Silencing of microRNA-132 reduces renal fibrosis by selectively inhibiting myofibroblast proliferation

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA+ cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132–treated mice displayed a reduction in the number of proliferating Ki67+ interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67+ epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-β signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy. Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA+ cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132–treated mice displayed a reduction in the number of proliferating Ki67+ interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67+ epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-β signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy. Chronic kidney disease affects about 1 in every 10 adults and is a leading cause of death due to premature cardiovascular disease.1Coresh J. Selvin E. Stevens L.A. et al.Prevalence of chronic kidney disease in the United States.JAMA. 2007; 298: 2038-2047Crossref PubMed Scopus (3894) Google Scholar, 2Foley R.N. Parfrey P.S. Sarnak M.J. Epidemiology of cardiovascular disease in chronic renal disease.J Am Soc Nephrol. 1998; 9: S16-S23PubMed Google Scholar Chronic kidney disease can be caused by various conditions including diabetes mellitus, inflammation, or exposure to toxic substances. Irrespective of the etiology, the common pathway in the pathophysiology of chronic kidney disease involves glomerular sclerosis and tubulointerstitial fibrosis characterized by myofibroblast proliferation and activation that is responsible for the excessive generation of extracellular matrix, the primary constituent of scar tissue in fibrosis.3Levey A.S. Coresh J. Chronic kidney disease.Lancet. 2012; 379: 165-180Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar Murine genetic lineage-tracing models have demonstrated that pericytes (perivascular cells), the stromal cells that cover and support capillary walls, are the major source of α-smooth muscle actin (α-SMA)–positive myofibroblasts in renal fibrosis.4Humphreys B.D. Lin S.L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar, 5Schrimpf C. Duffield J.S. Mechanisms of fibrosis: the role of the pericyte.Curr Opin Nephrol Hypertens. 2011; 20: 297-305Crossref PubMed Scopus (136) Google Scholar Understanding the molecular regulation of myofibroblast formation is important for the identification of factors that could serve as therapeutic targets to counteract the loss of kidney function. MicroRNAs (miRNAs) are small ∼22 nucleotide RNAs that constitute a class of highly conserved noncoding RNAs that control gene expression at the post-transcriptional level by inhibiting mRNA translation or promoting mRNA decay.6Guo H. Ingolia N.T. Weissman J.S. Bartel D.P. Mammalian microRNAs predominantly act to decrease target mRNA levels.Nature. 2010; 466: 835-840Crossref PubMed Scopus (3119) Google Scholar, 7Djuranovic S. Nahvi A. Green R. miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay.Science. 2012; 336: 237-240Crossref PubMed Scopus (648) Google Scholar MiRNAs are able to regulate multiple functionally related targets and thereby provide a means for the coordinated control of gene expression.8Baek D. Villen J. Shin C. et al.The impact of microRNAs on protein output.Nature. 2008; 455: 64-71Crossref PubMed Scopus (2986) Google Scholar, 9Selbach M. Schwanhausser B. 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McBride M.W. et al.miR-21 and miR-214 are consistently modulated during renal injury in rodent models.Am J Pathol. 2011; 179: 661-672Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Zarjou A. Yang S. Abraham E. et al.Identification of a microRNA signature in renal fibrosis: role of miR-21.Am J Physiol Renal Physiol. 2011; 301: F793-F801Crossref PubMed Scopus (213) Google Scholar, 14Zhong X. Chung A.C. Chen H.Y. et al.Smad3-mediated upregulation of miR-21 promotes renal fibrosis.J Am Soc Nephrol. 2011; 22: 1668-1681Crossref PubMed Scopus (347) Google Scholar and was demonstrated to promote renal fibrosis by silencing metabolic pathways.15Chau B.N. Xin C. Hartner J. et al.MicroRNA-21 promotes fibrosis of the kidney by silencing metabolic pathways.Sci Transl Med. 2012; 4: 121ra118Crossref Scopus (423) Google Scholar Also the miR-29 family has been shown to modulate organ fibrosis through its regulation of the synthesis of collagens.16Liu Y. Taylor N.E. 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Jenkins R. Luo D.D. et al.Loss of microRNA-192 promotes fibrogenesis in diabetic nephropathy.J Am Soc Nephrol. 2010; 21: 438-447Crossref PubMed Scopus (304) Google Scholar Here, we aimed to identify miRNAs that are specifically involved in the transition of perivascular cells into myofibroblasts. To that end, we used a FoxD1-GC;Z/Red mouse model4Humphreys B.D. Lin S.L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar to genetically label these perivascular cells and subsequently isolated them through fluorescence-activated cell sorter (FACS)-sorting from healthy kidneys and fibrotic kidneys that were exposed to unilateral ureteral obstruction. Following miRNA profiling of the isolated cells, we found miR-132 to be strongly increased. We demonstrated that silencing of miR-132 reduces the fibrotic response by selectively decreasing the proliferative capacity of pericyte-derived myofibroblasts. To follow the fate of pericytes in the pathogenesis of kidney fibrosis, we used a genetic mouse model expressing a GFP-Cre (GC) fusion protein driven by the FoxD1 promotor (FoxD1-GC). In FoxD1-GC;R26R adult mice, perivascular interstitial cells can be identified in kidney sections following LacZ staining. As shown in Figure 1a and confirming previous studies,4Humphreys B.D. Lin S.L. Kobayashi A. et al.Fate tracing reveals the pericyte and not epithelial origin of myofibroblasts in kidney fibrosis.Am J Pathol. 2010; 176: 85-97Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar 10 days after unilateral ureteral obstruction (UUO), we observed a marked expansion of LacZ-positive pericyte-derived myofibroblasts exclusively in the interstitium of fibrotic kidneys. In order to cell sort kidney cell suspensions by FACS sorting of fluorescently labeled pericyte-derived cells, we subsequently crossed the FoxD1-GC strain with the Z/Red (dsRed) reporter line, which shows an identical expression profile as the FoxD1-GC;R26R strain (Figure 1b). Fibrosis was induced in these FoxD1-GC;Z/Red mice by UUO and FoxD1-derivative interstitial cells (dsRed-positive) were isolated from enzymatically and mechanically dissociated cell suspensions of fibrotic kidneys (UUO) and contralateral kidneys (CLK) using FACS sorting. As expected, we observed a marked increase in dsRed-positive cells in the kidney after UUO, confirming a marked expansion of dsRed-positive pericyte-derived cells (∼10-fold more in UUO than in CLK) (Figure 1c). To identify miRNAs that are involved in the myofibroblastic response of pericytes in fibrotic kidney disease, we profiled miRNAs from FoxD1-derivative interstitial cells that were isolated from fibrotic and contralateral control kidneys. This provides a unique miRNA profile of a restricted cell population within an in vivo renal injury setting (Figure 1d, Table 1). Supplementary Table S1 contains complete miRNA profiling data. Among differentially regulated miRNAs, we observed a ∼21-fold increase of miR-132 expression in the myofibroblast, as compared to only a ∼2.5-fold up-regulation in total kidney (UUO vs. CLK) (Figure 1e). To further assess the association of increased miR-132 expression in renal injury, we sought to determine whether miR-132 would also be differentially regulated in a different renal injury model. Therefore, we induced ischemia-reperfusion injury in mice, isolated RNA from the kidneys 3 weeks after injury, which associates with longer-term fibrotic complications of ischemia-reperfusion injury ,24Kim J. Seok Y.M. Jung K.J. Park K.M. Reactive oxygen species/oxidative stress contributes to progression of kidney fibrosis following transient ischemic injury in mice.Am J Physiol Renal Physiol. 2009; 297: F461-F470Crossref PubMed Scopus (171) Google Scholar and observed significant up-regulation of miR-132 (Figure 1f).Table 1Differential miRNA expression in FoxD1-derivative interstitial cellsDescriptionParametricP valueMean of intensities in CLKMean of intensities in UUOFold changemmu-miR-146b8.80 × 10–06125,167.441,405,838.7511.2mmu-miR-2230.0005411300,290.574,273,538.814.3mmu-miR-342-3p0.0007948111,408.45658,123.935.9mmu-miR-1500.0036602462,549.953,083,917.856.7mmu-miR-574-3p0.004272345,198.89179,0914.0mmu-miR-2210.0045003382,274.52153,939.05–2.48mmu-miR-4550.00486481,697.2212,130.35–6.73mmu-miR-1320.005465338,732.3828,418.6421.3mmu-miR-2140.00602192,663.3244,306.5990.9mmu-miR-27b0.0078164343,928.67176,584.64–1.95mmu-miR-7410.0108911166.112.42–68.56mmu-miR-210.0137817582,439.881,837,951.13.1mmu-miR-199a-5p0.01481081.78308.9175.4mmu-miR-146a0.01520641,099,753.644,653,872.54.2mmu-miR-7080.019057537,212.54,528.03–8.22mmu-miR-3650.0202887729,981.29177,074.88–4.12mmu-miR-3830.020710612,843.49295.09–43.52mmu-miR-6850.029063445,829.841,348,570.5829.4mmu-miR-1340.03000525.893,216.63555.6mmu-miR-2940.03919343.89338.6590.9CLK, contralateral kidney; UUO, unilateral ureteral obstruction.The top 20 differentially expressed microRNAs (miRNAs) based on P value are represented. Mean intensity is an absolute value representing the abundance of the miRNA. Fold change of expression is change over contralateral kidney, – sign means down-regulation. Open table in a new tab CLK, contralateral kidney; UUO, unilateral ureteral obstruction. The top 20 differentially expressed microRNAs (miRNAs) based on P value are represented. Mean intensity is an absolute value representing the abundance of the miRNA. Fold change of expression is change over contralateral kidney, – sign means down-regulation. Given the strong increase of miR-132 in myofibroblasts, we next investigated the effect of miR-132 silencing in a mouse model of renal fibrosis. Previously we demonstrated that antagomirs can be used to specifically silence miRNA expression both in vitro as well as in vivo.25van Solingen C. Seghers L. Bijkerk R. et al.Antagomir-mediated silencing of endothelial cell specific microRNA-126 impairs ischemia-induced angiogenesis.J Cell Mol Med. 2009; 13: 1577-1585Crossref PubMed Scopus (241) Google Scholar, 26Bijkerk R. de Bruin R.G. van Solingen C. et al.MicroRNA-155 functions as a negative regulator of RhoA signaling in TGF-β-induced endothelial to mesenchymal transition.Microrna. 2012; 1: 2-10Crossref Scopus (41) Google Scholar, 27Bijkerk R. van Solingen C. de Boer H.C. et al.Silencing of miRNA-126 in kidney ischemia reperfusion is associated with elevated SDF-1 levels and mobilization of Sca-1+/Lin- progenitor cells.Microrna. 2014; 3: 144-149Crossref PubMed Google Scholar Wild-type mice were administrated antagomir-132 or scrambled control antagomirs (scramblemir; n = 7 per group, 40 mg/kg), exposed to UUO, and killed 5 or 10 days after the procedure. Antagomir silencing of miR-132 in the kidneys was confirmed by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (Figure 2a), although in the fibrotic kidneys a low level of expression was retained. To confirm silencing of miR-132 in myofibroblasts, we FACS sorted FoxD1-derivative interstitial cells in the UUO model (10 days) after antagomir-132 injection (Supplementary Figure S1). We used FoxD1-GC;tdTomato mice that show a similar fluorescent pattern (but stronger) as FoxD1-GC;Z/Red mice (Supplementary Figure S2). Subsequently, RNA was isolated from these cells. As illustrated in Figure 2b, we could confirm knockdown of miR-132 in these myofibroblasts. In addition, in situ hybridization for miR-132 was performed and demonstrates that miR-132 is widely expressed in the kidney in tubular epithelial cells, interstitial cells, and a subset of glomerular cells (Supplementary Figure S3A). Antagomir-132 treatment leads to a marked silencing of the miR-132 signal throughout the kidney. Furthermore, using an Alexa647-labeled antagomir, we analyzed where antagomir molecules accumulate in the kidney. The majority of antagomir accumulates in tubular epithelial cells. Nonetheless, antagomir is also found in the interstitial compartment (Supplementary Figure S3B). To determine the effect of miR-132 silencing on fibrosis, we performed picrosirius red staining to quantify the collagen content in the kidneys. Although no difference in kidney collagen content was observed 5 days after UUO, 10 days after UUO we did observe a significant protective effect of miR-132 silencing, amounting to ∼35% reduction in collagen deposition (P < 0.05) (Figure 2d and e). This result was consistent with the reduction of α1 type-1 collagen (col1α1) gene expression that was not different 5 days after UUO but was reduced in the antagomir-132–treated mice 10 days after UUO (P = 0.09) (Figure 2f). In concordance with reduced fibrosis, we found a reduction in the number of apoptotic tubular epithelial cells, as determined by terminal deoxynucleotidyl-transferase-mediated deoxyuridine 5-triphosphate nick end-labeling staining, in the antagomir-132–treated kidneys (Figure 2g and h). We confirmed this antagomir-132–dependent reduction in tubular apoptosis by staining for cleaved caspase-3 (Supplementary Figure S4). To assess the effect of antagomir-132 silencing on UUO-induced myofibroblast formation, we stained the kidney sections for α-SMA. We observed a marked reduction in the number of cells staining positive for α-SMA in kidneys from the mice treated with antagomir-132 as compared to the kidneys of scramblemir-treated mice 10 days after UUO (P < 0.05) (Figure 3a and b ). Also, the α-SMA mRNA levels were consistently decreased 10 days after UUO (P < 0.05) (Figure 3c). The decrease in α-SMA expression in the antagomir-132–treated group 10 days after surgery was confirmed with Western blot (P < 0.0005) (Figure 3d and e). No differences in α-SMA expression were observed 5 days after UUO. To confirm these in vivo observations, we assessed whether miR-132 silencing would also abrogate myofibroblast formation in vitro. To that end, we used mouse fibroblast cells (NIH3T3) that were stimulated with TGF-β to induce differentiation toward myofibroblasts. This was evidenced by a change in morphology (elongation; data not shown) and an increase in α-SMA protein expression. The addition of antagomir-132 led to abrogation of this transition as illustrated by decreased α-SMA protein expression in these cells (Figure 3f, g, and h). We confirmed this effect in a second C3H/10T1/2 fibroblast cell line that displayed decreased α-SMA mRNA levels upon antagomir-132 treatment (Figure 3i). As the loss of pericytes, and the subsequent transition into myofibroblasts, may be associated with regression of the peritubular capillary network, we next sought to determine the capillary density in both treatment groups. To achieve this, whole kidney sections were stained with mouse endothelial cell antigen. In contrast to our expectation, we did not observe a difference in capillary density between antagomir-132 and scramblemir-treated mice 10 days after UUO (Figure 3j and k). Given that silencing of miR-132 decreases fibrosis and attenuates myofibroblast formation, we next investigated the mechanism involved. Therefore, gene expression profiling was performed on the RNA we isolated from FACS-sorted FoxD1-derivative interstitial cells in the UUO model (10 days) after silencing miR-132 as described earlier. Supplementary Table S2 demonstrates differential gene expression in myofibroblasts in vivo as a result of inhibiting miR-132. By using ingenuity pathway analysis, this genome-wide differential gene expression data allowed for analysis of affected biological pathways as a result of silencing miR-132. As illustrated in Figure 4a, “cellular growth and proliferation” was the top most affected molecular and cellular function, which according to our gene expression data is inhibited. Furthermore, pathway analysis revealed that among potential upstream regulators, miR-132 silenced differentially regulated genes, which are under the control of TGF-β, suggesting the TGF-β signaling pathway is affected (Supplementary Table S3). Because our bioinformatic analysis implicated myofibroblast proliferation as a pathway regulated by miR-132, we next asked whether we could find evidence for regulation of cell proliferation in kidneys of antagomir-132– or scramblemir-treated mice. We examined expression of well-characterized regulators of cell cycle progression, including retinoblastoma-1 (RB1) and the active phosphorylated form of RB1 (p-RB1), RAS p21 protein activator 1, p21, and Cyclin-A. Surprisingly, in total kidney lysates, we found that silencing miR-132 resulted in an expression pattern of these regulatory proteins that suggested increased cell proliferation, not decreased, as reflected by increased phosphorylated RB1 and Cyclin-A and decreased RAS p21 protein activator 1and p21 levels, respectively (Figure 4b). By contrast, our pathway analysis of FACS-sorted interstitial myofibroblasts suggested a decrease in proliferation. We hypothesized that this discordance might be explained if miR-132 exerted different effects according to kidney cell type. We therefore performed Ki67 staining and compared the number of proliferating cells in either the tubular or the interstitial compartment in kidney sections from fibrotic kidneys that received antagomir-132 or scramblemir. Indeed, we observed an increased number of proliferating tubular epithelial cells in antagomir-132–treated kidneys (Figure 4e and f), which is consistent with our whole kidney lysate Western blot results. By contrast, we observed a significant decrease in the number of proliferating interstitial cells of these kidneys (Figure 4g and h), which is consistent with the aforementioned pathway analysis. Furthermore, in NIH3T3 and C3H/10T1/2 cells that were cultured with or without TGF-β and treated with antagomir-132 or scramblemir, we also observed that silencing miR-132 caused a significant reduction in cell proliferation using the thymidine incorporation assay (Figure 4c and d). We also investigated whether antagomir-132 could directly increase proximal tubular epithelial cell proliferation in vitro. Supplementary Figure S5 shows that with or without addition of TGF-β, the proliferative capacity of human kidney 2 cells is not increased by addition of antagomir-132. In fact, antagomir-132 induced a minor antiproliferative response, suggesting the observed effect on epithelial cells in vivo is indirect. These observations suggest that miR-132 regulates myofibroblast-specific cell proliferation during fibrosis. In addition, we performed a scratch assay on unstimulated or TGF-β–stimulated NIH3T3 cells and demonstrated that antagomir-132 addition resulted in a decreased wound healing capacity for TGF-β–stimulated cells (Figure 4i and j), further confirming the selective antiproliferative effect of miR-132 silencing on myofibroblasts. Lastly, we also investigated which direct, previously validated miR-132 targets could be involved in the proliferation of the myofibroblasts. Among others, Foxo3a,28Wong H.K. Veremeyko T. Patel N. et al.De-repression of FOXO3a death axis by microRNA-132 and -212 causes neuronal apoptosis in Alzheimer's disease.Hum Mol Genet. 2013; 22: 3077-3092Crossref PubMed Scopus (217) Google Scholar p300,29Lagos D. Pollara G. Henderson S. et al.miR-132 regulates antiviral innate immunity through suppression of the p300 transcriptional co-activator.Nat Cell Biol. 2010; 12: 513-519Crossref PubMed Scopus (283) Google Scholar RB1,30Park J.K. Henry J.C. Jiang J. et al.miR-132 and miR-212 are increased in pancreatic cancer and target the retinoblastoma tumor suppressor.Biochem Biophys Res Commun. 2011; 406: 518-523Crossref PubMed Scopus (154) Google Scholar and p120rasgap31Anand S. Majeti B.K. Acevedo L.M. et al.MicroRNA-132-mediated loss of p120RasGAP activates the endothelium to facilitate pathological angiogenesis.Nat Med. 2010; 16: 909-914Crossref PubMed Scopus (445) Google Scholar have been reported to be directly targeted by miR-132 and to have inhibitory (or stimulatory for RB1) effects on cell proliferation. We assessed the expression of these genes in the FACS-sorted FoxD1-derivative interstitial cells from antagomir-132 treated mice compared with scramblemir-treated mice, and found Foxo3a and p300 levels to be increased, providing a possible direct link between miR-132 silencing and decreased pericyte proliferation (Figure 5). Of note, RB1 levels did not change, whereas p120rasgap even showed a trend toward decreased levels. In addition, we checked gene expression levels, in the FACS-sorted FoxD1-derivative interstitial cells, of α-SMA, col1α1, Smad2, and Smad3, all TGF-β target genes that could be involved in proliferation and fibrosis. We confirmed down-regulation of α-SMA and col1α1 by antagomir-132 and found a trend toward a decrease in Smad2 and Smad3 gene expression (Figure 5a). In addition, we checked in vitro 3T3 fibroblasts to see whether we could confirm altered Smad2 and Smad3 gene expression and found that silencing miR-132 resulted in a TGF-β–dependent reduction in both Smad2 and Smad3 levels (Figure 5b). Together these data further confirm that silencing miR-132 affects TGF-β signaling, which we demonstrated by pathway analysis (Supplementary Table S3). In this study, we demonstrate that miR-132 expression is strongly increased in the pericyte-derivative myofibroblasts that accumulate during the fibrotic response. Silencing of this miRNA resulted in a decreased fibrotic phenotype of the kidneys 10 days after UUO as evidenced by a reduction in myofibroblast formation as well as a reduction in the buildup of collagen-rich scar tissue. In addition, blocking miR-132 inhibited the proliferative response of the myofibroblast. Therefore, our data support a facilitatory role for miR-132 in renal fibrosis by affecting the proliferative capacity of myofibroblasts. Pericyte (or perivascular fibroblast) to myofibroblast transition is considered to cause detachment of pericytes from the microvascular interface leaving unstable capillaries that would result in rarefaction.5Schrimpf C. Duffield J.S. Mechanisms of fibrosis: the role of the pericyte.Curr Opin Nephrol Hypertens. 2011; 20: 297-305Crossref PubMed Scopus (136) Google Scholar In fact, peritubular capillary rarefaction is a typical feature of renal fibrosis.32Ishii Y. Sawada T. Kubota K. et al.Injury and progressive loss of peritubular capillaries in the development of chronic allograft nephropathy.Kidney Int. 2005; 67: 321-332Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar However, we observed no difference in capillary density. Given that we did demonstrate miR-132 to regulate myofibroblast proliferation, and that reduced fibrosis due to miR-132 silencing appears to be a relatively late effect (as no effects were observed after 5 days), our findings suggest an initial miR-132–independent phase where pericytes detach from the vessels and transform into myofibroblasts, followed by a miR-132–modulated proliferation phase of these cells. Using pathway analysis, we identified miR-132 to be involved in cell proliferation, which interestingly was previously found to be among the Gene Ontology (GO) terms most highly associated with pericyte to myofibroblast transition.33Grgic I. Krautzberger A.M. Hofmeister A. et al.Translational profiles of medullary myofibroblasts during kidney fibrosis.J Am Soc Nephrol. 2014; 25: 1979-1990Crossref PubMed Scopus (59) Google Scholar However, in our study, individual genes were only moderately affected (Supplementary Table S1). Nonetheless, this is consistent with the ideas that (i) individual miRNAs could directly repress hundreds of genes, albeit to a modest degree, making miRNAs fine