-diamidino-2-phenylindole; FN: Fibronectin; IL: Interleukin; ILK: Integrin-linked kinase; KO: Knockout; LIMS: LIM and senescent cell antigen-like domain 1; miR: MicroRNA; SCr: Serum creatinine; SD: Standard deviation; α-SMA: α-smooth muscle actin.

To further clarify that the protective effect of exogenous miR-26a is mediated by inhibition of LIMS1 expression and ILK signaling, we injected miR-26a AAV in combination with LIMS1 AAV. As expected, overexpression of LIMS1 abrogated the protective effect of miR-26a AAV on cardiac and renal injury [Figure 6A–D, Supplementary Figure 3, http://links.lww.com/CM9/B854]. These results suggested that overexpression of miR-26a improved Ang-II-induced cardiorenal injury by negatively targeting LIMS1.

miR-26a attenuates Ang-II-induced inflammation and fibrosis in vitro

To broaden our in vivo results to the in vitro setting, AC16 cells were used for analysis. After Ang-II treatment, LIMS1 and ILK were increased in AC16 cells [Supplementary Figure 4A and B, http://links.lww.com/CM9/B854], accompanied by increased levels of the proinflammatory factors, ANP, BNP and β-MHC [Supplementary Figure 4A–C, http://links.lww.com/CM9/B854]. Additionally, miR-26a mimic treatment significantly reduced the expression of proinflammatory factors and fibrosis indicators; in contrast, miR-26a inhibitor aggravated these injuries induced by Ang-II [Supplementary Figure 4A–C, http://links.lww.com/CM9/B854]. Consistent with in vivo experiments, LIMS1 and ILK were increased in HK2 cells after Ang-II treatment, accompanied by increased levels of proinflammatory factors and fibrosis indicators [Supplementary Figure 4D and E, http://links.lww.com/CM9/B854]. Similar to the in vivo results, the miR-26a mimic treatment attenuated the upregulation of proinflammatory factors and fibrosis indicators, whereas the miR-26a inhibitor exacerbated these changes in Ang-II-induced HK2 cells [Supplementary Figure 4D and E, http://links.lww.com/CM9/B854].

Discussion

In this study, we provide evidence that miR-26a-mediated LIMS1 inhibition suppresses the following ILK signaling pathway, which is a common mechanism that is activated in both the heart and the kidney in CKD. Supplementation with exogenous miR-26a is a “one-stone-two-birds” approach that attenuates heart and kidney injury in the context of CKD via the inhibition of the LIMS1/ILK signaling pathway.

Increased cardiac inflammation and fibrosis are key features of cardiac remodeling and therefore contribute to cardiac dysfunction. Inflammation is an early event in tissue injury. When chronic inflammatory injury persists, organ fibrosis occurs.[19] Fibrosis is a key pathological driver of cardiac injury. Inhibiting fibrosis can effectively improve cardiac injury and inhibit cardiac remodeling.[20] It has been demonstrated that a range of molecules, such as matrix metalloprotein (MMPs), Ang-II and transforming growth factor-β (TGF-β), have the potential to regulate fibroblast proliferation and ECM turnover as well as myocyte hypertrophy.[21,22,23] Recent studies suggest that transient inhibition of the renin angiotensin system not only produces a sustained reduction in cardiac fibrosis but also results in protection against future collagen deposition.[24] In addition, non-coding RNA, especially miRNAs, also play important roles in the control of myocardial fibroblasts, such as ECM synthesis, cytokine secretion, and cell proliferation.[25] In kidney disease, inflammation and fibrosis are multifaceted, multilayered cellular responses that can be activated by multiple signaling pathways.[26] Signaling molecules such as TGF-β, phosphatidylinositol-3-hydroxykinase/protein kinase B (PI3K/Akt), nuclear factor-kappa B (NF-κB), Ang-II/reactive oxygen species (ROS), microRNAs, IL-6 and IL-18 play important roles in the process of renal inflammation and fibrosis.[26] However, little research has been conducted on the common pathways that simultaneously reduce inflammation and fibrosis in both the heart and the kidney. Because ILK signaling activation has previously been identified as a key mediator in various inflammation and fibrosis models,[27] it is not surprising that ILK signaling is overtly activated in the heart and kidney of Ang-II-infused mice. Of interest, ILK signaling is more activated in miR-26a-KO mice, and exogenous miR-26a treatment could inhibit this process and improve renal inflammation and fibrosis.

miR-26a-1 is localized on chromosome 3, miR-26a-2 is localized on chromosome 12, and the mature miRNAs for miR-26a-1 and miR-26a-2 have the same sequence.[28,29] miR-26a is embedded within the introns of genes encoding proteins of the carboxy-terminal domain of RNA polymerase II polypeptide A small phosphatase (CTDSP) family.[30] Our previous reports have revealed that knockout of miR-26a did not impact body weight, pregnancy rate, birth rate, or other factors.[18] Our research group has found that targeting miR-26a attenuated unilateral ureteral obstruction-induced renal fibrosis by modulating CTGF and TGF-β1, and exogenous miR-26a attenuated uraemic cardiomyopathy in CKD mice by limiting insulin resistance.[7,8] Chen et al[31] indicated that the miR-26a-5p/IL-6 axis can alleviate sepsis-induced acute kidney injury by inhibiting renal inflammation. In addition, miR-26a-5p plays an inhibitory role in cardiac hypertrophy and dysfunction by targeting ADAM metallopeptidase domain 17 (ADAM17).[32] Here, we confirmed that miR-26a decreases ILK signaling by directly targeting LIMS1, which binds with ILK and inhibits ILK degradation, thus improving renal injury. This tethering ensures that the reduction in miR-26a drives both cardiac and renal injury. Of note, the kidney and the heart showed the same changes in ILK signaling, inflammation, and fibrosis processes, as well as related targets of miR-26a, which indicated that both organs may share a common mechanism for inflammation and fibrosis in Ang-II-induced WT and miR-26a-KO mice. These results provide us with a new perspective, namely, the perspective of single-agent multiorgan therapies, indicating the possibility of applying miR-26a in the clinic to treat CKD patients.

Of interest, miR-26a could not regulate ILK directly in our model. Previous research has indicated that LIMS1 exerts its biological effects via its interaction with ILK.[9] Notably, our study has provided the evidence that miR-26a plays a role in regulating ILK activity by directly targeting LIMS1, ultimately contributing to the modulation of cardiac and renal injury in CKD. In accordance with prior findings, our investigation revealed that heightened levels of LIMS1 are associated with the progression of CKD injury, while inhibition of LIMS1 can significantly ameliorate CKD progression.[33]

In summary, our study has identified miR-26a as a crucial modulator of cardiac and renal injury in CKD. Our findings reveal that miR-26a can directly inhibit the ILK signaling pathway, highlighting a broader and more significant role of miR-26a in end-organ damage in CKD than previously recognized. As such, our study provides novel insights into potential therapeutic strategies for the treatment of CKD-induced end-organ injury.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of interest

None.

Funding

This research was supported by grants from the National Natural Science Foundation of China (Nos. 82200749, 82241047, 82070735, 82030024, 81720108007 and 81270725), Natural Science Foundation of Jiangsu Province (No. BK20221282), and National Key Research Programme of Ministry of Science and Technology (Nos. 2018YFC130046, 2018YFC1314000).

Supplementary Material

SUPPLEMENTARY MATERIAL

Footnotes

Weijie Ni and Yajie Zhao contributed equally to this work.

How to cite this article: Ni WJ, Zhao YJ, Shen JX, Yin Q, Wang Y, Li ZL, Tang TT, Wen Y, Zhang YL, Jiang W, Jiang LYZ, Wei JX, Gan WH, Zhang AQ, Zhou XY, Wang B, Liu BC. Therapeutic role of miR-26a on cardiorenal injury in a mice model of angiotensin-II induced chronic kidney disease through inhibition of LIMS1/ILK pathway. Chin Med J 2025;138:193–204. doi: 10.1097/CM9.0000000000002978

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Supplementary Materials

SUPPLEMENTARY MATERIAL
cm9-138-193-s001.doc (9MB, doc)

Data Availability Statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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