Basic Research

Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1

Eoin P. Brennan, Karen A. Nolan, Emma Börgeson, Oisín S. Gough, Caitríona M. McEvoy, Neil G. Docherty, Debra F. Higgins, Madeline Murphy, Denise M. Sadlier, Syed Tasadaque Ali-Shah, Patrick J. Guiry, David A. Savage, Alexander P. Maxwell, Finian Martin, Catherine Godson and on behalf of the GENIE Consortium
JASN April 2013, 24 (4) 627-637; DOI: https://doi.org/10.1681/ASN.2012060550

Abstract

Lipoxins, which are endogenously produced lipid mediators, promote the resolution of inflammation, and may inhibit fibrosis, suggesting a possible role in modulating renal disease. Here, lipoxin A4 (LXA4) attenuated TGF-β1–induced expression of fibronectin, N-cadherin, thrombospondin, and the notch ligand jagged-1 in cultured human proximal tubular epithelial (HK-2) cells through a mechanism involving upregulation of the microRNA let-7c. Conversely, TGF-β1 suppressed expression of let-7c. In cells pretreated with LXA4, upregulation of let-7c persisted despite subsequent stimulation with TGF-β1. In the unilateral ureteral obstruction model of renal fibrosis, let-7c upregulation was induced by administering an LXA4 analog. Bioinformatic analysis suggested that targets of let-7c include several members of the TGF-β1 signaling pathway, including the TGF-β receptor type 1. Consistent with this, LXA4-induced upregulation of let-7c inhibited both the expression of TGF-β receptor type 1 and the response to TGF-β1. Overexpression of let-7c mimicked the antifibrotic effects of LXA4 in renal epithelia; conversely, anti-miR directed against let-7c attenuated the effects of LXA4. Finally, we observed that several let-7c target genes were upregulated in fibrotic human renal biopsies compared with controls. In conclusion, these results suggest that LXA4-mediated upregulation of let-7c suppresses TGF-β1–induced fibrosis and that expression of let-7c targets is dysregulated in human renal fibrosis.

There is a growing appreciation of the role of endogenously produced lipid mediators including lipoxins, resolvins, and PGD synthase metabolites in promoting the resolution of inflammatory responses.1–4 We and others recently described distinct proresolution and antifibrotic properties of lipoxins in renal fibrosis.5,6 TGF-β1 is implicated in numerous fibrotic conditions including tubulointerstitial fibrosis and diabetic kidney disease. The development of fibrosis in this context may reflect activation of parenchymal fibroblasts, recruitment of circulating fibrocytes, and de-differentiation of epithelia and pericytes.7,8 In this study, we investigated the effect of lipoxin A4 (LXA4) on TGF-β1–induced fibrotic responses of renal epithelial cells and the mechanism underlying attenuation of this fibrotic injury pattern by lipoxins.

MicroRNAs (miRNAs) comprise a class of small noncoding RNAs that negatively regulate gene expression by base-pairing to partially complementary sites in the 3′ untranslated regions (UTRs) of target mRNA, preventing translation. miRNAs are implicated in the development and progression of a wide range of complex human diseases,9–12 including diabetic nephropathy.13–15 Here we report that LXA4 upregulates expression of the miRNA let-7c in HK-2 cells and attenuates response to TGF-β1. Lipoxins are protective in unilateral ureteral obstruction (UUO)–induced renal fibrosis and we report that this is associated with increased let-7c expression. Conversely, TGF-β1 decreases let-7c expression and this is associated with increased expression of let-7c targets, including TGFβ receptor type 1 (TGFβR1), collagens (COL1A1, COL1A2), and thrombospondin (THBS1). Overexpression of let-7c mimics the fibrosuppressant effects of LXA4, whereas suppression of let-7c mimics responses to TGF-β1. The importance of regulation of let-7c targets is further supported by evidence from human renal biopsy material where we report upregulation of several let-7c targets in CKD.

Results

LXA4 Attenuates TGF-β1 Responses of Renal Epithelia

We investigated the effect of LXA4 on TGF-β1–driven fibrotic responses of human proximal tubular epithelial (HK-2) cells. LXA4 is the predominant lipoxin generated in humans and binds to a G protein-coupled receptor designated ALX/FPR2.3,16 Using semi-quantitative PCR, we detected expression of ALX/FPR2 in HK-2 cells (Figure 1A), and ALX/FPR2 expression in human mesangial cells was used as a positive control.17,18 TGF-β1 (10 ng/ml; 24 hours) induced a loss of the epithelial marker E-cadherin (CDH1), gain of the mesenchymal markers N-cadherin (CDH2) and fibronectin (FN1), and upregulation of jagged-1 (JAG1) (Figure 1, B and C). Pretreatment of renal epithelial cells with LXA4 (1 nM; 30 minutes) attenuated TGF-β1–induced CDH2, FN1, and JAG1 protein expression (Figure 1, B and C). Notch signaling is an important driver of renal fibrosis,19,20 and it is noteworthy that LXA4 suppressed TGF-β1 driven expression of the Notch ligand JAG1 and its target gene, hairy and enhancer of split 1 (HES1), which is also associated with renal fibrosis (Figure 1D).21 LXA4 attenuated TGF-β1 induction of numerous profibrotic genes including FN1, COL1A1, COL1A2, and THBS1 (Figure 1D). The ALX/FPR2 receptor has been shown to couple via a pertussis toxin–sensitive G protein.22 Here we report that pertussis toxin blocked LXA4 attenuation of TGF-β1 responses including JAG1 (Figure 1, E and F) and CDH2 protein expression (Supplemental Figure 1). Using small interfering RNA (siRNA) targeted against ALX/FPR2, LXA4 no longer suppressed TGF-β1–induced JAG1 (Figure 1, G and H). These data indicate that LXA4 attenuation of the TGF-β1–driven profibrotic signal is mediated through ALX/FPR2.

Figure 1.
Figure 1.

LXA4 attenuates TGF-β1 signaling in HK-2 cells. (A) Semi-quantitative PCR detection of ALX/FPR2 in HK-2 cells. ALX/FPR2 expression in human mesangial cells (HMCs) is used as a positive control; NTC, non-template control. (B) Representative Western blot of epithelial (CDH1), mesenchymal (CDH2, FN1), and TGF-β1 signaling (JAG1) in HK-2 cells stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours). (C) Densitometry analysis of CDH1, CDH2, FN, and JAG1 expression in B (n=3, ± SEM). (D) TaqMan qRT-PCR measurement of CDH1, CDH2, JAG1, FN1, COL1A1, COL1A2, THBS1, and HES1 expression in HK-2 cells stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours). (E) Representative Western blot and (F) corresponding densitometry of JAG1 expression in HK-2 cells incubated with pertussis toxin (PTX) (50 ng/ml; 16 hours) followed by stimulation with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours). (G) ALX/FPR2 mRNA expression in HK-2 cells transfected with ALX/FPR2 siRNA. (H) TaqMan qRT-PCR analysis of JAG1 expression in HK-2 cells transfected with ALX/FPR2 siRNA in the presence of LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours). Expression was normalized to 18S (n=3, ± SEM). *P≤0.05; **P≤0.01.

let-7c Is Induced by Lipoxin and Suppressed by TGF-β1

miRNA expression in HK-2 cells pretreated with vehicle (0.1% ethanol) or LXA4 (1 nM) for 30 minutes and or TGF-β1 (10 ng/ml) stimulation for 24 hours was investigated by miRNA microarray (MRA-1001, miRBase V.14). miRNAs displaying high basal expression included let-7 and miR-200 family members. Several miRNAs previously identified as highly expressed in kidney23 were also detected (miR-192, miR-194, miR-215) (Supplemental Table 1). Multiple let-7 family members were prominent among the miRNAs induced 24 hours after LXA4 pretreatment (Figure 2A), and several of these (let-7a, let-7c) were subsequently validated by quantitative RT-PCR (qRT-PCR) (Figure 2B). let-7 family members are encoded in multiple regions of the human genome, and are highly conserved across multiple species with respect to sequence and function.24 Consistent with previous reports,25,26 we found downregulation of miR-192 in response to TGF-β1 (Supplemental Figure 2). Time-course analysis of let-7c expression (0–6 hours) revealed that let-7c was induced by LXA4 and repressed by TGF-β1 at early time points (Figure 2C). We investigated let-7c responses in primary human mesangial cells and rat renal fibroblasts (NRK49F) 2 hours after stimulation with LXA4 and/or TGF-β1. We observed significant elevation of let-7c levels in human mesangial cells in LXA4-treated cells, whereas in NRK49F cells we saw a significant reduction in let-7c expression in response to TGF-β1 (Figure 2D).

Figure 2.
Figure 2.

let-7c miRNA is induced by lipoxins and repressed by TGF-β1 and targets TGFβR1. (A) Normalized microarray signal intensities of miRs significantly regulated by LXA4 in HK-2 cells. (B) TaqMan qRT-PCR validation of let-7c and let-7a expression levels in HK-2 cells stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours), relative to unstimulated cells (24 hours), which are arbitrarily set to 1 (dashed line) (n=3, ± SEM). (C) Time-course of let-7c expression measured by TaqMan qRT-PCR in HK-2 cells stimulated with LXA4 (1 nM) and/or TGF-β1 (10 ng/ml) (n=3, ± SEM). (D) TaqMan qRT-PCR measurement of let-7c expression levels in human mesangial cells (hMCs) and rat renal fibroblast cells (NRK49F) stimulated with LXA4 (1 nM) and/or TGF-β1 (10 ng/ml) for 2 hours (n=3, ± SEM). (E) Benzo-LXA4 induces let-7c in a 3-day UUO model of renal fibrosis. Male Wistar rats are injected with vehicle (0.2% ethanol) or the benzo-lipoxin analog (15 µg/250-g rat) 15 minutes before surgery. After laparotomy, the left ureter is located and ligated (UUO). Animals are subsequently allowed to recover with or without obstruction for 3 days before kidneys were harvested. let-7c expression is measured by real-time TaqMan PCR assay (Applied Biosystems). RNU48 expression is selected as an endogenous control for normalization of let-7c. Relative miRNA expression is calculated using the ΔCt method of analysis (n=3 per group; ± SEM). NL–UUO, nonligated kidney; L–UUO, ligated kidney. *P≤0.05.

Finally, in an experimental animal model of renal fibrosis (i.e., UUO), we previously reported that the synthetic LXA4 analog exerts protective antifibrotic effects.5 Here we report that LXA4 analog is associated with increased renal let-7c expression in vivo (Figure 2E).

let-7c is located in an intron of C21orf34, a gene that is not expressed in HK-2 cells according to our published RNA-Seq data.27 This would suggest that let-7c expression is under the control of a unique let-7c promoter rather than a host gene promoter. Two studies indicate the let-7c transcription start site is approximately 6 kb upstream of let-7c.28,29 We selected an 8 kb region spanning let-7c and 2 kb upstream of the putative transcription start site and investigated conserved transcription factor binding sites between human and rodent genomes (Supplemental Table 2). Using this strategy, several conserved SMAD3/SMAD4 elements were identified, including a SMAD3 element adjacent to the let-7c coding sequence (Supplemental Figure 3).

let-7c Targets in Renal Epithelial Cells: TGFβR1 and HMGA2

Bioinformatic analysis of the cohort of miRNAs regulated by LXA4 identified multiple pathways predicted to be coordinately regulated (Table 1). Among the significantly regulated pathways (P≤0.05) were several implicated in renal fibrosis, including the TGF-β1, focal adhesion, mitogen-activated protein kinase signaling, and extracellular matrix–receptor interaction pathways. TGF-β1 signals through a heterodimeric serine-threonine kinase composed of two transmembrane polypeptides designated receptors type 1 (TGFβR1) and type 2 (TGFβR2). The 3′ UTR of human TGFβR1 gene contains an 8-mer (75–81: CUACCUCA) and 7-mer (3889–3895: UACCUCA) let-7c binding site conserved across multiple species (Supplemental Figure 4). We hypothesized that as LXA4 increases expression of let-7c, there would be a concomitant decreased expression of its targets including TGFβR1, which might underlie LXA4 modulation of the TGF-β1 response. We observed that TGF-β1 stimulated a 2-fold induction in TGFβR1 mRNA expression. However, in cells pretreated with LXA4 before TGF-β1 addition, TGFβR1 expression was attenuated (Figure 3A). In contrast to TGFβR1, the TGFβR2 3′ UTR does not contain let-7c recognition sites. Expression of TGFβR2 was unchanged by TGF-β1 or LXA4 stimulation (Figure 3B). We therefore propose that let-7c is a pivotal mediator of TGF-β1 responses in renal epithelia. To test this hypothesis, we transfected HK-2 cells with let-7c mimic or anti-miR, resulting in a 200-fold induction or 5-fold repression in let-7c expression levels, respectively (Supplemental Figure 5). We observed that transfection of let-7c mimic attenuates TGF-β1–driven TGFβR1 protein expression (Figure 3, E and F). These data suggest that downregulation of let-7c by TGF-β1 is necessary for subsequent upregulation of TGFβR1 expression. Conversely, we demonstrate that TGFβR1 protein expression is induced upon let-7c anti-miR transfection (Figure 3, G and H). Finally, luciferase reporter assays containing wild-type and mutant seed region let-7c binding sites of the TGFβR1 3′ UTR confirmed the interaction of let-7c mimic with these sites (Figure 3I).

View this table:
Table 1.

Pathways predicted to be targeted by LXA4 regulated miRNAs

Figure 3.
Figure 3.

let-7c targets TGFβR1. (A and B) TaqMan qRT-PCR and (C) Western blot measurement of TGFβR1 and TGFβR2 expression in HK-2 cells stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours) (n=3, ± SEM). (D) Densitometry analysis of TGFβR1 expression in C (n=3, ± SEM). (E and F) Western blot and corresponding densitometry analysis of TGFβR1 expression in HK-2 cells transfected with let-7c or control miRNA mimic and stimulated with TGF-β1 (10 ng/ml; 24 hours) (n=3, ±SEM). (G and H) Western blot and corresponding densitometry analysis of TGFβR1 expression in HK-2 cells transfected with let-7c or control anti-miR and stimulated with TGF-β1 (10 ng/ml; 24 hours) (n=3, ± SEM). (I) Luciferase/Renilla ratio results for HK-2 cells cotransfected with let-7c or control miRNA mimic together with pMIR-REPORT-TGFBR1 3′ UTR site 1 (wild-type or mutant) or pMIR-REPORT-TGFBR1 3′ UTR site 2 (wild-type or mutant) for 48 hours. *P≤0.05; **P≤0.01.

Other targets of let-7c implicated in renal fibrosis include HMGA2, which encodes an early phase transcription factor implicated in induction of transcriptional repressors of E-cadherin, including Snail, Slug, and Twist.30 HMGA2 contains six predicted let-7c binding sites within the 3′ UTR (Supplemental Figure 6). Consistent with our observations that TGF-β1 decreases let-7c, we observe that TGF-β1 increases HMGA2 gene expression, and this response is inhibited in cells overexpressing let-7c (Figure 4, A and B). LXA4 suppressed the upregulation of HMGA2 by TGF-β1 (Figure 4A). To determine whether this is mediated through let-7c upregulation, we transfected cells with the functional blocker of let-7c (i.e., let-7c anti-miR) (Figure 4C). In control cells, LXA4 significantly attenuated TGF-β1–induced HMGA2 expression. However, upon transfection with the let-7c anti-miR, LXA4 no longer reduced TGF-β1–stimulated HMGA2 levels at the mRNA or protein level (Figure 4, C and D). Luciferase reporter assays containing wild-type and mutant seed regions corresponding to the six let-7c binding sites of the HMGA2 3′ UTR identified sites 1 and 5 as functional miRNA-target interaction sites (Figure 4E).

Figure 4.
Figure 4.

let-7c targets HMGA2. (A) Time-course of HMGA2 expression measured by TaqMan qRT-PCR in HK-2 cells stimulated with LXA4 (1 nM) and/or TGF-β1 (10 ng/ml) (n=3, ± SEM). (B) TaqMan qRT-PCR measurement of HMGA2 expression in HK-2 cells transfected with control mimic or let-7c mimic, and stimulated with TGF-β1 (10 ng/ml; 6 hours) (n=3, ± SEM). (C) TaqMan qRT-PCR measurement of HMGA2 expression in HK-2 cells transfected with control anti-miR and let-7c anti-miR and stimulated with LXA4 (1 nM; 30 minutes) and TGF-β1 (10 ng/ml; 6 hours) (n=3, ± SEM). (D) Western blot analysis of HMGA2 expression in HK-2 cells transfected with let-7c or control anti-miR and stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours) (n=3, ± SEM). (E) Luciferase/Renilla ratio results for HK-2 cells cotransfected with let-7c or control miRNA mimic together with pMIR-REPORT-HMGA2 3′ UTR sites 1–6 (wild-type or mutant) for 48 hours. *P≤0.05; **P≤0.01.

Coregulated let-7c Targets in Human CKD Renal Biopsies

Given the foregoing evidence for let-7c as a regulator of fibrosis we investigated let-7c target gene expression in microarray datasets from human CKD and high-throughput sequence analysis of gene expression in TGF-β1–stimulated HK-2 cells.27 We identified 61 putative let-7c targets significantly upregulated by TGF-β1 (P≤0.05) in HK-2 cells (Supplemental Table 3). Among the top 20 let-7c targets significantly upregulated were COL1A1, THBS1, and TGFβR1 (Figure 5A). We interrogated CKD datasets (from diabetic kidney disease,31,32 IgA nephropathy,33 and FSGS34) for significantly upregulated genes containing 3′ UTR let-7c binding sites, and determined the overlap with let-7c targets upregulated by TGF-β1 in HK-2 cells (Figure 5B). Using this approach, 21 putative let-7c targets were identified as coregulated in TGF-β1–stimulated HK-2 cells and human CKD microarray datasets (Figure 5C). Greatest overlap was seen between tubulointerstitium-enriched diabetic kidney disease biopsies and the TGF-β1–stimulated HK-2 cells, and notable among these were multiple collagen encoding genes and THBS1.

Figure 5.
Figure 5.

Overlap of let-7c targets upregulated by TGF-β1 in HK-2 cells and also upregulated in renal biopsies of CKD patients versus control. (A) Top 20 let-7c targets significantly upregulated (P≤0.05) in HK-2 cells stimulated with TGF-β1 (5 ng/ml) for 48 hours versus unstimulated controls (n=3), ranked by fold-induction, as measured by RNA-Seq.27 (B) Overlap of significantly upregulated let-7c targets in HK-2 cells stimulated with TGF-β1 and also in renal biopsies from CKD patients versus control. Renal biopsy microarray datasets downloaded from Nephromine include: (1) DKD (n=11) versus minimal change disease (n=4) and living donor (n=3),31 (2) DKD (n=10) versus living donor (n=13),32 (3) IgAN (n=27) versus living donor (n=6),33 and (4) FSGS (n=8) versus normal kidney (n=9)34 (P≤0.05; fold-change ≥1.3). (C) Heatmap of expression of let-7c targets coregulated in HK-2 cells stimulated with TGF-β1 and also in human CKD renal biopsy microarray datasets (P≤0.05; fold-change ≥1.3). DKD, diabetic kidney disease; IgAN, IgA nephropathy.

let-7c Targets TGF-β1–Induced COL1A1, COL1A2, and THBS1 Expression

Sequence analysis of the 3′ UTRs of COL1A1, COL1A2, and THBS1 identified let-7c miRNA binding sites at positions 789–795, 378–385, and 1518–1524, respectively (Figure 6A). We previously demonstrated that LXA4 attenuates collagen deposition in an animal model for renal fibrosis, and attenuates the upregulation of let-7c target COL1A2.5 Minimal free energy calculations for let-7c pairing with 3′ UTR recognition sites indicated the formation of stable hybrid structures between let-7c and COL1A1 (ΔG = −20.8 kcal/mol−1), COL1A2 (ΔG = −20.3 kcal/mol−1), and THBS1 (ΔG = −26.9 kcal/mol−1). TGF-β1 stimulation of HK-2 cells (10 ng/ml; 24 hours) resulted in significant upregulation of COL1A1, COL1A2, and THBS1 gene expression, and transfection with let-7c miRNA mimic suppressed TGF-β1–mediated upregulation of these genes (Figure 6, B, D, and F). We conclude that suppression of let-7c miRNA by TGF-β1 is critical for TGF-β1–induced COL1A1, COL1A2, and THBS1 expression. In HK-2 cells transfected with let-7c anti-miR, TGF-β1 induction of COL1A1, COL1A2, and THBS1 remained unperturbed (Figure 6, C, E, and G), and let-7c miRNA silencing did not prevent LXA4-mediated suppression of these genes. Furthermore, protein levels of THBS1 in response to let-7c anti-miR transfection validated what we observed at the mRNA level (Supplemental Figure 7). Our data indicate that although let-7c miRNA upregulation may be a novel mechanism through which LXA4 suppresses COL1A1, COL1A2, and THBS1 expression, it is not the exclusive mechanism.

Figure 6.
Figure 6.

let-7c targets COL1A1, COL1A2, and THBS1. (A) Predicted let-7c target sites (red) of the human COL1A1, COL1A2, and THBS1 3′ UTRs, and minimal free energy (mfe) calculations measuring stability of miRNA/RNA hybrids. TaqMan qRT-PCR measurement of COL1A1 (B and C), COL1A2 (D and E), and THBS1 (F and G) expression in HK-2 cells transfected with let-7c mimic/anti-miR versus control miRNA mimic/anti-miR and stimulated with LXA4 (1 nM; 30 minutes) and/or TGF-β1 (10 ng/ml; 24 hours) (n=3, ± SEM). *P≤0.05; **P≤0.01.

Discussion

TGF-β1 is implicated in multiple fibrotic disorders. In this study, we assessed the role and mechanism of lipoxins in attenuating profibrotic responses to TGF-β1. We demonstrate that LXA4 potently represses the expression of key markers of renal fibrosis induced by TGF-β1. miRNA profiling identifies let-7c miRNA as being induced by LXA4 and repressed by TGF-β1 in renal epithelia. let-7c targets a key component of the TGF-β1 signaling pathway, namely TGFβR1, and several effectors and markers of renal fibrosis including HMGA2, COL1A1, COL1A2, and THBS1. We provide in vivo evidence that the protective effects of lipoxin that we previously observed in renal fibrosis5 are coupled to let-7c expression. Taken together, these data highlight the role of LXA4 in the resolution of renal fibrosis and identify a potential miRNA-mediated mechanism through which LXA4 and TGF-β1 act (Figure 7).

Figure 7.
Figure 7.

LXA4 attenuates TGF-β1–mediated fibrosis in HK-2 cells. In renal epithelia, LXA4 suppresses key mesenchymal and signaling markers driven by TGF-β1 (CDH2, FN1, JAG1, HES1) through a yet unknown mechanism. TGF-β1 suppresses let-7c miRNA expression, thereby upregulating multiple let-7c targets (COL1A1, COL1A2, HMGA2, TGFβR1, and THBS1). LXA4 opposes let-7c suppression by TGF-β1. Black arrow, upregulated/downregulated by TGF-β1; red arrow, upregulated/downregulated by LXA4.

LXA4 pretreatment of renal epithelial cells resulted in cellular resistance to the TGF-β1 fibrotic drive. LXA4 attenuated COL1A1, COL1A2, THBS1, CDH2, and FN1 expression. LXA4 also suppressed TGF-β1 induction of components of the Notch signaling pathway, the Notch ligand JAG1 and the downstream transcription factor HES1. We and others previously showed that JAG1 is upregulated in human diabetic kidney disease and renal fibrosis.21,35 The present data indicate that LXA4 suppresses the Notch pathway. The epithelial-to-neuronal cadherin switch is typically observed in epithelial to mesenchymal transition and tumor progression.36,37 Here, LXA4 stimulation attenuated CDH2 (N-cadherin) expression but did not restore the loss of CDH1 (E-cadherin) expression upon TGF-β1 stimulation. We also noted that LXA4 repressed CDH2 expression at the protein level but not at the RNA level, suggesting potential post-transcriptional silencing mechanisms.

Profiling of miRNA expression revealed a cluster of LXA4-responsive miRNAs. Prominent among these were several members of the let-7 family (let-7a, let-7c, let-7g). The let-7 miRNAs regulate cell proliferation and differentiation, and reduced let-7 miRNA expression has been implicated in epithelial to mesenchymal transition and enhanced cell migration/invasion.38,39 let-7 miRNAs inhibit the expression of multiple oncogenes, including RAS, MYC, and HMGA2.40,41 Time-course analysis of let-7c miRNA expression revealed that let-7c levels were rapidly downregulated by TGF-β1 and upregulated by LXA4. Analysis of the predicted let-7c promoter identified a large cohort of conserved transcription factor binding sites, including several putative SMAD sites. It is possible that LXA4 and TGF-β1 have opposing effects on several of these transcription factors, which may directly regulate let-7c transcription. We previously demonstrated that lipoxins reduce phospho-SMAD levels in a UUO model of fibrosis and in an in vitro cell model.5 Alternatively, LXA4 may regulate let-7c indirectly, through inhibition of the TGF-β1 profibrotic signal, which as a consequence may lead to derepression of let-7c.

Analysis of the cohort of miRNAs regulated by LXA4 identified co-ordinate regulation of the TGF-β1 pathway, with let-7c predicted to target TGFβR1. Renal expression of TGFβ receptors types 1 and 2 were previously shown to be elevated in human GN.42 Upon TGF-β1 stimulation, TGFβR1 expression was upregulated, and pretreatment with LXA4 prevented this. In contrast TGFβR2, which contains no let-7c 3′ UTR recognition sites, remained unperturbed by either TGF-β1 or LXA4. By transfecting a let-7c mimic into these cells, TGF-β1-induced TGFβR1 expression was repressed, suggesting that suppression of let-7c by TGF-β1 is a potential mechanism through which receptor type I expression is induced.

We provide evidence linking TGF-β1–mediated loss of let-7c with upregulation of HMGA2, COL1A1, COL1A2, and THBS1 genes in renal epithelia. HMGA2 encodes an early phase transcription factor previously shown to be upregulated by TGF-β130 containing multiple let-7c binding sites within the 3′ UTR. Lee and Dutta demonstrated that ectopic expression of let-7 miRNA reduces HMGA2 and cell proliferation in lung carcinoma.41 Here, we observed an induction of HMGA2 approximately 6 hours after TGF-β1 treatment, which was prevented by LXA4 pretreatment. Our data indicate that downregulation of let-7c miRNA by TGF-β1 is a necessary step for induction of HMGA2 expression. Furthermore, in the absence of let-7c, LXA4 suppression of TGF-β1–induced HMGA2 was attenuated, demonstrating the importance of this miRNA to LXA4 action on HMGA2 expression.

Using high-throughput sequencing technology, we recently investigated the transcriptomic response of HK-2 cells to TGF-β1.27 We identified upregulation of predicted let-7c targets from these data and determined the overlap with available human renal biopsy microarray datasets from CKD patients versus controls. Using this approach, we identified multiple collagen genes and THBS1 as let-7c targets upregulated in both in vitro and human disease datasets.

Our hypothesis that suppression of let-7c is important in TGF-β1–stimulated induction of COL1A1, COL1A2, and THBS1 is supported by attenuation of this response in cells transfected with let-7c mimic. However, let-7c anti-miR transfection failed to modulate COL1A1, COL1A2, or THBS1 levels, indicating that additional let-7 family members may be important in regulation of these targets. COL1A1, COL1A2, and THBS1 3′ UTRs contain recognition sites for multiple let-7 family members, including let-7a and let-7g whose expression is also upregulated by LXA4, and it is likely that upregulation of these miRs may act as a compensatory mechanism in let-7c silenced cells.

We extrapolated our in vitro findings to the in vivo setting by measuring let-7c miRNA expression in a UUO model of chronic renal fibrosis. UUO is an established model of progressive tubulointerstitial fibrosis relevant to CKD of diverse etiologies.43,44 We previously demonstrated amelioration of renal fibrosis by LXA4.5 Here we observed an upregulation of let-7c miRNA expression in kidneys of 3-day UUO rats treated with benzo-LXA4. To further substantiate our findings that let-7c contributes to the renoprotective effects of LXA4 in the context of UUO, it would be of interest to pharmacologically manipulate levels of let-7c with mimetics and anti-miRs in this experimental model. However, such work is beyond the scope of this study. Taken together, these novel data demonstrate that the LXA4 counter-regulatory signal in renal fibrosis may be mediated, in part by let-7c induction and that downregulation of let-7c miRNA by TGF-β1 is important in driving fibrotic responses. Future work will investigate the specific role of let-7c in human renal fibrosis.

Concise Methods

Cell Culture

A full list of abbreviations used in the manuscript is detailed in Supplemental Table 4. Immortalized human kidney epithelial cells (HK-2; ECACC, Porton Down, UK) were cultured at 37°C in a humidified atmosphere of 95% air/5% CO2, and maintained in DMEM-F12 (Sigma-Aldrich, Steinheim, Germany) supplemented with 2 mM l-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 10 ng/ml endothelial growth factor, 36 ng/ml hydrocortisone, 3 pg/ml triiodothyronine, and 5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml selenium (ITS) solution (Sigma-Aldrich). Primary human mesangial cells (Clonetics, Basel, Switzerland) were maintained in MCDB-131 medium (Gibco, Carlsbad, CA), supplemented with 10% (v/v) heat-inactivated FBS, 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Normal rat kidney fibroblasts (NRK-49F cells) were maintained in DMEM, supplemented with 10% (v/v) heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. After serum-starvation for 24 hours, cells were stimulated with vehicle (0.1% ethanol) or LXA4 (1.0×10−9 M; Merck, Calbiochem, Nottingham, UK) for 30 minutes and media was removed and replaced with media with or without TGF-β1 (10 ng/ml; PromoCell GmbH, Heidelberg, Germany) for 30 minutes to 48 hours. HK-2 cells were incubated with pertussis toxin (50 ng/ml; List Biologic Laboratories Inc, Campbell, CA) for 16 hours before LXA4 or TGF-β1 stimulation. For all cell stimulations, three independent experiments were performed.

Real-Time qPCR

RNA extraction from HK-2 cells was performed using an RNeasy RNA extraction kit according to the manufacturer’s protocol (Qiagen, Crawley, UK). RNA quality was assessed using a Bioanalyzer 2100 (Agilent Technologies, Berkshire, UK). Real-time TaqMan PCR was used to quantify relative gene expression levels of CDH1, CDH2, JAG1, TGFβR1, TGFβR2, COL1A1, COL1A2, THBS1, HES1, and FN1 using preoptimized gene expression assays (Applied Biosystems, Foster City, CA). 18s rRNA was used as an endogenous control for normalization. miRNA-enriched RNA was extracted from HK-2 cells using a miRNeasy RNA extraction kit (Qiagen). Real-time TaqMan PCR of miRNAs was used to validate let-7c, let-7a, and miR-192 expression using miRNA expression assays (Applied Biosystems). RNU48 expression was selected as an endogenous control for normalization of target miRNAs. Relative mRNA and miRNA expression was calculated using the ΔCt method of analysis (n=3; ± SEM), and P values of the t test were calculated (P≤0.05). Primers for ALX/FPR2 PCR were designed using Primer3: ALX/FPR2 forward, 5′-TTCCGGATGACACACACAGT-3′; and reverse, 5′-CTTTAGGGTCGTTGGTCCAG-3′.

Protein Extraction and Western Blot Analyses

Lysates were harvested in RIPA lysis buffer containing 50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, and 1 mM EDTA, supplemented with 1 mM PMSF, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and a protease inhibitor cocktail (1.0 µg/ml pepstatin, 1.0 µg/ml leupeptin, 1.0 µg/ml bestatin, and 1.0 µg/ml aprotinin) (Sigma-Aldrich). Total protein was estimated using the Bradford assay. For Western blot analysis, antibodies used included the following: β-actin (1:20,000; Sigma-Aldrich), CDH1 (1:1,000; BD Biosciences, Oxford, UK), CDH2 (1:1,000; BD Biosciences), JAG1 (1:2,000; Santa Cruz Biotechnology, Santa Cruz, CA), HMGA2 (1:500; Santa Cruz Biotechnology), FN1 (1:2,000; BD Biosciences), TGFβR1 (1:1000; Santa Cruz Biotechnology), TGFβR2 (1:1000; Abcam, Cambridge, MA), and THBS1 (1:1,000; Abcam, Cambridge, UK).

miRNA Microarray

miRNAs were extracted using the miRNeasy Mini Kit (Qiagen) and the miRNA microarray was performed using Micro Paraflo microfluidic chips (LC Sciences, Houston, TX). Small RNA fraction was labeled (Cy3, Cy5) and hybridized to the LC Sciences human miRNA microarray (MRA-1001, miRBase V.14, 894 miRNA probes). Data were analyzed by normalizing the signals using a LOWESS filter (locally weighted regression). Normalized microarray expression data are detailed in Supplemental Table 5. The ratio of the two sets of detected signals (log2 transformed) and P values of the t test were calculated (P≤0.05).

Bioinformatic Analyses of miRNAs: Target Prediction

TargetScan, miRanda, and PicTar were used to assess predicted targets sites for miRNAs. miRNA sequence was downloaded from the miRNA database (http://www.mirbase.org/). Transcription factor binding site prediction within the putative let-7c promoter was performed using both the UCSC genome browser TFBS Conserved track and the Footer algorithm,45 using default parameters (weighted average P<0.001). RNA Hybrid46 was used to predict the secondary structure of the RNA/miRNA duplex. Pathway targeting by miRNAs was performed using DIANA miRPath (http://diana.cslab.ece.ntua.gr/).47

siRNA/miRNA Mimic/Anti-miR: Transfections

siGENOME SMARTpool ALX/FPR2 siRNA and siGENOME RISC Free control siRNA were purchased from Dharmacon. siRNAs were transfected into HK-2 cells at 60% confluence using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) at a final concentration of 20 nM for 24 hours. Let-7c miScript miRNA mimic (20 nM) and All Stars negative control siRNA (20 nM) (Qiagen), and let-7c anti-miR (40 nM) and control anti-miR (40 nM) (Applied Biosystems) were transfected into HK-2 cells using Lipofectamine 2000 (Invitrogen). Cells were then stimulated with LXA4 and/or TGF-β1 as previously described.

Luciferase Reporter Assays

Synthetic oligonucleotides encompassing the putative let-7c recognition sequence (underlined), sticky ends for HindIII and SpeI, and a unique BlpI site to test for positive clones were synthesized and annealed. Oligonucleotide sequences for wild-type and mutant constructs are detailed in Supplemental Table 6. The annealed insert was then directly ligated into the HindIII and SpeI cloning sites of the pMIR-REPORT luciferase expression vector (Ambion, Austin, TX). Clones were selected after screening by restriction digestion with BlpI. Transfection of HK-2 cells was performed with Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. The cells were plated 24 hours before transfection in 24-well plates for luciferase assay. HK-2 cells were transfected with 200 ng of pMIR-REPORT vectors, together with Let-7c miScript miRNA mimic (20 nM) (Qiagen), or All Stars negative control (20 nM) (Qiagen) and 80 ng of pCMV-Renilla (internal control). Firefly and Renilla luciferase activities were measured consecutively by using Dual Luciferase Assay (Promega, San Luis Obispo, CA) 24 hours after transfection. Firefly luciferase values were normalized to Renilla, and the ratio of firefly/Renilla is presented.

UUO Animal Model

The UUO model of chronic renal fibrosis treated with LXA4 or benzo-lipoxin was previously described.5 Briefly, male Wistar rats weighing 250–350 g (2–3 months of age) were grouped as UUO and UUO benzo-LXA4. Vehicle (0.2% ethanol) or benzo-LXA4 (15 µg/250-g rat in 0.2% ethanol) was tail vein injected 15 minutes before surgery and 3-day ligation. After ligation, RNA was extracted from kidney tissue using TRIzol (Invitrogen) according to the manufacturer’s instructions.

HK-2 RNA-Seq and Human CKD Biopsy Microarray Analyses

We previously performed RNA-Seq analysis of HK-2 cells stimulated with TGF-β1 (5 ng/ml; 48 hr).27 Published human CKD microarray datasets31–34 were downloaded from Nephromine (http://www.nephromine.org). Cluster analysis of log-transformed normalized expression data of significantly upregulated genes (P≤0.05; fold-change ≥1.3) was performed using Hierarchical Clustering Explorer (http://www.cs.umd.edu/hcil/hce/).

Disclosures

None.

Acknowledgments

A complete list of the GENIE Consortium members is available in the Supplemental Material.

E.P.B. was supported by a Science Foundation Ireland North-South Research Partnership award (SFI 06/IN.1/B114NSs). O.S.G. is supported by a Molecular Medicine Ireland Studentship. D.F.H. is supported by an HRB Career Development Award. The GENIE Consortium is supported by a United States/Ireland R&D Partnership award funded by Science Foundation Ireland (SFI/08/US/B1517), the Northern Ireland Research and Development Office, and the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health (R010-DK081923).

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Lipoxins Attenuate Renal Fibrosis by Inducing let-7c and Suppressing TGFβR1
Eoin P. Brennan, Karen A. Nolan, Emma Börgeson, Oisín S. Gough, Caitríona M. McEvoy, Neil G. Docherty, Debra F. Higgins, Madeline Murphy, Denise M. Sadlier, Syed Tasadaque Ali-Shah, Patrick J. Guiry, David A. Savage, Alexander P. Maxwell, Finian Martin, Catherine Godson, on behalf of the GENIE Consortium
JASN Apr 2013, 24 (4) 627-637; DOI: 10.1681/ASN.2012060550
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