Seok Joon Shin

Preconditioning with erythropoietin protects against subsequent ischemia-reperfusion injury in rat kidney

Improving the ability of the kidney to tolerate ischemic injury has important implications. We investigated the effect of recombinant human erythropoietin (rHuEPO) treatment on subsequent ischemia/reperfusion (I/R) injury and evaluated the role of heat shock protein (HSP) 70 in rHuEPO‐induced renal protection. rHuEPO (3000 U/kg) was administered 24 h before I/R injury, and rats were killed at 24, 48, and 72 h after I/R injury. Pretreatment of rHuEPO resulted in the following: i) decreased serum creatinine level; ii) decreased tubular cell apoptosis and necrosis, measured by DNA fragmentation analysis and TUNEL staining and histomorphological criteria; iii) decreased tubular cell proliferation as determined by proliferating cell nuclear antigen expression; iv) increased bcl‐2 protein and decreased caspase 3 activity; and v) decreased JNK expression. rHuEPO treatment increased HSP70 expression in a dose‐dependent manner in normal rat kidneys, and inhibition of HSP70 expression by quercetin eliminated the renoprotective effect of rHuEPO in ischemic kidneys. Our study demonstrates that rHuEPO has a protective effect on subsequent I/R injury and that this effect is associated with induction of HSP70. Our study provides a new avenue for therapy to prevent renal damage after I/R injury.

Peroxisome proliferator-activated receptor-α activator fenofibrate prevents high-fat diet-induced renal lipotoxicity in spontaneously hypertensive rats

We investigated the effects of a high-fat (HF) diet and peroxisome proliferator-activated receptor (PPAR)-α activation on the intrarenal lipotoxicity associated with the renin–angiotensin system (RAS) and oxidative stress using spontaneously hypertensive (SHR) rats. Male SHR and Wistar–Kyoto (WKY) rats at 8 weeks of age were fed either a normal-fat diet or an HF diet without or with fenofibrate treatment for 12 weeks. Severe intrarenal lipid accumulation was noted in the SHR rats fed an HF diet than in WYK rats fed an HF diet (P<0.05). This lipid accumulation was associated with a 70% decrease in renal PPARα expression in SHR rats, whereas an HF diet increased the expression of PPARα in WKY rats by threefold. An HF diet also activated intrarenal, not systemic, RAS and induced oxidative stress associated with reduced nitric oxide (NO) bioavailability. By contrast, fenofibrate attenuated weight gain, fat mass and insulin resistance. Fenofibrate recovered HF diet-induced decreases in intrarenal PPARα expression and fat accumulation, and abolished intrarenal RAS activation and oxidative stress in SHR–HF animals (P<0.01). These activities conferred protection against increased blood pressure (BP), glomerulosclerosis and renal inflammation. Intrarenal free fatty acid and triglyceride concentrations were positively correlated with angiotensin II (γ=0.63, 0.36) and 24-h urinary 8-hydroxy-deoxyguanosine (γ=0.36, 0.39), and negatively correlated with PPARα contents (γ=−0.47, −0.44; P<0.05). An HF diet-induced lipotoxicity by depletion of intrarenal PPARα aggravated BP and renal inflammation as a result of intrarenal RAS activation and oxidative stress. Therefore, intervention with PPARα activators can effectively prevent diet-induced renal lipotoxicity in hypertensive rats.

Age-Associated Molecular Changes in the Kidney in Aged Mice

Background. Aging is a multifactorial process characterized by a progressive decline in physiological function. Decreased kidney function is associated with cardiovascular disease and mortality. Therefore, increasing our insight into kidney aging by understanding the anatomic, physiologic, and pathologic changes of aging in the kidney is important to prevent disastrous outcomes in elderly people. Methods. Male two-, 12-, and 24-month-old C57/BL6 mice were used in this study. We measured histological change, oxidative stress, and aging-related protein expression in the kidneys. Results. Twenty-four-month-old mice displayed increased albuminuria. Creatinine clearance decreased with aging, although this was not statistically significant. There were increases in mesangial volume and tubulointerstitial fibrosis in 24-month-old mice. There were also increases in F4/80 expression and in apoptosis detected by TUNEL assay. Urine isoprostane excretion increased with aging and SOD1 and SOD2 were decreased in 24-month-old mice. Oxidative stress may be mediated by a decrease in Sirt1, PGC-1α, ERR-1α, and PPARα expression. Klotho expression also decreased. Conclusions. Our results demonstrate that Sirt1 was decreased with aging and may relate to changed target molecules including PGC-1α/ERR-1α signaling and PPARα. Klotho can also induce oxidative stress. Pharmacologically targeting these signaling molecules may reduce the pathologic changes of aging in the kidney.

High-fat diet-induced renal cell apoptosis and oxidative stress in spontaneously hypertensive rat are ameliorated by fenofibrate through the PPARα–FoxO3a–PGC-1α pathway

BACKGROUND The peroxisome proliferator-activated receptor-α (PPARα) is a lipid-sensing transcriptional factor that has a role in gluco-oxidative stress and lipotoxicity. Forkhead box O (FoxO)s and peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α are also known to regulate cell metabolism, cell cycle arrest, apoptosis and oxidative stress during stressful conditions. We evaluated whether PPARα-FoxOs-PGC-1α signaling in overfed spontaneously hypertensive rats (SHR) has a protective role in the kidney. METHODS Male SHR and Wistar-Kyoto rats (WKY) fed a high-fat diet (HFD) received treatment with fenofibrate, PPARα agonist or tempol, antioxidants for 12 weeks and were evaluated about the PPARα-FoxOs-PGC-1α pathway. RESULTS The SHRs with an HFD had an elevated systolic pressure, plasma insulin, free fatty acid (FFA) and triglyceride (TGs) levels, and they had induced glucose intolerance as well as albuminuria, glomerular expansion and renal inflammation. An HFD caused the accumulation of intra-renal FFA and TGs and this was related to a decrease in the PPARα expression, the activation of phosphatidylinositol 3-kinase (PI3K)-Akt, phosphorylation of FoxO3a and decreases in the PGC-1α and estrogen-related receptor (ERR)-1α expressions, which suppressed the superoxide dismutase (SOD2) and Bcl-2 expressions and led to increases in oxidative stress and the number of apoptotic renal cells. Interestingly, administering fenofibrate or tempol to the HFD-induced SHRs reversed all of the renal phenotypes by increasing the PPARα expression with concomitant inactivation of the PI3K-Akt pathway, dephosphorylation of FoxO3a and activation of PGC-1α-ERR-1α signaling, and this all resulted in ameliorating the oxidative stress and apoptotic cell death. CONCLUSION Our results demonstrated that PPARα agonists or antioxidants are associated with improvement of the circulating FFA and TGs levels and this prevents HFD-induced renal lipotoxicity and hypertension by the activation of PPARα and its downstream signals of both FoxO3a and PGC-1α.

Tempol or candesartan prevents high-fat diet-induced hypertension and renal damage in spontaneously hypertensive rats

BACKGROUND Obesity has been strongly associated with the development and aggravation of hypertension and chronic kidney disease. To date, the systemic renin-angiotensin system (RAS) has been known to involve in obesity-induced tissue damage and hypertension. However, the intrarenal mechanism whereby obesity induces and aggravates hypertension and renal disease remains poorly understood. Therefore, we investigated the role of intrarenal RAS and oxidative stress in diet-induced hypertension and renal inflammation in spontaneously hypertensive rats (SHR) fed a high-fat diet. METHODS Male SHR and Wistar-Kyoto rats (WKY) were divided into eight groups: normal-fat diet-fed WKY (WKY-NF), high-fat diet-fed WKY (WKY-HF), high-fat diet-fed tempol-treated WKY (WKY-HF/T), high-fat diet-fed candesartan-treated WKY (WKY-HF/C), normal-fat diet-fed SHR (SHR-NF), high-fat diet-fed SHR (SHR-HF), high-fat diet-fed tempol-treated SHR (SHR-HF/T) and high-fat diet-fed candesartan-treated SHR (SHR-HF/C). After 12 weeks of treatment, haemodynamic measurements and histological assessment of the kidney were performed. RESULTS At the end of week 12, the high-fat fed SHR gained more body weight, their systolic blood pressure was further elevated and glucose intolerance induced. There was no significant difference in the insulin resistance index, serum lipid profile, plasma renin activity and serum aldosterone levels according to diet. However, the high-fat diet resulted in increases in immunohistochemical stains of renin and angiotensin II in the kidney. The real-time PCR also demonstrated significant increases in mRNA levels of renin, angiotensinogen and angiotensin-converting enzyme in the kidney, reflecting enhanced activation of the intrarenal RAS, which findings were also shown by Western blot analysis for renin and angiotensin II type 1 receptor. The expression of ED-1, osteopontin and TGF-beta1 in the renal cortex were prominently enhanced in the SHR-HF group with the increased intrarenal lipid concentrations and oxidative stress. Administration of tempol or candesartan in the high-fat diet-induced SHR inhibited the elevation of the systolic blood pressure, intrarenal lipid concentrations, oxidative stress and the degree of renal inflammation to the levels of, or more than, the SHR-NF with no differences in the body weight and periepididymal fat weight, compared to those in the SHR-HF group without such treatment. CONCLUSIONS Our study suggests that a high-fat diet induces fatty kidneys, aggravation of blood pressure and renal inflammation in the SHR. Blockade of oxidative stress by tempol or of RAS by candesartan ameliorates the increase in blood pressure and renal inflammation and improves intrarenal lipid accumulation. Therefore, antioxidants or angiotensin receptor blockers can prevent diet-induced hypertension and renal inflammation in hypertensive rats.

Vascular Endothelial Growth Factor Inhibition by dRK6 Causes Endothelial Apoptosis, Fibrosis, and Inflammation in the Heart via the Akt/eNOS Axis in db/db Mice

OBJECTIVE Vascular endothelial growth factor (VEGF), which is associated with the stimulation of angiogenesis and collateral vessel synthase, is one of the crucial factors involved in cardiac remodeling in type 2 diabetes. RESEARCH DESIGN AND METHODS We investigated VEGF inhibition by dRK6 on the heart in an animal model of type 2 diabetes. Male db/db and db/m mice either were treated with dRK6 starting at 7 weeks of age for 12 weeks (db/db-dRK6 and db/m-dRK6) or were untreated. RESULTS Cardiac dysfunction and hypertrophy were noted by echocardiogram and molecular markers in the db/db-dRK6 mice. The presence of diabetes significantly suppressed the expression of VEGF receptor (VEGFR)-1 and VEGFR-2, phospho-Akt, and phospho-endothelial nitric oxide synthase (eNOS) in the heart. In db/db-dRK6 mice, dRK6 completely inhibited VEGFR-2, phospho-Akt, and phospho-eNOS expression, whereas no effect on VEGFR-1 was observed. Cardiac fibrosis, microvascular scarcity associated with an increase in apoptotic endothelial cells, and inflammation were prominent, as well as increase in antiangiogenic growth factors. Cardiac 8-hydroxy-deoxyguanine and hypoxia-inducible factor-1α expression were significantly increased. No such changes were found in the other groups, including the db/m-dRK6 mice. The number of apoptotic human umbilical vein endothelial cells was increased by dRK6 in a dose-dependent manner only at high glucose concentrations, and this was associated with a decrease in phospho-Akt and phospho-eNOS related to oxidative stress. CONCLUSIONS Our results demonstrated that systemic blockade of VEGF by dRK6 had deleterious effects on the heart in an animal model of type 2 diabetes; dRK6 induced downregulation of the VEGFR-2 and Akt-eNOS axis and enhancement of oxidative stress.

Vascular endothelial growth factor-receptor 1 inhibition aggravates diabetic nephropathy through eNOS signaling pathway in db/db mice.

The manipulation of vascular endothelial growth factor (VEGF)-receptors (VEGFRs) in diabetic nephropathy is as controversial as issue as ever. It is known to be VEGF-A and VEGFR2 that regulate most of the cellular actions of VEGF in experimental diabetic nephropathy. On the other hand, such factors as VEGF-A, -B and placenta growth factor bind to VEGFR1 with high affinity. Such notion instigated us to investigate on whether selective VEGFR1 inhibition with GNQWFI hexamer aggravates the progression of diabetic nephropathy in db/db mice. While diabetes suppressed VEGFR1, it did increase VEGFR2 expressions in the glomerulus. Db/db mice with VEGFR1 inhibition showed more prominent features with respect to, albuminuria, mesangial matrix expansion, inflammatory cell infiltration and greater numbers of apoptotic cells in the glomerulus, and oxidative stress than that of control db/db mice. All these changes were related to the suppression of diabetes-induced increases in PI3K activity and Akt phosphorylation as well as the aggravation of endothelial dysfunction associated with the inactivation of FoxO3a and eNOS-NOx. In cultured human glomerular endothelial cells (HGECs), high-glucose media with VEGFR1 inhibition induced more apoptotic cells and oxidative stress than did high-glucose media alone, which were associated with the suppression of PI3K-Akt phosphorylation, independently of the activation of AMP-activated protein kinase, and inactivation of FoxO3a and eNOS-NOx pathway. In addition, transfection with VEGFR1 siRNA in HGECs also suppressed PI3K-Akt-eNOS signaling. In conclusion, the specific blockade of VEGFR1 with GNQWFI caused severe renal injury related to profound suppression of the PI3K-Akt, FoxO3a and eNOS-NOx pathway, giving rise to the oxidative stress-induced apoptosis of glomerular cells in type 2 diabetic nephropathy.

Oleanolic acid attenuates renal fibrosis in mice with unilateral ureteral obstruction via facilitating nuclear translocation of Nrf2

BackgroundRenal interstitial fibrosis is a common final pathological process in the progression of kidney disease. This is primarily due to oxidative stress, which contributes to renal inflammation and fibrosis. Nuclear factor-erythroid-2-related factor 2 (Nrf2) is known to coordinate induction of genes that encode antioxidant enzymes. We investigated the effects of oleanolic acid, a known Nrf2 activator, on oxidative stress-induced renal inflammation and fibrosis.MethodsOne day before unilateral ureteral obstruction (UUO) performed in C57BL/6 mice, oleanolic acid treatment was initiated and was continued until 3 and 7 days after UUO. Renal inflammation and fibrosis, markers of oxidative stress, and changes in Nrf2 expression were subsequently evaluated.ResultsIn the obstructed kidneys of UUO mice, oleanolic acid significantly attenuated UUO-induced collagen deposition and fibrosis on day 7. Additionally, significantly less inflammatory cell infiltration, a lower ratio of Bax to Bcl-2 expression, and fewer apoptotic cells on TUNEL staining were observed in the obstructed kidneys of oleanolic acid-treated mice. Oleanolic acid increased the expression of nuclear Nrf2, heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and heat shock protein 70, and decreased lipid peroxidation in the obstructed kidney of UUO mice. There were no changes in the expression of total Nrf2 and Kelch-like ECH-associated protein 1, indicating that oleanolic acid enhanced nuclear translocation of Nrf2.ConclusionsThese results suggest that oleanolic acid may exert beneficial effects on renal fibrosis by increasing nuclear translocation of Nrf2 and subsequently reducing renal oxidative stress.

Effect of Sodium-Glucose Co-Transporter 2 Inhibitor, Dapagliflozin, on Renal Renin-Angiotensin System in an Animal Model of Type 2 Diabetes

Background Renal renin-angiotensin system (RAS) activation is one of the important pathogenic mechanisms in the development of diabetic nephropathy in type 2 diabetes. The aim of this study was to investigate the effects of a sodium-glucose co-transporter 2 (SGLT-2) inhibitor, dapagliflozin, on renal RAS in an animal model with type 2 diabetes. Methods Dapagliflozin (1.0 mg/kg, OL-DA) or voglibose (0.6 mg/kg, OL-VO, diabetic control) (n = 10 each) was administered to Otsuka Long-Evans Tokushima Fatty (OLETF) rats for 12 weeks. We used voglibose, an alpha-glucosidase inhibitor, as a comparable counterpart to SGLT2 inhibitor because of its postprandial glucose-lowering effect without proven renoprotective effects. Control Long-Evans Tokushima Otsuka (LT) and OLETF (OL-C) rats received saline (n = 10, each). Changes in blood glucose, urine albumin, creatinine clearance, and oxidative stress were measured. Inflammatory cell infiltration, mesangial widening, and interstitial fibrosis in the kidney were evaluated by histological analysis. The effects of dapagliflozin on renal expression of the RAS components were evaluated by quantitative RT-PCR in renal tissue. Results After treatment, hyperglycemia and urine microalbumin levels were attenuated in both OL-DA and OL-VO rather than in the OL-C group (P < 0.05). The urine angiotensin II (Ang II) and angiotensinogen levels were significantly decreased following treatment with dapagliflozin or voglibose, but suppression of urine Ang II level was more prominent in the OL-DA than the OL-VO group (P < 0.05). The expressions of angiotensin type 1 receptor and tissue oxidative stress markers were markedly increased in OL-C rats, which were reversed by dapagliflozin or voglibose (P < 0.05, both). Inflammatory cell infiltration, mesangial widening, interstitial fibrosis, and total collagen content were significantly increased in OL-C rats, which were attenuated in OL-DA group (P < 0.05). Conclusion Dapagliflozin treatment showed beneficial effects on diabetic nephropathy, which might be via suppression of renal RAS component expression, oxidative stress and interstitial fibrosis in OLETF rats. We suggest that, in addition to control of hyperglycemia, partial suppression of renal RAS with an SGLT2 inhibitor would be a promising strategy for the prevention of treatment of diabetic nephropathy.

Long-term blockade of vascular endothelial growth factor receptor-2 aggravates the diabetic renal dysfunction associated with inactivation of the Akt/eNOS-NO axis

BACKGROUND Diabetic nephropathy is characterized by abnormal angiogenesis, and this is driven by several factors, including hyperglycaemia and ischaemia. We investigated the role of vascular endothelial growth factor receptor-2 (VEGFR-2) blockade and its effects on diabetic nephropathy. METHODS Male db/db and db/m mice received long-term treatment with dRK6, an arginine-rich anti-VEGF hexapeptide, for 12 weeks or short-term treatment for only the first 4 weeks, starting from 8 weeks of age. RESULTS The urinary albuminuria and VEGF excretion varied according to the duration of diabetes, and the urinary VEGF levels were strongly correlated with the levels of albuminuria. Diabetes increased the VEGFR-2 expression in the kidneys. At the end of the 12-week study, compared with the db/db control mice, the db/db mice with long-term dRK6 treatment, which selectively inhibited VEGFR-2, had more albuminuria, related to weak nephrin signalling and advanced renal phenotypes, which were associated with hypoxia-oxidative stress, and an increased number of apoptotic endothelial cells. Interestingly, these changes were related to a decrease in phospho-Akt/eNOS-NO bioavailability. On the in vitro study, dRK6 increased the number of apoptotic human umbilical vein endothelial cells (HUVECs) in the high glucose media by blocking phospho-Akt/eNOS-NO signalling, and this was related to the increased oxidative stress. The short-term inhibition of VEGFR-2 neither improved the albuminuria nor the renal phenotype induced by diabetes. CONCLUSIONS Long-term selective blockade of VEGFR-2 by dRK6 had deleterious renal effects, and this was associated with downregulation of the Akt/eNOS-NO axis in db/db mice. Short-term VEGFR-2 blockade did not improve the renal phenotypes and the albuminuria. These findings suggest that VEGF-A-VEGFR-2 inhibition, regardless of how long it may be, does not ameliorate diabetic nephropathy in type 2 diabetes.