Chronic kidney disease in a murine model of non-alcoholic steatohepatitis (NASH).

Clinical studies suggest that non-alcoholic steatohepatitis (NASH) is an independent risk factor for chronic kidney disease (CKD), but causality and mechanisms linking these two major diseases are lacking. To assess whether NASH can induce CKD, we have characterized kidney function, histological features, transcriptomic and lipidomic profiles in a well-validated murine NASH model. Mice with NASH progressively developed significant podocyte foot process effacement, proteinuria, glomerulosclerosis, tubular epithelial cell injury, lipid accumulation, and interstitial fibrosis. The progression of kidney fibrosis paralleled the severity of the histologic NASH-activity score. Significantly, we confirmed the causal link between NASH and CKD by orthotopic liver transplantation, which attenuated proteinuria, kidney dysfunction, and fibrosis compared with control sham operated mice. Transcriptomic analysis of mouse kidney cortices revealed differentially expressed genes that were highly enriched in mitochondrial dysfunction, lipid metabolic process, and insulin signaling pathways in NASH-induced CKD. Lipidomic analysis of kidney cortices further revealed that phospholipids and sphingolipids were the most significantly changed lipid species. Notably, we found similar kidney histological changes in human NASH and CKD. Thus, our results confirm a causative role of NASH in the development of CKD, reveal potential pathophysiologic mechanisms of NASH-induced kidney injury, and established a valuable model to study the pathogenesis of NASH-associated CKD. This is an important feature of fatty liver disease that has been largely overlooked but has clinical and prognostic importance.

Periodontitis aggravates renal inflammatory response in a mouse model of renal fibrosis.

OBJECTIVES: To assess the effects of periodontitis on renal interstitial fibrosis in a mouse model. MATERIALS AND METHODS: Thirty C57BL/6 male mice were divided into control, periodontitis (PD), unilateral ureteral ligation (UUO) and PD+UUO groups. Unilateral ureteral ligation was performed 6 days after periodontitis. After 2 weeks, all mice were sacrificed, and samples were collected for the assessment of gene expression, immune cells, biochemical indicators and renal pathology. RESULTS: Expression of tumour necrosis factor-α, interleukin-1ß, and Ly6G in the kidneys in the PD+UUO group was significantly greater than in the UUO group. The percentage of CD11b+ Ly6G+ cells was significantly higher in the PD+UUO than in the UUO group. Fibrotic areas in the kidneys in the PD+UUO group were slightly, but not significantly, greater than those in the UUO group. Kidneys from the PD+UUO group showed markedly higher gene expression of matrix metalloproteinase-9, but not α-smooth muscle actin or collagen I, than those in the UUO group. There were no significant differences in blood urea nitrogen, serum creatinine and uric acid between the PD+UUO and UUO groups. CONCLUSIONS: Periodontitis increases the renal inflammatory response without showing a significant influence on renal interstitial fibrosis or renal function in the UUO mouse model.

Molecular characterization and function analysis of grouper (Epinephelus coioides) Bruton's tyrosine kinase BTK.

Bruton's tyrosine kinase (BTK) is a Tec-family tyrosine kinase and plays a crucial role in B cell antigen receptor (BCR) signal pathway. Mutations in humans and mice BTK gene results in X-linked agammaglobulinemia (XLA) and X-linked immunodeficiency (XLD), respectively. To study the function of BTK in teleost, we cloned a BTK gene from orange-spotted grouper. Homology analysis showed that the grouper BTK (EcBTK) had a high amino acid identity with other vertebrates (63%-92%) and shared the highest amino acid identity with ballan wrasse Labrus bergylta BTK. EcBTK comprises a Bruton's tyrosine kinase pleckstrin homology (PH) domain, a Tec homology (TH) domain, a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain and a Protein Kinases, catalytic (PKc) domain. Tissue distribution analysis showed that EcBTK was mainly expressed in immune organs. EcBTK was uniform distributed throughout the cytoplasm of transfected HEK293T cells and overexpression of EcBTK slightly down-regulates NF-κB activity. Ibrutinib treatment can reduce the phosphorylation level of grouper's BTK. In groupers infected with Cryptocaryon irritans, up-regulation of EcBTK were not seen in the early stage of infected skin and gill until days 14-21. The phosphorylation level of grouper BTK was significantly increased in infected skin and gill.

Reduction in podocyte SIRT1 accelerates kidney injury in aging mice.

Both the incidence and prevalence of chronic kidney disease are increasing in the elderly population. Although aging is known to induce kidney injury, the underlying molecular mechanisms remain unclear. Sirtuin 1 (Sirt1), a longevity gene, is known to protect kidney cell injury from various cellular stresses. In previous studies, we showed that the podocyte-specific loss of Sirt1 aggravates diabetic kidney injury. However, the role of Sirt1 in aging-induced podocyte injury is not known. Therefore, in this study we sought to determine the effects of podocyte-specific reduction of Sirt1 in age-induced kidney injury. We employed the inducible podocyte-specific Sirt1 knockdown mice that express shRNA against Sirt1 (Pod-Sirt1RNAi) and control mice that express shRNA for luciferase (Pod-LuciRNAi). We found that reduction of podocyte Sirt1 led to aggravated aging-induced glomerulosclerosis and albuminuria. In addition, urinary level of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative stress, was markedly increased in aged Pod-Sirt1RNAi mice compared with aged Pod-LuciRNAi mice. Although podocyte-specific markers decreased in aged mice compared with the young controls, the decrease was further exacerbated in aged Pod-Sirt1RNAi compared with Pod-LuciRNAi mice. Interestingly, expression of cellular senescence markers was significantly higher in the glomeruli of Pod-Sirt1RNAi mice than Pod-LuciRNAi mice, suggesting that cellular senescence may contribute to podocyte loss in aging kidneys. Finally, we confirmed that Pod-Sirt1RNAi glomeruli were associated with reduced activation of the transcription factors peroxisome proliferator-activated receptor (PPAR)-α coactivador-1 (PGC1α)/PPARγ, forkhead box O (FOXO)3, FOXO4, and p65 NF-κB, through SIRT1-mediated deacetylation. Together, our data suggest that SIRT1 may be a potential therapeutic target to treat patients with aging-related kidney disease.

Cholesterol Crystal Embolism and Chronic Kidney Disease.

Renal disease caused by cholesterol crystal embolism (CCE) occurs when cholesterol crystals become lodged in small renal arteries after small pieces of atheromatous plaques break off from the aorta or renal arteries and shower the downstream vascular bed. CCE is a multisystemic disease but kidneys are particularly vulnerable to atheroembolic disease, which can cause an acute, subacute, or chronic decline in renal function. This life-threatening disease may be underdiagnosed and overlooked as a cause of chronic kidney disease (CKD) among patients with advanced atherosclerosis. CCE can result from vascular surgery, angiography, or administration of anticoagulants. Atheroembolic renal disease has various clinical features that resemble those found in other kidney disorders and systemic diseases. It is commonly misdiagnosed in clinic, but confirmed by characteristic renal biopsy findings. Therapeutic options are limited, and prognosis is considered to be poor. Expanding knowledge of atheroembolic renal disease due to CCE opens perspectives for recognition, diagnosis, and treatment of this cause of progressive renal insufficiency.

Nephrin Preserves Podocyte Viability and Glomerular Structure and Function in Adult Kidneys.

Nephrin is required during kidney development for the maturation of podocytes and formation of the slit diaphragm junctional complex. Because nephrin expression is downregulated in acquired glomerular diseases, nephrin deficiency is considered a pathologic feature of glomerular injury. However, whether nephrin deficiency exacerbates glomerular injury in glomerular diseases has not been experimentally confirmed. Here, we generated mice with inducible RNA interference-mediated nephrin knockdown. Short-term nephrin knockdown (6 weeks), starting after the completion of kidney development at 5 weeks of age, did not affect glomerular structure or function. In contrast, mice with long-term nephrin knockdown (20 weeks) developed mild proteinuria, foot process effacement, filtration slit narrowing, mesangial hypercellularity and sclerosis, glomerular basement membrane thickening, subendothelial zone widening, and podocyte apoptosis. When subjected to an acquired glomerular insult induced by unilateral nephrectomy or doxorubicin, mice with short-term nephrin knockdown developed more severe glomerular injury compared with mice without nephrin knockdown. Additionally, nephrin-knockdown mice developed more exaggerated glomerular enlargement when subjected to unilateral nephrectomy and more podocyte apoptosis and depletion after doxorubicin challenge. AKT phosphorylation, which is a slit diaphragm-mediated and nephrin-dependent pathway in the podocyte, was markedly reduced in mice with long-term or short-term nephrin knockdown challenged with uninephrectomy or doxorubicin. Taken together, our data establish that under the basal condition and in acquired glomerular diseases, nephrin is required to maintain slit diaphragm integrity and slit diaphragm-mediated signaling to preserve glomerular function and podocyte viability in adult mice.

Induction of retinol dehydrogenase 9 expression in podocytes attenuates kidney injury.

The intracellular concentration of retinoic acid is determined by two sequential oxidation reactions that convert retinol to retinoic acid. We recently demonstrated that retinoic acid synthesis is significantly impaired in glomeruli of HIV-1 transgenic mice (Tg26), a murine model of HIV-associated nephropathy. This impaired retinoic acid synthesis correlates with reduced renal expression of retinol dehydrogenase 9, which catalyzes the rate-limiting step of retinoic acid synthesis by converting retinol to retinal. Because retinoic acid has renal protective effects and can induce podocyte differentiation, we hypothesized that restoration of retinoic acid synthesis could slow the progression of renal disease. Herein, we demonstrate that overexpression of retinol dehydrogenase 9 in cultured podocytes induces the expression of podocyte differentiation markers. Furthermore, we confirm that podocyte-specific overexpression of retinol dehydrogenase 9 in mice with established kidney disease due to either HIV-associated nephropathy or adriamycin-induced nephropathy decreases proteinuria, attenuates kidney injury, and restores podocyte differentiation markers. Our data suggest that restoration of retinoic acid synthesis could be a new approach to treat kidney disease.

Nonlinear association between self-reported sleep duration and cognitive function among middle-aged and older adults in China: The moderating effect of informal care.

BACKGROUND: Cognitive impairment is a major public health problem urgently to be solved. This study aims to examine the association between sleep duration and cognitive function and its two subdimensions: episodic memory and mental status, and to explore the moderating effects of informal care on these associations among middle-aged and older adults in China. METHODS: Data was drawn from China Health and Retirement Longitudinal Study (CHARLS) 2011, 2013, 2015 and 2018 datasets. Sleep duration and informal care were self-reported. Cognitive function was measured using CHARLS Harmonized Cognitive Assessment Protocol. Effects of informal care on sleep duration-cognitive function were assessed using Generalized Estimating Equations models. RESULTS: The relationships between sleep duration and cognitive function, episodic memory, and mental status were all found to follow an inverted U-shaped pattern. Spouse care weakened the adverse effects of extreme sleep duration on cognitive function while the children care amplified them. Further, we only observed the moderating effects of spouse and children care on the association between sleep duration and episodic memory, but not mental status. CONCLUSIONS: The relationships between sleep duration and cognitive function, along with its different dimensions, are nonlinear in nature. The impacts of sleep duration on cognitive function and its dimensions are contingent upon the levels of informal care received and the sources of that care. We provide valuable insights into the complex interplay between sleep duration, informal care, and cognitive function.

Acyloxyacyl Hydrolase Protects against Kidney Injury via Inhibition of Tubular CD74-Macrophage Crosstalk.

Renal fibrosis is the common pathway in the progression of chronic kidney disease (CKD). Acyloxyacyl hydrolase (AOAH) is expressed in various phagocytes and is highly expressed in proximal tubular epithelial cells (PTECs). Research shows that AOAH plays a critical role in infections and chronic inflammatory diseases, although its role in kidney injury is unknown. Here, we found that AOAH deletion led to exacerbated kidney injury and fibrosis after folic acid (FA) administration, which was reversed by overexpression of Aoah in kidneys. ScRNA-seq revealed that Aoah-/- mice exhibited increased subpopulation of CD74+ PTECs, though the percentage of total PTECs were decreased compared to WT mice after FA treatment. Additionally, exacerbated kidney injury and fibrosis seen in Aoah-/- mice was attenuated via administration of methyl ester of (S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid (ISO-1), an inhibitor of macrophage inhibition factor (MIF) and CD74 binding. Finally, AOAH expression was found positively correlated with estimated glomerular filtration rate while negatively correlated with the degree of renal fibrosis in kidneys of CKD patients. Thus, our work indicates that AOAH protects against kidney injury and fibrosis by inhibiting renal tubular epithelial cells CD74 signaling pathways. Targeting kidney AOAH represents a promising strategy to prevent renal fibrosis progression.

Direct medical costs of ischemic heart disease in urban Southern China: a 5-year retrospective analysis of an all-payer health claims database in Guangzhou City.

Introduction: This study aimed to estimate the direct medical costs and out-of-pocket (OOP) expenses associated with inpatient and outpatient care for IHD, based on types of health insurance. Additionally, we sought to identify time trends and factors associated with these costs using an all-payer health claims database among urban patients with IHD in Guangzhou City, Southern China. Methods: Data were collected from the Urban Employee-based Basic Medical Insurance (UEBMI) and the Urban Resident-based Basic Medical Insurance (URBMI) administrative claims databases in Guangzhou City from 2008 to 2012. Direct medical costs were estimated in the entire sample and by types of insurance separately. Extended Estimating Equations models were employed to identify the potential factors associated with the direct medical costs including inpatient and outpatient care and OOP expenses. Results: The total sample included 58,357 patients with IHD. The average direct medical costs per patient were Chinese Yuan (CNY) 27,136.4 [US dollar (USD) 4,298.8] in 2012. The treatment and surgery fees were the largest contributor to direct medical costs (52.0%). The average direct medical costs of IHD patients insured by UEBMI were significantly higher than those insured by the URBMI [CNY 27,749.0 (USD 4,395.9) vs. CNY 21,057.7(USD 3,335.9), P < 0.05]. The direct medical costs and OOP expenses for all patients increased from 2008 to 2009, and then decreased during the period of 2009-2012. The time trends of direct medical costs between the UEBMI and URBMI patients were different during the period of 2008-2012. The regression analysis indicated that the UEBMI enrollees had higher direct medical costs (P < 0.001) but had lower OOP expenses (P < 0.001) than the URBMI enrollees. Male patients, patients having percutaneous coronary intervention operation and intensive care unit admission, patients treated in secondary hospitals and tertiary hospitals, patients with the LOS of 15-30 days, 30 days and longer had significantly higher direct medical costs and OOP expenses (all P < 0.001). Conclusions: The direct medical costs and OOP expenses for patients with IHD in China were found to be high and varied between two medical insurance schemes. The type of insurance was significantly associated with direct medical costs and OOP expenses of IHD.

5G Converged Network Resource Allocation Strategy Based on Reinforcement Learning in Edge Cloud Computing Environment.

Aiming at the problem that computing power and resources of Mobile Edge Computing (MEC) servers are difficult to process long-period intensive task data, this study proposes a 5G converged network resource allocation strategy based on reinforcement learning in edge cloud computing environment. n order to solve the problem of insufficient local computing power, the proposed strategy offloads some tasks to the edge of network. Firstly, we build a multi-MEC server and multi-user mobile edge system, and design optimization objectives to minimize the average response time of system tasks and total energy consumption. Then, task offloading and resource allocation process is modeled as Markov decision process. Furthermore, the deep Q-network is used to find the optimal resource allocation scheme. Finally, the proposed strategy is analyzed experimentally based on TensorFlow learning framework. Experimental results show that when the number of users is 110, final energy consumption is about 2500 J, which effectively reduces task delay and improves the utilization of resources.

Indoor air pollution from solid fuels use, inflammation, depression and cognitive function in middle-aged and older Chinese adults.

BACKGROUND: Few studies have focused on the influence of indoor air pollution on depression and cognitive impairment; besides, the underlying mechanism is not well-established. OBJECTIVE: This study aimed to fill the above gaps by exploring the underlying influence mechanism of solid fuel use, the major cause of indoor air pollution, with the risk of depression and cognitive impairment. METHODS: This data came from China Health and Retirement Longitudinal Study (CHARLS) 2015 dataset. Self-reported household cooking fuels were collected and categorized as clean fuels and solid fuels. High-sensitivity C-reactive protein (CRP) and white blood cells (WBC) were used to measure inflammation. Depression and cognitive function were assessed by using standardized questionnaires. RESULTS: Respondents had an average Center for Epidemiologic Studies Depression Scale (CESD-10) scores of 7.68 (SD = 6.14) and cognitive function scores of 15.97 (SD = 4.84). In the whole sample, 36.4 % of respondents used solid fuels use, but this proportion was much greater among those living in rural areas (78.38 %). Compared with clean fuel users, solid fuel users had more depression and worse cognitive function. After adjusting for confounders, indoor air pollution was significantly associated with depression and cognitive function respectively (ß = -0.444, p < 0.001; ß = 0.656, p < 0.001). Indoor air pollution was significantly related to the WBC (ß = 0.170, p < 0.01), but not for the CRP. The WBC mediated the association between indoor air pollution and depression (ß = 0.026, p < 0.01). CONCLUSION: In conclusion, solid fuel use was significantly associated with a higher risk of depression and cognitive impairment. Furthermore, we found that solid fuel use influences depression partly via the inflammatory profile.

[Extract of Ginkgo biloba and alpha-lipoic acid attenuate advanced glycation end products accumulation and RAGE expression in diabetic nephropathy rats].

OBJECTIVE: To investigate the accumulation of advanced glycation end products (AGEs) and expression of receptor for AGEs (RAGE) in streptozocin (STZ)-induced diabetic nephropathy in rats, and the role of antioxidants on the AGEs-RAGE signaling. METHODS: Diabetic rats were induced by once intraperitoneal injection of STZ at the dose of 60 mg/kg, and randomly divided into the DN group (n=12, treated with normal saline by intraperitoneal injection, once daily), the extract of Ginkgo biloba (EGb) group (n=14, treated with EGb 300 mg/kg by oral administration, once every other day), and the alpha-lipoic acid (ALA) group (n=12, treated with ALA at the dose of 35 mg/kg by intraperitoneal injection, once every other day). Rats of the normal control group (n=10) were given vehicle citrate buffer at the dose of 60 mg/kg. Rats were sacrificed at the 12th week and the 20th week of this study. The four groups were compared in terms of body weight, blood glucose, renal function, 24-h urine protein. Renal pathological changes were observed by PAS staining. Oxidative stress indices were detected using spectrophotometry. The concentrations of AGEs were measured using fluoro spectrophotometry, and the expressions of RAGE were detected by Real-time PCR and Western blot. RESULTS: Compared with the normal control group, the 24-h urine protein quantitation was higher and the glomerular filtration rate increased in rats at the 12th week and the 20th week. The pathological tissue staining showed dilated glomerular mesangium, proliferated glomerular matrix, vacuolar degeneration of the renal tubular epithelium. Malonaldehyde (MDA) levels and 8-hydroxide radical guanine deoxyriboside (8-OHdG) levels increased, and catalase (CAT) and reduced glutathione hormone (GSH) levels decreased. The AGEs contents in serum and renal tissue homogenate increased. The expressions of RAGE mRNA and protein increased in the DN group at the 12th and the 20th week. The 24-h urine protein quantitation was reduced in the EGb group and the ALA group, with alleviated pathological changes, lowered MDA and 8-OHdG levels, increased CAT and GSH levels, decreased AGEs contents, and down-regulated RAGE expressions. CONCLUSIONS: AGEs contents increased and RAGE expression up-regulated in the circulation and local renal tissues in DN rats. EGb and ALA could inhibit AGEs production and down-regulate RAGE expressions by reducing oxidative stress, thus further improving the renal tissue structure and renal functions of DN rats. It had better application prospect in treatment and prevention of DN.

The complete mitochondrial genome of two-belt cardinal and striped cardinalfish Apogonichthyoides taeniatus (Cuvier, 1828).

The complete mitochondrial DNA sequence of Apogonichthyoides taeniatus (Cuvier, 1828) is determined. The mitochondrial genome is 17,050 in length and has the same composition and gene order like most other vertebrates. The phylogenetic analysis based on 13 concatenated PCGs nucleotide sequences among 20 species showed that this species has high support with the sister branch Jaydia lineata. Our findings provide useful information for phylogenetic and evolutionary research of Kurtiformes species.

Highly effective remediation of high-arsenic wastewater using red mud through formation of AlAsO4@silicate precipitate.

High-arsenic wastewater derived from the metallurgical industry of nonferrous minerals is one of the most dangerous arsenic (As) sources that usually follow the emission of massive hazardous arsenic-bearing wastes. Considering the properties of red mud (RM), we propose an alternative and environmentally friendly method for the efficient remediation of high-arsenic wastewater using RM through formation of AlAsO4@silicate precipitate, aiming at ''zero-emission of hazardous solid waste''. The results show nearly 100% of arsenic could be stepwisely removed from high-arsenic wastewater and reduce the arsenic concentration from 6100 mg/L to 40 µg/L using RM at room temperature. The highest arsenic removal capacity of RM reaches 101.5 mg/g at a RM-to-wastewater ratio of 40 g/L due to the superior arsenic adsorption and the co-precipitation of arsenate and Al3+ to form insoluble aluminum arsenate. The silicate shell of arsenic-loaded RM created at an alkaline condition acts as an arsenic stabilizer, resulting in a leached arsenic concentration of 1.2 mg/L in TCLP tests. RM acts as a highly effective arsenic remover and stabilizer for the disposal of high-arsenic wastewater. It shows great potential for the remediation of wastewater containing heavy metals with varying concentrations to produce clean water available for industrial purpose.

Molecular Analysis of the Kidney From a Patient With COVID-19-Associated Collapsing Glomerulopathy.

Recent case reports suggest that coronavirus disease 2019 (COVID-19) is associated with collapsing glomerulopathy in African Americans with apolipoprotein L1 gene (APOL1) risk alleles; however, it is unclear whether disease pathogenesis is similar to HIV-associated nephropathy. RNA sequencing analysis of a kidney biopsy specimen from a patient with COVID-19-associated collapsing glomerulopathy and APOL1 risk alleles (G1/G1) revealed similar levels of APOL1 and angiotensin-converting enzyme 2 (ACE2) messenger RNA transcripts as compared with 12 control kidney samples downloaded from the GTEx (Genotype-Tissue Expression) Portal. Whole-genome sequencing of the COVID-19-associated collapsing glomerulopathy kidney sample identified 4 indel gene variants, 3 of which are of unknown significance with respect to chronic kidney disease and/or focal segmental glomerulosclerosis. Molecular profiling of the kidney demonstrated activation of COVID-19-associated cell injury pathways such as inflammation and coagulation. Evidence for direct severe acute respiratory syndrome coronavirus 2 infection of kidney cells was lacking, which is consistent with the findings of several recent studies. Interestingly, immunostaining of kidney biopsy sections revealed increased expression of phospho-STAT3 (signal transducer and activator of transcription 3) in both COVID-19-associated collapsing glomerulopathy and HIV-associated nephropathy as compared with control kidney tissue. Importantly, interleukin 6-induced activation of STAT3 may be a targetable mechanism driving COVID-19-associated acute kidney injury.

Acute respiratory distress syndrome induced by H9N2 virus in mice.

H9N2 avian influenza viruses have repeatedly caused infections in swine and humans in some countries. The purpose of the present study was to evaluate the pulmonary pathology caused by H9N2 viral infection in mice. Six- to eight-week-old BALB/c mice were infected intranasally with 1 x 10(4) MID(50) of A/Chicken/Hebei/4/2008(H9N2) virus. Clinical signs, pathological changes and viral replication in lungs, arterial blood gas, and cytokines in bronchoalveolar lavage fluid (BALF) were observed at different time points after infection. A control group was infected intranasally with noninfectious allantoic fluid. H9N2-infected mice exhibited severe respiratory syndrome, with a mortality rate of 60%. Gross observations showed that infected lungs were highly edematous. Major histopathological changes in infected lungs included diffuse pneumonia and alveolar damage, with neutrophil-dominant inflammatory cellular infiltration, interstitial and alveolar edema, hemorrhage, and severe bronchiolitis/peribronchiolitis. In addition, H9N2 viral infection resulted in severe progressive hypoxemia, lymphopenia, and a significant increase in neutrophils, tumor necrosis factor-alpha and interleukin-6 in BALF. The features described above satisfy the criteria for acute respiratory distress syndrome (ARDS). Our data show that H9N2 viral infection resulted in ARDS in mice, and this may facilitate studies of the pathogenesis of future potential H9N2 disease in humans.

Effects of advanced glycation end products on renal fibrosis and oxidative stress in cultured NRK-49F cells.

BACKGROUND: Advanced glycation end products (AGEs) play a critical role in the development of diabetic nephropathy. Reactive oxygen species (ROS) may play a critical role in AGEs induced growth factor expression. In this study, the effects of AGEs on transforming growth factor beta1 (TGF-beta1), connective tissue growth factor (CTGF) and fibronectin (Fn) mRNA expression and oxidative stress in cultured NRK-49F cells were examined. METHODS: NRK-49F cells were incubated with medium containing different doses of AGEs (50, 100 or 200 microg/ml) for 24 hours, or with AGEs 100 microg/ml for different times (0, 12, 24 or 48 hours). Cells in the serum-free medium or medium containing 25 mmol/L glucose were controls. Cells were treated with 25 mmol/L glucose and 100 microg/ml AGEs for 24 hours to determine the effects between AGEs and glucose. We clarified the role of antioxidant by pretreating cells with N-acetylcysteine (10 mmol/L), ginkgo biloba extract (50 or 100 mg/L) for 24 hours and with 100 microg/ml AGEs for further 24 hours. Alamarblue dye assay was used to analyze cell growth; intracellular ROS generation was measured by flow cytometry; intracellular glutathione by fluorescence spectrophotometry; expressions of TGF-beta1, CTGF and Fn mRNA by semiquantitative RT-PCR. RESULTS: AGEs significantly increased the expressions of TGF-beta1, CTGF, Fn mRNA and intracellular ROS generation, and decreased the glutathion level in NRK-49F cells in dose- and time-dependent manners. High glucose and AGEs together significantly increased the expression of TGF-beta1, CTGF and Fn mRNA, compared with AGEs and high glucose separately. Preincubation with N-acetylcysteine or ginkgo biloba extract increased GSH level, suppressed AGEs-induced oxidative stress and TGF-beta1, CTGF and Fn mRNA overexpression. CONCLUSIONS: AGEs can significantly increase expression of TGF-beta1, CTGF, Fn mRNA in NRK-49F cells through enhancement of oxidative stress. The accumulation of AGEs may play a pivotal role in the pathogenesis of tubulointerstitial fibrosis in diabetic nephropathy. Suppression of AGEs induced TGF-beta1, CTGF and Fn mRNA overexpression in renal fibroblasts through inhibition of oxidative stress may be a mechanism underlying effect of ginkgo biloba extract in diabetic nephropathy. In addition, antioxidant therapy may help prevent AGEs accumulation and its induced damage.