Acta Diabetol DOI 10.1007/s00592-013-0492-8
ORIGINAL ARTICLE
Regulation of TIMP3 in diabetic nephropathy: a role for microRNAs Loredana Fiorentino • Michele Cavalera • Maria Mavilio • Francesca Conserva • Rossella Menghini • Loreto Gesualdo Massimo Federici
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Received: 9 June 2013 / Accepted: 11 June 2013 Ó Springer-Verlag Italia 2013
Abstract Diabetic nephropathy (DN) is the major cause of chronic kidney disease in developed countries and contributes significantly to increased morbidity and mortality among diabetic patients. Morphologically, DN is characterized by tubulo-interstitial fibrosis, thickening of the glomerular basement membrane and mesangial expansion mainly due to accumulation of extracellular matrix (ECM). ECM turnover is regulated by metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) activities. In diabetic conditions, TIMP3 expression in kidney is strongly reduced, but the causes of this reduction are still unknown. The aim of this study was to elucidate at least one of these mechanisms which relies on differential expression of TIMP3-targeting microRNAs (miRs) in a hyperglycemic environment either in vitro (MES13 cell line) or in vivo (mouse kidney and human biopsies). Among the TIMP3-targeting miRs, miR-21 and miR-221 were significantly upregulated in kidneys from diabetic mice compared to control littermates, and in a mesangial cell line grown in high glucose conditions. In human samples, only miR-21 expression was increased in kidney
Communicated by Renato Lauro. L. Fiorentino M. Cavalera M. Mavilio R. Menghini M. Federici (&) Department of Systems Medicine, University of Rome ‘‘Tor Vergata’’, Via Montpellier 1, 00133 Rome, Italy e-mail:
[email protected] F. Conserva L. Gesualdo Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy M. Federici Center for Atherosclerosis, University Hospital Tor Vergata, Rome, Italy
biopsies from diabetic patients compared to healthy controls. The expression of miR-217, which targets TIMP3 indirectly through downregulation of SirT1, was also increased in diabetic kidney and MES13 cell line. In agreement with these result, SirT1 expression was reduced in mouse and human diabetic kidneys as well as in MES13 mesangial cell line. TIMP3 deficiency has recently emerged as a hallmark of DN in mouse and human. In this study, we demonstrated that this reduction is due, at least in part, to increased expression of certain TIMP3-targeting miRs in diabetic kidneys compared to healthy controls. Unveiling the post-transcriptional mechanisms responsible for TIMP3 downregulation in hyperglycemic conditions may orient toward the use of this protein as a possible therapeutic target in DN. Keywords Diabetic Nephropathy MicroRNA TIMP3 SirT1 Abbreviations TIMP Tissue inhibitor of metalloproteinases DN Diabetic nephropathy STZ Streptozotocin miR MicroRNA PCR Polymerase chain reaction
Introduction Diabetic nephropathy (DN) is the leading cause of endstage renal disease in Western world and affects one-third of patients with both type 1 and type 2 diabetes. The main histological features of the disease are glomerular basement membrane thickening and mesangial extracellular
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matrix expansion which leads to fibrosis. The integrity of extracellular matrix is regulated by the balance between metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs). TIMP3 is the most highly expressed TIMP in the kidney, and its loss has been associated with inflammation, renal fibrosis and tubular interstitial injury in mice [1–5]. Studies in human and mouse models linked downregulation of TIMP3 to diabetes complications and metabolic inflammation [6–16]. We have previously shown that, in a hyperglycemic environment, TIMP3 expression was strongly reduced in both mouse and human kidneys with features of DN [17]. Genome-wide mRNA profiling of kidneys from streptozocin-treated mice revealed that the deletion of TIMP3 under diabetic condition reduced the expression of FoxO1 transcription factor and blunted the activation of the autophagy machinery through a FoxO1-mediated downregulation of a number of autophagic genes. Rescue of TIMP3 expression reverted the molecular phenotype. Analysis of human biopsies confirmed that TIMP3 is lost in subjects with DN in association with reduced autophagic genes expression [17]. Despite these evidences, the mechanisms that lie behind this reduction are still unknown. MicroRNAs (miRs) play key roles in regulating the expression of mammalian genes relevant to cancer and other diseases [18–20]. Recently, they have been shown to be functionally important in modulating the renal response to hyperglycemia and the progression of DN [21, 22]. TIMP3 expression is regulated by a number of miRs such as miR-21, miR-181b, miR-182, miR-216 and miR-221/2, expressed in a tissue or cell specific manner [23, 24]. In this study, we investigated the role of miRs in glucotoxicityinduced TIMP3 downregulation, and pointed at their potential role as sensitive, non-invasive biomarkers of kidney disease and progression.
Methods Mice Induction of diabetes by streptozotocin (STZ) injection in TIMP3 knockout mice used in this study has been described elsewhere [17]. Briefly, STZ was dissolved in sterile sodium citrate buffer (pH 4.5) and used within 15 min to prevent degradation. Mice were starved for 5 h and then injected intraperitoneally with 50 mg/kg of STZ (Sigma, St. Louis, MO, USA) for 5 consecutive days. After each injection, a solution of 10 % glucose was supplied to mice for 2 h to avoid hypoglycemia. Blood glucose levels were monitored at baseline and then once a week for 12 weeks to follow the development of hyperglycemia. Mice were considered diabetic with a blood glucose level above 250 mg/dl.
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Age-matched littermates were injected with sodium citrate buffer alone and used as controls. After 12 weeks, mice were euthanized and kidneys analyzed. Animal studies were approved by the University of Tor Vergata Animal Care and Use Committee. All animals received human care according to the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy Sciences and published by the National Institutes of Health (NIH publication 86-23 revised 1985). Cells SV40 MES 13 mouse mesangial cells (ATCC CRL 1927; Manassas, VA, USA) were grown in low-glucose DMEM. To induce hyperglycemia, 20 mM glucose was added to the culture medium (25 mM final concentration) for 24–48 h. Mannitol (25 mM final concentration) was used for osmotic control. RNA extraction was carried out using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA). Extraction, retro-transcription and quantitative real-time PCR on human RNA Human kidney biopsies were collected for diagnostic purpose from 8 patients with biopsy-proven DN, increased proteinuria and decreased glomerular filtration rate, and 4 control kidney tissues from patients with no histological signs of nephropathy. Informed consent was obtained from all subjects, and all the experiments conformed to the principles set out in the WMA Declaration of Helsinki [http://www.wma.net/en/30publications/10policies/b3/] and the NIH Belmont Report [http://ohsr.od.nih.gov/guidelines/ belmont.html]. Total RNA was extracted using the miRNeasy FFPE Kit (Qiagen, Valencia, CA, USA) according to manufacturer’s instructions. First-strand cDNA synthesis and real-time polymerase chain reaction (PCR) were performed according to the miRCURY LNA Universal RT miRs PCR protocol (EXIQON, Vedbaek, Denmark). Data were normalized to SNORD38B and miR103. Fold change expression was calculated according to the 2 DDCT method. microRNA and mRNA reverse transcription and quantitative polymerase chain reaction analysis Real-time quantification of miRs was performed with the TaqMan miRNA reverse transcription kit and miRNA assay according to the manufacturer’s protocol. Data were normalized to 202 small nucleolar RNA (sno202). For mRNA analysis, single-strand complementary DNA (cDNA) was synthesized from 1 lg of total RNA with a high-capacity cDNA archive kit (Applied Biosystems,
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Foster City, CA, USA), according to the standard protocol. Fifty nanograms of cDNA were amplified by real-time PCR. Actin was used as housekeeping reference gene. Fold change expression was calculated according to the 2 DDCT method.
As this could indicate very low expression in normoglycemic condition, we hypothesize a regulation similar to the one observed in mice.
Statistical analysis Results of the experimental studies are mean ± SD, as indicated. Statistical analysis was performed using unpaired Student’s t test on Graph Pad Prism 5. Values of p \ 0.05 were considered statistically different.
Results Expression analysis of mice and human Timp3targeting microRNAs C57Bl6 mice were treated with STZ for 12 weeks to induce hyperglycemia and diabetes. As we have recently shown, under these conditions, TIMP3 expression was significantly reduced [17]. RNA from kidneys of healthy and diabetic mice was used to analyze the expression of a number of miRs shown to be involved in the progression of DN [21] and which are also known to target Timp3 mRNA inducing protein downregulation. In particular, miR-21, miR-181b, miR-182, miR-192, miR-216, miR-221 and miR-222 were assayed by quantitative PCR. We also evaluated miR-217 expression, as it has been recently shown that this miRs can modulate Timp3 indirectly, through SirT1 inhibition [9, 25]. As shown in Fig. 1a, miR-21, miR-217 and miR-221 were significantly increased in murine diabetic kidneys compared to controls; this could explain the previously reported decrease in Timp3 expression in STZ mice compared to their control littermates [17]. We observed that SirT1 expression was also reduced in diabetic kidneys. Interestingly, we did not detect a significant change in the expression of miR-192 and miR-216, which have been implicated in DKD. Additionally, the analysis performed on kidney RNA from eight diabetic patients and four healthy controls revealed a significant increase in miR-21 in the diabetic subjects (Fig. 1b), which could explain the decrease in TIMP3 expression previously reported in the same patients [17]. Real-time PCR analysis on miR-217 and SirT1 in these biopsies revealed a significant reduction in SirT1 expression in diabetic subjects compared to healthy controls (Fig. 1b). As far as miR-217 was concerned, we were not able to measure its transcript in 3 out of 4 control samples, while 50 % of diabetic patients showed a detectable level of this miRNA (data not shown)
Fig. 1 a Real-time PCR analysis on kidney RNA from diabetic (STZ-treated) mice and control littermates. n = 6, **p \ 0.005; *p \ 0.05. b Real-time PCR analysis on kidney RNA extracted from human kidney biopsies collected for diagnostic purpose from 8 patients with biopsy-proven diabetic nephropathy (DN), increased proteinuria and decreased glomerular filtration rate, and 4 control kidney tissues from patients with no histological signs of nephropathy. **p \ 0.005; *p \ 0.05
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Acta Diabetol Fig. 2 Real-time PCR analysis on RNA extracted from MES13 mouse mesangial cells grown in high glucose condition (25 mM glucose) for 24 or 48 h. n = 3, **p \ 0.005; *p \ 0.05
microRNAs expression in mesangial cells To validate the results obtained in mice and humans, we investigated the expression of the Timp3-targeting miRs in mouse mesangial cells (MES13) grown in low- or high glucose conditions for 24 or 48 h. As shown in Fig. 2, glucose treatment led to a significant reduction in both Timp3 and SirT1 transcripts, compared to untreated cells. These data seem to correlate with a concomitant increase in miR-21 and miR-217 following 24 h of treatment. Interestingly, both miRs were upregulated after 24 h of high glucose and disappeared at 48 h. The effects on the target mRNAs (Timp3 and SirT1, respectively) were instead mainly observed at the latest time point. In all the experiments, mannitol was used as osmotic control, but it did not affect miRs expression (data not shown).
Discussion We have previously shown that TIMP3 expression was reduced in the kidney of STZ-treated mice, a well-established model of hyperglycemia and glucotoxicity, and in kidney biopsies from diabetic patients [17]. Works from other labs have also clearly demonstrated the involvement of TIMP3 in kidney pathology [1–5, 14–16] The present
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study highlights the potential involvement of miRs in the regulation of TIMP3 expression under diabetic conditions. Kidney from diabetic mice showed an increased expression of miR-21 and miR-221 (already known to target TIMP3 [23, 24]), while other Timp3-targeting miRs such as miR181b, miR-182, miR-216 and miR-222 remained unchanged. miR-21 was also found significantly overexpressed in human kidney biopsies from diabetic patients, and in mesangial cells grown under high glucose conditions. These results seem to strictly correlate with a decrease in Timp3 mRNA, confirming the hypothesis that a glycemic overload can induce miR-21 expression leading to Timp3 messenger RNA destruction. Notably, miR-21 increase could be detected as early as after 24 h of high glucose treatment; at the same time point, Timp3 mRNA showed only a slight decrease, that became significant after 48 h, when miR-21 transcript was hardly detectable. This timing is in keeping with the general mechanism of action of miRs, i.e., induction by a stimulus such as glucose precedes target downregulation. We found that miR-217 was also significantly upregulated in diabetic mouse kidneys and high glucose treatedmesangial cells; it was detectable only in one out of 4 normal kidney biopsies and in 50 % of diabetic patients. Even though this molecule does not have Timp3 as a direct target, we have previously shown that miR-217 can
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modulate Timp3 expression in vascular smooth muscle cells and in monocyte/macrophage through downregulation of the SirT1 deacetylase [9, 25]. SirT1 expression was indeed reduced in kidneys of both STZ-treated mice and human diabetic patients as well as in cellular models of hyperglycemia, suggesting that SirT1-mediated TIMP3 downregulation could represent a more general mechanism of TIMP3 modulation that takes place in different tissues. Taken together, our results shed some light on the complexity of DN by investigating the possible mechanisms of TIMP3 post-transcriptional regulation through a set of miRs known to be involved in disease progression. miRs have recently become a central part of the puzzle in this pathology as they appear to be the downstream regulator molecules. Finally, it is a recent finding that miRs can circulate, protected from RNase-dependent degradation, in biological fluids like serum and urine. The assessment of miRNAs in biofluids seems extremely appealing when aiming to set up an alternative, non-invasive technique for early diagnosis and intervention.
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14. Acknowledgments This work was supported by JDRF RRG 1-2007-665 and Fondazione Roma 2008, PRIN 2009FATXW3_002 (all to M.F.) and Ministero della Salute-Ricerca Finalizzata 2008 to L.G. Conflict of interest The authors declare that there is no duality of interest associated with this manuscript.
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