Anti-Fibrotic Effect of Losartan, an Angiotensin II Receptor Blocker, Is Mediated through Inhibition of ER Stress via Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin

Endoplasmic reticulum (ER) stress is increasingly identified as modulator of fibrosis. Losartan, an angiotensin II receptor blocker, has been widely used as the first choice of treatment in chronic renal diseases. We postulated that anti-fibrotic effect of losartan is mediated through inhibition of ER stress via SIRT1 (silent mating type information regulation 2 homolog 1) hemeoxygenase-1 (HO-1)/thioredoxin pathway. Renal tubular cells, tunicamycin (TM)-induced ER stress, and unilateral ureteral obstruction (UUO) mouse model were used. Expression of ER stress was assessed by Western blot analysis and immunohistochemical stain. ER stress was induced by chemical ER stress inducer, tunicamycin, and non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin. Losartan suppressed the TM-induced ER stress, as shown by inhibition of TM-induced expression of GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α), through up-regulation of SIRT1 via HO-1 and thioredoxin. Losartan also suppressed the ER stress by non-chemical inducers. In both animal models, losartan reduced the tubular expression of GRP78, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis. These findings provide new insights into renoprotective effects of losartan and suggest that SIRT1, HO-1, and thioredoxin may be potential pharmacological targets in kidney diseases under excessive ER stress condition.


Introduction
Renal fibrosis, particularly tubulointerstitial fibrosis, is considered to be a central event in the progression of chronic kidney diseases, regardless of the original cause of the kidney disease. Therefore, renal fibrosis is a reliable predictor and a major determinant of renal insufficiency. However, the underlying mechanism has not been completely understood. The endoplasmic reticulum (ER) stress refers to physiological or pathological states that result in accumulation of misfolded proteins in the ER, which leads to cell stress conditions (ER stress). To mitigate protein misfolding stress, cells activate a homeostatic intracellular signaling network cumulatively called the unfolded protein response (UPR). UPR reestablishes the homeostasis of ER through the activation of three major sensors known as PERK (PKR-like ER kinase), IRE-1 (inositol requiring enzyme-1), and ATF-6 (activating transcription factor 6). The ER chaperone GRP78 (glucose related protein 78) is bound to these three transmembrane

Losartan Suppressed the ER Stress Induced by Tunicamycinin in Tubular Epithelial Cells
To determine whether losartan suppressed the tunicamycin-induced ER stress in tubular HK-2 cells, we examined the change of two ER stress biomarkers, the up-regulation of GRP78 and p-eIF2α. Losartan (10 µM) suppressed the tunicamycin-induced ER stress, as shown by inhibition of tunicamycin-induced up-regulation of GRP78 and p-eIF2α (p < 0.05). Losartan itself had no effects on the expression of GRP78 and p-eIF2α ( Figure 1). To determine whether losartan suppressed the tunicamycin-induced ER stress in tubular HK-2 cells, we examined the change of two ER stress biomarkers, the up-regulation of GRP78 and p-eIF2α. Losartan (10 µM) suppressed the tunicamycin-induced ER stress, as shown by inhibition of tunicamycin-induced up-regulation of GRP78 and p-eIF2α (p < 0.05). Losartan itself had no effects on the expression of GRP78 and p-eIF2α ( Figure 1).

Figure 1.
Inhibition of tunicamycin-induced endoplasmic reticulum (ER) stress by losartan. Proximal tubular cells (HK-2 cells) were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) for 24 h. Expression of GRP78 (A) and p-eIF2α (B) was examined by Western blot analysis. The relative densities of the bands for GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α) were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control), ##: p < 0.05 vs. TM.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Up-Regulation of SIRT1
Losartan induced the expression of SIRT1 in a dose dependent manner (1-10 µM) (Figure2A). To determine whether up-regulation of SIRT1 by losartan was directly involved in losartan's inhibitory effect on the tunicamycin-induced ER stress, we examined the effect of SIRT1 inhibitor (sirtinol, 10 µM). Western blot analysis revealed that sirtinol blocked the inhibitory effect of losartan on the tunicamycin-induced ER stress (p < 0.05), suggesting that the inhibitory effect of losartan on the ER stress was mediated through up-regulation of SIRT1 ( Figure 2B). Proximal tubular cells (HK-2 cells) were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) for 24 h. Expression of GRP78 (A) and p-eIF2α (B) was examined by Western blot analysis. The relative densities of the bands for GRP78 (glucose related protein 78) and p-eIF2α (phosphospecific-eukaryotic translation initiation factor-2α) were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control), ##: p < 0.05 vs. TM.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Up-Regulation of SIRT1
Losartan induced the expression of SIRT1 in a dose dependent manner (1-10 µM) (Figure 2A). To determine whether up-regulation of SIRT1 by losartan was directly involved in losartan's inhibitory effect on the tunicamycin-induced ER stress, we examined the effect of SIRT1 inhibitor (sirtinol, 10 µM). Western blot analysis revealed that sirtinol blocked the inhibitory effect of losartan on the tunicamycin-induced ER stress (p < 0.05), suggesting that the inhibitory effect of losartan on the ER stress was mediated through up-regulation of SIRT1 ( Figure 2B).

Figure 2.
Induction of SIRT1 (silent mating type information regulation 2 homolog 1) by losartan (A) and reversal of inhibitory effect of losartan on the tunicamycin-induced ER stress by sirtinol (B). Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 1-10 µM) for 24 h. Expression of SIRT1 was examined by Western blot analysis (A); Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) and sirtinol (SIRT1 inhibitor, 10 µM) for 24 h. Expression of GRP78 and p-eIF2α was examined by Western blot analysis (B). The relative densities of the bands for SIRT1, GRP78, and p-eIF2α were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losar.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Induction of Heme Oxygenase-1 and Thioredoxin
Losartan (10 µM) increased the expression of heme oxygenase (HO-1) and thioredoxin ( Figure  3A). To determine whether up-regulation of HO-1 and thioredoxin by losartan was directly involved in losartan's inhibitory effect on the tunicamycin-induced ER stress, we examined the effect of HO-1 inhibitor (Zinc protoporphyrin IX, Zn(II)PPIX, 10 µM) and thioredoxin inhibitor (PX12, 20 µM). Western blot analysis revealed that both Zn(II)PPIX and PX12 reversed losartan's inhibitory effect on the tunicamycin-induced ER stress (p < 0.05), suggesting that the inhibitory effect of losartan on the ER stress was mediated through induction of HO-1 and thioredoxin ( Figure 3B).

Figure 2.
Induction of SIRT1 (silent mating type information regulation 2 homolog 1) by losartan (A) and reversal of inhibitory effect of losartan on the tunicamycin-induced ER stress by sirtinol (B). Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 1-10 µM) for 24 h. Expression of SIRT1 was examined by Western blot analysis (A); Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) and sirtinol (SIRT1 inhibitor, 10 µM) for 24 h. Expression of GRP78 and p-eIF2α was examined by Western blot analysis (B). The relative densities of the bands for SIRT1, GRP78, and p-eIF2α were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losar.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Induction of Heme Oxygenase-1 and Thioredoxin
Losartan (10 µM) increased the expression of heme oxygenase (HO-1) and thioredoxin ( Figure 3A). To determine whether up-regulation of HO-1 and thioredoxin by losartan was directly involved in losartan's inhibitory effect on the tunicamycin-induced ER stress, we examined the effect of HO-1 inhibitor (Zinc protoporphyrin IX, Zn(II)PPIX, 10 µM) and thioredoxin inhibitor (PX12, 20 µM). Western blot analysis revealed that both Zn(II)PPIX and PX12 reversed losartan's inhibitory effect on the tunicamycin-induced ER stress (p < 0.05), suggesting that the inhibitory effect of losartan on the ER stress was mediated through induction of HO-1 and thioredoxin ( Figure 3B).  . The relative densities of the bands for HO-1, thioredoxin, GRP78, and p-eIF2α were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as nfold increase over control as mean ± S.E. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losar.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin
To determine whether SIRT1 could induce the expression of HO-1 and thioredoxin, we examined the effect of SRT1720 (SIRT1 inducer, 2.5 µM) with or without sirtinol (SIRT1inhibitor, 10 µM). Western blot analysis showed that SRT1720 increased the expression of HO-1 and thioredoxin, which were prevented by sirtinol (p < 0.05) ( Figure 4A).
To confirm whether induction of HO-1 and thioredoxin by losartan was mediated through upregulation of SIRT1, we examined the effect of sirtinol (SIRT1 inhibitor, 10 µM). Western blot analysis revealed that sirtinol blocked the losartan-induced expression of HO-1 and thioredoxin (p < 0.05) (Figure4B). These data indicated that the inhibitory effect of losartan on the tunicamycin-induced ER stress was mediated through up-regulation of SIRT1, followed by induction of HO-1 and thioredoxin. Expression of GRP78 and p-eIF2α was examined by Western blot analysis (B). The relative densities of the bands for HO-1, thioredoxin, GRP78, and p-eIF2α were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losar.

Inhibitory Effect of Losartan on the Tunicamycin-Induced ER Stress Was Mediated through Up-Regulation of SIRT1, Followed by Induction of HO-1 and Thioredoxin
To determine whether SIRT1 could induce the expression of HO-1 and thioredoxin, we examined the effect of SRT1720 (SIRT1 inducer, 2.5 µM) with or without sirtinol (SIRT1inhibitor, 10 µM). Western blot analysis showed that SRT1720 increased the expression of HO-1 and thioredoxin, which were prevented by sirtinol (p < 0.05) ( Figure 4A).
To confirm whether induction of HO-1 and thioredoxin by losartan was mediated through up-regulation of SIRT1, we examined the effect of sirtinol (SIRT1 inhibitor, 10 µM). Western blot analysis revealed that sirtinol blocked the losartan-induced expression of HO-1 and thioredoxin (p < 0.05) ( Figure 4B). These data indicated that the inhibitory effect of losartan on the tunicamycin-induced ER stress was mediated through up-regulation of SIRT1, followed by induction of HO-1 and thioredoxin. . Expression of heme oxygenase-1 (HO-1) and thioredoxin was examined by Western blot analysis. The relative densities of the bands for HO-1 and thioredoxin were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control) or TM; ##: p < 0.05 vs. TM + SRT or TM + losar.

Losartan also Inhibited the Tunicamycin-Induced Epithelial-Mesenchymal Transition (EMT)
To determine whether losartan inhibited the tunicamycin-induced EMT, we examined the change of two EMT biomarkers, the up-regulation of α-smooth muscle actin and the down-regulation of E-cadherin. Losartan (10 µM) suppressed the tunicamycin-induced EMT, as evidenced by the inhibition of tunicamycin-induced up-regulation of α-smooth muscle actin and the down-regulation of E-cadherin (p < 0.05). Losartan itself had no effects on the expression of α-smooth muscle actin and Ecadherin ( Figure 5). with or without SRT1720 (SRT, SIRT1 inducer, 2.5 µM) and sirtinol (SIRT1 inhibitor, 10 µM) for 24 h (A); Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) and sirtinol (SIRT1 inhibitor, 10 µM) for 24 h (B). Expression of heme oxygenase-1 (HO-1) and thioredoxin was examined by Western blot analysis. The relative densities of the bands for HO-1 and thioredoxin were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control) or TM; ##: p < 0.05 vs. TM + SRT or TM + losar.

Losartan also Inhibited the Tunicamycin-Induced Epithelial-Mesenchymal Transition (EMT)
To determine whether losartan inhibited the tunicamycin-induced EMT, we examined the change of two EMT biomarkers, the up-regulation of α-smooth muscle actin and the down-regulation of E-cadherin. Losartan (10 µM) suppressed the tunicamycin-induced EMT, as evidenced by the inhibition of tunicamycin-induced up-regulation of α-smooth muscle actin and the down-regulation of E-cadherin (p < 0.05). Losartan itself had no effects on the expression of α-smooth muscle actin and E-cadherin ( Figure 5).

Figure 5.
Inhibition of tunicamycin-induced epithelial mesenchymal transition by losartan. Proximal tubular cells were incubated with tunicamycin (TM, 0.2 µM) with or without losartan (losar, 10 µM) for 24 h. Expression of α-SMA (α-smooth muscle actin) and E-cadherin was examined by Western blot analysis. The relative densities of the bands for α-SMA and E-cadherin were normalized to those for actin for standardization. Representative blots and quantitative analysis from three independent experiments were shown. Results were expressed as n-fold increase over control as mean ± S.E. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM.

Treatment with Losartan Reduced the Renal Tubular Expression of GRP78 and Increased the Expression of HO-1 and Thioredoxin through the Up-Regulation of SIRT1 in a Mouse Model of Tunicamycin-Induced ER Stress
To demonstrate in vivo inhibitory the effect of losartan on the tunicamycin-induced ER stress, we performed an animal study using a mouse model of tunicamycin-induced ER stress. Consistent with the results of in vitro culture study, treatment with losartan reduced the renal tubular expression of GRP78 and increased HO-1, thioredoxin, and SIRT1, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor, 10 µM) in a mouse model of tunicamycin-induced ER stress (p < 0.05) ( Figure  6A). Western blot of renal cortical tissue showed the same results ( Figure 6B). To demonstrate in vivo inhibitory the effect of losartan on the tunicamycin-induced ER stress, we performed an animal study using a mouse model of tunicamycin-induced ER stress. Consistent with the results of in vitro culture study, treatment with losartan reduced the renal tubular expression of GRP78 and increased HO-1, thioredoxin, and SIRT1, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor, 10 µM) in a mouse model of tunicamycin-induced ER stress (p < 0.05) ( Figure 6A). Western blot of renal cortical tissue showed the same results ( Figure 6B). Tunicamycin-induced ER stress mouse model was induced by a single intraperitoneal injection of tunicamycin (TM, 2 mg/kg). Mice were randomly divided into four groups: control mice (n = 4), mice with TM injection (TM mouse, n = 4), TM mice with losartan (n = 4), and TM mice with losartan plus sirtinol (SIRT1 inhibitor, 5 mg/kg intraperitoneally). (n = 4). Losartan was administered by gavage at 10 mg/kg/day. Immunohistochemical staining (A) and Western blot of renal cortical tissue (B) for GRP78, heme oxygenase-1 (HO-1), thioredoxin, and SIRT1 were performed. Representative microscopic scans and quantitative analysis were shown. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losartan (losar); scale bars = 20 µm. Tunicamycin-induced ER stress mouse model was induced by a single intraperitoneal injection of tunicamycin (TM, 2 mg/kg). Mice were randomly divided into four groups: control mice (n = 4), mice with TM injection (TM mouse, n = 4), TM mice with losartan (n = 4), and TM mice with losartan plus sirtinol (SIRT1 inhibitor, 5 mg/kg intraperitoneally). (n = 4). Losartan was administered by gavage at 10 mg/kg/day. Immunohistochemical staining (A) and Western blot of renal cortical tissue (B) for GRP78, heme oxygenase-1 (HO-1), thioredoxin, and SIRT1 were performed. Representative microscopic scans and quantitative analysis were shown. #: p < 0.05 vs. con (control); ##: p < 0.05 vs. TM; ###: p < 0.05 vs. TM + losartan (losar); scale bars = 20 µm.

Losartan also Suppressed the ER Stress Induced by Non-Chemical ER Stress Inducers Such as Tumor
Growth Factor-β (TGF-β), Angiotensin II, High Glucose, and Albumin through Induction of SIRT1 To determine whether losartan suppressed the TGF-β-, angiotensin II-, high glucose-, and albumin-induced ER stress, we examined the change of two ER stress biomarkers, the up-regulation of GRP78 and p-eIF2α. Losartan (10 µM) suppressed the TGF-β-, angiotensin II-, high glucose-, and albumin-induced ER stress, as shown by inhibition of up-regulation of GRP78 and p-eIF2α expression, which were abolished by pretreatment with sirtinol (SIRT1 inhibitor, 10 µM) (p < 0.05) (Figure 7).

Treatment with Losartan Reduced the Renal Tubular Expression of GRP78 and Renal Fibrosis through the Up-Regulation of SIRT1 in a Mouse Model of Unilateral Ureteral Obstruction (UUO)
To demonstrate the in vivo inhibitory effect of losartan on the UUO-induced ER stress and renal fibrosis, we performed an animal study using a mouse model of UUO. Treatment with losartan reduced the renal tubular expression of GRP78 and increased the expression of HO-1, thioredoxin, and SIRT1, which were abolished by sirtinol in a mouse model of UUO (p < 0.05). Furthermore, sirtinol blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis (p < 0.05) ( Figure 8A). Western blot of renal cortical tissue and collagen deposit score showed the same results ( Figure 8B). 0.05 vs. con (control); ##: p < 0.05 vs. TGF-β; AgII, glu or alb; ###: p < 0.05 vs. TGF-β + losar, AgII + losar, glu + losar, or alb + losar.

Treatment with Losartan Reduced the Renal Tubular Expression of GRP78 and Renal Fibrosis through the Up-Regulation of SIRT1 in a Mouse Model of Unilateral Ureteral Obstruction (UUO)
To demonstrate the in vivo inhibitory effect of losartan on the UUO-induced ER stress and renal fibrosis, we performed an animal study using a mouse model of UUO. Treatment with losartan reduced the renal tubular expression of GRP78 and increased the expression of HO-1, thioredoxin, and SIRT1, which were abolished by sirtinol in a mouse model of UUO (p < 0.05). Furthermore, sirtinol blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis (p < 0.05) ( Figure 8A). Western blot of renal cortical tissue and collagen deposit score showed the same results ( Figure 8B).

Discussion
The present study demonstrated that losartan suppressed the tunicamycin (TM)-induced ER stress through up-regulation of SIRT1, followed by induction of heme oxygenase-1 and thioredoxin in tubular epithelial cells. Losartan also reduced the ER stress induced by non-chemical agents such as TGF-β, angiotensin II, high glucose, and albumin. In addition, losartan inhibited the TM-induced epithelial mesenchymal transition. Consistent with the results of the cell culture study, losartan reduced the renal tubular expression of GRP78 and increased the expression of SIRT1, HO-1, and thioredoxin in a mouse model of tunicamycin-induced ER stress and unilateral ureteral obstruction (UUO), which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis, suggesting that the anti-fibrotic effect of losartan might be mediated at least in part through inhibition of ER stress via up-regulation of SIRT1, followed by induction of HO-1 and thioredoxin (Figure 9).

Discussion
The present study demonstrated that losartan suppressed the tunicamycin (TM)-induced ER stress through up-regulation of SIRT1, followed by induction of heme oxygenase-1 and thioredoxin in tubular epithelial cells. Losartan also reduced the ER stress induced by non-chemical agents such as TGF-β, angiotensin II, high glucose, and albumin. In addition, losartan inhibited the TM-induced epithelial mesenchymal transition. Consistent with the results of the cell culture study, losartan reduced the renal tubular expression of GRP78 and increased the expression of SIRT1, HO-1, and thioredoxin in a mouse model of tunicamycin-induced ER stress and unilateral ureteral obstruction (UUO), which were abolished by pretreatment with sirtinol (SIRT1 inhibitor). Sirtinol also blocked the inhibitory effect of losartan on the UUO-induced renal fibrosis, suggesting that the anti-fibrotic effect of losartan might be mediated at least in part through inhibition of ER stress via up-regulation of SIRT1, followed by induction of HO-1 and thioredoxin (Figure 9). Tubulointerstitial fibrosis plays a most important role in the progression of chronic renal disease to end-stage renal failure. Losartan is an angiotensin II receptor blocker (ARB) that is widely used to delay the progression of chronic renal disease. However, the mechanism underlying the renoprotective effects of losartan has not been revealed completely.
Although a role of ER stress and UPR activation in kidney disease is increasingly convincing, the potential effect of losartan on the ER stress in renal tubular epithelial cells has not been analyzed in detail.
In this study, we found that losartan suppressed the ER stress induced by chemical ER stress inducers (tunicamycin) as well as non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin in renal tubular epithelial cells. Consistent with the results of the cell culture study, animal studies showed that losartan reduced the tubular expression of ER stress protein (GRP78) in a mouse model of tunicamycin-induced ER stress and unilateral ureteral obstruction (UUO). In agreement with our study, it has been reported that losartan is able to reduce the glucose-induced ER stress in pancreatic β cells [3]. Similarly, animal studies also showed that treatment with other ARBs such as candesartan and olmesartan could prevent the progression of dilated cardiomyopathy in rats through the inhibition of ER stress and reduce the ER stress-induced apoptosis in streptozotocin-induced diabetic animal model, respectively [25,26]. Together with these reports, our data suggested that inhibition of ER stress could be one of the possible mechanisms underlying the renoprotective effect of ARBs. SIRT1, a NAD-dependent protein deacetylase, contributes to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance [9]. In kidney, SIRT1 is reported to exhibit renoprotection through the reduction of fibrosis, anti-apoptotic effect, anti-inflammatory effect, and induction of autophagy [10]. In addition, SIRT1 is known to suppress the ER stress in renal tubular cells [11]. However, the effect of losartan on the expression of SIRT1 in proximal tubular cells has not been explored so far.
We found that losartan could induce the expression of SIRT1 in a dose dependent manner in renal tubular cells. Animal studies also showed that treatment with losartan induced the tubular expression of SIRT1. In support of our results, it has been reported that treatment with other ARBs, such as telmisartan and valsartan, increased the expression of SIRT1 in skeletal muscle cells of obese db/db mice and in myocardial cells of type-2 diabetes mellitus mice, respectively [27,28]. In addition, Figure 9. Proposed signaling pathways involved in anti-fibrotic effect of losartan. ↑ represents up-regulation; ↓ represents down-regulation.
Tubulointerstitial fibrosis plays a most important role in the progression of chronic renal disease to end-stage renal failure. Losartan is an angiotensin II receptor blocker (ARB) that is widely used to delay the progression of chronic renal disease. However, the mechanism underlying the renoprotective effects of losartan has not been revealed completely.
Although a role of ER stress and UPR activation in kidney disease is increasingly convincing, the potential effect of losartan on the ER stress in renal tubular epithelial cells has not been analyzed in detail.
In this study, we found that losartan suppressed the ER stress induced by chemical ER stress inducers (tunicamycin) as well as non-chemical inducers such as TGF-β, angiotensin II, high glucose, and albumin in renal tubular epithelial cells. Consistent with the results of the cell culture study, animal studies showed that losartan reduced the tubular expression of ER stress protein (GRP78) in a mouse model of tunicamycin-induced ER stress and unilateral ureteral obstruction (UUO). In agreement with our study, it has been reported that losartan is able to reduce the glucose-induced ER stress in pancreatic β cells [3]. Similarly, animal studies also showed that treatment with other ARBs such as candesartan and olmesartan could prevent the progression of dilated cardiomyopathy in rats through the inhibition of ER stress and reduce the ER stress-induced apoptosis in streptozotocin-induced diabetic animal model, respectively [25,26]. Together with these reports, our data suggested that inhibition of ER stress could be one of the possible mechanisms underlying the renoprotective effect of ARBs. SIRT1, a NAD-dependent protein deacetylase, contributes to regulate intracellular metabolism and attenuate reactive oxidative species (ROS)-induced apoptosis leading to longevity and acute stress resistance [9]. In kidney, SIRT1 is reported to exhibit renoprotection through the reduction of fibrosis, anti-apoptotic effect, anti-inflammatory effect, and induction of autophagy [10]. In addition, SIRT1 is known to suppress the ER stress in renal tubular cells [11]. However, the effect of losartan on the expression of SIRT1 in proximal tubular cells has not been explored so far.
We found that losartan could induce the expression of SIRT1 in a dose dependent manner in renal tubular cells. Animal studies also showed that treatment with losartan induced the tubular expression of SIRT1. In support of our results, it has been reported that treatment with other ARBs, such as telmisartan and valsartan, increased the expression of SIRT1 in skeletal muscle cells of obese db/db mice and in myocardial cells of type-2 diabetes mellitus mice, respectively [27,28]. In addition, we also found that the inhibitory effect of losartan on the ER stress was mediated through the up-regulation of SIRT1. Heme oxygenase-1 (HO-1) is the key rate-limiting enzyme in heme degradation, which produces biliverdin, carbon monoxide (CO), and iron. Clearance of excess free heme by HO-1 is critical in preventing heme-induced production of reactive oxygen species (ROS). Furthermore, CO and biliverdin is known to have anti-oxidant, anti-apoptotic, and anti-inflammatory properties, resulting in cytoprotection. CO also attenuates the ER stress-induced activation of IRE1, ATF6, and CREBH [10].
Thioredoxin is an important endogenous antioxidant and is ubiquitously expressed [13]. Thioredoxin is also known as an endogenous inhibitor of ASK1, which mediates apoptosis on ER stress [29]. Zeng et al. demonstrated that thioredoxin could suppress the 1-methyl-4-phenylpyridinium ion-induced ER stress by inhibiting the activation of IRE1α, TRAF2, JNK, caspase-12, and CHOP [30].
Therefore, we postulated that the inhibitory effect of losartan on the ER stress might be mediated through the induction of HO-1 and thioredoxin. We found that losartan increased the expression of HO-1 and thioredoxin, and also that the inhibitor of HO-1 and thioredoxin blocked the inhibitory effect of losartan on the tunicamycin-induced ER stress in tubular epithelial cells, thereby suggesting that the inhibitory effect of losartan on the ER stress was mediated through the induction of HO-1 and thioredoxin. In line with our results, it has been reported that the inhibition of 6-hydroxydopamine-mediated ER stress-induced apoptosis by insulin-like growth factor-1 was mediated through the regulation of HO-1 in PC12 cells [15]. Similarly, it has been suggested that the inhibition of tunicamycin-or thapsigargin-induced ER stress by AMP-activated protein kinase was mediated via the induction of HO-1 and thioredoxin [14].
It is well known that SIRT1 is able to induce the expression of HO-1 by the activation of Nrf2 as well as thioredoxin through FoxO3a and PGC-1α [31]. Therefore, we postulated that the inhibitory effect of losartan on ER stress might be mediated through the up-regulation of SIRT1, followed by the induction of HO-1 and thioredoxin. We found that treatment with SRT1720 (SIRT1 inducer) induced the expression of HO-1 and thioredoxin, which were prevented by sirtinol (SIRT1 inhibitor). Sirtinol also blocked the losartan-induced expression of HO-1 and thioredoxin, suggesting that the inhibitory effect of losartan on the ER stress was mediated through the SIRT1-HO-1/thioredoxin pathway.
Epithelial mesenchymal transition (EMT) is a process in which primary epithelial cells lead to morphological changes to fibroblastoid morphology, the up-regulation of mesenchymal markers including α-smooth muscle actin, vimentin, and fibroblast-specific protein-1 and the down-regulation of epithelial markers such as E-cadherin, ZO-1, and cytokeratin [32]. It has been suggested that renal tubular epithelial cells are able to become fibroblasts through EMT and participate in the pathogenesis of tubulointerstitial fibrosis [33].
ER stress is also a known cause of EMT and we previously reported that ER stress was able to induce EMT in renal tubular cells [34]. In this study, we found that losartan could suppress the tunicamycin-induced EMT as well. Consistent with our results, it has been reported that losartan suppresses the high-glucose-induced EMT in renal proximal tubular cells [35].
It is well proven that losartan ameliorates renal fibrosis in the animal model of 5/6 nephrectomy, unilateral ureteral obstruction, diabetic nephropathy, ischemia-reperfusion injury, and cyclosporine-induced fibrosis [36][37][38][39][40]. In this study, we found that sirtinol (SIRT1 inhibitor) could block the inhibitory effect of losartan on the UUO-induced renal fibrosis, suggesting that the anti-fibrotic function of losartan might be mediated at least in part through the inhibition of ER stress via the up-regulation of SIRT1.
ER stress is known to play important roles in renal pathology during nephrotoxicity, ischemia-reperfusion injury, viral infections, glomerulonephritis, podocytopathies, albuminuria, and kidney ageing [41]. Therefore, our data suggest that inducing SIRT1, HO-1, and thioredoxin may have clinical therapeutic potential in kidney diseases under excessive ER stress conditions.

Cell Culture and Conditioning
All experiments were performed using HK-2 cells, a human proximal tubular cell line [42]. HK-2 cells were obtained from the American Type Culture Collection. The media were changed every three days until confluent. Cells were growth-arrested in serum-free medium for 24 h before being used in experiments. To determine whether losartan suppressed the tunicamycin (TM)-induced ER stress, cells were incubated with TM (0.2 µM) with or without losartan (10 µM) for 24 h. To determine whether losartan suppressed the TGF-β-, angiotensin II-, high glucose-, and albumin-induced ER stress, cells were incubated with TGF-β (10 ng/mL), angiotensin II (1 µM), high glucose (30 mM), and albumin (5 mg/mL) for three days and then treated with losartan (10 µM) for two days. Concentrations of TM, TGF-β, angiotensin II, high glucose, and albumin used in our experiments were based on a previous study [14].

Western Blot Analysis
Proteins of whole-cell lysates were fractionated by 10% SDS polyacrylamide gels, and transferred onto a nylon membrane. The membranes were blocked with Tris-buffered saline containing 5% non-fat milk, probed with primary antibody for 2 h, followed by peroxidase-conjugated secondary antibody. The blots were developed by using enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington, IL, USA). The band intensities were quantified by using a GS-710 densitometer and Quantity One software (version 4.2.1, Bio-Rad, Hercules, CA, USA). The results were normalized to β-actin as an internal control for standardization.

Experimental Mouse Model of Tunicamycin-Induced ER Stress
Male mice (C57BL/6), weighing about 20 g, were kept on a 12 h light/dark cycle in a temperature-controlled room, with free access to water and standard chow. A mouse model of tunicamycin-induced ER stress was induced by a single intraperitoneal injection of tunicamycin (TM, 2 mg/kg). In this model, it has been reported that male mice show high induction of ER stress markers such as GRP78 and CHOP with proximal tubular damages in the outer cortex [43]. Mice were randomly divided into four groups: control mice (n = 4), mice with TM injection (TM mouse, n = 4), TM mice with losartan (n = 4), and TM mice with losartan plus sirtinol (5 mg/kg i.p.) (n = 4). Losartan was administered by gavage at 10 mg/kg/day. Mice were euthanized after three days.

Experimental Mouse Model of Unilateral Ureteral Obstruction (UUO)-Induced Progressive Kidney Injury
UUO model is characterized by interstitial inflammatory cell infiltration, oxidative stress, apoptosis, and fibrosis. It has been known that angiotensin II and TGF-β contribute to UUO-induced renal fibrosis [44]. Furthermore, it has been reported that activation of ER stress is associated with UUO-induced renal apoptosis and fibrosis [18]. Therefore, we used a mouse model of UUO to test the hypothesis that the anti-fibrotic effect of losartan was mediated through the inhibition of ER stress via up-regulation of SIRT1. For induction of unilateral ureteral obstruction, male mice (C57BL/6) weighing about 20 g were anesthetized using evertin (125 mg/kg) and the left ureter was ligated through flank incision. Mice were randomly divided into four groups: control mice (n = 4), mice with UUO (n = 4), mice with UUO plus losartan (n = 4), and mice with UUO plus losartan and sirtinol (5 mg/kg i.p.) (n = 4). Losartan was administered by gavage at 10 mg/kg/day. On the 14th day after UUO surgery, the mice were sacrificed by using CO 2 . The kidney was fixed in 4% buffered formalin and embedded in paraffin for histological evaluation. Tubulointerstitial fibrosis was assessed by degree of interstitial collagen deposition using Masson trichrome stain [18]. All animal protocols were approved by Institutional Animal Care and Use Committee of Asan Institute for Life Sciences.

Immunohistochemical Staining
Paraffin embedded tissues were cut into 4 um sections and were deparaffinized in xylene and rehydrated in graded ethanol. To block endogenous peroxidase activity, sections were immersed in 0.3% hydrogen peroxide in PBS (phosphate-buffered saline) for 30 min. A microwave-based antigen retrieval method with 10 mmol/L citrate buffer (pH 6.0) for 10 min was done.
Nonspecific binding was blocked in 1% BSA (bovine serum albumin). Sections were then incubated with primary antibodies for GRP78, HO-1, and thioredoxin for 2 h and biotinylated secondary antibodies for 1 h and horseradish peroxidase-streptavidin conjugate for 30 min, followed by detection using DAB (3,3'-diaminobenzidine) stain (Dako, Glostrup, Denmark). The sections were counterstained with hematoxylin.

Statistical Analysis
Data were expressed as mean ± S.E. Statistical analysis was performed using Kruskall-Wallis, followed by a Mann-Whitney U-test using SPSS for Window 10.0 (SPSS Inc., Chicago, IL, USA). p < 0.05 value was considered statistically significant.

Conclusions
In conclusion, our study provides new information about the renoprotective effects of losartan, which may serve as the foundation for the targeted therapies designed to reduce ER stress.