Downregulation of uncoupling protein-2 by genipin exacerbates diabetes-induced kidney proximal tubular cells apoptosis.

Renal tubular epithelial cell injury is a major pathological event that contributes to the development of diabetic kidney disease (DKD). Uncoupling protein-2 (UCP2), a mitochondrial membrane protein, has been reported to participate in the regulation of reactive oxygen species (ROS) generation, which contributes to tubular cell apoptosis induced by hyperglycemia. In this study, we found that genipin, a UCP2 inhibitor, dramatically boosted oxidative stress, attenuated antioxidative capacity, and exacerbated cell apoptosis accompanied with caspase-3 activation in rat renal proximal tubular cells (NRK-52E) incubated with high glucose. The present study results suggest that manipulation of UCP2 could be important in the prevention of oxidative stress damage in renal tubular epithelial cells induced by hyperglycemia in vitro.

Dendrimer-encapsulated copper as a novel oligonucleotides label for sensitive electrochemical stripping detection of DNA hybridization.

This paper describes the synthesis and characterization of a novel electrochemical label for sensitive electrochemical stripping detection of DNA hybridization based on dendrimer-encapsulated copper. The generation 4.5 (G 4.5) carboxyl-terminated poly(amidoamine) dendrimer with a trimesyl core was used as a template for synthesis of Cu²âº/dendrimer nanocomposites (Cu-DNCs). Ratios of Cu²âº/dendrimer were optimized in order to obtain stable nanocomposites with maximal copper loading in the interior of a polymeric shell. Cu-DNCs labeled DNA probe was employed for determining a target ssDNA immobilized on multi-walled carbon nanotubes-modified glassy carbon electrode (GCE) based on a specific hybridization reaction. The hybridization events were monitored by electrochemical detection of Cu anchored on the hybrids after the release in a diluted nitric acid by anodic stripping differential pulse voltammetry (ASDPV). The results showed that only a complementary sequence could form a dsDNA with the Cu-DNCs DNA probe and give an obvious electrochemical signal. The non-complementary sequence exhibited negligible signal change compared with the blank measurement (means: the electrode containing no target DNA incubating in hybridization buffer solution containing Cu-DNCs DNA probe for a certain time). The use of Cu encapsulated-dendrimer as tags and ASDPV for the detection of the released Cu ions could enhance the hybridization signal, and result in the increase of the sensitivity for the target DNA. Under the conditions employed here, the detection limit for measuring the full complementary sequence is down to pM level.

Amelioration of rhabdomyolysis-induced renal mitochondrial injury and apoptosis through suppression of Drp-1 translocation.

INTRODUCTION: Mitochondrial dysfunction plays an important role in acute kidney injury (AKI). Mitochondrial fission regulated by dynamin-related protein 1 (Drp-1) impairs the function of the mitochondria and the survival of cells. This study was conducted to explore the effects of suppression of Drp-1 accumulation in the mitochondria, on mitochondrial function and renal tubular cell apoptosis in rhabdomyolysis (RM)-induced AKI. METHODS: An RM model was induced by intramuscular injection of glycerol in Sprague Dawley rats. Twenty-four and 48 hours after intraperitoneal injections of mitochondrial division inhibitor 1 (Mdivi-1), we observed the functions of the kidney, changes in pathology, expressions of Drp-1 in tubular tissues (by immunohistochemistry and Western blot) and accumulation of Drp-1 and mitofusin 2 in tubular mitochondria (by Western blot). Mitochondrial function (ATP and ROS) and tubular epithelial cell apoptosis (by TUNEL) were also measured. RESULTS: RM induced Drp-1 accumulation, decreased ATP production and increased ROS in mitochondria. With increasing cytochrome c expression, cell apoptosis increased, whereas kidney function decreased. These changes were time-dependent. At different time points, despite not significantly influencing the overall expression of Drp-1, Mdivi-1 suppressed the accumulation of Drp-1, inhibited the insertion of proapoptotic Bax in mitochondria and inhibited the release of cytochrome c, thus ameliorating cell apoptosis. CONCLUSIONS: To conclude, in RM-induced AKI, suppression of Drp-1 accumulation in mitochondria favors the maintenance of mitochondrial function and reduces the apoptosis of tubular cells. Regulation of the mitochondrial fusion-fission balance may offer a novel strategy for the prevention and treatment of RM-induced AKI.

[Relation between bone matrix proteins and monocyte chemoattractant protein-1 in renal small artery in diabetic nephropathy rats].

OBJECTIVE: To observe the expressions of bone matrix proteins and monocyte chemoattractant protein-1 ((MCP-1) in the renal arteriole of diabetic nephropathy (DN) rats and analyze their correlations and roles in diabetic nephropathy. METHODS: Adult Sprague-Dawley male rats were used to establish the animal model of diabetic nephropathy induced by peritoneal injection of 55 mg/kg of streptozocin. Calcium deposit around the renal arteriole was observed by alizarin red staining. The protein and mRNA levels of core-bind factor alpha 1 (cbfalpha1), bone morphogenetic protein 2 (BMP-2) and matrix Gla protein (MGP) in renal arteriole of DN rats were detected by immunohistochemistry, in-situ hybridization and real-time PCR. The biochemical indices were detected by routine test. RESULTS: 1. Blood glucose and Urine protein of 24 h were significantly increased in the renal arteriole of DN rats versus the control rats (P < 0.05), serum creatinine (SCr) and phosphorus were significantly increased from 12 weeks. 2. Little deposit of calcium salt was observed in the renal arteriole of DN rats at the 4th week and a large amount of deposit was observed at 24th week, but no calcium deposit was observed in control rats. 3. Cbfalpha1 and BMP-2 expressions were significantly increased in the renal arteriole of DN rats from 4 to 24 weeks vs. the control rats. MGP mRNA expression in the renal arteriole of DN rats was significantly decreased from 4 to 24 weeks. MCP-1 expression was obviously upregulated in the renal arteriole of DN rats at 24th week versus that at 4th and 12th week. No MCP-1 expression was observed in the renal arterioles of control rats. MCP-1 were positively correlated with the expression of cbfalpha1 and BMP-2. CONCLUSION: Bone matrix proteins has already expressed in renal arteriole before the formation of vascular calcification. MCP-1 can affect the expression of cbfalpha1, BMP-2; cbfalpha1, BMP-2, MGP and MCP-1 may be involved in the formation of vascular lesions of DN.

[Association of diabetes and early failure of arteriovenous fistula in the end stage of renal diseases].

OBJECTIVE: To determine the association of diabetes and glycemic control with early failure of native arteriovenous fistula(AVF). METHODS: 266 patients with end stage renal diseases(ESRD) were recruited and divided into non-diabetic group (165), HbA1C < 7% group (51) and HbA1C > or = 7% group (50). Clinical indicators and early failure of AVF were examined. RESULTS: In total, 63 (23.7%) patients had AVF early failure. The AVF early failure occurred in 18. 1% of patients in the non-diabetic group and 21.6% of patients in the HbA1C < 7% group, significantly less than that in the HbA1C > or = 7% group (44%). The COX regression model showed that increased HbA1C, total cholesterol (TC) and decreased high-density lipoprotein (HDL)increased the risk of AVF failure. CONCLUSION: The levels of glycemic and serum lipid subfractions are associated with AVF early failure in ESRD patients. Good control of glycemic and lipid can lower the rates of AVF early failure.

Application of hybrid blood purification treatment for severe acute arsine poisoning.

OBJECTIVE: Severe acute arsine poisoning (SAAP) complicated by multiple organ dysfunction syndrome is a critical clinical illness. The limited efficacy of conventional drug therapy prompted us to investigate the application of hybrid blood purification treatment (HBPT) to improve the prognosis in critically ill patients. The present manuscript describes a series of cases treated with HBPT. METHODS: Eleven SAAP subjects were enrolled. The study did not include a control group, because of ethical issues. On the basis of conventional therapy, HBPT (plasma exchange [PE] + continuous venovenous hemofiltration [CVVH]) was used to treat SAAP. PE was performed once a day for 5 days, and CVVH was performed after each session of PE for 7 days or more; HBPT treatment duration amounted to an average of 10 days (range 7-18 days). Arsenic was detected in blood and discarded liquid. Clinical indicators, laboratory parameters, and prognostic indicators were assessed. RESULTS: HBPT was smoothly implemented without obvious adverse reaction. It can continuously remove arsenic and terminate hemolysis in a time-dependent manner. HBPT also significantly improved the poor clinical manifestations and laboratory indicators of SAAP, leading to a low mortality. Ten patients were discharged because of improved conditions, and only 1 patient died. CONCLUSIONS: The early application of HBPT can improve the prognosis of SAAP. The advantage of HBPT is that it can integrate the characteristics of different blood purification technologies to maximize treatment efficacy.

Early protective effect of mitofusion 2 overexpression in STZ-induced diabetic rat kidney.

Diabetic nephropathy (DN) is a serious complication of diabetes with a poorly defined etiology and limited treatment options. Early intervention is key to preventing the progression of DN. Mitofusin 2 (Mfn2) regulates mitochondrial morphology and signaling, and is involved in the pathogenesis of numerous diseases. Furthermore, Mfn2 is also closely associated with the development of diabetes, but its functional roles in the diabetic kidney remain unknown. This study investigated the effect of Mfn2 at an early stage of DN. Mfn2 was overexpressed by adenovirus-mediated gene transfer in streptozotocin-induced diabetic rats. Clinical parameters (proteinuria, albumin/creatinine ratio), pathological changes, ultra-microstructural changes in nephrons, expression of collagen IV and phosph-p38, ROS production, mitochondrial function, and apoptosis were evaluated and compared with diabetic rats expressing control levels of Mfn2. Endogenous Mfn2 expression decreased with time in DN. Compared to the blank transfection control group, overexpression of Mfn2 decreased kidney weight relative to body weight, reduced proteinuria and ACR, and improved pathological changes typical of the diabetic kidney, like enlargement of glomeruli, accumulation of ECM, and thickening of the basement membrane. In addition, Mfn2 overexpression inhibited activation of p38, and the accumulation of ROS; prevented mitochondrial dysfunction; and reduced the synthesis of collagen IV, but did not affect apoptosis of kidney cells. This study demonstrates that Mfn2 overexpression can attenuate pathological changes in the kidneys of diabetic rats. Further studies are needed to clarify the underlying mechanism of this protective function. Mfn2 might be a potential therapeutic target for the treatment of early stage DN.

Combined blood purification for treating acute fatty liver of pregnancy complicated by acute kidney injury: a case series.

Acute fatty liver of pregnancy (AFLP) complicated by acute kidney injury (AKI) is serious and life-threatening for the mother. The present study aimed to determine the clinical efficacy of combined blood purification treatment (CBPT) in patients with AFLP complicated by AKI. The CBPT involves plasma exchange (PE) combined with continuous venovenous hemofiltration (CVVH). The subjects were 17 patients with AFLP complicated by AKI. The CBPT was implemented based on the timely termination of pregnancy and general treatment. Changes in clinical manifestations, laboratory tests, liver ultrasounds, as well as Sequential Organ Failure Assessment (SOFA) and Glasgow scores were evaluated. The efficacy and adverse reactions of the CBPT were also assessed. The CBPT was smoothly performed without any obvious adverse reaction. After treatment, the clinical manifestations, laboratory examinations, and liver ultrasonography significantly improved. Therefore, the SOFA scores correspondingly decreased 1 week after treatment [9 (range 5-11) vs. 3 (range 0-10), P = 0.002], and the median was close to normal by the second week. The clearance rate of the total bilirubin in PE was significantly higher than that in CVVH (37.2 vs. 7.9%, P = 0.000). The incidence of acute pulmonary edema in CVVH was less than that in PE (0 vs. 41.2%, P = 0.007). Finally, the maternal mortality was 5.88% (95% CI: 0-29%). Overall, we think that CBPT aids in the recovery of liver and kidney function. Different blood purification methods may be combined to integrate and maximize their advantages to improve the prognoses of patients with serious AFLP.

[Influence of mannose binding lectin to expression of TGF-beta1 and NF-kappaB in high concentration of glucose cultured human renal glomerular endothelial cells].

OBJECTIVE: To investigate the effect of mannose binding lectin (MBL) complement pathway on expression of transforming growth factor-beta1 (TGF-beta1) and NF-kappaB in cultured human renal glomerular endothelial cells (HRGECs) stimulated by high concentration of glucose. METHODS: Human glomerular endothelial cells in culture were randomly divided into 5 groups according to different managements: normal concentration of glucose as controlled group, MBL + normal concentration of glucose group, high concentration of glucose, MBL + high glucose and MBL + high glucose + MBL blocker respectively. Flow cytometry was used to detect the depositions of MBL and C3 on the surfaces of HRGECs. Real-time PCR method was used to detect the mRNA levels of TGF-beta1. Human TGF-beta1 ELISA kit was used to detect the concentration of TGF-beta1 in supernatant fluid. ESMA was used to detect the activity of NF-kappaB in HRGECs. RESULTS: Compared with the normal glucose group and high glucose group, the depositions of MBL, C3 were apparently increased in MBL + high glucose group (P < 0.05). Expression of TGF-beta1 were significantly higher (P < 0.05) in MBL + high concentration of glucose groups than the normal glucose group and the high concentration of glucose group. Compared with the high glucose group, the activity of NF-kappaB in HRGECs was apparently increased in MBL + high glucose group, which could be significantly downregulated by MBL blocking antibody. CONCLUSION: High concentration of glucose can increase the expression of TGF-beta1 of cultured human glomerular endothelial cells. At the same time, high glucose together with MBL can up regulate the expression of TGF-beta1 and the activity of NF-kappaB in HRGECs.

[Influence of high glucose and mannose binding lectin complement pathway activation to IL-6 and TNF-alpha's expression by human renal glomerular endothelial cells].

OBJECTIVE: To investigate the effect of high glucose and mannose binding lectin (MBL) complement pathway activation's effect on expression of Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-alpha) from human renal glomerular endothelial cells (HRGEC), to explore unknown pathogenesy of diabetic nephropathy. METHODS: Normal HRGEC was divided randomly into normal glucose group(5 mmol/L D-glucose), manicol group (5 mmol/L D-glucose+25 mmol/L manicol) and high glucose group (30 mmol/L D-glucose). Real-time PCR was used to detect IL-6 and TNF-alpha mRNA expression in each group, Euzymelinked Immunosorbent Assay (ELISA) was performed to examine the protein expression of IL-6 and TNF-alpha in supernatant after 24 hours' culture. HRGEC was then randomly divided into two groups: single high glucose group and high glucose + MBL group. After 24 hours' culture with 30 mmol/L D-glucose, 30% MBL deficiency human serum was added into two groups, 1 microg/mL MBL was only added into high glucose + MBL group, continued the culturation for another 4 hours. Flow cytometry and immunofluorescence technique were applied to evaluate MBL, C3 and membrane attacks complex (MAC) deposition on cell surface respectively. Real-time PCR and ELISA were performed to examine mRNA and protein expression of both IL-6 and TNF-alpha in each group. RESULTS: Compared with normal glucose group and manicol group, the mRNA and protein expression of IL-6 and TNF-alpha in high glucose group were increased (P < 0.05). Flow cytometry confirmed obvious MBL and C3 co-deposition and Immunofluorescence confirmed obvious MAC deposition on cell surface in high glucose+ MBL group. Compared with single high glucose group, the mRNA and protein expression of IL-6 and TNF-alpha in high glucose+ MBL group were significantly higher (P < 0.05). CONCLUSION: High glucose can bring inflammatory factors' overexpression from cultured HRGEC; high glucose together with MBL can bring MBL complement pathway activation and inflammatory factors' overexpression, this indicates that the activation of MBL complement pathway may be a potential unknown pathogenesy of diabetic nephropathy and its proinflammatory status.

[Effects of high glucose on expression of core binding factor alpha1 and osteocalcin in vascular smooth muscle cells].

OBJECTIVE: To investigate the effects of high glucose on expression of core binding factor alpha1 (cbfalpha-1) and osteocalcin (OC) in vascular smooth muscle cells (VSMCs), and discuss the mechanism of small vessels calcification induced by high glucose (GS) in vitro. METHODS: The primary cultured VSMCs from rats' aortic segments were divided into three groups, including normal control group (5 mmol/L D-glucose), high glucose group (25 mmol/L D-glucose) and mannitol group (5 mmol/L D-glucose plus 25 mmol/L mannitol). We measured quantitatively the calcium deposition in VSMCs and investigated the calcium extent of VSMCs by alizarin red stain in each group. The mRNA levels of cbfalpha-1 and OC were measured by real-time PCR, and the protein expression levels of cbfalpha-1 and OC were examined by Western blot. The activity of alkaline phosphatase was measured by alkaline phosphatase activity testing kit, and the protein level of alpha-smooth muscle actin (a-SMA) was detected by immunohistochemistry. RESULTS: When compared with the normal group and mannitol group, the high glucose group showed that the calcium deposition and calcium extent of VSMCs increased obviously, the mRNA and protein levels of cbfalpha-1 and OC also increased significantly (P < 0.05), while the protein level of alpha-SMA decreased (P < 0.05), which were in a dose-dependent manner. The level of alkaline phosphatase activity of VSMCs was approximately doubled in high glucose group. CONCLUSION: The mechanism of high glucose induced calcification in VSMCs may be due to the increased expression of cbfalpha-1 and OC. High glucose decrease the expression of alpha-SMA in VSMCs, which could induce the transdifferentiation from RVSMCs to osteoblast-like cells.

[Effects of valsartan on the renal vascular endothelial growth factor and its receptor flk-1 of diabetic rats].

OBJECTIVE: To explore effects of valsartan on expression changes of vascular endothelial growth factor (VEGF) and its receptor Flk-1 in diabetic rat kidney. METHODS: To establish the diabetic nephropathy (DN) rat models and divide the experiment rats into three groups--the DN group, the valsartan group and the control group, and use the reverse transcriptase polymerase chain reaction (RT-PCR), Western Blotting and immunohistochemical techniques to detect the expression of VEGF and Flk-1, and detect the urine protein and glomerular area and volume, then analyze the relationship of the data. RESULTS: The mRNA and protein expression of VEGF and Flk-1, the urine protein and glomerular area and volume in the DN were higher than those in the control group and valsartan group (P < 0.05). VEGF and Flk-1 were positively correlated with the urine protein and glomerular area and volume (P < 0.05). CONCLUSION: VEGF and Flk-1 play an important role in the pathogenesis of DN, of which over-expression may lead to the damage of kidney. The angiotensin II receptor antagonist--valsartan can protect kidney through the non-hemodynamic mechanism of inhibiting the abnormal expression of VEGF and Flk-1.

[Expression level of cubilin in the rat model of diabetic nephropathy].

OBJECTIVE: To observe the expression level of Cubilin in the renal tubules of rats with STZ-induced diabetic nephropathy, to assess its correlation with 24 hours' albuminuria, and to investigate the mechanisms of tubular dysfunction at the early stage of diabetic nephropathy. METHODS: Diabetic nephropathy was induced in Sprague-Dalwley rats by intraperitoneal injection of STZ, while the rats of normal group were injected with normal saline. Biochemical indices of blood and urine specimens were observed in both groups at weeks 2, 4 and 6 respectively. The renal expression levels of Cubilin in the two groups were determined by immunohistochemistry and RT-PCR. RESULTS: The expression level of Cubilin in the diabetic nephropathy group was significantly decreased at week 2 after operation (P < 0.05), and it continued to decrease from week 2 to week 6. Also there was significant difference between each two time-points (P < 0.05), and the Cubilin expression level was negatively correlated with albuminuria (P < 0.01). CONCLUSION: The decreased expression level of Cubilin in early-stage diabetic nephropathy rats may partly contribute to the development of microalbuminuria. Cubilin can be regarded as one of the early markers when tubular dysfunction develops in the case of diabetic nephropathy.

[Effect of matrine on regulating renal tubulointerstitial expression of MMP-3, TIMP-1 and FN].

OBJECTIVE: To explore the effect and mechanism of matrine on regulating renal tubulointerstitium expression of MMP-3, TIMP-1 and FN. METHODS: Seventy male SD rats were randomly allocated to five groups: normal group, shame UUO group, UUO group, UUO group treated with fusinopril (F group), UUO group treated with large dose of matrine (E group) and UUO group treated with small dose of matrine (D group). The expression levels of MMP-3, TIMP-1 and FN of the rats were determined with immunohistochemistry at 7, 14 days of the experiment. RESULTS: The expression levels of MMP-3 of the rats in the UUO group and treatment groups decreased significantly compared with the normal group and shame UUO group (P<0.05). The treatment groups had higher expression of MMP-3 than that of the UUO group (P< 0.05). The expression levels of TIMP-1 and FN of the rats in the normal group and shame UUO group were amongst the lowest. The treatment groups had lower expression of TIMP-1 and FN than that of the UUO group (P<0.05). No significant difference of expression of MMP-3, FN and TIMP-1 were found between E group and F group (P>0.05). The expression level of MMP-3 was negatively correlated with those of TIMP-1 and FN in the UUO group (P < 0.01). CONCLUSION: Matrine decreases the expression of TIMP-1 and FN in the tubulointerstitium and increases the expression of MMP-3, which would delay the progression of renal tubulointerstitial fibrosis.