240 Endocrine Research
Authors
K-C. Huang1*, Y-G. Cherng2*, L-J. Chen3, C-T. Hsu4, J-T. Cheng5, 6
Affiliations
Affiliation addresses are listed at the end of the article
Key words
Abstract
▶ diabetic nephropathy ● ▶ TZD ● ▶ PPARγ ● ▶ FGFR ● ▶ klotho ● ▶ STZ rat ●
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A marked decrease of klotho expression was observed in the kidney of streptozotocin-induced diabetic rats (STZ rats) showing diabetic nephropathy. It has been documented that klotho is the target gene of PPARγ. However, the effect of PPARγ agonist on klotho expression in kidney of STZ rats remains obscure. Thus, we used rosiglitazone (TZD) as PPARγ agonist to investigate the effect on renal dysfunction in STZ rats. Treat-
Introduction
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received 17.08.2013 accepted 12.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357161 Published online: October 17, 2013 Horm Metab Res 2014; 46: 240–244 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Prof. J.-T. Cheng Institute of Medical Science Chang Jung Christian University Guei-Jen Tainan City 71101 Taiwan Tel.: + 886/6/331 8516 Fax: + 886/6/331 7532 [email protected]
The Klotho gene, named after a Greek goddess who spins the thread of life, was identified in 1997 as a gene mutated in the Klotho mouse, which exhibited multiple disorders resembling human premature-aging syndrome [1]. It is expressed in a limited numbers of tissues, most notably in distal convoluted tubules of kidney and choroid plexus of brain [1]. Fibroblast growth factor 23 (FGF23) is primarily produced in osteocytes and acts on kidney to induce phosphate excretion [2]. The major receptor for FGF23 named as FGFR1 plays an important role in the FGF23-klotho axis [3] where 3 partners (klotho, FGF23, and FGFR1) are involved, and klotho formed a complex with FGFR to increase the affinity of FGF23 [4]. Change of klotho expression under hyperglycemic environment has been documented [5]. However, serum level of FGF23 was the same as normal in patients with renal disease [6]. It has been demonstrated that an activation of FGF23-klotho axis could suppress the renal fibrosis in mice [7]. Clinically, chronic kidney disease (CKD) defined as the loss of kidney function more than 3 months is characterized pathologically by glomerulosclerosis, interstitial fibrosis, tubular * Equal contribution in this study.
Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
ment of TZD reversed the lower levels of PPARγ, klotho, and FGFR1 expressions in kidneys of STZ rats without the correction of hyperglycemia. Also, renal functions and structural defeats were improved by TZD treatment. Taken together, oral administration of TZD may improve STZ-induced diabetic nephropathy due to restoration of the expression of klotho axis through an increase in PPARγ expression without changing blood glucose in rats.
atrophy, peritubular capillary rarefaction, and inflammation. Also, the most commonly reported pathological changes in progressive renal failure is chronic interstitial fibrosis and tubular atrophy, which is accompanied by the vascular changes and glomerulosclerosis [8]. In our previous report, klotho expression is reduced in the kidney of rats showing diabetic nephropathy [5]. Thiazolidinediones (TZDs), including rosiglitazone and pioglitazone, are widely used as insulin-sensitizing agents for the treatment of type 2 diabetes [9, 10]. These agents have been identified as the agonists of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor involved in regulating cell differentiation and metabolism [11, 12]. It has also been documented that klotho is the target gene of PPARγ [13]. Thus, we were interested to know whether TZD can increase the expression of klotho to help the improvement of renal dysfunctions in diabetic animals.
Materials and Methods
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Animal model Male Wistar rats, aged 6 weeks, were obtained from the Animal Center of National Cheng Kung University Medical College. Diabetes were
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Rosiglitazone is Effective to Improve Renal Damage in Type-1-like Diabetic Rats
Endocrine Research
induced in rats by intravenously (i. v.) injecting streptozotocin (65 mg/kg) (Sigma-Aldrich Inc., USA) into fasting rats as described previously [14]. Animals were considered to be diabetic if they had elevated plasma glucose concentrations of 350 mg/dl. Then, these type 1-like diabetic rats, known as STZ rats, were considered to have diabetic nephropathy as their blood urea nitrogen (BUN) and creatinine values were elevated. All experiments were carried out at 9 weeks after diabetes induction. The animal experiment was approved and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in Chi-Mei Medical Center and followed the Guide for the Care and Use of Laboratory Animals as well as the guidelines of the Animal Welfare Act. The age-matched rats were divided into 3 groups (n = 6): vehicletreated normal rats (Wistar); vehicle-treated STZ rats (STZ); and TZD-treated STZ rats (STZ + TZD) through oral intake of 10 mg/kg rosiglitazone (Avandia) daily for 28 days as described previously [15, 16]. The levels of plasma glucose, BUN, and creatinine, were measured in blood samples collected from the femoral vein of anesthetized rats (pentobarbital, 30 mg/kg, i. p.) 1 h before treatment. Body temperature was also monitored during the experiment. At the end of treatment, animals were sacrificed and kidneys were dissected, washed with saline, weighed for analysis, and frozen in liquid nitrogen to store at − 80 °C for future analysis. Blood samples from rats were centrifuged at 12 000 g for 3 min. Samples were then analyzed using glucose, BUN, and creatinine kit reagents (Applied Bio assay kits; Hercules, CA, USA). The levels of plasma glucose, BUN, and serum creatinine were then estimated by an auto-analyzer (Quik-Lab, USA) run in duplicate.
Table 1 Effects of TZD on renal functions and plasma glucose in STZdiabetic rats.
Histological analysis
After 9 weeks of diabetes induction, the serum levels of glucose, BUN, and creatinine in STZ rats were markedly higher than in ▶ Table 1); marked difference in these age-matched normal rats (● levels between 2 groups were observed (p < 0.05). HE (hematoxylin and eosin stain) staining also showed the structural defects of ▶ Fig. 1). glomerosclerosis and tubular thickness in STZ rats (● After treatment with TZD for 28 days, the serum glucose was not significantly changed as compared with the vehicle-treated STZ ▶ Table 1). But the BUN and creatinine levels in TZD-treated rats (● ▶ Table 1). These treatments STZ rats were markedly reduced (● ameliorated glomerosclerosis and improved tubular thickness, ▶ Fig. 1). as shown in HE staining (●
Western blotting analysis Protein levels of PPARγ, klotho, and FGFR1 in rat kidneys were determined by Western blot. RIPA buffer (25 mM Tris · HCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % SDS) was used to extract total protein. Protein extracts were separated by SDS-PAGE, electrotransferred, and immobilized on a nitrocellulose membrane. The membrane was blocked with 5 % nonfat milk in phosphate-buffered saline containing 0.1 % Tween 20 (PBS-T) and incubated for 2 h. The membrane was then washed in PBS-T and hybridized with primary antibodies, which were diluted to a suitable concentration in PBS-T for 16 h. Specific antibodies for klotho and FGFR1 were purchased from Abcam (Cambridge, MA, USA) and for PPARγ from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Dilution of 1:1 000 was used. Incubation with secondary antibodies and detection of the antigen-antibody complex were performed using an ECL kit (Amersham Biosciences, UK). Immunoblot densities were quantified using a laser densitometer.
Statistical analysis Results were expressed as mean ± SE of each group at indicated sample size (n). Statistical analysis was carried out using ANOVA
BUN (mg/dl) Creatinine (mg/dl) Glucose (mg/dl)
13.76 ± 0.12 0.38 ± 0.02 108.24 ± 5.71
STZ 29.04 ± 1.14** 0.63 ± 0.03** 387.86 ± 9.56**
STZ + TZD 14.78 ± 0.83 0.41 ± 0.02 376.89 ± 11.62
The streptozotocin-induced diabetic rats (STZ) were orally treated with TZD (10 mg/kg) per day for 28 day The values represent the mean ± SEM of 6 animals. **p < 0.01 compared with the control (Wistar rats)
Fig. 1 Histological pictures for changes in kidneys of STZ-diabetic rats treated with TZD. The response in normal rats showed as Wistar is used to compare with that of diabetic rats (STZ) receiving TZD (STZ + TZD). Difference was characterized using hematoxylin/eosin (HE) staining.
analysis and Newman-Keuls post-hoc analysis. Statistical significance was achieved if the p-values were < 0.05.
Results
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Effect of TZD on rats with diabetic nephropathy
Effect of TZD on the expression of PPARγ in kidney of diabetic rats Changes of PPARγ expression in kidneys from diabetic rats with ▶ Fig. 2 nephropathy were quantified using Western blots. ● shows the decrease of PPARγ expressions in diabetic nephropathy as compared to the control group. After treatment with TZD for 28 days, in addition to the recovery of BUN and creatinine levels, PPARγ expression was reversed in kidneys of these dia▶ Fig. 2). betic rats (●
Effect of TZD on the expression of klotho in kidney of diabetic rats Changes of klotho expression in kidneys from diabetic rats with nephropathy were also quantified using Western blots. The ▶ Fig. 3) were markedly reduced in kidneys klotho expressions (● of diabetic rats showing nephropathy as compared to the control group. After treatment with TZD for 28 days, klotho expression
Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
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The kidney was removed from each rat, fixed in PBS containing 10 % formaldehyde, and embedded in paraffin. Each sample was then cut into 5-lm thick sections and were mounted on glass slides and depleted of paraffin with xylene. Then sections are subjected to standard hematoxylin/eosin (HE) staining. The sections were observed with a light microscope.
Control
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Fig. 2 PPARγ protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using PPARγ over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
Fig. 4 FGFR1 protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using FGFR1 over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
expressions in the kidneys of diabetic rats showing nephropathy ▶ Fig. 4) as compared to control group. were markedly reduced (● After treatment with TZD for 28 days, FGFR1 expression was ▶ Fig. 4). reversed in kidneys of these diabetic rats (●
Discussion
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Fig. 3 Klotho protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using klotho over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
was also significantly restored in kidneys of these diabetic rats ▶ Fig. 3). (●
Effect of TZD on the expression of FGFR1 in kidney of diabetic rats Changes of FGFR1 expression in kidneys from diabetic rats with nephropathy were also quantified using Western blots. FGFR1
In the present study, we found that rosiglitazone (TZD) at 10 mg/ kg/day for 28 days has an ability to improve diabetic nephropathy in STZ-diabetic rats. In addition, TZD decreased BUN and creatinine to improve diabetic nephropathy via the restoration of renal PPARγ, klotho, or FGFR1 in STZ-diabetic rats without correction of hyperglycemia. It seems that TZD has an effect on klotho expression to improve the diabetic nephropathy but not through the correction of glucose level. The available TZDs – pioglitazone, rosiglitazone, and troglitazone – are widely used to treat the type 2 diabetes by increasing insulin sensitivity [17]. These agents were known as the agonists of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor involved in regulating cell differentiation and metabolism [11]. It has also been observed to inhibit the inflammation resulting from ischemia and decrease the inflammatory markers without changing glucose level [18, 19]. Actually, pioglitazone has been mentioned to activate PPARα [20]. But, other TZDs including rosiglitazone that used in this study are known to activate PPARγ only [21–24]. In the present study, we employed STZ-diabetic rats to investigate the effects of TZD on the diabetic nephropathy. This animal model belonged to type-1 like diabetes and has widely been used to evaluate the diabetic nephropathy [25, 26]. Plasma BUN and creatinine levels are the popular indicators to assess renal functions [27, 28]. STZ-induced diabetic nephropathy causes a marked increase in plasma BUN and creatinine levels as shown ▶ Table 1. Moreover, histological changes in kidney were also in ●
Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
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242 Endocrine Research
Endocrine Research
thy. This action of TZD is not associated with altering blood glucose. Thus, TZD seems helpful in the improvement of diabetic nephropathy via an increase in klotho and FGFR1 expressions.
Acknowledgements
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We thank Shan-Yuan Liang and Yi-Zhi Chen for their assistance in this study. The present study was supported in part by a grant from the Chi-Mei Medical Center (CLFHR10004) in Taiwan, the Republic of China.
Conflict of Interest
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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Affiliations Department of Nephrology, Chi-Mei Medical Center, Liou Ying, Tainan City, Taiwan 2 Department of Anesthesiology, Shuang Ho Hospital, New Taipei City, and Department of Anesthesiology, College of Medicine, Taipei Medical University, Taiwan 3 Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 4 Department of Pathology, Edah University Medical Center, Yanchao, Kaohsiung City, Taiwan 5 Department of Medical Research, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan 6 Institute of Medical Science, Chang Jung Christian University, Guei-Jen, Tainan City, Taiwan 1
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Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
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observed in STZ-diabetic rats showing the diabetic nephropathy ▶ Fig. 1). TZD significantly decreased the plasma BUN and cre(● ▶ Table 1). In the present atinine levels in STZ-diabetic rats (● study, TZD was orally administered into STZ-diabetic rats at 10 mg/kg once daily for 28 days as described previously [15, 16]. As compared to vehicle-treated STZ-diabetic rats, TZD produced a marked protective action against the diabetic nephropathy as ▶ Fig. 1. Thus, the beneficial effects of TZD on STZshown in ● induced renal damage were identified. In clinics, TZDs ameliorate diabetic nephropathy in type 2 diabetic patients through correction of both hyperlipidemia and hyperglycemia [29, 30]. Since klotho has been found as the target of PPARγ [13], the upregulation of klotho expression by PPARγ may result in the merits for renal dysfunctions of diabetes. The clinically applied TZDs, including pioglitazone, rosiglitazone and troglitazone, have been demonstrated to increase the expressions of klotho [13]. Then, we used troglitazone, the most effective one of TZDs [13], to investigate the influence on klotho in this study. Actually, our result showed that TZD has an ability to improve renal dysfunctions at the dose showing no alteration of ▶ Table 1). Failure of TZD in the correction of hyperglycemia (● hyperglycemia in STZ rats is consistent with the previous reports [31] and it seems mainly related to the absence of insulin and other mechanisms that should be clarified in the future. Previous study showed that overexpression of klotho in a mouse model of glomerulonephritis restored the mitochondrial function and suppressed the mitochondrial DNA damage in the kidney [32]. It also suppressed the accelerated cellular senescence and apoptosis induced by glomerulonephritis, resulting in preservation of renal function and improvement of survival [32]. Klotho plays an important role in the protection of kidney due to antioxidation [32, 33]. Klotho expression was reduced under hyperglycemic condition in kidney and renal tubular cells [5]. Thus, decrease of klotho in kidney seems related to the process of diabetic nephropathy. Because klotho has been mentioned as the target of PPARγ [13], TZD shows an increase of klotho expression in kidneys of type-1 like diabetic rats. This seems related to the improvement of renal dysfunctions while klotho has renal protective action as described previously [34–37]. PPARγ dimerizes with retinoid X receptor to activate the gene expression after binding to the cognate PPRE within the regulatory region of target genes [38, 39]. Although no apparent consensus exists, PPRE has been found within the regulatory region of klotho gene, and noncanonical motifs linked with klotho gene for PPARγ binding have been identified [40]. These motifs are often located at a distal region upstream of the transcription initiative site [41, 42] and they are functionally activated by rosiglitazone to play the role in regulation of klotho gene expression [13]. FGFR1 is a major receptor for FGF23 to play an important role in the fibroblast growth factor 23 (FGF23)-klotho axis by interacting with its co-receptor klotho [3]. It has also been demonstrated that activation of FGF23-klotho axis could improve the renal function in mice [10]. This is consistent with our data that expressions of FGFR1 were restored by TZD in parallel with an improvement of renal dysfunctions in STZ rats. Taken together, it can be speculated that an increase of FGF23-klotho axis is induced by TZD for improvement of renal dysfunctions in STZ rats. But this view needs more investigations in the future. In conclusion, the present study is the first one to indicate that TZD is effective to reverse the decreased expression of PPARγ, klotho or FGFR1 in kidneys of rats showing diabetic nephropa-
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Authors
K-C. Huang1*, Y-G. Cherng2*, L-J. Chen3, C-T. Hsu4, J-T. Cheng5, 6
Affiliations
Affiliation addresses are listed at the end of the article
Key words
Abstract
▶ diabetic nephropathy ● ▶ TZD ● ▶ PPARγ ● ▶ FGFR ● ▶ klotho ● ▶ STZ rat ●
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A marked decrease of klotho expression was observed in the kidney of streptozotocin-induced diabetic rats (STZ rats) showing diabetic nephropathy. It has been documented that klotho is the target gene of PPARγ. However, the effect of PPARγ agonist on klotho expression in kidney of STZ rats remains obscure. Thus, we used rosiglitazone (TZD) as PPARγ agonist to investigate the effect on renal dysfunction in STZ rats. Treat-
Introduction
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received 17.08.2013 accepted 12.09.2013 Bibliography DOI http://dx.doi.org/ 10.1055/s-0033-1357161 Published online: October 17, 2013 Horm Metab Res 2014; 46: 240–244 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence Prof. J.-T. Cheng Institute of Medical Science Chang Jung Christian University Guei-Jen Tainan City 71101 Taiwan Tel.: + 886/6/331 8516 Fax: + 886/6/331 7532 [email protected]
The Klotho gene, named after a Greek goddess who spins the thread of life, was identified in 1997 as a gene mutated in the Klotho mouse, which exhibited multiple disorders resembling human premature-aging syndrome [1]. It is expressed in a limited numbers of tissues, most notably in distal convoluted tubules of kidney and choroid plexus of brain [1]. Fibroblast growth factor 23 (FGF23) is primarily produced in osteocytes and acts on kidney to induce phosphate excretion [2]. The major receptor for FGF23 named as FGFR1 plays an important role in the FGF23-klotho axis [3] where 3 partners (klotho, FGF23, and FGFR1) are involved, and klotho formed a complex with FGFR to increase the affinity of FGF23 [4]. Change of klotho expression under hyperglycemic environment has been documented [5]. However, serum level of FGF23 was the same as normal in patients with renal disease [6]. It has been demonstrated that an activation of FGF23-klotho axis could suppress the renal fibrosis in mice [7]. Clinically, chronic kidney disease (CKD) defined as the loss of kidney function more than 3 months is characterized pathologically by glomerulosclerosis, interstitial fibrosis, tubular * Equal contribution in this study.
Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
ment of TZD reversed the lower levels of PPARγ, klotho, and FGFR1 expressions in kidneys of STZ rats without the correction of hyperglycemia. Also, renal functions and structural defeats were improved by TZD treatment. Taken together, oral administration of TZD may improve STZ-induced diabetic nephropathy due to restoration of the expression of klotho axis through an increase in PPARγ expression without changing blood glucose in rats.
atrophy, peritubular capillary rarefaction, and inflammation. Also, the most commonly reported pathological changes in progressive renal failure is chronic interstitial fibrosis and tubular atrophy, which is accompanied by the vascular changes and glomerulosclerosis [8]. In our previous report, klotho expression is reduced in the kidney of rats showing diabetic nephropathy [5]. Thiazolidinediones (TZDs), including rosiglitazone and pioglitazone, are widely used as insulin-sensitizing agents for the treatment of type 2 diabetes [9, 10]. These agents have been identified as the agonists of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor involved in regulating cell differentiation and metabolism [11, 12]. It has also been documented that klotho is the target gene of PPARγ [13]. Thus, we were interested to know whether TZD can increase the expression of klotho to help the improvement of renal dysfunctions in diabetic animals.
Materials and Methods
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Animal model Male Wistar rats, aged 6 weeks, were obtained from the Animal Center of National Cheng Kung University Medical College. Diabetes were
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Rosiglitazone is Effective to Improve Renal Damage in Type-1-like Diabetic Rats
Endocrine Research
induced in rats by intravenously (i. v.) injecting streptozotocin (65 mg/kg) (Sigma-Aldrich Inc., USA) into fasting rats as described previously [14]. Animals were considered to be diabetic if they had elevated plasma glucose concentrations of 350 mg/dl. Then, these type 1-like diabetic rats, known as STZ rats, were considered to have diabetic nephropathy as their blood urea nitrogen (BUN) and creatinine values were elevated. All experiments were carried out at 9 weeks after diabetes induction. The animal experiment was approved and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in Chi-Mei Medical Center and followed the Guide for the Care and Use of Laboratory Animals as well as the guidelines of the Animal Welfare Act. The age-matched rats were divided into 3 groups (n = 6): vehicletreated normal rats (Wistar); vehicle-treated STZ rats (STZ); and TZD-treated STZ rats (STZ + TZD) through oral intake of 10 mg/kg rosiglitazone (Avandia) daily for 28 days as described previously [15, 16]. The levels of plasma glucose, BUN, and creatinine, were measured in blood samples collected from the femoral vein of anesthetized rats (pentobarbital, 30 mg/kg, i. p.) 1 h before treatment. Body temperature was also monitored during the experiment. At the end of treatment, animals were sacrificed and kidneys were dissected, washed with saline, weighed for analysis, and frozen in liquid nitrogen to store at − 80 °C for future analysis. Blood samples from rats were centrifuged at 12 000 g for 3 min. Samples were then analyzed using glucose, BUN, and creatinine kit reagents (Applied Bio assay kits; Hercules, CA, USA). The levels of plasma glucose, BUN, and serum creatinine were then estimated by an auto-analyzer (Quik-Lab, USA) run in duplicate.
Table 1 Effects of TZD on renal functions and plasma glucose in STZdiabetic rats.
Histological analysis
After 9 weeks of diabetes induction, the serum levels of glucose, BUN, and creatinine in STZ rats were markedly higher than in ▶ Table 1); marked difference in these age-matched normal rats (● levels between 2 groups were observed (p < 0.05). HE (hematoxylin and eosin stain) staining also showed the structural defects of ▶ Fig. 1). glomerosclerosis and tubular thickness in STZ rats (● After treatment with TZD for 28 days, the serum glucose was not significantly changed as compared with the vehicle-treated STZ ▶ Table 1). But the BUN and creatinine levels in TZD-treated rats (● ▶ Table 1). These treatments STZ rats were markedly reduced (● ameliorated glomerosclerosis and improved tubular thickness, ▶ Fig. 1). as shown in HE staining (●
Western blotting analysis Protein levels of PPARγ, klotho, and FGFR1 in rat kidneys were determined by Western blot. RIPA buffer (25 mM Tris · HCl pH 7.6, 150 mM NaCl, 1 % NP-40, 1 % sodium deoxycholate, 0.1 % SDS) was used to extract total protein. Protein extracts were separated by SDS-PAGE, electrotransferred, and immobilized on a nitrocellulose membrane. The membrane was blocked with 5 % nonfat milk in phosphate-buffered saline containing 0.1 % Tween 20 (PBS-T) and incubated for 2 h. The membrane was then washed in PBS-T and hybridized with primary antibodies, which were diluted to a suitable concentration in PBS-T for 16 h. Specific antibodies for klotho and FGFR1 were purchased from Abcam (Cambridge, MA, USA) and for PPARγ from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Dilution of 1:1 000 was used. Incubation with secondary antibodies and detection of the antigen-antibody complex were performed using an ECL kit (Amersham Biosciences, UK). Immunoblot densities were quantified using a laser densitometer.
Statistical analysis Results were expressed as mean ± SE of each group at indicated sample size (n). Statistical analysis was carried out using ANOVA
BUN (mg/dl) Creatinine (mg/dl) Glucose (mg/dl)
13.76 ± 0.12 0.38 ± 0.02 108.24 ± 5.71
STZ 29.04 ± 1.14** 0.63 ± 0.03** 387.86 ± 9.56**
STZ + TZD 14.78 ± 0.83 0.41 ± 0.02 376.89 ± 11.62
The streptozotocin-induced diabetic rats (STZ) were orally treated with TZD (10 mg/kg) per day for 28 day The values represent the mean ± SEM of 6 animals. **p < 0.01 compared with the control (Wistar rats)
Fig. 1 Histological pictures for changes in kidneys of STZ-diabetic rats treated with TZD. The response in normal rats showed as Wistar is used to compare with that of diabetic rats (STZ) receiving TZD (STZ + TZD). Difference was characterized using hematoxylin/eosin (HE) staining.
analysis and Newman-Keuls post-hoc analysis. Statistical significance was achieved if the p-values were < 0.05.
Results
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Effect of TZD on rats with diabetic nephropathy
Effect of TZD on the expression of PPARγ in kidney of diabetic rats Changes of PPARγ expression in kidneys from diabetic rats with ▶ Fig. 2 nephropathy were quantified using Western blots. ● shows the decrease of PPARγ expressions in diabetic nephropathy as compared to the control group. After treatment with TZD for 28 days, in addition to the recovery of BUN and creatinine levels, PPARγ expression was reversed in kidneys of these dia▶ Fig. 2). betic rats (●
Effect of TZD on the expression of klotho in kidney of diabetic rats Changes of klotho expression in kidneys from diabetic rats with nephropathy were also quantified using Western blots. The ▶ Fig. 3) were markedly reduced in kidneys klotho expressions (● of diabetic rats showing nephropathy as compared to the control group. After treatment with TZD for 28 days, klotho expression
Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
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The kidney was removed from each rat, fixed in PBS containing 10 % formaldehyde, and embedded in paraffin. Each sample was then cut into 5-lm thick sections and were mounted on glass slides and depleted of paraffin with xylene. Then sections are subjected to standard hematoxylin/eosin (HE) staining. The sections were observed with a light microscope.
Control
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Fig. 2 PPARγ protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using PPARγ over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
Fig. 4 FGFR1 protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using FGFR1 over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
expressions in the kidneys of diabetic rats showing nephropathy ▶ Fig. 4) as compared to control group. were markedly reduced (● After treatment with TZD for 28 days, FGFR1 expression was ▶ Fig. 4). reversed in kidneys of these diabetic rats (●
Discussion
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Fig. 3 Klotho protein levels in kidneys of STZ-diabetic rats treated with TZD. The upper shows the protein level or the corresponding β-actin (actin) level as internal control in kidneys isolated from STZ-diabetic rats receiving TZD or not. The treatments are described in Materials and Methods. Quantification of protein levels using klotho over β-actin to show the means with SE (n = 6 per group) in each column are indicated in the lower panel. ***p < 0.001 compared to control.
was also significantly restored in kidneys of these diabetic rats ▶ Fig. 3). (●
Effect of TZD on the expression of FGFR1 in kidney of diabetic rats Changes of FGFR1 expression in kidneys from diabetic rats with nephropathy were also quantified using Western blots. FGFR1
In the present study, we found that rosiglitazone (TZD) at 10 mg/ kg/day for 28 days has an ability to improve diabetic nephropathy in STZ-diabetic rats. In addition, TZD decreased BUN and creatinine to improve diabetic nephropathy via the restoration of renal PPARγ, klotho, or FGFR1 in STZ-diabetic rats without correction of hyperglycemia. It seems that TZD has an effect on klotho expression to improve the diabetic nephropathy but not through the correction of glucose level. The available TZDs – pioglitazone, rosiglitazone, and troglitazone – are widely used to treat the type 2 diabetes by increasing insulin sensitivity [17]. These agents were known as the agonists of peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor involved in regulating cell differentiation and metabolism [11]. It has also been observed to inhibit the inflammation resulting from ischemia and decrease the inflammatory markers without changing glucose level [18, 19]. Actually, pioglitazone has been mentioned to activate PPARα [20]. But, other TZDs including rosiglitazone that used in this study are known to activate PPARγ only [21–24]. In the present study, we employed STZ-diabetic rats to investigate the effects of TZD on the diabetic nephropathy. This animal model belonged to type-1 like diabetes and has widely been used to evaluate the diabetic nephropathy [25, 26]. Plasma BUN and creatinine levels are the popular indicators to assess renal functions [27, 28]. STZ-induced diabetic nephropathy causes a marked increase in plasma BUN and creatinine levels as shown ▶ Table 1. Moreover, histological changes in kidney were also in ●
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242 Endocrine Research
Endocrine Research
thy. This action of TZD is not associated with altering blood glucose. Thus, TZD seems helpful in the improvement of diabetic nephropathy via an increase in klotho and FGFR1 expressions.
Acknowledgements
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We thank Shan-Yuan Liang and Yi-Zhi Chen for their assistance in this study. The present study was supported in part by a grant from the Chi-Mei Medical Center (CLFHR10004) in Taiwan, the Republic of China.
Conflict of Interest
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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported. Affiliations Department of Nephrology, Chi-Mei Medical Center, Liou Ying, Tainan City, Taiwan 2 Department of Anesthesiology, Shuang Ho Hospital, New Taipei City, and Department of Anesthesiology, College of Medicine, Taipei Medical University, Taiwan 3 Institute of Basic Medical Science, College of Medicine, National Cheng Kung University, Tainan City, Taiwan 4 Department of Pathology, Edah University Medical Center, Yanchao, Kaohsiung City, Taiwan 5 Department of Medical Research, Chi-Mei Medical Center, Yong Kang, Tainan City, Taiwan 6 Institute of Medical Science, Chang Jung Christian University, Guei-Jen, Tainan City, Taiwan 1
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Huang K-C et al. Merit of TZD in Diabetic Nephropathy … Horm Metab Res 2014; 46: 240–244
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observed in STZ-diabetic rats showing the diabetic nephropathy ▶ Fig. 1). TZD significantly decreased the plasma BUN and cre(● ▶ Table 1). In the present atinine levels in STZ-diabetic rats (● study, TZD was orally administered into STZ-diabetic rats at 10 mg/kg once daily for 28 days as described previously [15, 16]. As compared to vehicle-treated STZ-diabetic rats, TZD produced a marked protective action against the diabetic nephropathy as ▶ Fig. 1. Thus, the beneficial effects of TZD on STZshown in ● induced renal damage were identified. In clinics, TZDs ameliorate diabetic nephropathy in type 2 diabetic patients through correction of both hyperlipidemia and hyperglycemia [29, 30]. Since klotho has been found as the target of PPARγ [13], the upregulation of klotho expression by PPARγ may result in the merits for renal dysfunctions of diabetes. The clinically applied TZDs, including pioglitazone, rosiglitazone and troglitazone, have been demonstrated to increase the expressions of klotho [13]. Then, we used troglitazone, the most effective one of TZDs [13], to investigate the influence on klotho in this study. Actually, our result showed that TZD has an ability to improve renal dysfunctions at the dose showing no alteration of ▶ Table 1). Failure of TZD in the correction of hyperglycemia (● hyperglycemia in STZ rats is consistent with the previous reports [31] and it seems mainly related to the absence of insulin and other mechanisms that should be clarified in the future. Previous study showed that overexpression of klotho in a mouse model of glomerulonephritis restored the mitochondrial function and suppressed the mitochondrial DNA damage in the kidney [32]. It also suppressed the accelerated cellular senescence and apoptosis induced by glomerulonephritis, resulting in preservation of renal function and improvement of survival [32]. Klotho plays an important role in the protection of kidney due to antioxidation [32, 33]. Klotho expression was reduced under hyperglycemic condition in kidney and renal tubular cells [5]. Thus, decrease of klotho in kidney seems related to the process of diabetic nephropathy. Because klotho has been mentioned as the target of PPARγ [13], TZD shows an increase of klotho expression in kidneys of type-1 like diabetic rats. This seems related to the improvement of renal dysfunctions while klotho has renal protective action as described previously [34–37]. PPARγ dimerizes with retinoid X receptor to activate the gene expression after binding to the cognate PPRE within the regulatory region of target genes [38, 39]. Although no apparent consensus exists, PPRE has been found within the regulatory region of klotho gene, and noncanonical motifs linked with klotho gene for PPARγ binding have been identified [40]. These motifs are often located at a distal region upstream of the transcription initiative site [41, 42] and they are functionally activated by rosiglitazone to play the role in regulation of klotho gene expression [13]. FGFR1 is a major receptor for FGF23 to play an important role in the fibroblast growth factor 23 (FGF23)-klotho axis by interacting with its co-receptor klotho [3]. It has also been demonstrated that activation of FGF23-klotho axis could improve the renal function in mice [10]. This is consistent with our data that expressions of FGFR1 were restored by TZD in parallel with an improvement of renal dysfunctions in STZ rats. Taken together, it can be speculated that an increase of FGF23-klotho axis is induced by TZD for improvement of renal dysfunctions in STZ rats. But this view needs more investigations in the future. In conclusion, the present study is the first one to indicate that TZD is effective to reverse the decreased expression of PPARγ, klotho or FGFR1 in kidneys of rats showing diabetic nephropa-
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