22-Oxacalcitriol prevents progression of endothelial dysfunction through antioxidative effects in rats with type 2 diabetes and early-stage nephropathy.

BACKGROUND: Vitamin D deficiency is associated with endothelial dysfunction in type 2 diabetes patients, but the effectiveness of vitamin D supplementation remains controversial. We assessed whether 22-oxacalcitriol (OCT) could prevent endothelial dysfunction in type 2 diabetes mellitus (DM) rats. METHODS: DM rats with early-stage nephropathy were treated for 10 weeks with OCT (0.2 µg/kg) three times per week or by an implanted insulin pellet. Endothelial dysfunction was assessed by femoral flow-mediated dilation (FMD). RESULTS: Insulin significantly improved FMD as blood glucose levels normalized. OCT also improved FMD without hypercalcemia or hyperphosphatemia and without affecting blood glucose or blood pressure. In femoral arteries, OCT significantly suppressed the elevated expression of p22(phox), a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit, and improved the endothelial nitric oxide synthase (eNOS) dimer-to-monomer ratio. In cultured endothelial cells, OCT significantly inhibited high-glucose (HG)-induced reactive oxygen species (ROS) production. Simultaneously, OCT significantly suppressed HG-induced p22(phox) expression and improved eNOS uncoupling as was observed in the in vivo study. CONCLUSION: In DM rats, OCT improved endothelial dysfunction, at least in part, by suppressing ROS generation through p22(phox) expression, which might contribute to improving eNOS uncoupling.

Renoprotection by continuous erythropoietin receptor activator in puromycin aminonucleoside-induced nephrotic syndrome.

BACKGROUND/AIMS: Recent studies have demonstrated that erythropoiesis-stimulating agents (ESAs) induce a tissue-protective effect in the kidney. In this study, we examined whether continuous erythropoietin receptor activator (CERA), a long-acting ESA, could prevent kidney injury, especially podocyte damage, in a rat model of nephrotic syndrome induced by puromycin aminonucleoside (PAN). METHODS: Rats were injected with CERA (30 µg/kg) or vehicle 4 h before the injection of PAN (50 mg/kg). Renal function, kidney injury, and podocyte damage were assessed at 7 days. RESULTS: The levels of proteinuria, BUN, and plasma creatinine significantly increased in rats with PAN-induced nephrosis. Treatment with CERA significantly prevented these deteriorations induced by PAN. Glomerular lesions, especially vacuolation of podocytes, and the increase of desmin expression in PAN-treated rats were significantly ameliorated by treatment with CERA. Treatment with CERA also significantly prevented the decrease in the protein productions of nephrin and podocin in the kidneys of PAN-treated rats. We found persistent activation of the Akt signaling pathway in the kidneys of CERA-treated rats. CONCLUSION: CERA could ameliorate renal dysfunction in PAN-induced nephrosis, which might be due to the amelioration of podocyte injury. CERA inhibited the depletion of nephrin and podocin, key components of the glomerular filtration barrier, and alleviated proteinuria. Activation of the Akt signaling pathway might be involved in the renoprotective effect of CERA.

Alteration of mRNA expression of molecules related to iron metabolism in adenine-induced renal failure rats: a possible mechanism of iron deficiency in chronic kidney disease patients on treatment.

BACKGROUND: Recombinant human erythropoietin (rHuEpo) is a definitive treatment for anaemia in chronic kidney disease (CKD). During long-term rHuEpo treatment most patients develop and show persistent iron deficiency in spite of oral iron supplementation. Abnormalities of iron absorption and transport in the duodenum may contribute to this deficiency. METHODS: To investigate changes in iron absorption and transport in CKD and iron deficiency against the background of rHuEpo treatment, we used severely anaemic rats with adenine-induced renal failure (adenine rats) and sham-treated control rats given only the vehicle. After 4 weeks on adenine or the vehicle, the rats were divided into four groups according to whether or not they received rHuEpo for the next 4 weeks: rHuEpo(-)-adenine, rHuEpo(-)-control, rHuEpo(+)-adenine and rHuEpo(+)-control. We evaluated the effects of rHuEpo treatment on iron balance, duodenal mRNA expression of molecules related to iron absorption and transport and hepatic mRNA expression of hepcidin. RESULTS: Treatment with rHuEpo improved anaemia and induced iron deficiency only in the adenine rats, in whom the expression of mRNAs for ferroportin 1 and hephaestin 1 increased and for divalent metal transporter 1 (DMT1) was unchanged. In contrast, control rats treated with rHuEpo showed no changes. Hepcidin mRNA expression was greater in adenine rats than in control rats. CONCLUSIONS: In the adenine rats, rHuEpo treatment improved renal anaemia and induced persistent iron deficiency. An alteration of mRNA expression of molecules related to iron metabolism in renal insufficiency may be one of the reasons for this iron deficiency.

Erythropoietin-induced proliferation of gastric mucosal cells.

AIM: To analyze the localization of erythropoietin receptor on gastric specimens and characterize the effects of erythropoietin on the normal gastric epithelial proliferation using a porcine gastric epithelial cell culture model. METHODS: Erythropoietin receptor was detected by RT-PCR, Western blotting and immunohistochermistry. Growth stimulation effects of erythropoietin on cultured gastric mucosal cells were determined by ELISA using bromodeoxyuridine (BrdU). RESULTS: Erythropoietin receptor was detected on cultured porcine gastric mucosal epithelial cells. Erythropoietin receptor was also detected histochemically at the base of gastric mucosal epithelium. BrdU assay demonstrated a dose-dependent increase in growth potential of cultured porcine gastric mucosal epithelial cells by administration of erythropoietin, as well as these effects were inhibited by administration of anti- erythropoietin antibody (P<0.01). CONCLUSION: These findings indicate that erythropoietin has a potential to proliferate gastric mucosal epithelium via erythropoietin receptor.

Hepcidin production in response to iron is controlled by monocyte-derived humoral factors.

Hepcidin, which is mainly produced by the liver, is the key regulator in iron homeostasis. Hepcidin expression is up-regulated by iron loading in vivo, but the mechanism underlying this process is not completely understood. In the present study, we investigated the mechanism, following the hypothesis that hepcidin production in response to iron loading is regulated by extra-hepatic iron sensors. We measured serum hepcidin concentrations and iron indices in Wistar rats treated with saccharated ferric oxide (SFO). Human hepatoma-derived HepG2 cells were stimulated using SFO-administered rat sera, and co-cultured with rat spleen cells, human monocyte-derived THP-1 cells, or human monocytes with diferric transferrin (holo-Tf), and hepcidin concentrations in the conditioned media were measured. SFO elevated rat serum hepcidin concentrations. SFO-treated rat sera increased hepcidin production from HepG2 cells, and this induction correlated with serum hepcidin levels, but not with iron indices. Holo-Tf up-regulated hepcidin concentrations in media from HepG2 cells co-cultured with rat spleen cells, THP-1 cells, or human monocytes with or without cell-to-cell contacts, while holo-Tf did not up-regulate hepcidin from HepG2 cells alone. Our results suggest the existence of humoral factors capable of inducing hepcidin production that are secreted by extra-hepatic cells, such as reticuloendothelial monocytes, in response to iron.

Left ventricular hypertrophy is associated with inflammation in sodium loaded subtotal nephrectomized rats.

The pathological influences of inflammation on left ventricular hypertrophy (LVH) were studied in subtotal nephrectomized (SNx) rats after 0.3% NaCl loading for 5 weeks. We found that mild hypertension, increased plasma levels of creatinine, inorganic phosphate, asymmetric dimethylarginine (ADMA), and parathyroid hormone (PTH) were observed in the present SNx rats without LVH. In the present study, the NaCl-loaded SNx (SNx + NaCl) rats were characterized by significant LVH and hypertension with aggravated values of all the parameters. We further confirmed that glomerular sclerosis, tubulointerstitial fibrosis, and inflammatory cell infiltration into the tubulointerstitial area, observed in the SNx rats, were more severely caused in the SNx + NaCl rats. In addition, plasma interleukin-6 (IL-6) levels in the SNx + NaCl rats were significantly increased compared to those in the SNx rats. These findings indicated that NaCl-loaded SNx rats developed LVH and hypertension, which were accompanied with increased plasma levels of PTH, creatinine, inorganic phosphorus, ADMA, and IL-6. Thus, these results suggest that inflammation as well as endothelial dysfunction would be correlated with LVH as non-traditional risk factors at the early stage in the present renal failure model.

Effect of erythropoietin on human tumor growth in xenograft models.

Recombinant human erythropoietin (rhEPO) has been used in the EU and the United States for the treatment of anemia in cancer patients after myelosuppressive chemotherapy or radiotherapy. However, several conflicting results have been reported concerning the detrimental effect of rhEPO on survival benefit in cancer patients. In experimental studies, contradictory results were also reported in in vitro tumor cell proliferation studies and in vivo tumor growth studies using tumor cells expressing EPO-receptor (EPO-R). Therefore, we tried to clarify the effect of epoetin ß, a product of rhEPO, on tumor growth in xenograft models using five EPO-R-positive human cancer cell lines, namely the MCF7 breast, 786-O renal, SCH gastric, A549 lung and SK-OV-3 ovarian cancer cell lines. Epoetin ß was administered once a week for 3 weeks at doses of 1,000, 3,000 and 10,000 IU/kg in accordance with the clinical administration schedule and dosages. As a result, no enhancement of tumor growth from the administration of epoetin ß was observed in any of the xenograft models throughout the experiment duration. The effect of epoetin ß on the antitumor activity of bevacizumab, an anti-angiogenic agent, was additionally examined using A549 and MCF7 xenograft models, since rhEPO reportedly stimulates tumor neovascularization. Epoetin ß showed no significant effect on the antitumor activity of bevacizumab in either xenograft model. These findings suggest that epoetin ß is not involved in in vivo tumor growth promotion.

Erythropoietin receptor signaling mitigates renal dysfunction-associated heart failure by mechanisms unrelated to relief of anemia.

OBJECTIVES: We examined the effect of asialoerythropoietin (asialoEPO), a nonerythrogenic derivative of erythropoietin (EPO), on renal dysfunction-associated heart failure. BACKGROUND: Although EPO is known to exert beneficial effects on cardiac function, the clinical benefits in patients with chronic kidney disease are controversial. It remains to be addressed whether previously reported outcomes were the result of relief of the anemia, adverse effects of EPO, or direct cardiovascular effects. METHODS: Mice underwent 5/6 nephrectomy to cause renal dysfunction. Eight weeks later, when renal dysfunction was established, anemia and cardiac dysfunction and remodeling were apparent. Mice were then assigned to receive saline (control), recombinant human erythropoietin (rhEPO) at 5,000 IU (714 pmol)/kg, or asialoEPO at 714 pmol/kg, twice/week for 4 weeks. RESULTS: Although only rhEPO relieved the nephrectomy-induced anemia, both rhEPO and asialoEPO significantly and similarly mitigated left ventricular dilation and dysfunction. The hearts of rhEPO- or asialoEPO-treated mice showed less hypertrophy, reflecting decreases in cardiomyocyte hypertrophy and degenerative subcellular changes, as well as significant attenuation of fibrosis, leukocyte infiltration, and oxidative deoxyribonucleic acid damage. These phenotypes were accompanied by restored expression of GATA-4, sarcomeric proteins, and vascular endothelial growth factor and decreased inflammatory cytokines and lipid peroxidation. Finally, myocardial activation was observed of extracellular signal-regulated protein kinase and signal transducer and activator of transcription pathways in the treated mice. CONCLUSIONS: EPO receptor signaling exerts direct cardioprotection in an animal model of renal dysfunction-associated heart failure, probably by mitigating degenerative, pro-fibrosis, inflammatory, and oxidative processes but not through relief of anemia.

Nicorandil improves glomerular injury in rats with mesangioproliferative glomerulonephritis via inhibition of proproliferative and profibrotic growth factors.

Recent clinical studies on chronic kidney disease (CKD) reported that renal dysfunction was a critical risk factor for cardiovascular events (CVE), which lead us to reconsider the effect of cardioprotective agents on the kidney. Glomerulonephritis, which is the major cause of CKD, is characterized by mesangial cell proliferation and extracellular matrix deposition. Nicorandil, a therapeutic drug for angina and acute heart failure, have been reported to show antiproliferative activity in mesangial cells. In this study, we first investigated the in vivo effects of nicorandil in anti-Thy1 nephritis rats. In male F344 rats, anti-Thy1 nephritis was induced by the injection of an anti-Thy1 antibody. From three days before induction, nicorandil (10, 30 mg/kg per day) was administered in the drinking water for 12 consecutive days. Anti-Thy1 nephritis resulted in a significant increase in proteinuria and glomerular mesangial cell proliferation. In nephritis rats, nicorandil (30 mg/kg per day) significantly suppressed increase in proteinuria, mesangial cell proliferation (the number of glomerular cell and glomerular area), and renal hypertrophy without affecting blood pressure. Nicorandil significantly prevented the overexpression of type I collagen, fibronectin, transforming growth factor (TGF)-beta, and platelet-derived growth factor (PDGF) mRNA. These results suggest that nicorandil may have renoprotective effects in mesangioproliferative glomerulonephritis.

Improvement of impaired calcium and skeletal homeostasis in vitamin D receptor knockout mice by a high dose of calcitriol and maxacalcitol.

Vitamin D plays a major role in mineral and skeletal homeostasis through interaction with the nuclear vitamin D receptor (VDR) of target cells. Recent reports have indicated that some cellular effects of vitamin D may occur via alternative signaling pathways, but concrete evidence for mineral homeostasis has not been shown in vivo. To investigate this issue, the actions of calcitriol (1,25D) and maxacalcitol (OCT), which were developed for treatment of uremia-induced secondary hyperparathyroidism, were analyzed in VDR knockout (VDR(-/-)) mice. The VDR(-/-) mice were fed a rescue diet immediately after weaning. 1,25D, OCT or a control solution was administered intraperitoneally to these mice three times a week for eight weeks. Biological markers and bone growth were measured and bone histomorphometric analysis of the calcein-labeled tibia was performed 24 h after the final administration. Significantly higher levels of serum Ca(2+) were observed in 1,25D- and OCT-treated mice, but the serum parathyroid hormone level was unchanged by both agents. Impaired bone growth, enlarged and distorted cartilaginous growth plates, morphological abnormalities of cancellous and cortical bones; a morbid osteoid increase, lack of calcein labeling, and thinning of cortical bone, were all significantly improved by 1,25D and OCT. The significance of these effects was confirmed by bone histomorphometrical analysis. Upregulation of the calbindin D(9k) mRNA expression level in the duodenum may explain these findings, since this protein is a major modulator of Ca transport in the small intestine. We conclude that 1,25D and OCT both at a high dose exert significant effects on Ca and skeletal homeostasis with the principal improvement of Ca status in VDR(-/-) mice, and some of these effects may occur through an alternative vitamin D signaling pathway.

Severe hyperparathyroidism with bone abnormalities and metastatic calcification in rats with adenine-induced uraemia.

BACKGROUND: Marked parathyroid hyperplasia with bone diseases and vascular calcification are unsolved issues in dialysis patients. In this study, we made azotemic model rats by adenine feeding and analyzed the development and progression of the abnormalities. METHODS: Renal failure was induced in 8-week-old male Wistar rats by feeding 0.75% adenine-containing diet for 6 weeks. Serum parameters, parathyroid hyperplasia, bone changes and metastatic calcification were examined at 2, 4 and 6 weeks. RESULTS: Progressive increase of serum creatinine and inorganic phosphate, and decreased levels of serum calcium and 1,25(OH)2D3 were confirmed. Markedly enlarged parathyroid glands and extremely high PTH levels were observed in all adenine-fed rats compared with the control (PTH: 199.3+/-58.0 vs 10.5+/-3.0 pmol/l, P<0.01, respectively, at 6 weeks). In cortical bone of the femur, the morphometric parameters showed increased bone resorption with increased fibrosis, whereas in the trabecular bone, bone resorption decreased and bone volume increased with a larger amount of osteoid compared with the control. Metastatic calcification in aorta, coronary artery and other soft tissues were also found in adenine-fed rats. CONCLUSIONS: Uraemic rats made by adenine diet developed severe abnormalities of calcium metabolism in a relatively short period and therefore they may serve as a useful model for the analysis of parathyroid hyperplasia and vascular calcification in chronic renal failure.