| Background:Klotho was first identified in close association with premature onset of aging phenotype in mice. Klotho homozygous mutant mice (kl/kl) exhibit a shortened life span, infertility, ectopic calcification, skin atrophy, pulmonary emphysema, osteoporosis and so on, which resemble aged state of human. Mouse klotho is predominantly expressed in the kidney and slightly in the brain. Two forms of Klotho have been identified:membrane Klotho and secreted Klotho. The membrane Klotho functions as a co-receptor of FGF23 receptor. FGF23, a member of the fibroblast growth factor (FGF) family, is a bone derived hormone exerting its function in the kidney where it helps to maintain homeostasis in phosphate and vitamin D metabolism by regulating the sodium phosphate co-transporter and the key vitamin D-metabolizing enzymes CYP27B1 and CYP24A1. Similar to FGF23-deficient mice, kl/kl mice also exhibit hyperphosphatemia and elevated serum 1,25 (OH)2 vitamin D3, which are suspected to be the primary cause of premature aging because restrict vitamin D intake improves kl/kl phenotype. Secreted Klotho is formed via either gene alternative splicing or ectodomain shedding of extracellular domain into the extracellular space and subsequently the circulation by the membrane Klotho.Renal tubulointerstitial fibrosis (RTF) is the final common pathological condition of chronic kidney disease (CKD) regardless of the underlying causes. It is characterized by excessive accumulation of extracellular matrix (ECM), fibroblast activation along with a loss of functioning nephrons. Recent studies suggest that Klotho plays important roles in RTF. Kidney injuries, such as ischemia-reperfusion, peritoneal cisplatin injection, or Angtensin II administration induce Klotho deficiency, whereas exogenous Klotho protein attenuates kidney injury. In mice model of CKD, unilateral ureteral obstruction (UUO)-induced RTF is exaggerated in klotho heterozygous mutant mice and is ameliorated in Klotho over-expressing mice. Secreted Klotho is proved to interfer with TGF-β signaling and Wnt/β-catenin signaling to suppress UUO-induced RTF.Klotho homozygous mutant mice, on the other hand, exhibit phenotypes very different from wild-type or heterozygous mutant mice. Hyperphosphatemia, high 1, 25(OH)2 vitamin D3 and FGF23 are most noticeable features in blood index. ECM deposition is increased in kl/kl mice even without UUO which are thought to be Klotho deficiency. However, activated form of vitamin D and its analogues have been shown to protect the kidney from fibrosis against various kidney injuries.1,25(OH)2 vitamin D3 reduces gene expression related to TGF-β-induced fibrosis in human uterine leiomyoma cells. Active vitamin D3 analogue maxacalcitol recruits PPM1A/VDR complex to pSmad3 to accelerate its dephosphorylation. Thus, the outcome of UUO-induced RTF become uncertain because of opposite function of elevated vitamin D and Klotho deficiency. Since all the existing studies on the effect of Klotho on UUO-induced RTF are carried out using klotho heterozygous mice, whether the disturbed homeostasis in kl/kl mice have an effect is not know.Objectives:By comparing UUO-induced RTF among wild-type, heterozygous and homozygous mutant mice and in vivo experiments, we analyzed how individual disturbed homeostasis factors, such as high levels of inorganic phosphate, FGF23, and 1,25(OH)2 vitamin D3, affect fibrosis in in vitro experiments.Methods:1. Animals:klotho/Jcl mice used in this study were purchased from CLEA Japan. Mice were raised in standard facilitates with free access to food and water.2. UUO animal models:Six to eight week-old male wild-type (WT), heterozygous (HT), and kl/kl mice from both standard diet and D-diet groups were used to perform UUO. Sham groups (n=3) received the same procedure except ureteral ligation.3. Tissue preparation:At the time of sacrifice, the ligated kidneys (or sham kidneys) were removed and cut transversely. Tissues were fixed with 4% paraformaldehyde for histopathological examination, lysed by denaturing lysis buffer or Trizol reagent for RNA isolation.4. Cell culture:1) Primary proximal tubular epithelia cells were generated from the kidneys of wt, k1/+ and kl/k1 mice and cultured in culture medium (REBM bullet kit) and incubated at 37℃ in a 5% CO2 incubator with medium changes every 2 days until-80% confluent.2) NRK52E cells were obtained from ATCC and cultured in DMEM with 5% FBS (Gibco) without supplement of antibiotics.5. TGF-β1-induced EMT:For TGF-β1 stimulation, cells were underwent 12 hrs serum starvation and then were exposed to 5 ng/ml TGF-β1 (cell signaling) for 12 hrs or 24 hrs. Cells were collected for RNA isolation or protein lysis.6. Disturbed homeostasis simulation:NRK52E cells were grown in normal culture medium (DMEM with 5% FBS), in high Pi culture medium (DMEM with 5% FBS supplemented with 2mM or 4mM NaH2PO4), in hight FGF23 culture medium (DMEM with 5% FBS supplemented with 0.2 μg/ml or 2 μg/ml murine FGF23) or in hight active vitamin D3 medium (DMEM with 5% FBS supplemented with 0.1 μg/ml,1 μg/ml Calcitriol).7. Immunohistochemistry and immunofluorescent staining was performed on paraffin sections using a microwave-based antigen retrieval technique to detect the expression of Collagen I, aSMA and pSmad3.8. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze mRNAs of Collagen I, aSMA TGF-β 1 and E-cadherin.9. Tissue or cell lysate (20μg) was separated by SDS-PAGE and blotted on polyvinylidene difluoride membranes. Primary antibodies were rabbit anti-pSmad3, rat anti-E-cadherin, mouse anti-aSMA, rabbit anti-Smad3, mouse anti-(3-actin and goat anti-GAPDH. Siganls were developed with ECL Plus detection system.10. ELISA assay were performed to assess the content of total TGF-β1 protein in the tissue lysate.11. Blood was collected from inferior vena cava and the serum was separated. The serum levels of 1,25(OH)2 vitamin D3 were measured by RIA.12. Body weight werr measured.13. Rescue experiment:Klotho hetero female mice were maintained on vitamin D3 free diet after pregnancy, The offsprings of D-diet klotho hetero females were also fed on D-diet all through the experiments.14. Statistical Analysis:The data were analyzed with a Student’s t-test and a one-way ANOVA with a Student-Newman-Keuls test (SPSS,13.0) and expressed as mean ± SD. P<0.05 were considered to be statistically significant.Results:1. UUO-induced RTF is aggravated in mice with Klotho haploinsufficiency whereas suppressed in kl/kl mice6 wk-old wild-type (wt), k1/+, and kl/kl mice were subjected to UUO for 3 days or 1 week. Expression of RTF markers, Collagen I and aSMA was assessed by immunohistochemistry and quantitative real-time PCR. RTF developed after UUO operation in a time-dependent course in wt and k1/+ mice compared with sham-operated control groups. The expression of RTF markers are much higher in kl/+mice than in wt mice which is paralleled with previous reports. In sham-operated groups, the expression of RTF markers were comparable between wt and k1/+ mice, but was increased by more than 2-fold in k1/k1 mice. Unexpectedly, kl/kl mice showed only slight increase in the expression of fibrosis markers after UUO.2. TGF-β/Smad3 signaling is suppressed in kl/kl miceThe phosphorylation of SMAD3 and TGF-β1 expression was assayed. In parallel with RTF marker, after UUO, phosphorylated Smad3 and TGF-β1 expression were highest in k1/+ mice and were significantly suppressed in kl/k1 mice. It is interesting to note that in sham operated group, kl/kl mice harbor higher percentage of pSmad3 positive staining cells than the other two group, however, the TGF-β1 protein content was the lowest, suggesting that Smad3 phospharylation in sham operated kl/kl mice was not a result of increased TGF-β1.3. No suppression of TGF-β1-induced epithelial-mesenchymal transition is observed in primary proximal tubular epithelial culture cells derived from kl/kl miceWe compared the expression levels of molecules that are involved in epithelial-mesenchymal transition (EMT) using cultured proximal tubular epithelial cells (PTECs) from wt, kl/+ and kl/kl mice. Cells were cultured in the presence of 5 ng/ml TGF-β1 for 12h or 24h. TGF-β1-induced EMT was enhanced in k1/+cells compared with in wt cells manifested by higher aSMA expression and lower E-cadherin expression at both mRNA and protein levels at time point indicated. Smad3 activation was also enhanced in k1/+cells. Unlike UUO kidneys, kl/kl cells showed no suppression of TGF-β1-induced EMT and Smad3 phosphorylation compared with k1/+ cells. These results suggest that the suppressed TGF-β/Smad3 signaling was a result of disturbed homeostasis but not complete Klotho depletion.4. TGF-β1 induced EMT is suppressed not by high phosphate or FGF23 but by 1,25(OH)2 vitamin D3 in dose-dependent courseWe used normal kidney epithelial cells (NRK52E) cultured in the presence of high phosphate (2 mM,4 mM), FGF23(0.2 μg/ml,2 μg/ml) or 1,25(OH)2 vitamin D3 (0.1 μg/ml,1 μg/ml) to evaluate their effect on TGF-β1-induced EMT. Cells treated with TGF-β1 together with phosphate or FGF23 for 12h or 24h, TGF-β1-induced EMT was not affected at neither mRNA nor protein levels compared with cells treated with TGF-β1 alone. By contrast,1,25(OH)2 vitamin D3 ameliorated TGF-β1-induced Smad3 phosphorylation, aSMA expression, and E-cadherin suppression in a dose-dependent manner at both mRNA and protein levels. Interestingly, cells cultured in high phosphate (4mM) for 48h have more Smad3 activation and higher aSMA expression compared with cells cultured in normal DMEM even in the absence of TGF-β1.5. Vitamin D restriction normalizes serum 1,25(OH)2 vitamin D3 levels and restores the expression of UUO-induced RTF markers in k1/kl miceVitamin D free diet (D-diet) normalized serum 1,25(OH)2 vitamin D3 levels and increased the body weight of kl/kl mice that indistinguishable from wt littermates.After UUO operation, the expression of Collagen I and aSMA was restored by D-diet, which is increased by 3-fold and 2-fold, respectively, compared with kl/kl mice fed on standard diet, evaluated by IHC staining positive areas. The mRNAs showed a similar tendency. In addition, D-diet restored the activation TGF-β/Smad3 signaling. Percentage of pSmad3-positive cells increased approximately 2.5-fold as a result of vitamin D withdraw. UUO-induced TGF-β1 expression was increased approximately 2-fold by D-diet at both protein and mRNA levels.Conclusions:In this study, we demonstrated that UUO-induced RTF was greatly suppressed in kl/kl mice compared with HT mice, albeit already existing mild renal fibrosis in sham operated kl/kl mice. The suppression of RTF marker expression may be due to decreased levels of TGF-β/Smad3 signaling activation. Disturbed humoral homeostasis in kl/kl mice plays an important role in the onset of renal fibrosis in sham-operated mice and in the suppression of RTF in UUO-operated mice... |
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