Angiotensin II blockade upregulates the expression of Klotho, the anti-ageing gene, in an experimental model of chronic cyclosporine nephropathy.

BACKGROUND: The Klotho gene plays a role in suppressing ageing-related disorders. It is suggested that activation of renin-angiotensin system (RAS) or oxidative stress suppresses Klotho in the kidney. This study evaluated the association between Klotho expression and RAS in cyclosporine (CsA)-induced renal injury. METHODS: Chronic CsA nephropathy was induced by administering CsA (30 mg/kg) to mice on a low-salt diet (LSD) for 4 weeks. A normal-salt diet (NSD) was used as the control. Reverse transcription-polymerase chain reaction, western blot and immunohistochemistry were performed for Klotho and intrarenal RAS activity was measured using immunohistochemistry for angiotensinogen and renin. Oxidative stress was measured with urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). RESULTS: CsA treatment decreased Klotho mRNA and protein in mouse kidney in a dose-dependent and time-dependent manner, but a concurrent treatment with losartan, an angiotensin II type 1 (AT1) receptor blocker, reversed the decrease in Klotho expression with histological improvement. This finding was more marked in the LSD than the NSD. Klotho expression was correlated with angiotensinogen and renin expression, tubulointerstitial fibrosis score and urinary 8-OHdG excretion. CONCLUSIONS: Angiotensin II may play a pivotal role in regulating Klotho expression in CsA-induced renal injury. AT1 receptor blocker may inhibit the ageing process by decreasing oxidative stress caused by CsA.

Early and delayed effects of AST-120 on chronic cyclosporine nephropathy.

BACKGROUND: Removal of uraemic toxins by AST-120 (Kremezin(®)) decreases the progression of chronic kidney disease by reducing oxidative stress. We performed this study to evaluate whether AST-120 has a similar effect on progression of cyclosporine (CsA)-induced renal injury. METHODS: Two separate studies were performed in adult Sprague-Dawley rats. First, AST-120 was administered with CsA (15 mg/kg) for 4 weeks (early treatment). Second, AST-120 was administered to the rats for 3 weeks after treatment with CsA for 3 weeks (delayed treatment). Uraemic toxin and oxidative stress were evaluated with plasma indoxyl sulphate (IS) levels and urinary 8-OHdG excretion. The effects of AST-120 on CsA-induced renal injury were evaluated in terms of renal function, interstitial fibrosis, inflammation, and apoptotic cell death. RESULTS: CsA treatment for 4 weeks showed 2-fold increase in plasma IS and urinary 8-OHdG levels compared with the VH group. Early treatment with AST-120 significantly decreased both parameters, and this was accompanied by improved renal function and decreased interstitial inflammation, fibrosis, and apoptotic cell death compared with those of rats that received CsA alone. Delayed treatment with AST-120 also decreased the plasma IS and urinary 8-OHdG levels, and reduced the progression of chronic CsA nephropathy. Furthermore, delayed AST-120 treatment decreased the epithelial-mesenchymal transition in chronic CsA nephropathy. CONCLUSIONS: Removal of uraemic toxins with AST-120 treatment is effective in decreasing the progression of CsA-induced renal injury by reducing oxidative stress.

Effect of sirolimus on calcineurin inhibitor-induced nephrotoxicity using renal expression of KLOTHO, an antiaging gene.

BACKGROUND: The aim of this study was to observe the effect of sirolimus (SRL) on calcineurin inhibitor (CNI)-induced nephrotoxicity in the aging process by using renal expression of KLOTHO, an antiaging gene. METHODS.: Mice were treated with vehicle (VH; 1 mL/kg/day of olive oil), cyclosporine A (CsA; 30 mg/kg/day), or tacrolimus (FK; 1 mg/kg/day) with or without SRL (0.3 mg/kg/day) for 2 weeks. KLOTHO expression was evaluated by using reverse-transcriptase polymerase chain reaction, immunoblotting, and immunohistochemistry. Oxidative stress was evaluated by using immunohistochemistry and urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). The calcium metabolism was evaluated by using renal ectopic calcification, serum intact parathyroid hormone level, and renal fibroblast factor 23 (FGF23) expression. RESULTS: Treatment with CsA or FK alone significantly decreased KLOTHO expression and increased urinary 8-OHdG excretion compared with VH treatment but SRL treatment did not. Treatment SRL+CsA or SRL+FK further decreased KLOTHO expression and increased urinary 8-OHdG excretion compared with treatment of CsA or FK alone. There was a strong correlation between KLOTHO expression and urinary 8-OHdG excretion (r=-0.893; P<0.001). Treatment of CsA or FK alone increased renal ectopic calcification and serum intact parathyroid hormone level and decreased renal FGF23 expression compared with VH treatment (P<0.05) but SRL treatment did not. Treatment with SRL+CNI aggravated these parameters compared with CNI alone. CONCLUSIONS: SRL accelerates the CNI-induced oxidative process by down-regulating the renal antioxidant KLOTHO expression in the kidney.

Upregulation of hyaluronan and its binding receptors in an experimental model of chronic cyclosporine nephropathy.

AIM: Hyaluronan (HA) is an important extracellular matrix (ECM) proteoglycan. The localization of HA and its binding receptors, CD44 and LYVE-1, was evaluated in an experimental model of chronic cyclosporine A (CsA)-induced nephropathy. METHODS: Sprague-Dawley rats maintained on a low-salt diet (0.05% sodium) received an s.c. injection of vehicle (1 mL/kg per day olive oil; VH groups) or CsA (15 mg/kg per day; CsA groups) for 1 or 4 weeks. Induction of chronic CsA nephropathy was evaluated according to renal function and pathology and expression of HA, CD44, LYVE-1, ED-1 and alpha-smooth muscle actin (alpha-SMA). RESULTS: CsA treatment for 4 weeks caused renal dysfunction, which was accompanied by typical striped interstitial fibrosis. In the VHroup, HA immunoreactivity was observed only in the inner medulla. However, the area of HA immunoreactivity increased with the duration of CsA treatment: CsA treatment for 1 week extended HA immunoreactivity to the outer medulla, and CsA treatment for 4 weeks caused a further extension of HA immunoreactivity to the cortex, which was vulnerable to CsA-induced renal injury. HA binding receptor, CD44 and LYVE-1 expression were also upregulated in the CsA groups, and were localized to the area of fibrosis and the peritubular capillaries of the cortex. In the CsA groups, ED-1 and alpha-SMA were predominantly expressed in fibrotic areas in which HA had accumulated. CONCLUSION: These findings suggest that upregulation of HA and its binding receptors are involved in interstitial fibrosis in chronic CsA-induced renal injury.

Clinical significance of monitoring circulating CD4+CD25+ regulatory T cells in kidney transplantation during the early posttransplant period.

The CD4(+)CD25(+) T regulatory cells (Tregs) play an important role in immune tolerance in experimental transplantation but the clinical significance of circulating Tregs in the peripheral blood is undetermined. In 50 kidney transplant (KT) recipients, 29 healthy controls and 32 liver transplant (LT) recipients, the frequency of Tregs was measured with flow cytometry before and after transplantation. In the KT recipients, IL-10 secretion was measured with an enzyme-linked immunospot (ELISPOT) assay. The median frequency of circulating Tregs before KT was similar to that in healthy controls but significantly lower than that in LT patients before transplantation. The frequency of Tregs was significantly decreased in patients with subclinical acute rejection compared with those without subclinical acute rejection. Calcineurin inhibitors (CNIs) and anti-CD25 antibody decreased the frequency of Tregs but mTOR inhibitor did not. The frequency of donor-specific IL-10 secreting cells did not correlate with the number of Tregs. The frequency of circulating Tregs in KT recipients is strongly affected by CNIs and anti-CD25 antibody, and a low frequency of Tregs is associated with subclinical acute rejection during the early posttransplant period.

Expression of ammonia transporters, Rhbg and Rhcg, in chronic cyclosporine nephropathy in rats.

BACKGROUND/AIMS: Cyclosporine (CsA)-induced renal injury causes renal tubular acidosis. The current study was performed to evaluate the influence of CsA-induced renal injury on the ammonia transporter family members, Rh B-glycoprotein (Rhbg) and Rh C-glycoprotein (Rhcg). METHODS: Rats were treated daily for 1 or 4 weeks with vehicle (VH) or CsA. Induction of chronic CsA-induced nephropathy was confirmed by demonstrating impaired renal function and characteristic histopathology. Rhbg and Rhcg expression was evaluated with immunoblot, immunohistochemistry, real-time RT-PCR and electron microscopy. RESULTS: CsA treatment for 4 weeks developed mild metabolic acidosis and decreased urinary ammonia excretion. Rhcg mRNA expression was unchanged in both the cortex and outer medulla, but Rhcg protein expression in the CsA group was significantly reduced in the cortex and outer medulla. There were no significant differences in Rhbg mRNA and protein expression between the CsA and VH group. CONCLUSION: Long-term treatment with CsA in rats results in decreased urinary ammonia excretion accompanied by decreased expression of Rhcg; these changes are likely to mediate the CsA-induced defect in ammonium excretion in the collecting duct.

The role of macrophage in the pathogenesis of chronic cyclosporine-induced nephropathy.

BACKGROUND: Macrophages play diverse roles in tissue injury. We evaluated their role in cyclosporine (CsA)-induced renal injury by depletion with liposomal clodronate (CL). METHODS: Male Sprague Dawley rats were treated with CsA with or without CL treatment for 28 days. We assessed responses from the pathology and by measuring renal functions and levels of a proinflammatory cytokine (osteopontin), a profibrotic cytokine (betaig-h3), innate immune response markers (toll-like receptor 2 and MHC class II molecules), apoptotic cell death (deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling staining and caspase-3 expression) and oxidative stress (8-hydroxy-2'-deoxyguanosine, 8-OHdG). RESULTS: Macrophage depletion with CL improved not only renal function but also histopathology compared with the CsA-treated rats. Osteopontin and betaig-h3 levels increased significantly in CsA-treated rat kidneys, but CL treatment decreased both markers. Enhanced innate immune response and apoptotic cell death in CsA-treated rat kidney were decreased with CL. The increased rates of urinary 8-OHdG excretion and the tubular expression of 8-OHdG produced by CsA treatment were reversed with CL treatment. CONCLUSIONS: Thus, infiltrating macrophages were involved in both nonimmunologic and immunologic injury and led to apoptotic cell death in this rat model of chronic CsA nephropathy.

Expression and regulation of osteoprotegerin in adipose tissue.

PURPOSE: Osteoprotegerin (OPG), a potent inhibitor of osteoclastic bone resorption, has a variety of biological functions that include anti-inflammatory effects. Adipocytes and osteoblasts share a common origin, and the formation of new blood vessels often precedes adipogenesis in developing adipose tissue microvasculature. We examined whether OPG is secreted from adipocytes, therefore contributing to the prevention of neovascularization and protecting the vessels from intimal inflammation and medial calcification. MATERIALS AND METHODS: The mRNA expression of OPG and receptor activator of NF-kappaB ligand (RANKL) was measured in differentiated 3T3L1 adipocytes and adipose tissues. RESULTS: OPG mRNA expression increased with the differentiation of 3T3L1 adipocytes, while RANKL expression was not significantly altered. OPG mRNA was expressed at higher levels in white adipose tissue than in brown adipose tissue and was most abundant in the epididymal portion. In differentiated 3T3L1 adipocytes, Rosiglitazone and insulin reduced the OPG/RANKL expression ratio in a dose- and time- dependent manner. In contrast, tumor necrosis factor-alpha (TNF-alpha) increased the expression of both OPG and RANKL in a time-dependent manner. The OPG/RANKL ratio was at a maximum two hours after TNF-alpha treatment and then returned to control levels. Furthermore, OPG was abundantly secreted into the media after transfection of OPG cDNA with Phi C31 integrase into 3T3L1 cells. CONCLUSION: Our results indicate that OPG mRNA is expressed and regulated in the adipose tissue. Considering the role of OPG in obesity-associated inflammatory changes in adipose tissue and vessels, we speculate that OPG may have both a protective function against inflammation and anti-angiogenic effects on adipose tissue.

Peripheral effect of alpha-melanocyte-stimulating hormone on fatty acid oxidation in skeletal muscle.

To study the peripheral effects of melanocortin on fuel homeostasis in skeletal muscle, we assessed palmitate oxidation and AMP kinase activity in alpha-melanocyte-stimulating hormone (alpha-MSH)-treated muscle cells. After alpha-MSH treatment, carnitine palmitoyltransferase-1 and fatty acid oxidation (FAO) increased in a dose-dependent manner. A strong melanocortin agonist, NDP-MSH, also stimulated FAO in primary culture muscle cells and C2C12 cells. However, [Glu6]alpha-MSH-ND, which has ample MC4R and MC3R agonistic activity, stimulated FAO only at high concentrations (10(-5) M). JKC-363, a selective MC4R antagonist, did not suppress alpha-MSH-induced FAO. Meanwhile, SHU9119, which has both antagonistic activity on MC3R and MC4R and agonistic activity on both MC1R and MC5R, increased the effect of alpha-MSH on FAO in both C2C12 and primary muscle cells. Small interference RNA against MC5R suppressed the alpha-MSH-induced FAO effectively. cAMP analogues mimicked the effect of alpha-MSH on FAO, and the effects of both alpha-MSH and cAMP analogue-mediated FAO were antagonized by a protein kinase A inhibitor (H89) and a cAMP antagonist ((Rp)-cAMP). Acetyl-CoA carboxylase activity was suppressed by alpha-MSH and cAMP analogues by phosphorylation through AMP-activated protein kinase activation in C2C12 cells. Taken together, these results suggest that alpha-MSH increases FAO in skeletal muscle, in which MC5R may play a major role. Furthermore, these results suggest that alpha-MSH-induced FAO involves cAMP-protein kinase A-mediated AMP-activated protein kinase activation.