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• This study reports the development of a rat model of tacrolimus-induced nephropathy with renal interstitial fibrosis via 2 weeks administration of tacrolimus following renal ischemia reperfusion. • The TAC + IRI group, which received tacrolimus daily for 2 weeks, had significantly lower renal function and higher levels of Scr, BUN compared to the other groups. • The TAC + IRI group exhibited noticeable kidney injury, such as vacuolization and glomerulosclerosis, as well as increased α-SMA, TGF-β, and KIM-1 expression in their renal tubulointerstitium compared with the Sham, IRI, and TAC groups. Nephrotoxicity is the major adverse reaction to tacrolimus; however, the underlying mechanisms remain to be fully elucidated. Although several tacrolimus-induced nephrotoxicity animal models have been reported, most renal injury rat models contain factors other than tacrolimus. Here, we report the development of a new nephrotoxicity with interstitial fibrosis rat model induced by tacrolimus administration. Thirty Wistar rats were randomly divided into four groups: sham-operated (Sham), vehicle-treated ischemia reperfusion (I/R) injury (IRI), tacrolimus treated (TAC) and tacrolimus treated I/R injury (TAC + IRI). Rats subjected to IR injury and treated with tacrolimus for 2 weeks showed higher serum creatinine (Scr), blood urea nitrogen (BUN), serum magnesium (Mg) and serum potassium (K), indicating decreased renal function. In addition, tacrolimus treatment combined with IR injury increased histological injury (tubular vacuolation, glomerulosclerosis and interstitial fibrosis), as well as α-smooth muscle actin (α-SMA), transforming growth factor-β (TGF-β), and kidney injury molecule-1 (KIM-1) expression in the renal cortex. In summary, we have developed a tacrolimus-induced kidney injury rat model with interstitial fibrosis within 2 weeks by creating conditions mimicking renal transplantation via tacrolimus administration following ischemia-reperfusion. [ABSTRACT FROM AUTHOR]

Purpose: Angiotensin II (ANG II) has been shown to affect iron metabolism through alteration of iron transporters, leading to increased cellular and tissue iron contents. Serum ferritin, a marker of body iron storage, is elevated in various cardiovascular diseases, including hypertension. However, the associated changes in iron absorption and the mechanism underlying increased iron content in a hypertensive state remain unclear. Methods: The C57BL6/J mice were treated with ANG II to generate a model of hypertension. Mice were divided into three groups: (1) control, (2) ANG II-treated, and (3) ANG II-treated and ANG II receptor blocker (ARB)-administered (ANG II-ARB) groups. Results: Mice treated with ANG II showed increased serum ferritin levels compared to vehicle-treated control mice. In ANG II-treated mice, duodenal divalent metal transporter-1 and ferroportin (FPN) expression levels were increased and hepatic hepcidin mRNA expression and serum hepcidin concentration were reduced. The mRNA expression of bone morphogenetic protein 6 and CCAAT/enhancer-binding protein alpha, which are regulators of hepcidin, was also down-regulated in the livers of ANG II-treated mice. In terms of tissue iron content, macrophage iron content and renal iron content were increased by ANG II treatment, and these increases were associated with reduced expression of transferrin receptor 1 and FPN and increased expression of ferritin. These changes induced by ANG II treatment were ameliorated by the administration of an ARB. Conclusions: Angiotensin II (ANG II) altered the expression of duodenal iron transporters and reduced hepcidin levels, contributing to the alteration of body iron distribution. [ABSTRACT FROM AUTHOR]

Tacrolimus—a widely used immunosuppressant to prevent allograft rejection after organ transplantation—is nephrotoxic, increasing the risk of kidney injury accompanied by kidney fibrosis. The mammalian target of rapamycin (mTOR) inhibitor, everolimus, is an immunosuppressant used together with tacrolimus. Although mTOR signaling inhibition has been demonstrated to exhibit antifibrotic effects, the efficacy of everolimus against tacrolimus-induced kidney fibrosis has not been explored. Therefore, we evaluated the protective effects of everolimus against tacrolimus-induced kidney fibrosis. To assess antifibrotic effect of everolimus against tacrolimus-induced kidney fibrosis, male Wistar rats were subcutaneously administered vehicle or tacrolimus (5 mg/kg per day) and/or everolimus (0.2 mg/kg per day) for 2 weeks after bilateral renal ischemia for 45 min. The antifibrotic effect of everolimus was also assessed using rat kidney fibroblast cell line (NRK-49F). Tacrolimus administration increased predominant profibrotic cytokine transforming growth factor-β (TGF-β) and fibroblast activation marker α-smooth muscle actin (α-SMA) expression and promoted the infiltration of macrophages in the kidney cortex, resulting in renal interstitial fibrosis in rats. Tacrolimus increased serum creatinine, blood urea nitrogen, kidney injury molecule-1 (KIM-1), and kidney injuries, such as tubular dilation, vacuolization, and glomerular atrophy. Everolimus administration attenuated tacrolimus-induced kidney fibrosis and the associated abnormalities. Everolimus strongly suppressed TGF-β-induced kidney fibroblast activation and extracellular matrix protein expression by the mTOR signaling inhibition. We demonstrated that everolimus attenuates tacrolimus-induced renal interstitial fibrosis in rats. Owing to its protective effect against tacrolimus-induced kidney fibrosis, everolimus may be useful when used concomitantly with tacrolimus. • Tacrolimus-induced kidney fibrosis is a major complication after organ transplantations. • Everolimus attenuated tacrolimus-induced kidney fibrosis and dysfunction in rats. • Everolimus suppressed TGF-β induced kidney fibroblasts activation by its mTOR inhibition. • Antifibrotic effect of everolimus may be useful when used concomitantly with tacrolimus. [ABSTRACT FROM AUTHOR]

Hepcidin, a liver-derived iron regulatory protein, plays a crucial role in iron metabolism. It is known that gender differences exist with respect to iron storage in the body; however, the effects of sex steroid hormones on iron metabolism are not completely understood. We focused on the effects of the female sex hormone estrogen on hepcidin expression. First, ovariectomized (OVX) and sham-operated mice were employed to investigate the effects of estrogen on hepcidin expression in an in vivo study. Hepcidin expression was decreased in the livers of OVX mice compared to the sham-operated mice. In OVX mice, bone morphologic protein-6 (BMP6), a regulator of hepcidin, was also found to be downregulated in the liver, whereas ferroportin (FPN), an iron export protein, was upregulated in the duodenum. Both serum and liver iron concentrations were elevated in OVX mice relative to their concentrations in sham-operated mice. In in vitro studies, 17β-estradiol (E2) increased the mRNA expression of hepcidin in HepG2 cells in a concentration-dependent manner. E2-induced hepatic hepcidin upregulation was not inhibited by ICI 182720, an inhibitor of the estrogen receptor; instead, hepcidin expression was increased by ICI 182720. E2 and ICI 182720 exhibit agonist actions with G-protein coupled receptor 30 (GPR30), the 7-transmembrane estrogen receptor. G1, a GPR30 agonist, upregulated hepcidin expression, and GPR30 siRNA treatment abolished E2-induced hepcidin expression. BMP6 expression induced by E2 was abolished by GPR30 silencing. Finally, both E2 and G1 supplementation restored reduced hepatic hepcidin and BMP6 expression and reversed the augmentation of duodenal FPN expression in the OVX mice. In contrast, serum hepcidin was elevated in OVX mice, which was reversed in these mice with E2 and G1. Thus, estrogen is involved in hepcidin expression via a GPR30-BMP6-dependent mechanism, providing new insight into the role of estrogen in iron metabolism. [ABSTRACT FROM AUTHOR]

Iron is an essential trace metal for most organisms. However, excess iron causes oxidative stress through production of highly toxic hydroxyl radicals via the Fenton/Haber-Weiss reaction. Iron storage in the body is reported to be associated with fat accumulation and type 2 diabetes mellitus. We investigated the role of iron in adiposity by using KKAy mice and obese and diabetic model mice. Eight-week-old KKAy mice were divided into two groups and treated with deferoxamine (DFO), an iron chelator agent, or a vehicle for 2 wk. DFO treatment diminished fat iron concentration and serum ferritin levels in KKAy mice. Fat weight and adipocyte size were reduced significantly in DFO-treated mice compared with vehicle-treated mice. Macrophage infiltration into fat was also decreased in DFO-treated mice compared with vehicle-treated mice. Superoxide production and NADPH oxidase activity in fat, as well as urinary 8-hydroxy-2′-deoxyguanosine excretion, were decreased in KKAy mice after DFO treatment while p22phox expression in adipose tissue was diminished in such mice. Ferritin expression in the fat of DFO-treated KKAy mice was decreased. In addition, F4/80-positive cells also presented through both p22phox and ferritin expression. The mRNA expression levels of inflammatory cytokines were also reduced in fat tissue of DFO-treated mice. These findings suggest that reduction of iron levels ameliorates adipocyte hypertrophy via suppression of oxidative stress, inflammatory cytokines, and macrophage infiltration, thereby breaking a vicious cycle in obesity. [ABSTRACT FROM AUTHOR]

Abstract: Background: Deferoxamine (DFO), an iron chelator for disorders of excess iron, upregulates the expression of angiogenic factors, such as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2), indicating that it affects angiogenesis. Herein, we clarify the effect and mechanism of action of DFO on angiogenesis. Methods and results: In an in vitro study, DFO increased endothelial nitric oxide synthesis (eNOS) phosphorylation in human aortic endothelial cells (HAECs), which were inhibited by the phosphatidylinositol 3-kinase inhibitor LY294002. Tube formation, cell proliferation, and cell migration in HAECs were promoted by DFO, which were significantly reduced by LY294002. In an in vivo study, DFO promoted blood flow recovery in response to the hindlimb ischemia in mice with unilateral hindlimb surgery. The density of capillaries and arterioles in ischemic muscle was higher in DFO-treated mice compared to vehicle-treated mice. Endothelial cell proliferation increased and oxidative stress and apoptosis decreased in ischemic muscles of DFO-treated mice. The phosphorylation of Akt and eNOS on the ischemic side was elevated and urinary nitric oxide/nitric dioxide (NOx) excretion was higher in DFO-treated mice compared to vehicle-treated mice. The effect of DFO on angiogenesis was abolished in eNOS-deficient mice with hindlimb ischemia. Conclusion: These findings indicate that DFO promotes revascularization via the activation of vascular endothelial cell function by an Akt–eNOS-dependent mechanism. [Copyright &y& Elsevier]

Several biomarkers are used to monitor organ damage caused by drug toxicity. Traditional markers of kidney function, such as serum creatinine and blood urea nitrogen are commonly used to estimate glomerular filtration rate. However, these markers have several limitations including poor specificity and sensitivity. A number of serum and urine biomarkers have recently been described to detect kidney damage caused by drugs such as cisplatin, gentamicin, vancomycin, and tacrolimus. Neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid-binding protein (L-FABP), kidney injury molecule-1 (KIM-1), monocyte chemotactic protein-1 (MCP-1), and cystatin C have been identified as biomarkers for early kidney damage. Hy's Law is widely used as to predict a high risk of severe drug-induced liver injury caused by drugs such as acetaminophen. Recent reports have indicated that glutamate dehydrogenase (GLDH), high-mobility group box 1 (HMGB-1), Keratin-18 (k18), MicroRNA-122 and ornithine carbamoyltransferase (OCT) are more sensitive markers of hepatotoxicity compared to the traditional markers including the blood levels of amiotransferases and total bilirubin. Additionally, the rapid development of proteomic technologies in biofluids and tissue provides a new multi-marker panel, leading to the discovery of more sensitive biomarkers. In this review, an update topics of biomarkers for the detection of kidney or liver injury associated with pharmacotherapy. [ABSTRACT FROM AUTHOR]

Kidney transplantation is the treatment of choice for patients with advanced chronic kidney disease (CKD) and end stage renal disease (ESRD). However, acute rejection (AR) is a common complication in kidney transplantation and is associated with reduced graft survival. Current diagnosis of AR relies mainly on clinical monitoring including serum creatinine, proteinuria, and confirmation by histopathologic assessment in the biopsy specimen of graft kidney. Although an early protocol biopsy is indispensable for depicting the severity of pathologic lesions in subclinical acute rejection (subAR), it is not acceptable in some cases and cannot be performed because of its invasive nature. Therefore, we examined the detection of noninvasive biomarkers that are closely related to the pathology of subAR in protocol biopsies three months after kidney transplantation. In this study, the urinary level of microtubule-associated protein 1 light chain 3 (LC3), monocyte chemotactic protein-1 (MCP-1), liver-type fatty acid-binding protein (L-FABP), neutrophil gelatinase-associated lipocalin (NGAL), and human epididymis secretory protein 4 (HE4) were measured three months after kidney transplantation. Urine samples of 80 patients undergoing kidney transplantation between August 2014 to September 2016, were prospectively collected after three months. SubAR was observed in 11 patients (13.8%) in protocol biopsy. The urinary levels of LC3, MCP-1, NGAL, and HE4 were significantly higher in patients with subAR than in those without, while those of L-FABP did not differ between the two groups. Multivariate regression models, receiver-operating characteristics (ROC), and areas under ROC curves (AUC) were used to identify predicted values of subAR. Urinary HE4 levels were able to better identify subAR (AUC = 0.808) than the other four urinary biomarkers. In conclusion, urinary HE4 is increased in kidney transplant recipients of subAR three months after kidney transplantation, suggesting that HE4 has the potential to be used as a novel clinical biomarker for predicting subAR. [ABSTRACT FROM AUTHOR]

Renal fibrosis plays an important role in the onset and progression of chronic kidney diseases (CKD). Although several mechanisms underlying renal fibrosis and candidate drugs for its treatment have been identified, the effect of iron chelator on renal fibrosis remains unclear. In the present study, we examined the effect of an iron chelator, deferoxamine (DFO), on renal fibrosis in mice with surgically induced unilateral ureter obstruction (UUO). Mice were divided into 4 groups: UUO with vehicle, UUO with DFO, sham with vehicle, and sham with DFO. One week after surgery, augmented renal tubulointerstitial fibrosis and the expression of collagen I, III, and IV increased in mice with UUO; these changes were suppressed by DFO treatment. Similarly, UUO-induced macrophage infiltration of renal interstitial tubules was reduced in UUO mice treated with DFO. UUO-induced expression of inflammatory cytokines and extracellular matrix proteins was abrogated by DFO treatment. DFO inhibited the activation of the transforming growth factor-β1 (TGF-β1)-Smad3 pathway in UUO mice. UUO-induced NADPH oxidase activity and p22phox expression were attenuated by DFO. In the kidneys of UUO mice, divalent metal transporter 1, ferroportin, and ferritin expression was higher and transferrin receptor expression was lower than in sham-operated mice. Increased renal iron content was observed in UUO mice, which was reduced by DFO treatment. These results suggest that iron reduction by DFO prevents renal tubulointerstitial fibrosis by regulating TGF-β-Smad signaling, oxidative stress, and inflammatory responses. [ABSTRACT FROM AUTHOR]

Excess iron causes oxidative stress through hydroxyl-radical production via Fenton/ Haber-Weiss reactions. Recently, body iron reduction has been found to ameliorate diabetes. In the present study, we examined the protective effect of dietary iron restriction against diabetic nephropathy in the db/db mouse model of diabetic nephropathy using db/m mice as controls. The db/db mice were divided into two groups and fed a normal diet (ND) or a low-iron diet (LID). Increasing urinary albumin excretion was observed in the ND db/db mice, but this was suppressed in db/db mice with LID. Histologically, the db/db mice in the ND group had increased glomerular volume and mesangial area compared with the LID group. Augmented deposition of extracellular matrixes was decreased in db/db mice with LID. In terms of oxidative stress, increased superoxide production observed in the kidneys of the ND db/db mice was diminished in the LID group. NADPH oxidase activity and renal expression of NADPH oxidase components p22phox and NADPH oxidase 4 (NOX4) were augmented in the ND group, and this was abolished by LID. There were no differences in expression of renal iron importers, transferrin receptor, or divalent metal transporter-1 between db/m mice and db/db mice. The level of ferroportin, an iron exporter, increased in the kidneys of the db/db mice. Urinary iron excretion was significantly higher in ND db/db mice and was reduced in the LID group. These findings suggest that dietary iron restriction exerts a preventive effect on the progression of diabetic nephropathy partly due to the reduction of oxidative stress. [ABSTRACT FROM AUTHOR]

Tacrolimus is widely used as an immunosuppressant in liver transplantation, and tacrolimus-induced acute kidney injury (AKI) is a serious complication. The urinary neutrophil gelatinase-associated lipocalin (NGAL) level has been linked to tacrolimus-induced AKI in patients starting tacrolimus treatment the morning after liver transplantation. Here we tested this association using a different immunosuppression protocol: Mycophenolate mofetil administration beginning on Postoperative Day 1 and tacrolimus administration beginning on Postoperative Day 2 or 3. Urine samples were collected from 26 living donor liver transplant recipients before (Postoperative Day 1) and after (Postoperative Day 7 or 14) tacrolimus administration. NGAL levels were measured via enzyme-linked immunosorbent assays, as were those of three additional urinary biomarkers for kidney diseases: Monocyte chemotactic protein-1 (MCP-1), liver-type fatty acid-binding protein (L-FABP), and human epididymis secretory protein 4 (HE4). HE4 levels after tacrolimus administration were significantly higher in patients who developed AKI (n = 6) than in those who did not (n = 20), whereas NGAL, MCP-1, and L-FABP levels did not differ significantly before or after tacrolimus administration. These findings indicate that NGAL may not be a universal biomarker of AKI in tacrolimus-treated liver transplant recipients. To reduce the likelihood of tacrolimus-induced AKI, our immunosuppression protocol is recommended. [ABSTRACT FROM AUTHOR]

We previously clarified that heparin cofactor II (HCII), a serine proteinase inhibitor, exerts various protective actions on cardiovascular diseases in both experimental and clinical studies. In the present study, we aimed to clarify whether HCII participates in the regulation of angiogenesis. Male heterozygous HCII-deficient (HCII+/-) mice and male littermate wild-type (HCII+/+) mice at the age of 12-16 weeks were subjected to unilateral hindlimb ligation surgery. Laser speckle blood flow analysis showed that blood flow recovery in response to hindlimb ischemia was delayed in HCII+/- mice compared with that in HCII+/+ mice. Capillary number, arteriole number, and endothelial nitric-oxide synthase (eNOS), AMP-activated protein kinase (AMPK), and liver kinase B1 (LKB1) phosphorylation in ischemic muscles were decreased in HCII+/- mice. Human purified HCII (h-HCII) administration almost restored blood flow recovery, capillary density, and arteriole number as well as phosphorylation levels of eNOS, AMPK, and LKB1 in ischemic muscles of HCII+/- mice. Although treatment with h-HCII increased phosphorylation levels of eNOS, AMPK, and LKB1 in human aortic endothelial cells (HAECs), the h-HCIIinduced eNOS phosphorylation was abolished by compound C, an AMPK inhibitor, and by AMPK siRNA. In a similar fashion, tube formation, proliferation, and migration of HAECs were also promoted by h-HCII treatment and were abrogated by pretreatment with compound C. HCII potentiates the activation of vascular endothelial cells and the promotion of angiogenesis in response to hindlimb ischemia via an AMPK-eNOS signaling pathway. These findings suggest that HCII is a novel therapeutic target for treatment of patients with peripheral circulation insufficiency. [ABSTRACT FROM AUTHOR]