Hypermethylation and suppression of microRNA219a-2 activates the ALDH1L2/GSH/PAI-1 pathway for fibronectin degradation in renal fibrosis

Qingqing Wei; Xiao Xiao; Emily Huo; Chunyuan Guo; Xiangjun Zhou; Xiaoru Hu; Charles Dong; Huidong Shi; Zheng Dong

doi:10.1016/j.ymthe.2024.09.020

. 2024 Sep 17;33(1):249–262. doi: 10.1016/j.ymthe.2024.09.020

Hypermethylation and suppression of microRNA219a-2 activates the ALDH1L2/GSH/PAI-1 pathway for fibronectin degradation in renal fibrosis

Qingqing Wei ^1,^∗, Xiao Xiao ^1,⁸, Emily Huo ^1,², Chunyuan Guo ^1,⁹, Xiangjun Zhou ³, Xiaoru Hu ^4,¹⁰, Charles Dong ⁵, Huidong Shi ⁶, Zheng Dong ^1,^7,^∗∗

PMCID: PMC11764320 PMID: 39295147

Abstract

Epigenetic regulations, such as DNA methylation and microRNAs, play an important role in renal fibrosis. Here, we report the regulation of microRNA219a-2 by DNA methylation in fibrotic kidneys, unveiling the crosstalk between these epigenetic mechanisms. Through genome-wide DNA methylation analysis and pyrosequencing, we detected the hypermethylation of microRNA219a-2 in renal fibrosis induced by unilateral ureteral obstruction (UUO) or renal ischemia/reperfusion, which was accompanied by a significant decrease in microRNA-219a-5p expression. Functionally, overexpression of microRNA219a-2 enhanced fibronectin induction during hypoxia or TGF-β1 treatment of cultured renal cells. In mice, inhibition of microRNA-219a-5p suppressed fibronectin accumulation in UUO and ischemic/reperfused kidneys. Aldehyde dehydrogenase 1 family member L2 (ALDH1L2) was identified to be the direct target gene of microRNA-219a-5p in renal fibrotic models. MicroRNA-219a-5p suppressed ALDH1L2 expression in cultured renal cells, while inhibition of microRNA-219a-5p prevented the decrease of ALDH1L2 in injured kidneys. Knockdown of ALDH1L2 enhanced plasminogen activator inhibitor-1 (PAI-1) induction during TGF-β1 treatment of renal cells, which was associated with fibronectin expression. In conclusion, the hypermethylation of microRNA219a-2 in response to fibrotic stress may attenuate microRNA-219a-5p expression and induce the upregulation of its target gene ALDH1L2, which reduces fibronectin deposition by suppressing PAI-1.

Keywords: DNA methylation, microRNA, oxidative stress, fibronectin, kidney fibrosis

Graphical abstract

Wei, Dong, and their colleagues have found that, in response to fibrotic stress, hypermethylation leads to the downregulation of microRNA-219, resulting in the activation of the ALDH1L2/glutathione/PAI-1 axis for the expression of fibronectin, unveiling a novel crosstalk between two epigenetic mechanisms in renal fibrosis.

Introduction

Chronic kidney disease (CKD) is a highly prevalent renal disease that affects about 15% of adults in the USA (https://nccd.cdc.gov/CKD/Default.aspx). CKD is featured by a gradual loss of renal function with various risk factors including hypertension, diabetes, autoimmune disorders, and acute kidney injury transition to CKD. Renal fibrosis, a pathological hallmark of CKD, is characterized by excessive accumulation of extracellular matrix (ECM) proteins including fibronectin and collagens. Currently, effective therapy that can reverse or delay renal fibrosis is lacking. Various signaling pathways, including the TGF-β signaling pathway and the hypoxia-inducible factor (HIF) pathway, play pivotal roles in renal fibrosis by promoting ECM protein production.¹^,²^,³ The ECM is continuously undergoing degradation and remodeling. The imbalance between ECM protein expression and degradation leads to excessive ECM deposition and fibrosis.⁴ However, the understanding of dysregulation of ECM remodeling in renal fibrosis remains incomplete.

Epigenetic regulation includes the mechanisms that modulate gene expression inheritably without changing the primary DNA sequence. DNA methylation is one of the epigenetic mechanisms that broadly affect gene expression by adding a methyl group to the cytosine at a CpG site through a reaction catalyzed by DNA methyltransferases (DNMTs).⁵ Functionally, the methylation of CpG sites in the promotor region of a gene usually suppresses the transcription of the gene. Aberrant DNA methylation has been implicated in various diseases, including CKD.⁶^,⁷^,⁸^,⁹^,¹⁰^,¹¹^,¹²^,¹³ However, it remains poorly understood how DNA methylation contributes to the development and progression of CKD.

Non-coding RNAs, including microRNAs, represent another major mechanism of epigenetic regulation. MicroRNAs are a group of small non-coding RNAs of 21–25 nucleotides. By binding to the target sequence in the 3′ untranslated region (UTR) of mRNAs, microRNAs can suppress gene expression by inhibiting mRNA translation and/or inducing mRNA degradation. Multiple microRNAs, such as microRNA-21, microRNA-192, microRNA-214, and microRNA-29, have been shown to play important roles in CKD and renal fibrosis¹⁴^,¹⁵^,¹⁶^,¹⁷^,¹⁸^,¹⁹ MicroRNA-219a (mir-219a) is encoded by two separate genes (mir219a-1 and mir219a-2), but both genes produce the same leading strand mir-219a-5p. Mir-219a-5p has been reported to regulate neurodegeneration, nerve system development, and cancer progression.²⁰^,²¹^,²²^,²³ There is no report about mir-219a in kidney diseases including CKD and renal fibrosis. Furthermore, the mechanism that controls mir-219a gene expression is unclear.

In this study, we examined DNA methylation changes in mouse models of renal ischemia/reperfusion and unilateral ureteral obstruction (UUO). Our genome-wide DNA methylation analysis identified the hypermethylation of mir219a-2 at its gene promotor region in these models, which was associated with a decrease in mir-219a-5p expression. Functionally, mir-219a-5p was shown to directly target and repress ALDH1L2, leading to the suppression of reduced glutathione (GSH) production, induction of plasminogen activator inhibitor-1 (PAI-1), and accumulation of fibronectin. These findings unveil hypermethylation of mir219a-2 as a novel anti-fibrotic mechanism that is activated in renal tubular cells in renal fibrosis.

Results

Hypermethylation of mir219a-2 is accompanied by decreased mir-219a-5p expression in fibrotic kidneys

We analyzed genome-wide DNA methylation changes by reduced representation bisulfite sequencing in models with significant renal fibrosis (Figure S1), including 25 min of bilateral renal ischemia followed by 1 week reperfusion (I25/1wk) or 1 month reperfusion (I25/1M), and UUO for 7 days (UUO7D). Interestingly, in addition to protein-coding genes (data not shown), we identified multiple microRNA genes with differential methylation in fibrotic kidneys compared with control kidneys (Figure 1A). Among them, mir219a-2 was the only one showing significant hypermethylation (>20%) (mean methylation values of 5 CpG sites) at the 5′-end promotor region of the gene in all models tested (Figures 1A and 1B; Table S1). We further confirmed the hypermethylation of two specific CpG sites in the mir219a-2 promotor region in these kidneys by pyrosequencing (Figure 1B; Table S1).

Mir-219a is encoded by two genes, i.e., mir219a-1 and mir219a-2, leading to the expression of the same highly conserved leading strand mir-219a-5p in various species including human, mouse, and rat (Figure S2). We speculated that hypermethylation at mir219a-2 promotor region would attenuate mir-219a-5p expression in renal fibrosis. Indeed, qPCR analysis showed that mir-219a-5p significantly decreased in fibrotic kidneys after UUO7D and I25/1wk (Figures 1C and 1D). In situ hybridization further verified the decrease of mir-219a-5p in renal tubular cells after UUO7D (Figure 1E). In vitro, hypoxia for 48 and 72 h significantly inhibited mir-219a-5p expression in mouse proximal tubular cells (BUMPT) (Figure 1F). However, TGF-β1 treatment did not induce significant changes in mir-219a-5p expression (Figure 1G). In addition, mir-219a-5p was significantly reduced in human kidney tubules with obstructive nephropathy (Figure S3). Collectively, these results demonstrate that the hypermethylation of mir219a-2 mainly under hypoxic stress is accompanied by the suppression of mir-219a-5p expression during renal fibrosis.

Mir219a-2 enhances fibronectin expression during hypoxia or TGF-β1 treatment of renal tubular cells

Renal interstitial fibrosis involves the accumulation or deposition of multiple ECM components such as fibronectin and collagens in the interstitium space. To understand the role of mir-219a-5p in renal fibrosis, we established a mir219a-2 gene-overexpressing BUMPT cell line. Mir219a-2 overexpression in this cell line was confirmed by qPCR analysis (Figure S4). Mir219a-2-overexpressing cells and their control cells were treated with hypoxia (Figures 2A and 2B) or TGF-β1 (Figures 2C and 2D) to induce fibrotic changes, shown by significant accumulation of fibronectin in immunoblots. Mir219a-2-overexpressing cells showed significantly more fibronectin accumulation. Consistently, mir-219a-5p mimics induced fibronectin accumulation in rat renal proximal tubular cells (RPTCs) during hypoxia treatment (Figures S5A and S5B) and in human HK2 renal proximal tubular cells during TGF-β1 treatment (Figures S5C and S5D). Similar effects of mir-219a-5p mimics were shown in HEK293 (human embryonic kidney) cells (Figures S5E and S5F) during TGF-β1 treatment. These cell culture results suggest a pro-fibrotic role of mir-219a-5p in kidney.

Antagonism of mir-219a-5p suppresses fibronectin accumulation in fibrotic kidneys

To examine the role of mir-219a-5p in renal fibrosis in vivo, we used anti-mir-219a-5p locked nucleic acids (LNAs) to treat mice and then subjected them to UUO for 2 weeks (Figure 3A). The inhibition of mir-219a-5p by LNA treatment in mouse kidneys was confirmed by qPCR (Figure 3B). UUO induced significant tubulointerstitial fibronectin, shown by the increase of total protein expression in immunoblots (Figures 3C and 3D) and immunohistochemical staining (Figure 3E). Notably, anti-mir-219a-5p LNAs reduced the accumulation of interstitial fibronectin as shown by both immunoblots (Figures 3C and 3D) and immunohistochemical staining (Figure 3E).

Antagonism of mir-219a-5p suppresses fibronectin expression in UUO-induced renal fibrosis

C57BL/6J male mice were treated with negative control (NC) or anti-mir-219a-5p LNAs, and then subjected to unilateral UUO for 2 weeks. The whole lysates of the contralateral (control) and obstructive kidneys (UUO 2 weeks) were examined by immunoblotting. (A) Schematic diagram showing the mouse treatment procedure. (B) qPCR analysis of mir-219a-5p in mouse kidneys. n = 4, ∗unpaired t test, p < 0.0001, t = 108.8, df = 6. (C) Representative immunoblots of fibrotic proteins. A219, anti-mir-219a-5p. (D) Densitometry analysis of fibronectin in mouse kidneys. The value was normalized by cyclophilin b. n = 5, two-way ANOVA with uncorrected Fisher’s LSD for multiple comparisons. ∗p = 0.0001, t = 5, df = 16. (E) Immunohistochemical staining of fibronectin in control and UUO kidneys (UUO 2 weeks). Scale bar, 0.1 mm.

The effect of anti-mir-219a-5p LNAs was further examined in a mouse model of 25 min of unilateral kidney ischemia followed by 2 weeks of reperfusion (UIR 2 weeks) (Figure S6). To avoid the influence of LNAs during the acute kidney injury phase, LNAs were administered 3 days after kidney ischemia, and their effects were assessed during the kidney recovery/fibrosis development period (Figure S6A). As expected, mir-219a-5p levels decreased in fibrotic kidneys with UIR 2 weeks injury, and LNA treatment completely suppressed mir-219a-5p in both control and UIR 2 weeks kidneys (Figure S6B). Anti-mir-219a-5p LNA may promote kidney recovery, showing improved renal function after UIR 2 weeks injury (Figure S6C). In addition, the induction of fibronectin by UIR 2 weeks was significantly suppressed (Figures S6D, S6E, and S6G).

Interestingly, anti-mir-219a-5p did not affect the expression of other renal fibrosis marker proteins, such as col IV and α-smooth muscle actin (α-SMA) (Figures 3C, S7C, and S7D). Consistently, histological examinations did not show noticeable effects of anti-mir-219a-5p on α-SMA induction (Figure S7A) or overall collagen deposition in UUO kidneys (Figures S7B and S7E). Similarly, anti-mir-219a-5p did not significantly affect collagen deposition or α-SMA induction in UIR kidneys (Figure S8). Thus, mir-219a-5p may contribute to renal fibrosis mainly by regulating fibronectin expression.

Mir-219a-5p targets ALDH1L2 in renal fibrosis

MicroRNAs repress target genes by binding to their mRNAs in the RNA-induced silencing complex (RISC) to suppress translation. In our experiments, mir-219a-5p enhanced fibronectin expression (Figures 2 and 3), indicating that fibronectin was not a direct target of mir-219a-5p. To elucidate the underlying mechanism, we systematically identified the direct targets of mir-219a-5p in renal cells (Figure 4A). Specifically, we performed RISC-immunoprecipitation (RISC-IP) to pull down the potential target mRNAs of mir-219a-5p for deep sequencing, revealing 197 mRNAs (Table S2). We then analyzed the potential mir-219a-5p targeting sites in the 3′ UTR of these mRNAs by an online database (http://www.microrna.org)²⁴ and identified 10 mRNAs with conserved mir-219a-5p targeting sites in both human and mouse (Figure 4B). We further searched the online database (The Human Protein Atlas, https://www.proteinatlas.org) to examine the protein expression and function of these 10 potential target genes.²⁵ Five of these genes mainly showed expression and function in neurons and were excluded from further study. For the other five genes (CANX, ALDH1L2, SMG1, NR2C2, CRTC1), we verified that mir-219a-5p induced significant accumulation of these five gene mRNAs in RISC, while it did not induce obvious changes in cellular levels of these mRNAs (Figures 4C and 4D). Thus, we considered them as the potential direct targets of mir-219a-5p in renal cells. To further identify the target gene that is responsible for the effect of mir-219a-5p in renal fibrosis, we examined the protein expression of these potential targets. Mir-219a-5p did not change CANX protein expression, although it induced the highest mRNA accumulation of CANX in RISC (Figure S9). The second most accumulated mRNA induced by mir-219a-5p in RISC was ALDH1L2 (Figure 4B). In BUMPT cells, mir-219a-2 overexpression significantly suppressed ALDH1L2 protein expression (Figures 5A and 5B). Similarly, in HEK293 cells, mir-219a-5p mimics inhibited ALDH1L2 expression (Figures 5D and 5E). In mouse kidneys, ALDH1L2 was mainly localized in renal tubular cells (Figure 5C). Renal fibrosis in UUO was associated with a remarkable loss of ALDH1L2 in a large portion of renal tubules, which was prevented by anti-mir-219a-5p (Figures 5C and S10). Similarly, UIR 2 weeks led to ALDH1L2 loss in the kidney, while anti-mir-219a-5p obviously preserved its renal expression (Figures S6D, S6F, and S6H). In luciferase microRNA target assay, the luciferase expression was significantly suppressed by mir-219a-5p in the presence of the predicted binding sequence at 3′ UTR, whereas it had no inhibitory effect when the binding sequence was mutated (Figures 5F and S11). Together, these data suggest that ALDH1L2 is a direct target of mir-219a-5p in renal fibrosis.

Identification of potential targets of mir-219a-5p

HEK293 cells were co-transfected with Flag-Ago2 plasmid and mir-219a-5p mimics or its negative control oligos (NC). RNA samples were extracted from whole cells (total mRNA) or Ago2 immunoprecipitates (RISC mRNA) for deep RNA sequencing (RNA-seq), n = 3. (A) Schematics indicating the procedure to identify potential mir-219a-5p targets. (B) The list of potential targets with predicted mir-219a-5p binding sites in both human and mouse. (C) qPCR to confirm the mRNA levels of potential targets in RISC. n = 3, p values (vs. NC): ∗p = 0.0013, t = 8.100, df = 4; ∗∗p = 0.0030, t = 6.414, df = 4; #p = 0.0088, t = 4.776, df = 4; ##p = 0.0027, t = 6.651, df = 4; Δp = 0.0008, t = 9.178, df = 4. Unpaired t test. (D) qPCR analysis of total RNA samples. The mRNA levels were compared between the cells transfected with mir-219a-5p mimics and those with negative control oligos.

Confirmation of ALDH1L2 as a mir-219a-5p direct target

(A) Representative immunoblots of ALDH1L2 in BUMPT cells stably transfected with mir-219a-2 or empty vector (control) with GAPDH as internal loading control. (B) Densitometry analysis of ALDH1L2 in BUMPT cells (normalized by GAPDH). ∗Unpaired t test, p = 0.0013, t = 4.423, df = 10, n = 6. (C) Immunohistochemical staining of ALDH1L2 in mouse kidney samples with negative control (NC) or anti-mir-219-5p LNA (anti-mir-219) treatment (three repeats). Scale bar, 0.1 mm. (D) Immunoblots of ALDH1L2 in HEK293 cells transiently transfected with negative control (NC) or mir-219a mimics with cyclophilin b as internal control. (E) Densitometry analysis of ALDH1L2 in HEK293 cells normalized with cyclophilin b level. ∗Unpaired t test, p = 0.0024, t = 4.361, df = 8, n = 5. (F) Luciferase assay to confirm the binding of mir-219a-5p to the predicted binding site in ALDH1L2 3′ UTR. HEK293 cells were co-transfected with RNA oligos (negative control or mir-219a-5p mimics) and luciferase plasmids without insertion (empty vector) or with insertion (predicted mir-219a-5p binding site of ALDH1L2 [ALDH1L2], or mutated binding site [mutated]) in the 3′ UTR of luciferase. The luciferase ratio between mir-219a-5p vs. negative control was calculated for each group and then normalized by values from empty vector group. One-way ANOVA (F = 5.474) with uncorrected Fisher’s LSD for multiple comparisons. n = 4; ∗p = 0.0173, t = 2.910, df = 9; ∗∗p = 0.0201, t = 2.819, df = 9.

ALDH1L2 promotes GSH accumulation and inhibits PAI-1

To determine the role of ALDH1L2 in regulating fibronectin expression, we knocked down ALDH1L2 with specific siRNAs or shRNAs in HEK293 cells or BUMPT cells, respectively (Figures 6A, 6B, 6F, and 6G). ALDH1L2 knockdown cells had significantly higher levels of fibronectin expression during TGF-β1 treatment than negative control sequence-transfected cells in both cell lines(Figures 6C, 6D, 6F, and 6H), indicating an anti-fibrotic role of ALDH1L2. Interestingly, ALDH1L2 knockdown increased fibronectin protein without changing fibronectin mRNA expression (Figure 6E), suggesting that ALDH1L2 regulates fibronectin at the level of protein turnover. ALDH1L2 is a mitochondrial folate metabolic enzyme that mediates fatty acid metabolism, and its depletion leads to impaired production of GSH and enhanced oxidative stress.²⁶^,²⁷ Reduction of GSH can upregulate PAI-1 to suppress plasmin-mediated fibronectin degradation.²⁸ Therefore, we examined the possible connections between mir-219a-5p, ALDH1L2, and fibronectin. Mir-219a-2 overexpression or mir-219a-5p mimics led to significant decreases in GSH in BUMPT cells and HEK293 cells, respectively (Figures 7A and 7B). Similarly, the knockdown of ALDH1L2 decreased GSH in HEK293 cells (Figure 7C). TGF-β1 induced PAI-1 as reported previously,²⁸ and this induction was markedly higher in ALDH1L2 knockdown cells (Figure 7D). We also measured GSH levels in mouse kidneys. GSH levels were markedly higher in UUO kidneys compared with control kidneys, while anti-mir-219a-5p LNA treatment significantly promoted the GSH increase in UUO kidneys (Figure 7E). Meanwhile, UUO induced PAI-1 in specific renal tubules, which was suppressed by anti-mir-219a-5p, confirming the ALDH1L2/GSH/PAI-1/fibronectin regulation in the UUO condition (Figures 7F and 7G). However, we did not detect a significant change in GSH levels in UIR 2 weeks kidneys (Figure S6I), although anti-mir-219a-5p LNA did suppress PAI-1 induction (Figures S6J and S6K), suggesting the existence of potential alternative regulation.

ALDH1L2 suppresses fibronectin protein induction

(A–E) HEK293 cells were transfected with ALDH1L2 siRNA (SiRNA) or negative control oligos (NC). (A) Immunoblot analysis verifying ALDH1L2 knockdown in siRNA-transfected cells. (B) Densitometry analysis of ALDH1L2 normalized by cyclophilin b. ∗Unpaired t test, p = 0.0024, t = 4.358, df = 8. (C–E) ALDH1L2 siRNA or negative control oligo-transfected HEK293 cells were treated with 20 ng/mL TGF-β1 for 24 h. (C) Representative immunoblots of fibronectin with cyclophilin b as loading control. (D) Densitometry analysis of fibronectin normalized by cyclophilin b or β-actin. Two-way ANOVA with uncorrected Fisher’s LSD for multiple comparisons. n = 6; ∗p = 0.0021, t = 3.530, df = 20. (E) qPCR analysis of fibronectin mRNA. n = 3. (F–H) BUMPT stable cell lines were established by transfection with negative control (NC) or ALDH1L2 knockdown shRNAs (shRNA). The cells were treated with (tgfb1) or without (NT) 10 ng/mL TGF-β1 for 24 h. (F) Representative immunoblots of ALDH1L2 and fibronectin with cyclophilin b or GAPDH as loading control, respectively. (G) Densitometry analysis of ALDH1L2 in NT condition normalized by cyclophilin b, n = 3. ∗p = 0.0017, t = 23.90, df = 2, paired t test. (H) Densitometry analysis of fibronectin normalized by GAPDH, n = 4. ∗Two-way ANOVA with uncorrected Fisher’s LSD for multiple comparisons. p = 0.0111, t = 3.621, df = 6.

ALDH1L2 promotes GSH accumulation and inhibits PAI-1

(A) GSH concentrations in BUMPT cells stably transfected with mir-219-2 or empty vector (control). ∗Unpaired t test, p = 0.0003, t = 7.681, df = 5.754, n = 4. (B) GSH concentrations in HEK293 cells with negative control (NC) or mir-219a-5p mimic transfection. ∗Unpaired t test, p = 0.0334, t = 3.079, df = 4.311, n = 4. (C) GSH concentrations in HEK293 cells with negative control (NC) or ALDH1L2 siRNA transfection. ∗Unpaired t test, p = 0.0351, t = 2.889, df = 4.895, n = 4. (D) HEK293 cells were transfected with ALDH1L2 siRNA (SiRNA) or negative control sequence (NC). PAI-1 mRNA was examined by qPCR. Two-way ANOVA with uncorrected Fisher’s LSD for multiple comparisons, n = 3. p = 0.004, t = 5.805, df = 8. (E–G) Mice were treated with negative control (NC) or anti-mir-219a-5p LNA and subjected to UUO for 2 weeks. (E) GSH concentrations in mouse kidney tissue, n = 3. Two-way ANOVA with uncorrected Fisher’s LSD for multiple comparisons. ∗p = 0.0055, t = 3.769, df = 8. (F) Percentage of the renal tubular area with significant PAI-1 induction after 2 weeks of UUO. Unpaired t test, p = 0.0498, t = 2.781, df = 4, n = 3. (G) Immunohistochemical images of PAI-1 in mouse kidney samples with or without UUO. Scale bar, 0.2 mm. ∗Renal tubules with significant induction of PAI-1.

Discussion

In this study, we deciphered a novel crosstalk between two epigenetic mechanisms, DNA methylation and microRNA, in renal fibrosis. The genome-wide DNA methylation sequencing revealed mir219a-2 as the only hypermethylated microRNA gene in mouse models with renal fibrosis, including ureter obstruction and maladaptive repair after renal ischemia-reperfusion injury (Figures 1A and 1B). Furthermore, this mir219a-2 hypermethylation is associated with the suppression of mir-219a-5p expression (Figures 1C–1F) both in vivo and in vitro. All these results indicate the importance of mir219a-2 hypermethylation in renal fibrosis. Although various aberrant epigenetic regulations, including both DNA methylation and microRNA regulation, have been noted as critical pathological mechanisms, the crosstalk between different epigenetic mechanisms is not well understood. DNA methylation has been reported to regulate microRNA biogenesis²⁹ and DNA methylation-related microRNA expression change has been profiled in diabetic nephropathy.³⁰ However, a detailed functional analysis of such crosstalk is lacking. Although miR-219a-5p can be derived from both miR219a-1 and miR219a-2 genes, we did not detect hypermethylation of miR219a-1 in our experimental models. A study using miR219a-1 and/or miR219a-2 knockout mouse models has shown that miR219a-2 contributes approximately 95% of miR-219a-5p expression in the optic nerve.³¹ While we cannot conclusively determine that miR219a-2 is the dominant gene for miR-219a-5p expression in the kidney, it is plausible that the hypermethylation of miR219a-2 is responsible for the dramatic suppression of miR-219a-5p during renal fibrosis. Overall, our study elucidated a crosstalk between DNA methylation and microRNA regulation by comprehensive examinations of the mir219a-2 promotor hypermethylation, mir-219a-5p inhibition, and the downstream protein factors regulated by mir-219a-5p in renal fibrosis.

Both HIF (activated by hypoxia) and TGF-β signaling pathways are activated in UUO and ischemia/reperfusion-induced renal fibrosis.³²^,³³^,³⁴ However, it is not clear which pathway plays the major role in DNA methylation regulation in the kidney during this process. In other organs, hypoxia has been shown to induce HIF and further influence the expression of DNMTs and demethylases.³⁵^,³⁶^,³⁷ DNMT expression can also be regulated by the TGF-β signaling pathway.³⁸^,³⁹ In this study, we have found that the expression of miR-219a-5p is mainly affected by hypoxia but not TGF-β1 treatment (Figures 1F and 1G). However, we cannot exclude the effect of the TGF-β pathway on DNA methylation of other genes.

Moreover, the new pro-fibrotic function of mir-219a-5p was identified in renal fibrosis. We examined BUMPT cells with mir219a-2 overexpression and identified that this overexpression led to more fibronectin expression in vitro (Figure 2). The pro-fibrotic effect of mir-219a-5p was further confirmed in a few other renal cell lines including rat RPTCs, human proximal tubular cells (HK2), and HEK293 cells (Figure S5). In vivo, the inhibition of mir-219a-5p was mainly localized in renal tubules in UUO kidneys (Figure 1E). If mice were treated with anti-mir-219a-5p LNAs, the interstitial fibronectin accumulation in the kidneys after UUO injury was relieved (Figure 3). Meanwhile, we did not detect an obvious effect on α-SMA (the fibroblast marker). Similar results were observed in kidneys with UIR injury (Figure S6). Although renal fibroblast is one of the major contributors to ECM accumulation in renal fibrosis, emerging evidence indicates that renal tubular cells are playing critical roles in renal fibrosis initiation and progression.⁴⁰ Of note, even though mir-219a-5p is pro-fibrotic, we detected its downregulation during renal fibrosis development (Figures 1C, 1D, 1F, and S6B). The results indicate that the downregulation of miR219a-2 is a protective response of kidney tissues against fibrotic stress. Specifically, it may act as a compensatory response to counterbalance other pro-fibrotic pathways. Therefore, the suppression of mir219a-2 gene expression by DNA hypermethylation may be considered as a self-protection mechanism for the kidney to prevent fibrosis development in CKD, and renal tubular cells are the major functional sites of mir-219a-5p.

The function of mir-219a-5p has been examined previously in neurons and cancer cells.²⁰^,²¹^,²²^,²³ In those studies, multiple pathways were reported to be suppressed by mir-219a-5p, including the NMDA receptor pathway, Tau and Hedgehog signaling pathway, and Wnt/β-catenin pathway. Therefore, mir-219a-5p may target different genes in different tissues or organs. To identify the target genes in renal fibrosis, we performed Ago2-IP with deep sequencing, followed by the examination of putative target protein expression and the luciferase microRNA target assay. These analyses identified ALDH1L2 as a direct target of mir-219a-5p in renal fibrosis, especially fibronectin expression (Figures 4 and 5). ALDH1L2 is a mitochondrial homolog of 10-formyltetrahydrofolate dehydrogenase, which controls the β-oxidation in fatty acid metabolism.²⁶^,²⁷ There is no report about ALDH1L2 in renal pathophysiology. Fatty acid oxidation is a major energy source in kidney proximal tubular cells, and its dysregulation is closely involved in renal fibrosis.⁴¹^,⁴²^,⁴³ In our experiment, the knockdown of ALDH1L2 significantly enhanced fibronectin expression during TGF-β1 treatment (Figure 6), suggesting that ALDH1L2 is a key downstream target of mir-219a-5p to regulate renal fibrosis. In vivo, anti-mir-219a-5p prevented the decrease of ALDH1L2 in UUO and UIR injured kidneys, which was associated with less fibronectin induction (Figures 5C, S6D, S6F, and S10). Altogether, these results demonstrate the important role of ALDH1L2 as a mir-219a-5p target in renal fibrosis.

How does ALDH1L2 suppression lead to renal fibrosis? In HEK293 cells, ALDH1L2 knockdown increased fibronectin protein but not its mRNA (Figure 6), suggesting the regulation of fibronectin protein stability by ALDH1L2. Fibronectin is one of the major components of the ECM that contributes to fibrosis.⁴ Especially, fibronectin polymerization is an essential step for other ECM proteins to deposit in the fibrotic niche.⁴⁴ The degradation of fibronectin and related ECM remodeling involves two major families of metalloproteinases, matrix metalloproteinases (MMPs) and plasmin.⁴ MMPs degrade a variety of ECM proteins, whereas plasmin is more specific for degrading fibronectin, fibrin, and laminin to regulate ECM remodeling. Our results showed that PAI-1 expression was induced after ALDH1L2 knockdown (Figure 7D). PAI-1 is the principal plasminogen activator inhibitor to promote plasminogen truncation to be plasmin,⁴⁵ and its expression can be regulated by GSH level.²⁸ In this regard, ALDH1L2 promotes the production of GSH in cells,²⁷ which may suppress PAI-1 expression and induce plasminogen to plasmin conversion for fibronectin degradation.²⁸ Consistently, in our present study ALDH1L2 knockdown attenuated GSH production and induced PAI-1 in kidney cells (Figures 7A–7C). Moreover, in UUO kidneys, we detected an increase in GSH levels and suppression of PAI-1 (Figures 7E–7G) associated with anti-miR-219a-5p treatment and ALDH1L2 preservation (Figures 5C and S10). However, even though anti-miR-219a-5p treatment enhanced ALDH1L2 and suppressed PAI-1 in UIR kidneys, the difference in GSH levels was not obvious (Figure S6), suggesting the existence of alternative pathways.

In our study, inhibition of mir-219a-5p mainly suppressed fibronectin expression, and its effect on other ECM proteins, such as collagens, was marginal (Figures 3, S7, and S8). Even though plasmin mainly mediates fibronectin degradation and remodeling,⁴ global knockout of PAI-1 attenuated both fibronectin and collagen deposition in kidney fibrosis.⁴⁶ In addition, PAI-1 inhibitors including TM5441 reduced overall ECM accumulation in diabetic nephropathy.⁴⁷ There are a few possibilities that may explain why mir-219a-5p did not have significant effects on collagen deposition in our study. First, according to the histological examinations of mir-219a-5p and ALDH1L2 (Figures 1E and 5C), mir-219a-5p inhibition to regulate ALDH1L2 and fatty acid oxidation is mainly localized in renal tubules. In kidney, because of the high energy requirement, proximal tubule is the major renal compartment using fatty acid as a fuel source.⁴⁸ Therefore, the effect of mir-219a-5p inhibition in ECM remodeling is relatively limited in renal proximal tubules. However, global PAI-1 deficiency or PAI-1 inhibitor treatment can function on all renal cells, such as myofibroblasts and macrophages, to regulate ECM deposition. In fact, both PAI-1 knockout and inhibitor treatment were shown to suppress the inflammation as well as the transcription of ECM proteins in kidney.⁴⁶^,⁴⁷ Second, mir-219a-5p may have multiple potential target genes according to our microRNA target prediction and the Ago2-IP/RNA sequencing (RNA-seq) result (Figure 4). Although we confirmed ALDH1L2 as a direct target of mir-219a-5p in renal fibrosis, other target genes of mir-219a-5p may modulate ECM remodeling and collagen deposition as well. Finally, we only examined renal fibrosis up to two weeks after UUO or UIR injury. It is unclear whether the changes in fibronectin may affect collagen deposition at later time points or in the long term.

In conclusion, this study has demonstrated the regulation of mir-219a-2 by DNA methylation in renal fibrosis and its association of mir-219a-5p suppression, unveiling a novel crosstalk between epigenetic mechanisms. Specifically, the mechanism may include that fibrotic stress leads to hypermethylation of the mir219a-2 gene, causing the downregulation of mir-219a-5p and a consequent increase in its target ALDH1L2 expression. Upon expression, ALDH1L2 promotes GSH to suppress PAI-1 expression, resulting in fibronectin degradation and less renal fibrosis.

Materials and methods

Animal models

C57BL/6J mice were originally from The Jackson Laboratory (Bar Harbor, ME), and bred and housed in Charlie Norwood VA Medical Center animal facility. Male mice, 8–12 weeks old, were used for bilateral kidney ischemia surgery or UUO surgery. All animal experiments were approved by the Institutional Animal Care and Use Committee in Charlie Norwood VA Medical Center and performed following all the relevant regulatory standards.

Kidney ischemia-reperfusion was conducted as described previously.⁴⁹ In brief, mice were anesthetized with 60 mg/kg pentobarbital or 110 mg/kg ketamine/11 mg/kg xylazine and kept on a homoeothermic blanket to maintain body temperature at 36.5°C. For bilateral kidney ischemia, both kidney pedicles were clamped with micro-aneurysm clips for 25 min to induce kidney ischemia. For unilateral kidney ischemia, only the left kidney pedicle was clamped. The clips were released for kidney reperfusion and the mice were kept for 1 week, 2 weeks, or 1 month to collect kidneys. Sham operation was performed without renal pedicle clamping.

UUO was induced in mice as previously.⁵⁰ In brief, mice were anesthetized with 60 mg/kg pentobarbital and kept on a homoeothermic blanket for body temperature maintenance. The left ureter was ligated at two points with 4-0 silk sutures to block urine and a cut was made between these two ligations. Sham operation was performed without ureter ligation and cut for comparison in in situ hybridization experiment. The contralateral kidneys were used in other experiments as the control for comparison purposes.

Genome-wide DNA methylation sequencing

The genomic DNA samples were extracted from mouse kidney cortex and outer medulla using a QIAmp DNA Blood Mini kit from QIAGEN (Germantown, MD) according to the manufacturer’s manual. The reduced representative bisulfite sequencing and reads alignment were conducted in the Cancer Center Genomic Core Facility at Augusta University, as described before.⁵¹ The data have been deposited to GEO for public sharing (GEO: GSE236018, GEO: GSE236019).

Pyrosequencing

The differentially methylated regions associated with the mir-219a-2 gene from genomic sequencing analysis were examined by pyrosequencing conducted by EpigenDx (Hopkinton, MA) to confirm the methylation levels. The sequencing information is shown in Table S3.

RNA extraction

Total RNA from kidneys or cultured cells was extracted using miVana miRNA isolation kit (Thermo Fisher Scientific, Carlsbad, CA) microRNA analysis or a GeneJet RNA purification kit (Thermo Fisher Scientific) for mRNA analysis following the manufacturer’s instructions.

Reverse transcription and quantitative real-time PCR

The analysis of mRNA and microRNA expression was conducted as described previously.⁴⁹^,⁵² To quantify mRNA expression, 1 μg of total RNAs was reversely transcribed into cDNA using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). qPCR was performed using iTaq Universal SYBR Green Supermix (Bio-Rad) with 18s rRNA as internal normalization control and the quantification was done using ΔCt values. The primers were synthesized by Integrated DNA Technologies (Coralville, IA), and the sequence is shown in Table S4. To examine microRNA expression, 40 ng of total RNAs was reversely transcribed using a TaqMan MicroRNA Reverse Transcription Kit (Thermo Fisher Scientific) and qPCR was performed using TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) with detecting primers from TaqMan MicroRNA Assays (Thermo Fisher Scientific). Small nuclear RNA 202 was used as internal normalization control and the quantification was done using ΔCt values. All qPCR was performed using The Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific).

In situ hybridization

In situ hybridization of mir-219a-5p in mouse kidneys was conducted following a protocol modified from our previous publications.⁴⁹^,⁵³ Mouse kidney samples were fixed in 4% paraformaldehyde overnight at 4°C and balanced in 20% sucrose in PBS. Fresh cryo-sections of 7 μm were air-dried for 10 min and briefly fixed in 4% paraformaldehyde for 20 min. After PBS wash, the slides were treated with proteinase K (0.5 μg/mL) for 10 min at room temperature and rinsed with PBS. The hybridization was then performed using an IsHyb in situ hybridization kit (Biochain Institute, Newark, CA) following the manufacturer’s instructions. In brief, the slides were incubated with a pre-hybridization solution at 63°C for 3 h. The hybridization mixture with 0.5 nM DIG-labeled LNA probe (Exiqon, Germantown, MD) in 100 μL hybridization solution was heated at 65°C for 5 min to linearize probes and then chill on ice. The slides were incubated with a hybridization mixture at 58°C overnight. After the SSC solution washes and blocking, the slides were exposed to anti-DIG alkaline phosphatase-conjugated antibody. Finally, the signal was developed with NBT/BCIP solution.

Human kidney samples were collected in Renmin Hospital of Wuhan University following the Institution Human Sample Collection Regulation with patients’ consent. Kidneys were fixed using 4% paraformaldehyde, and the expression of mir-219a-5p was detected using an FITC-labeled probe through in situ hybridization conducted by Servicebio (Wuhan, Hubei, China).

In vitro renal fibrosis models

The following renal cells were used for in vitro treatment to induce fibrosis: (1) the mouse proximal tubular cell (BUMPT) line was originally obtained from W. Lieberthal and J.H. Shwartz at Boston University and maintained in DMEM culture medium with 10% FBS.⁵⁴ BUMPT cells were stably transfected with empty vectors (pCMV-MIR, OriGene, Rockville, MD) or mir219a-2 overexpression plasmids (OriGene) to examine the role of mir-219a-5p in renal fibrosis. To examine the role of ALDH1L2, negative control plasmid (MISSION shRNA, TRC1 control, Sigma, St. Louis, MO) or ALDH1L2 shRNA (MISSION shRNA, TRCN0000174996, Sigma) were stably transfected in BUMPT cells and selected by 1.25 μg/mL puromycin. (2) RPTCs were originally from Dr. Ulrich Hopfer.⁵⁵ RPTCs were maintained in DMEM/F12 medium with 10% FBS and transiently transfected with miRIDIAN miRNA mimics negative control no. 1 or miRIDIAN mir-219a-5p mimics (Dharmacon, Lafayette, CO) to examine the role of mir-219a-5p. (3) The NRK-49F cell line was a renal fibroblast cell line purchased from ATCC (Manassas, VA). NRK-49F cells were maintained in DMEM with 10% FBS and transiently transfected with negative control or mir-219a-5p mimics to examine the role of mir-219a-5p. Cells were treated at 24 h after transfection to induce fibrosis. (4) The HEK293 cell line was from ATCC and maintained in MEM medium with 10% FBS. The cells were transiently transfected with negative control vs. mir-219a-5p mimics, or ON-TARGETplus non-targeting siRNAs negative control pool vs. ALDH1L2 siRNA (targeting sequence: AGAAAGAGCCACUCGGUGU) (Horizon, Lafayette, CO) to examine the role of mir-219a-5p or ALDH1L2. (5) Human kidney proximal tubular cell line (HK2) was from ATCC and maintained in DMEM/F12 medium with 10% FBS. The cells were transiently transfected with negative control or mir-219a-5p mimics.

For hypoxia-induced fibrosis, the cells were incubated in a full culture medium in hypoxia (1% O₂) for 24–72 h to induce fibrosis. Cells cultured in normoxia were used for comparison. To induce fibrosis by TGF-β1 (EMD Millipore, Burlington, MA), cells were cultured in a serum-free medium with 10 or 20 ng/mL TGF-β1 for 24–72 h. Cells without TGF-β1 treatment were used for comparison.

Immunoblotting

Cultured cells were lysed in 1× SDS buffer (72.5 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol) with proteinase inhibitor cocktail (Sigma-Aldrich) and benzonase (EMD Millipore) supplement for immunoblots. Kidney tissue was homogenized in 1× SDS buffer to obtain lysates for immunoblots. The lysates were separated by 10% SDS-PAGE gels and transferred to PVDF membrane. After blocking with 5% milk in TBS with 0.05% Tween 20, the membranes were incubated with specific primary antibodies: anti-fibronectin (ab2413, Abcam, Waltham, MA), anti-α-SMA (Abcam, ab5694), anti-col IV (Abcam, ab6586);, anti-calnexin (ThermoFisher, PA5-34757), anti-ALDH1L2 (gift from Dr. Sergey Krupenko in University of North Carolina, Chapel Hill, NC), anti-ALDH1L2 (21391-1-AP, Proteintech, Rosemont, IL), anti-ALDH1L2 (ab113496, Abcam), anti-GAPDH (Cell Signaling, no. 2118), and anti-cyclophilin b (Cell Signaling, no. 43603)] diluted in 5% milk at 4°C overnight. The signal was detected by HRP-labeled secondary antibodies (Thermo Fisher, Madison, WI) followed by enhanced luminol-based chemiluminescent substrate exposure (Bio-Rad). The signal was recorded by MyECL Imager (Thermo Fisher) or KwikQuant imager (Kindle Biosciences), or X-ray film exposure. ImageJ 1.53e (http://imagej.nih.gov/ij) was used for densitometry calculation and the densitometry value of target protein was normalized by internal loading control for comparison purposes.

In vivo LNA delivery

LNAs of negative control sequence or anti-mir-219a-5p (Exiqon/QIAGEN, Germantown, MD) were injected into mice through the tail vein. LNAs were dissolved in nuclease-free PBS at a concentration of 5 mg/mL. Two injections of 20 mg/kg of LNAs were delivered 2 days before UUO surgery and 3 days after UUO surgery, respectively. Animals were sacrificed at 2 weeks after UUO surgery. One injection of 20 mg/kg of LNAs was delivered at 3 days after unilateral kidney ischemia surgery. In this experiment, the contralateral kidneys were used as a control for comparison with kidneys with ureter obstruction or kidney ischemia.

Immunohistochemical staining and Masson’s trichrome staining

Mouse kidneys were harvested at the end of experiments and fixed in 4% paraformaldehyde overnight. The kidneys were dehydrated and embedded in paraffin. Cross-sections of 5 μm were used for immunohistochemical staining and Masson’s trichrome staining.

For immunohistochemical staining, kidney sections were deparaffinized and rehydrated. The sections were steamed for 1 h in 10 mM sodium citrate (pH 6.0) or 1 mM EDTA (pH 8.0) with 0.05% Tween 20 for antigen retrieval and incubation in hydrogen peroxide to inhibit endogenous peroxidase. Then the sections were blocked in a blocking buffer containing 2% BSA, 0.2% non-fat milk, 0.8% Triton X-100 in PBS, and incubated with primary antibody (anti-fibronectin [Abcam, ab2413], anti-α-SMA [Agilent, M085129-2], anti-ALDH1L2 [a gift from Dr. Sergey Krupenko, or ab113496, Abcam], anti-PAI-1 [Thermo Fisher, PA5-115715]) diluted in blocking buffer at 4°C overnight. HRP polymer-labeled secondary antibodies (Vector Laboratories, Burlingame, CA) were used to amplify the signal. Finally, the signal was developed using a DAB kit from Vector Laboratories. For the fibronectin-staining experiment, the nuclei were counterstained by hematoxylin. For quantification of PAI-1 signaling, 10–20 images (200×) were randomly taken in the cortical area of each specimen. The tubules with significant induction of PAI-1 were selected and the percentage of the tubular area was analyzed by ImageJ 1.53e (http://imagej.nih.gov/ij).

A Trichrome Stain (Masson) Kit from Sigma-Aldrich was used for Masson’s trichrome staining. The kidney sections were stained following the manufacturer’s instructions and examined by bright-field microscopy. Minimally, 30 images were taken to cover the whole cortical and outer medulla area for collagen deposition analysis. ImageJ 1.53e was used to calculate the percentage of area with a positive collagen signal.

RISC-IP and RNA-seq

HEK293 cells were co-transfected with Flag-Ago2 overexpression plasmids (Addgene, Watertown, MA)⁵⁶ and 100 nM RNA oligos from Dharmacon (Negative control vs. mir-219 mimics). The cells were lysed with lysis Buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM EDTA, 0.5% NP-40, 5 mM DTT) containing proteinase cocktail from Sigma-Aldrich and 100 U/mL SUPERase⋅In RNase Inhibitor (Thermo Fisher Scientific). RISC complexes were pulled down by anti-Flag-agarose (Sigma-Aldrich) and the RNAs in the RISC complex were extracted using an miVana miRNA isolation kit for RNA-seq. The high-throughput mRNA deep sequencing with a minimum of 6G raw data/sample and standard quantification data analysis were performed by Novogene (Durham, NC). The data are available at Figshare (Figshare: https://doi.org/10.6084/m9.figshare.23508423) and GEO (GEO: GSE235842, GEO: GSE236019).

MicroRNA target luciferase assay

HEK293 cells were co-transfected with luciferase DNA plasmids and 100 nM RNA oligos from Dharmacon. Three DNA plasmids were used for comparison: empty vector (pMIR-REPORT Luciferase from Thermo Fisher Scientific), ALDH1L2 (pMir-REPORT luciferase plasmid inserted with predicted mir-219a-5p binding site in the 3′ UTR of human ALDH1L2), and mutated (pMIR-REPORT luciferase plasmid inserted with mutated mir-219a-5p binding site) (Figure S11). At 24 h after transfection, the cell lysates were collected with reporter lysis buffer (Promega, Madison, WI) and the luciferase activity was examined using a Luciferase Assay System (Promega) with a Tecan plate reader.

GSH measurement

Cells were plated into a 96-well plate and GSH level was measured using a GSH-Glo glutathione assay from Promega following the assay manual. For tissue GSH measurement, the kidney samples were homogenized in PBS with 2 mM EDTA (1 mg/0.1 mL) on ice. The homogenate was centrifuged at 12,000 × g for 5 min at 4°C to collect supernatant for GSH measurement according to the glutathione assay manual.

Statistics

Data were expressed as mean ± SD and analyzed with Microsoft Excel and/or GraphPad Prism 9. Student’s t test was used to show the significant difference between two groups. One-way or two-way ANOVA analysis with multiple comparisons specified in the figure legends was used for multigroup difference analysis. A p value less than 0.05 was considered statistically significant.

Data and code availability

The DNA methylation sequencing data has been deposited to GEO for public sharing (GEO: GSE236018, GEO: GSE236019). The RNA deep sequencing data of RISC-IP products is available at Figshare (Figshare: https://doi.org/10.6084/m9.figshare.23508423) and GEO (GEO: GSE235842, GEO: GSE236019). All the data supporting the finding of this article are available upon request to the corresponding authors.

Acknowledgments

Q.W. was supported by a grant from NIH/NIDDK (1R01 DK126763-01). Z.D. was a recipient of the Senior Research Career Scientist award from the Department of Veterans Affairs of USA and was supported by grants from NIH/NIDDK (5R01DK087843, 2R01DK058831) and the Department of Veterans Administration of USA (5I01BX000319, 5TK6BX005236). X.Z. was supported by a grant from National Natural Science Foundation of China (82270802). We thank Dr. Sergey A Krupenko for his generous gift of the ALDH1L2 antibody. We thank Augusta University Histology Core for tissue processing.

Author contributions

Experiment design, conducting experiments, data collection and analysis, and manuscript preparation, Q.W. Genomic DNA sample preparation for sequencing and DNA methylation data analysis, X.X. Conducting experiments for data collection, E.H. DNA methylation sequencing and pyrosequencing data analysis, C.G. Human kidney sample collection and data collection of FISH, X.Z. Data collection and analysis for FISH, X.H. Conducting experiments for data collection, C.D. Data collection and analysis of DNA methylation sequencing, H.S. Experiment design, data analysis, and manuscript preparation, Z.D.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2024.09.020.

Contributor Information

Qingqing Wei, Email: qwei@augusta.edu.

Zheng Dong, Email: zdong@augusta.edu.

Supplemental information

Document S1. Figures S1–S11 and Tables S1, S3, and S4

mmc1.pdf^{(18MB, pdf)}

Table S2. RNA-seq data of mRNA induced in RISC and p < 0.05

mmc2.xlsx^{(16.2KB, xlsx)}

Document S2. Article plus supplemental information

mmc3.pdf^{(21.9MB, pdf)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Figures S1–S11 and Tables S1, S3, and S4

mmc1.pdf^{(18MB, pdf)}

Table S2. RNA-seq data of mRNA induced in RISC and p < 0.05

mmc2.xlsx^{(16.2KB, xlsx)}

Document S2. Article plus supplemental information

mmc3.pdf^{(21.9MB, pdf)}

Data Availability Statement

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PERMALINK

Hypermethylation and suppression of microRNA219a-2 activates the ALDH1L2/GSH/PAI-1 pathway for fibronectin degradation in renal fibrosis

Qingqing Wei

Xiao Xiao

Emily Huo

Chunyuan Guo

Xiangjun Zhou

Xiaoru Hu

Charles Dong

Huidong Shi

Zheng Dong

Abstract

Graphical abstract

Introduction

Results

Hypermethylation of mir219a-2 is accompanied by decreased mir-219a-5p expression in fibrotic kidneys

Figure 1.

Mir219a-2 enhances fibronectin expression during hypoxia or TGF-β1 treatment of renal tubular cells

Figure 2.

Antagonism of mir-219a-5p suppresses fibronectin accumulation in fibrotic kidneys

Figure 3.

Mir-219a-5p targets ALDH1L2 in renal fibrosis

Figure 4.

Figure 5.

ALDH1L2 promotes GSH accumulation and inhibits PAI-1

Figure 6.

Figure 7.

Discussion

Materials and methods

Animal models

Genome-wide DNA methylation sequencing

Pyrosequencing

RNA extraction

Reverse transcription and quantitative real-time PCR

In situ hybridization

In vitro renal fibrosis models

Immunoblotting

In vivo LNA delivery

Immunohistochemical staining and Masson’s trichrome staining

RISC-IP and RNA-seq

MicroRNA target luciferase assay

GSH measurement

Statistics

Data and code availability

Acknowledgments

Author contributions

Footnotes

Contributor Information

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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RESOURCES

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