Metformin prevents renal interstitial fibrosis in mice with unilateralureteral obstructionRita C. Cavaglieri 1, Robert T. Day, Denis Feliers *, Hanna E. Abboud †

Department of Medicine/Nephrology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA

A R T I C L E I N F O

Article history:Received 2 April 2015Received in revised form 4 June 2015Accepted 4 June 2015Available online 9 June 2015

Keywords:MetforminAdenosine monophosphate-activatedkinaseKidneyFibrosisInflammation

A B S T R A C T

Unilateral ureteral obstruction causes important tubulo-interstitial fibrosis in the kidney. Metformin reducesfibrosis in mice with diabetic nephropathy. We examined the effects of metformin in a mouse model ofunilateral ureteral obstruction (UUO). Expression of inflammation and fibrosis markers was studied byimmunohistochemistry, immunoblot and quantitative real-time polymerase chain reaction. Seven daysafter UUO, kidneys presented dilated tubules, expansion of the tubulo-interstitial compartment, and sig-nificant infiltration of inflammatory cells. Macrophage infiltration and inflammation markers expressionwere increased in obstructed kidneys and reduced by metformin. Metformin reduced expression ofextracellular matrix proteins and profibrotic factor TGFβ in obstructed kidneys, measured byimmunohistochemistry. Interstitial fibroblast activation was evident in obstructed kidneys andameliorated by metformin. UUO did not affect adenosine monophosphate-activated kinase (AMPK) ac-tivity, but metformin activated AMPK. Our results show that metformin prevents or slows down the onsetof renal inflammation and fibrosis in mice with UUO, an effect that could be mediated by activation ofAMPK.

© 2015 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Obstructive nephropathy can occur in both children and adults.In adults, renal function usually recovers after elimination of theobstruction, but in children obstruction can have long term con-sequences. Fetal and neonatal obstruction result in reduced nephronnumber, with the magnitude of reduction being dependent on theseverity and duration of obstruction (Chevalier et al., 2009).Obstruction is accompanied by changes in kidney structure, in-flammation and tubulointerstitial fibrosis, leading to decrease andeventually loss of renal function if the obstruction persists. Inter-stitial fibrosis is a major prognostic indicator of end-stage renaldisease (Eddy, 2000). Inhibition of interstitial fibrosis is thereforecritical to preserve kidney function (Eddy, 2005). Unilateral ure-teral obstruction (UUO) is an experimental model for tubulo-interstitial fibrosis (Chevalier et al., 2009). UUO causes renal

hemodynamic and metabolic changes, leading to tubular injury andrenal inflammation, characterized by macrophage infiltration. Ac-tivation of interstitial fibroblasts causes their differentiation intomyofibroblasts, which contribute to accumulation of the extracel-lular matrix proteins, such as fibronection, collagens I and II andlead eventually to renal fibrosis (for a review, see Chevalier et al.,2009). Upon recruitment and activation, macrophages producevarious pro-inflammatory cytokines, such as TNFα and TGFβ whichin turn promote expression of adhesion molecules, such as vascu-lar cell adhesion molecule 1 (VCAM1), and contribute to furtherrecruitment of circulating inflammatory cells (see Chevalier et al.,2010 for a review).

Metformin is a biguanide compound that has been used asan oral anti diabetic drug for over 50 years. It is the first-line drugof choice for the treatment of type 2 diabetes, in particular, inoverweight and obese people and those with normal kidney func-tion. Metformin has pleiotropic actions, including inhibition of themitochondrial respiratory chain complex I (El-Mir et al., 2000) andactivation of the adenosine monophosphate-activated kinase (AMPK)(Zhou et al., 2001). In addition to its glycemia-lowering effects,metformin has been shown to reduce inflammation and fibrosis ina model of non-alcoholic steato-hepatitis (Kita et al., 2012). However,the effect of metformin on inflammation and fibrosis in the kidneyhas not been studied.

In this study, we studied the effect of treatment with metformin,initiated 1 day before surgery, on kidney structure, inflammationand fibrosis in mice with unilateral ureteral obstruction.

* Corresponding author. Department of Medicine/Nephrology, MC 7882, TheUniversity of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, SanAntonio, TX 78229, USA. Tel.: +1 210 567 6140; fax: +1 210 567 4712.

E-mail address: feliers@uthscsa.edu (D. Feliers).1 Present address: Laboratório de Investigação Médica LIM-29, Universidade de

Sao Paulo, Av. Dr. Arnaldo, 455 – 4° andar – sala 4304 –, Cerqueira César, São Paulo,SP, Brazil.

† This manuscript is dedicated to the memory of our dear friend and colleagueHanna E. Abboud who passed away shortly before the completion of the manuscript.

http://dx.doi.org/10.1016/j.mce.2015.06.0060303-7207/© 2015 Elsevier Ireland Ltd. All rights reserved.

Molecular and Cellular Endocrinology 412 (2015) 116–122

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Molecular and Cellular Endocrinology

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2. Materials and methods

2.1. Mouse model of unilateral ureteral obstruction (UUO)

Five- to 7-month-old adult male C57Bl/6 mice, weighting 25–35 g,were anesthetized with isoflurane/oxygen and UUO was per-formed using 4-0 silk in the midline abdominal incision and sham-operated mice had their ureters exposed and manipulated but notligated (Cachat et al., 2003). After 7 or 14 days of obstruction, animalswere sacrificed by cervical dislocation and kidneys were decapsu-lated and weighed. One piece of a kidney was removed for histologicand immunohistochemical studies and the remaining was frozenin liquid nitrogen and stored at −80 °C for protein and RNA extrac-tion. Three groups were used: sham (n = 9), sham-operated micereceiving vehicle (water) by gavage, UUO (n = 9), mice subjected tounilateral ureter obstruction model and received vehicle by gavage;and UUO + metformin (n = 10), UUO mice receiving metformin bygavage (200 mg/kg/day). All experiments were approved by the Uni-versity of Texas Health Science Center at San Antonio InstitutionalAnimal Care and Use Committee.

2.2. Renal histology and Sirius Red staining

Briefly, kidneys were fixed in 10% formaldehyde and embed-ded in paraffin, and 4-μm sections were cut. Staining with PeriodicAcid Schiff (PAS) and Masson’s Trichrome were performed by theCancer Therapy and Research Center at the University of Texas HealthScience Center San Antonio. Images were obtained with an OlympusAX70 microscope using the DP image acquisition software.

2.3. Immunohistochemistry

Immunohistochemistry was performed as described by Day et al.(2013).

2.3.1. Fibronectin stainingAntigen retrieval from paraffin-embedded sections (4 μm) was

performed in citrate buffer at 100 °C for 6 minutes, slides werequenched in 3% hydrogen peroxide for 6 minutes. After blocking withBackground Sniper for 20 minutes, slides were incubated with theprimary antibody (listed in Supplementary Fig. S1) overnight at 4 °Cin a humidified chamber. After rinsing, slides were incubated withgoat anti-rabbit polymer-horseradish peroxidase (Biocare, Concord,CA) for 20 minutes at room temperature. Immunoreactivity was vi-sualized with 3-3′-diaminobenzidine (DAB, Biocare, CA).

2.3.2. F4/80, α-SMA and E-cadherin stainingAntigen retrieval from paraffin-embedded sections (4 μm) was

performed in citrate buffer at 100 °C for 6 minutes. Primary anti-bodies (listed in Supplementary Fig. S1) were incubated overnightat 4 °C in a humidified chamber. After rinsing, slides were incu-bated with biotinylated secondary antibody for 20 minutes at roomtemperature. Streptavidin–peroxidase complex (LASB®+ System-AP, DAKO) was used for amplification and immunoreactivity wasvisualized with Fast (DAKO).

2.4. Morphometric analysis

Measure of tubular lumen area was performed on slides stainedwith E-cadherin. Images were imported in ImageJ64 and the lumencontour was traced using the free hand tool. Perimeter and surfacearea of the trace were determined by the software. Both perime-ter and surface area show similar results, and we chose to presentonly the lumen surface area data, expressed in percentage of lumenarea in sham-operated mice. We measured at least 15 tubules in3–4 different mice for each group.

2.5. Reverse transcription and quantitative real-time polymerasechain reaction (RT-qPCR)

RNA was extracted from kidney cortex (~50 μg of tissue) usingTrizol (Invitrogen), treated with nuclease I and used for reverse tran-scription (RT) using iScript RT SuperMix (BioRad). The resultingcDNAs were used for quantitative PCR using the RT2 qPCR MasterMix and primers listed in Supplementary Fig. S2. The qPCR was runin a MasterCycler RealPlex4 (Eppendorf). Quantitation of the mRNAswas performed using the 2−ΔΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping gene.

2.6. Immunoblot analysis

Slices of kidneys were homogenized in lysis buffer (Invitrogen,#FNN-0071) supplemented with protease inhibitor mix (Sigma,P-2714), 1mM PMSF, and 5 mM orthovanadate. Protein concentra-tion was measured and 10–20 μg of whole-cell lysates was separatedon SDS–PAGE, transferred to nitrocellulose membranes and probedovernight at 4 °C with the antibodies listed in Supplementary Fig. S1.IRDye800- or IRDye700-coupled secondary antibodies were usedfor detection using Odyssey Infrared Imaging System (LiCor Bio-sciences, Lincoln, NE).

2.7. AMPK activity

Briefly, AMPKα was immunoprecipitated from kidneyhomogenates using a specific antibody. Immunocomplexes were col-lected by centrifugation after incubation with protein A/G-agarosebeads. After washes in lysis buffer and washes in PBS, the beads wereresuspended in kinase buffer consisting of 50 mM HEPES (pH 7.4),10 mM MgCl2, 5% glycerol, 1 mM DTT, 1 mM orthovanadate and 0.05% Triton X100. Reaction was initiated by addition of 50 μM ATP, 1 μgSAMS peptide and 10 μCi γ[32P]-ATP and lasted 10 min. An aliquotof the reaction mixture was spotted on P81 paper disks which werethen washed three times in 0.5% phosphoric acid and once in acetone.Radioactivity incorporated in the SAMS peptide was counted in aBeckman 6000IC scintillation counter. Antibodies used are listed inSupplementary Fig. S1.

2.8. Statistics

Data were expressed as mean ± standard error of the mean (SEM)and analyzed by analysis of variance (ANOVA) for comparison amongmultiple groups using the Tukey post-test analysis for compari-son, using the GraphPad Prism 5.0 software. Values of p < 0.05 (aftermultiple comparison adjustment as needed) were consideredsignificant.

3. Results

3.1. Renal morphology

Unilateral ureter obstruction was performed in 5- to 7-month-old mice. Kidney morphology was studied after 1 week ofobstruction. Renal histology was studied after Periodic Acid Schiffstaining of paraffin-embedded kidney sections (Fig. 1A). Ob-structed kidneys displayed dilated tubules with areas of infiltratingcells (arrow), suggesting inflammation. Glomeruli were histologi-cally normal. Metformin treatment significantly reduced the numberof infiltrating cells in obstructed kidneys. E-cadherin is a trans-membrane protein involved in cell–cell contact and expressed inepithelial cells (Okada, 1988). In sham-operated kidneys, E-cadherinwas highly expressed in some tubules, and its expression was sig-nificantly lower in obstructed kidneys (Fig. 1B), indicating loss oftubular structure. Decrease of E-cadherin expression was attenuated

117R.C. Cavaglieri et al./Molecular and Cellular Endocrinology 412 (2015) 116–122

Fig. 1. Effect of metformin on renal histology and extracellular matrix expansion. (A) Renal histology was studied by Periodic Acid Schiff staining on section of paraffin-embedded kidneys. 200× magnification. (B) Extracellular matrix expansion was studied by Masson’s Trichrome staining. 100× magnification. N = 4 mice per group.

Fig. 2. Effect of metformin on inflammation in obstructed kidneys. (A) F4/80-positive cells were detected by immunohistochemistry on sections of paraffin-embedded kidneys.Positive cells stain brown. 200× magnification. N = 4 mice per group. (B) TNFα and VCAM1 expression was measured by immunoblot on homogenates of the left kidneyfrom sham-operated, UUO and UUO mice treated with metformin. The lower panels show the results of densitometric analysis of the immunoblots. TNFα mRNA (C) andVCAM1 mRNA (D) expression was measured by RT-qPCR and normalized using GAPDH as a housekeeping gene. N = 6-9 mice per group. *p < 0.05, **p < 0.01 and ***p < 0.001vs sham by ANOVA, ##p < 0.01 vs UUO by ANOVA.

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by treatment with metformin in obstructed kidneys (Fig. 1B). Next,we measured the tubular lumen area by morphometry. We foundthat the lumen area increased about 5-fold in mice with UUO.Metformin treatment tended to decrease the lumen surface, but thedifference did not reach statistical significance. These data show thatmetformin slows down infiltration of circulating cells and tubulardamage, with a minimal effect on tubular dilatation in obstructedkidneys.

3.2. Renal inflammation

The PAS staining detected inflammatory cells inside the paren-chyma of obstructed kidneys. To assess whether these cells containmacrophages, staining for F4/80 was performed. Fig. 2A shows thatfew F4/80 positive cells were present in kidneys from sham-operated mice. The number of F4/80 positive cells was significantlyincreased in obstructed kidneys, mostly in the tubulo-interstitialspace. Treatment with metformin prevented the increase of F4/80positive cells in obstructed kidneys. These data suggested thatmetformin slows down macrophage infiltration in obstructedkidneys.

TNFα plays an important role in renal inflammation in severalmodels of kidney injury, including UUO (Guo et al., 1999). TNFα ex-pression was increased at the protein (Fig. 2B) and the mRNA level(Fig. 2C) in kidneys from mice with UUO compared to sham-operated mice. Metformin treatment prevented increased TNFαprotein expression, which remained higher than that in sham-operated mice. TNFα mRNA levels were completely normalized bymetformin treatment (Fig. 2C). These data suggest that metformin

prevents the increase in TNFα gene transcription, but not a post-transcriptional regulation of TNFα protein in obstructed kidneys.Infiltration of inflammatory cells in the kidney requires coordi-nated expression of several pro-inflammatory molecules, such asVCAM1 which allows adhesion to the endothelium and infiltra-tion into the renal parenchyma. VCAM1 expression was increasedat the protein (Fig. 2B) and mRNA level (Fig. 2D) in obstructedkidneys. Metformin attenuated the increase in VCAM1 expressionin obstructed kidneys, but both mRNA and protein were still higherin kidneys from mice UUO-metformin that in sham-operated mice.

3.3. Renal fibrosis

Unilateral ureteral obstruction causes progressive accumula-tion of extracellular matrix in the kidney, leading to tubulo-interstitial fibrosis. Masson’s Trichrome stain allows detection ofextracellular matrix in tissues, as blue staining. Fig. 3A shows thatobstructed kidneys have significant tubulo-interstitial and glomeru-lar accumulation of extracellular matrix. Metformin treatmentreduced accumulation of extracellular matrix in obstructed kidneys(Fig. 3A).

Collagens and fibronectin are two major constituents of extra-cellular matrix in the kidney. Fibronectin deposition was assessedby immunohistochemistry: basal staining of fibronectin in the glom-eruli and the tubulo-interstitial compartment in kidneys from sham-operated mice was significantly increased in obstructed kidneys(Fig. 3B). Treatment with metformin reduced accumulation offibronectin deposition in obstructed kidneys. Fibronectin expres-sion was quantified by immunoblot (Fig. 3C) and RT-qPCR (Fig. 3D).

Fig. 3. Effect of metformin on collagen expression in obstructed kidneys. Collagen deposition was measured by Sirius red staining under direct light (A) or indirect light(B). 100× magnification. N = 4 mice per group. Expression of collagen Iα2 mRNA (C) and collagen IIIα1 mRNA (D) was measured by RT-qPCR and normalized to GAPDH mRNA.N = 6–9 mice per group. **p < 0.01 and ***p < 0.001 vs sham by ANOVA, #p < 0.01 vs UUO by ANOVA.

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Fibronectin protein and mRNA were upregulated in obstructedkidneys, and treatment with metformin reduce attenuated the in-creased fibronectin expression in obstructed kidneys.

Collagen deposition was assessed by Sirius Red staining usingdirect light, which allows detection of collagens I, III and IV (Fig. 4A),and polarized light, which allows detection of cross-linked colla-gens I and III (Fig. 4A). Collagen deposition was increased aroundthe glomeruli and in the tubulo-interstitial compartment of ob-structed kidneys. No increase staining inside the glomeruli wasobserved. Treatment with metformin attenuated deposition of col-lagens in obstructed kidneys. Expression of collagen Iα2 (Fig. 4B)and IIIα1 mRNA (Fig. 4C), measured by RT-qPCR, was increased inobstructed kidneys and normalized after treatment with metformin.

Transforming Growth Factor β (TGFβ) is an important regula-tor of fibrosis in several models of kidney injury (Kaneto et al., 1993;Sato et al., 2003), including the UUO. Its expression was quanti-fied by immunoblot (Fig. 5A) and RT-qPCR (Fig. 5B). TGFβ proteinand mRNA were upregulated in obstructed kidneys compared tosham-operated kidneys. Treatment with metformin prevented in-creased expression of TGFβ protein and mRNA.

Activation of interstitial fibroblasts plays a role in the develop-ment of tubulo-interstitial fibrosis in mice with UUO (Chevalier,1999; Grande and López-Novoa, 2009). Fibroblast activation is ac-companied by upregulation of α-smooth muscle actin (α-SMA)(Grande and López-Novoa, 2009). In kidneys from sham-operatedmice, α-SMA was localized mainly in the blood vessels, and aroundthe glomeruli some tubules (Fig. 5C). In obstructed kidneys, α-SMAwas significantly increased in the tubulo-interstitial compart-ment, and treatment with metformin prevented this increase. The

expression of α-SMA was quantified by immunoblot. Fig. 5D showsthat α-SMA was increased 3.7-fold in obstructed kidneys com-pared to sham-operated kidneys. Metformin reduced the increasein α-SMA expression in obstructed kidneys.

Together, these data show that metformin prevents the devel-opment of renal fibrosis in obstructed kidneys, most likely throughinhibition of TGFβ expression.

3.4. Adenosine monophosphate-activated kinase (AMPK)

Metformin is an indirect activator of AMPK (Viollet et al., 2012).We measured AMPKα activation by in vitro kinase assay using theSAMS peptide as a substrate (Fig. 6). Renal AMPKα activity was un-changed in obstructed kidneys, but treatment with metforminresulted in a significant increase in AMPKα activity.

4. Discussion

Our study shows for the first time that metformin treatment, ini-tiated 1 day before surgery, attenuates development of renalinflammation and fibrosis but not of renal structural changes in micewith unilateral ureteral obstruction. The latter is a consequence ofurine reflux into the kidney due to the obstruction, and is logical-ly not affected by metformin. The former complications are due tothe release of soluble factors by the injured kidney tissue. Our datasuggest that this is antagonized by metformin. This is the case forTNFα, which is involved in inflammation and TGFβ, involved infibrosis.

Fig. 4. Effect of metformin on fibronectin expression in obstructed kidneys. (A) Fibronectin was detected by immunohistochemistry in sections of paraffin-embedded kidneys.Positive staining is brown. 100× magnification. N = 4 mice per group. (B) Fibronectin (FN) protein expression was measured by immunoblot on homogenates of the left kidneyfrom sham-operated, UUO and UUO mice treated with metformin. The lower panels show the results of densitometric analysis of the immunoblots. (C) FN mRNA expres-sion was measured by RT-qPCR and normalized using GAPDH as a housekeeping gene. N = 6–9 mice per group. **p < 0.01 and ***p < 0.001 vs sham by ANOVA, #p < 0.05 vsUUO by ANOVA.

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Metformin has been shown to have anti-inflammatory and anti-fibrotic effects in other tissues. The anti-inflammatory effects ofmetformin have been demonstrated in vivo in the liver and lung.Metformin reduced expression of pro-inflammatory and pro-fibrotic genes in a mouse model of non-alcoholic hepatic steatosis

(Kita et al., 2012), and reduced inflammation in the airways of asth-matic mice (Park et al., 2012). The anti-inflammatory effect ofmetformin could be due to inhibition of the NF-κB pathway, as evi-denced in cultured cancer cells (Hirsch et al., 2013), vascular cells(Isoda et al., 2006), monocytes (Arai et al., 2010) and macrophages(Hyun et al., 2013). The anti-fibrotic effect of metformin has beencharacterized mostly in the cardiovascular system. Metforminreduced expression of pro-fibrotic genes in hearts of mice with pres-sure overload (Xiao et al., 2010), in hearts of obese rats (Burlá et al.,2013), and in hearts from spontaneously hypertensive rats (Cittadiniet al., 2012). The mechanism is likely through antagonism of TGFβsignaling, as demonstrated in cultured cardiac fibroblasts (Xiao et al.,2010) and canine kidney epithelial cells (Cufí et al., 2010). Our studyshows for the first time that metformin exerts similar anti-inflammatory and anti-fibrotic effects in the kidney from mice withUUO.

Our data show that amelioration of inflammation and fibrosisin kidneys from metformin-treated mice was accompanied by in-creased activity of AMPK. Metformin has been shown to activateAMPK in several organs (Viollet et al., 2012), and although there areinstances when the effects of metformin are independent of AMPK(Ben Sahra et al., 2011; Kalender et al., 2010), activation of AMPKis widely believed to mediate the effects of metformin (Viollet et al.,2012). It is therefore likely that metformin attenuates inflamma-tion and fibrosis in obstructed kidneys through activation of AMPK.In support of this hypothesis, it has been shown that pharmaco-logical activation of AMPK reduces fibrosis in rats with kidneyablation and infarction (Satriano et al., 2013). Our data show thatAMPK activity is unchanged in obstructed kidneys, suggesting that

Fig. 5. Effect of metformin on TGFβ and α-SMA expression in obstructed kidneys. (A) TGFβ protein expression was measured by immunoblot on left kidney homogenatesfrom the indicated groups of mice. The lower panels show the results of densitometric analysis of the immunoblots. (B) TGFβ mRNA expression was measured by RT-qPCRand normalized to GAPDH. N = 6–9 mice per group. (C) α-SMA was detected by immunohistochemistry on sections of paraffin-embedded kidneys. Positive staining is brown.100× magnification. N = 4 mice per group. (D) α-SMA protein expression was measured by immunoblot on left kidney homogenates. The lower panels show the results ofdensitometric analysis of the immunoblots. The lower panel shows the results of densitometric analysis of the immunoblots. N = 6–9 mice per group. **p < 0.01 and ***p < 0.001vs sham by ANOVA, #p < 0.05 vs UUO by ANOVA.

Fig. 6. Effect of metformin on AMPK activity in obstructed kidneys. AMPK activitywas measured by in vitro kinase assay using SAMS peptide as a substrate, after im-munoprecipitation of AMPKα using a specific antibody. N = 6 mice per group. **p < 0.01and ***p < 0.001 vs sham by ANOVA, #p < 0.05 vs UUO by ANOVA.

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inflammation and fibrosis in these kidneys are due to an inhibi-tion of AMPK activity, in contrast with diabetic nephropathy. Inkidneys from rats with early diabetes, AMPK activity is decreasedand pharmacological activation of AMPK reduces extracellular matrixprotein expression (Lee et al., 2007).

Acknowledgements

This study was supported by the JDRF (1-2010-141) to DF andthe NIDDK (DK-R01-078971) and a VA Merit Review to HEA. Peri-odic Acid Schiff and Sirius Red staining were performed by the CancerTherapy and Research Center at the University of Texas HealthScience Center San Antonio, through the NCI Cancer Center SupportGrant (2 P30 CA054174-17).

Appendix: Supplementary material

Supplementary data to this article can be found online atdoi:10.1016/j.mce.2015.06.006.

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