MOLECULAR MEDICINE REPORTS 13: 901-908, 2016 Lefty1 alleviates renal tubulointerstitial injury in mice with unilateral ureteral obstruction CHANGGENG XU1, MINGWEI XU2, WEI WANG3 and JIE ZHANG3,4 1Department of Urology, Wuhan Central Hospital, Wuhan, Hubei 430014; 2Department of Urology, Xiangyang Central Hospital, Xiangyang, Hubei 441021; 3Department of Urology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060; 4Department of Urology, Huangshi Central Hospital, Hubei Polytechnic University, Huangshi, Hubei 435000, P.R. China Received December 7, 2014; Accepted August 27, 2015 DOI: 10.3892/mmr.2015.4631 Abstract. Lefty is a member of the transforming growth Lefty1 may therefore by a putative therapeutic agent in the factor (TGF) β superfamily, which is implicated in left-right treatment of renal injury. patterning during embryogenesis. Previous studies revealed that lefty attenuates the epithelial-mesenchymal transition Introduction in tubular epithelial cells. In the present study, the protec- tive effect of lefty1 on renal interstitial injury was further Chronic renal diseases (CKDs), irrespective of the means of assessed. Mice with a unilateral ureteral obstruction (UUO) the initial insult, relentlessly progress to end-stage kidney were sacrificed on days 3, 5 and 7 following surgery, and the disease, with an irreversible loss of renal architecture and association between the expression of lefty1 and the degree of function (1). Renal tubulointerstitial injury is considered to be interstitial fibrosis was investigated. Subsequently, mice with a the hallmark of progressive kidney disease, and the extent of UUO were administered recombinant lefty1 (300 µg/kg body the injury is inversely correlated with renal function. Renal weight) or vehicle (0.9% saline solution; 100 µl) through tubulointerstitial injury is histopathologically characterized tail-vein injection every other day for 6 days. The effects of by an accumulation of myofibroblasts, the marked deposition lefty1 were assessed by measuring the degree of tubuloin- of extracellular matrix and tubular atrophy (2). Although the terstitial fibrosis, tubular injury and atrophy, and also by molecular mechanisms underlying interstitial injury have been monitoring the expression levels of α-smooth muscle actin partly elucidated (3), only a few therapies are currently used (α-SMA), TGF-β1, phosphorylated (p)-Smad2/3, kidney clinically. Therefore, specific treatments to target interstitial injury molecular-1 and endogenous lefty1. The expression of injury are urgently required. lefty1 in the kidney decreased in a time-dependent manner Among the signaling pathways associated with renal tubu- in mice with a UUO, which was inversely correlated with the lointerstitial injury, the Smad-mediated transforming growth degree of renal interstitial fibrosis. Furthermore, compared factor (TGF)-β1 signaling pathway occupies a central role in with vehicle treatment, lefty1 attenuated renal interstitial this process (4). In experimental models and human kidney fibrosis. Ureteral ligation induced increased expression levels diseases, TGF-β1 is markedly upregulated, and Smad2/3 is of α-SMA, TGF-β1 and p-Smad2/3. However, these effects highly activated in the fibrotic kidney (5). were reduced following treatment with lefty1. The UUO also The underlying mechanisms by which TGF-β1 mediate induced tubular injury and atrophy, whereas lefty1 treat- renal injury are as follows: i) TGF-β1 markedly induces the ment exerted a marked suppressive effect on tubular injury. proliferation and activation of renal fibroblasts during renal In addition, exogenous lefty1 administered to mice restored fibrosis (5,6); ii) TGF‑β1 elicits the production of the extracel- the endogenous expression levels of lefty1. The present study lular matrix (ECM) through Smad-dependent mechanisms (7); demonstrated that lefty1 attenuated renal interstitial injury by iii) TGF-β1 inhibits ECM degradation by suppressing matrix inhibiting the Smad-dependent TGF-β1 signaling pathway. metalloproteinases and inducing the tissue inhibitor of metalloproteinase (8); and iv) TGF-β1 is involved in tubular deletion (9,10). Lefty is a member of the TGF-β superfamily with two Correspondence to: Professor Jie Zhang, Department of Urology, variants, which are designated as leftyA and leftyB in Renmin Hospital of Wuhan University, 99 Zhangzhidong Road, humans (11), and lefty1 and lefty2 in mice (12). Initially, a Wuhan, Hubei 430060, P.R. China study by Meno et al (13) revealed that lefty was involved in E-mail: [email protected] the formation of embryonic lateral patterning. Ulloa et al (14) demonstrated that leftyA inhibited the phosphorylation of Key words: renal interstitial fibrosis, lefty1, TGF-β1 Smad2/3, inhibited the heterodimerization of R-Smads with Smad4, and also inhibited the nuclear translocation of the Smad complex (14). In addition, it was demonstrated that 902 XU et al: LEFTY1 ALLEVIATES RENAL TUBULOINTERSTITIAL INJURY leftyA markedly reduced the quantity of collagen deposition, anti-human collagen I (cat. no. ab34710; 1:100), rabbit poly- and promoted collagenolysis (15). It was also demonstrated clonal anti-human fibronectin (cat. no. ab2413; 1:400) and that leftyA or lefty1 attenuated the epithelial-mesenchymal rabbit polyclonal anti-human anti-kidney injury molecular-1 transition (EMT) by inhibiting TGF-β1 signaling in the mouse (Kim-1; cat. no. ab47635; 1:100) antibodies were purchased HK2 tubular epithelial (16), or the rat NRK52E tubular epithe- from Abcam (Cambridge, UK). Rabbit polyclonal anti-human lial (17) cell lines, respectively. Due to the active involvement α-smooth muscle actin (SMA) antibody (cat. no. BM0002; of the TGF-β1 signaling pathway in renal injury, it was 1:50) was purchased from Boshide Bioengineering Co., Ltd. hypothesized that lefty1 may alleviate renal interstitial injury. (Wuhan, China). The primary antibodies were incubated Using a mouse model of unilateral ureteral obstruction for 1 h at room temperature, or at 4˚C overnight. Goat (UUO), a well-established experimental model of renal injury polyclonal anti-rabbit IgG (cat. no. BA1055; 1:200; Boshide characterized by progressive tubulointerstitial fibrosis and Bioengineering Co., Ltd.) was used as the secondary antibody. tubular atrophy, the present study aimed to assess the ability The antigen-specific, positive cells were visualized using of lefty1 to protect against renal injury, and to determine the streptavidin peroxidase reagents purchased from Boshide possible underlying mechanisms. Bioengineering Co., Ltd. Brown staining taken up by the cells was indicative of a positive result. Materials and methods Semi‑quantitative analysis for the histological studies. A total Experimental design. Male C57BL/6 mice (weighing, ~20 g) of 20 random, non‑overlapping fields (magnification, x400) were obtained from the Experimental Animal Center of from each animal was selected for assessing PSR staining. Wuhan University (Wuhan, China). The mice were granted These were quantified by assessing the size of the positive free access to water and chow, and were kept under a 12-h area as a percentage of the total area. A total of 10 random, light/dark cycle, at 24˚C and 50% humidity. The UUO was non‑overlapping fields (magnification, x400) of each animal performed using a previously described method (18). Briefly, were assessed for evaluating tubular Kim-1 staining. under sodium pentobarbital anesthesia (60 mg/kg body These were quantified by counting the positive tubules as a weight; Merck, Darmstadt, Germany), a complete ureteral percentage of the total. A total of 10 random, non-overlapping obstruction was performed by ligating the left ureter with fields (magnification, x200) of each animal were selected for 4-0 silk (Tianhou Medical Co., Jinan, China). Sham-operated evaluating the tubular atrophy. Tubular atrophy was scored on mice had their ureters exposed and manipulated, although they a scale between 0 and 3, as previously reported (19), although were not ligated. To study the correlation between the expres- with certain modifications: 0, absent (no atrophy); 1, mild sion of lefty1 and the degree of interstitial fibrosis, 20 mice (percentage of atrophied tubules, 1-15%); 2, moderate were randomly assigned to four groups: i) Sham-operation; (percentage of atrophied tubules, 16-30%); 3, severe ii) UUO for 3 days; iii) UUO for 5 days; iv) UO for 7 days (percentage of atrophied tubules, >30%). (n=5/group). The mice were subsequently sacrificedon days 3, 5 and 7 under anesthesia and were fixed by perfusion through Western blot analysis. The kidney tissues were homogenized in the left ventricle. To assess the effects of lefty1 on renal tubu- a lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, lointerstitial injury, 18 mice were divided into three groups: 1% Triton X 100, sodium pyrophosphate, β-glycerophosphate, i) Sham-operation; ii) UUO plus vehicle treatment; iii) UUO EDTA, Na3VO4 and leupeptin (Biyuntian, Wuhan, China) plus lefty1 treatment. On the day following surgery, the mice on ice. The lysates were centrifuged at 12,000 x g at 4˚C for were administered recombinant mouse lefty1 (R&D Systems, 20 min and the protein concentration was determined using Inc., Minneapolis, MN, USA) through a tail-vein injection at a bicinchoninic acid (BCA) protein assay kit. A total of a dose of 300 µg/kg body weight, and subsequently on every 40 µg protein was separated by 12 or 8% SDS-PAGE (Boshide, other day for up to 6 days. The control mice received