Shenkang injection (SKI) is a classic prescription composed of Radix Astragali, rhubarb, Astragalus, Safflower, and Salvia. This treatment was approved by the State Food and Drug Administration of China in 1999 for treatment of chronic kidney diseases based on good efficacy and safety. This study aimed to investigate the protective effect of SKI against high glucose (HG)-induced renal tubular cell senescence and its underlying mechanism. Primary renal proximal tubule epithelial cells were cultured in (1) control medium (control group), medium containing 5 mmol/L glucose; (2) mannitol medium (mannitol group), medium containing 5 mmol/L glucose, and 25 mmol/L mannitol; (3) HG medium (HG group) containing 30 mmol/L glucose; (4) SKI treatment at high (200 mg/L), medium (100 mg/L), or low (50 mg/L) concentration in HG medium (HG + SKI group); or (5) 200 mg/L SKI treatment in control medium (control + SKI group) for 72 h. HG-induced senescent cells showed the emergence of senescence associated heterochromatin foci, up-regulation of P16 and cyclin D1, increased senescence-associated β-galactosidase activity, and elevated expression of membrane decoy receptor 2. SKI treatment potently prevented these changes in a dose-independent manner. SKI treatment prevented HG-induced up-regulation of pro-senescence molecule mammalian target of rapamycin and p66Shc and down-regulation of anti-senescence molecules klotho, sirt1, and peroxisome proliferator-activated receptor-g in renal tubular epithelial cells. SKI may be a novel strategy for protecting against HG-induced renal tubular cell senescence in treatment of diabetic nephropathy.
Animals ; Cells, Cultured ; Cellular Senescence ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Diabetic Nephropathies ; drug therapy ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; drug effects ; metabolism ; Glucose ; Kidney Tubules, Proximal ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL

OBJECTIVE: To investigate the effect of blocking pannexin-1 against acute kidney injury induced by cisplatin. METHODS: Twenty-six male C57BL/6 mice aged 6-8 weeks were randomly divided into control group, cisplatin model (Cis) group and cisplatin + carbenoxolone treatment group (Cis + CBX). In Cis group and Cis + CBX group, the mice were injected intraperitoneally with 20 mg/kg of cisplatin and with CBX (20 mg/kg) at 30 min before and 24 and 48 h after cisplatin inhjection, respectively. All the mice were sacrificed at 72 h after cisplatin injection, and plasma and kidney samples were collected for testing mRNA and protein expression levels of pannexin-1 in the renal tissue using RT-qPCR and Western blotting and for detecting plasma creatinine and BUN levels; the pathological changes in the renal tissues were observed using Periodic Acid-Schiff staining. The expression of kidney injury molecule 1 (KIM-1) was examined using immunohistochemistry and the mRNA expressions of KIM-1 and neutrophil gelatinase- related lipid transport protein (NGAL) were detected by RT-qPCR to evaluate the injuries of the renal tubules. The infiltration of F4/80-positive macrophages and CD4-positive T cells were observed by immunofluorescence. In the experiment, human proximal tubule epithelial cell line HK-2 was stimulated with 50 μmol/L cisplatin to establish a cell model of acute kidney injury, and the mRNA and protein expressions of pannexin-1 were detected by RT-qPCR and Western blotting at 4, 6, 12, 18 and 24 h after the stimulation. RESULTS: Compared with the control mice, the cisplatin-treated mice showed significantly up-regulated protein levels ( < 0.05) and mRNA levels ( < 0.005) of pannexin-1 in the kidney tissue. Cisplatin stimulation also caused significant increases in the protein levels ( < 0.005) and mRNA levels ( < 0.005) of pannexin-1 in cultured HK-2 cells. Compared with cisplatin-treated mice, the mice treated with both cisplatin and the pannexin-1 inhibitor CBX showed obviously lessened kidney pathologies and milder renal tubular injuries with significantly reduced plasma BUN and Scr levels ( < 0.01), expressions of KIM-1 and NGAL in the kidney ( < 0.05), and infiltration of F4/80-positive macrophages ( < 0.01) and CD4- positive T cells ( < 0.05) in the kidney tissues. CONCLUSIONS In cisplatin induced acute kidney injury mice model, Pannexin-1 expression is up-regulated in the kidneys tissue, and blocking pannexin-1 alleviates the acute kidney injury reducing renal inflammatory cell infiltration.
Acute Kidney Injury ; drug therapy ; metabolism ; Animals ; Cisplatin ; pharmacology ; Connexins ; drug effects ; metabolism ; Cross-Linking Reagents ; pharmacology ; Humans ; Kidney ; Kidney Tubules ; Male ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; drug effects ; metabolism ; Random Allocation

Animals ; Cell Line ; Diabetic Nephropathies ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Eukaryotic Initiation Factor-2 ; metabolism ; Glucose ; pharmacology ; Humans ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Tubules, Proximal ; drug effects ; metabolism ; Rats ; Signal Transduction ; drug effects

To discover the nephroprotective substances from Huangkui capsule.The components of Huangkui capsule were isolated by preparative liquid chromatography, and the active components were screened by LC/MS and identified. The adriamycine-injured HK-2 cells were treated with various active components with different concentrations, and the malonaldehyde (MDA) content, adenosine triphosphate (ATP) level and mitochondrial oxygen consumption rate were measured to verify the protective activity of the compounds.Four active components in Huangkui capsule were identified to exert nephroprotective effects. Fifteen flavanoids from these four components were tentatively identified by LC/MS, and hyperin, myricetin, quercetin, rutin and isoquercetin were confirmed. Hyperin, myricetin quercetin and rutin showed dose-dependent protective effects on injured HK-2 cells. Espacially, hyperin significantly reduced MDA content, quercetin and rutin significantly increased ATP level, and myricetin significantly increased mitochondrial oxygen consumption rate.Hyperin, myricetin, querctein and rutin might be the potential nephroprotective compounds in Huangkui capsule, their effects may be related to the inhibition of lipid peroxidation and the alleviation of mitochondrial damage.
Abelmoschus ; chemistry ; drug effects ; Adenosine Triphosphate ; metabolism ; Cell Line, Transformed ; Chromatography, Liquid ; Doxorubicin ; Drugs, Chinese Herbal ; Epithelial Cells ; drug effects ; Flavonoids ; pharmacology ; Kidney Diseases ; chemically induced ; drug therapy ; prevention & control ; Kidney Tubules, Proximal ; drug effects ; Lipid Peroxidation ; drug effects ; Malondialdehyde ; metabolism ; Mass Spectrometry ; Mitochondria ; drug effects ; Oxygen Consumption ; drug effects ; Protective Agents ; chemistry ; pharmacology ; Quercetin ; analogs & derivatives ; pharmacology ; Rutin ; pharmacology

OBJECTIVETo investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells.

METHODSThe gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups: control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA.

RESULTSpCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P<0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P<0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P<0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P<0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P<0.05).

CONCLUSIONSmiR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.

Cells, Cultured ; Epithelial Cells ; drug effects ; pathology ; Epithelial-Mesenchymal Transition ; Humans ; Kidney Tubules, Proximal ; drug effects ; pathology ; MicroRNAs ; physiology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo investigate the effects of Huaiqihuang Granules (, HQH), a mixture of Chinese herbs including Trametes robiniophila Murr, Fructus Lycii and Polygonatum sibiricum, on adriamycininduced nephropathy (ADRN) in rats and its underlying mechanisms.

METHODSRats with ADRN were divided into four groups: the sham group, the model group (distilled water), the low-dose HQH-treated (2 g/kg) group, and the high-dose HQH-treated (4 g/kg) group. Body weight and 24-h urinary protein (Upro) were checked every week. After 5-week intervention, at the end of the study, the rats were sacrificed and blood samples were collected for examination of biochemical parameters, including glomerular morphological makers, podocyte shape, cellular apoptosis, expressions of nephrin, inflammatory and apoptosis markers.

RESULTSHQH ameliorated the rat's general status, proteinuria, renal morphological appearance and glomerulosclerosis. The decreased expression of nephrin in ADRN rats was increased by HQH, as well as the impaired podocyte foot process fusion. Cytosolic levels of p65 and inhibitor of nuclear factor κBα (IκBα) were decreased in ADRN rats, and recovered by the treatment of HQH. Consistently, the induced expression of tumor necrosis factor α (TNF-α), phosphorylated nuclear factor κB p65 (p-NFκB p65) and IκBα in ADRN were markedly suppressed by HQH. In addition, induction of Bax, cleaved caspase-3 and cytochrome C in ADRN rats were suppressed by HQH, indicating the amelioration of apoptosis.

CONCLUSIONHQH could ameliorate renal impairments in ADRN rats by increasing nephrin expression, inhibiting NF-κB signaling pathway via the down-regulation of p-NF-κB p65 and p-IκBα, and suppression of glomerular and tubular apoptosis.

Animals ; Apoptosis ; drug effects ; Body Weight ; drug effects ; Caspase 3 ; metabolism ; Chromatography, High Pressure Liquid ; Cytochromes c ; metabolism ; Doxorubicin ; adverse effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Kidney ; drug effects ; pathology ; Kidney Diseases ; blood ; chemically induced ; complications ; drug therapy ; Kidney Glomerulus ; drug effects ; pathology ; ultrastructure ; Kidney Tubules ; drug effects ; pathology ; ultrastructure ; Male ; Membrane Proteins ; metabolism ; NF-KappaB Inhibitor alpha ; metabolism ; NF-kappa B ; metabolism ; Organ Size ; drug effects ; Proteinuria ; blood ; complications ; drug therapy ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; bcl-2-Associated X Protein ; metabolism

BACKGROUND: Dapagliflozin, a selective sodium-glucose co-transporter 2 (SGLT2) inhibitor, lowers blood glucose by reducing glucose reabsorption at the proximal renal tubule in an insulin-independent manner. We aimed to evaluate the efficacy and safety of dapagliflozin and to identify the risk factors of adverse drug events in patients with type 2 diabetes. METHODS: As an institutional pharmacovigilance review activity, we reviewed data from medical records of 455 patients with type 2 diabetes who received dapagliflozin therapy from July 2014 to August 2015 in Seoul National University Hospital. We analyzed the changes in laboratory data and examined the characteristics of dapagliflozin users who showed adverse effects. RESULTS: Mean changes in HbA1c and fasting serum glucose level from baseline to second visit were −0.42% (8.07 ± 1.51% to 7.65 ± 1.31%, P < 0.001) and −22.9 mg/dL (167.8 ± 48.5 mg/dL to 144.9 ± 37.6 mg/dL, P < 0.001), respectively. Adverse drug events observed during this study were lower urinary tract symptoms (7.7%), dehydration-related symptoms (6.1%), ketonuria (3.4%), hypoglycemia (3.4%), and urogenital infection (4.2%). Thiazide use, age, insulin use, number of anti-diabetic drugs, gender and history of urogenital infection were the risk factors for adverse drug events (P < 0.05). CONCLUSION: Dapagliflozin significantly improved hyperglycemia in patients with type 2 diabetes without serious adverse drug events. The incidences of adverse drug events were was similar to those ofthat in the previous studies.
Blood Glucose ; Diabetes Mellitus ; Drug-Related Side Effects and Adverse Reactions ; Fasting ; Glucose ; Humans ; Hyperglycemia ; Hypoglycemia ; Incidence ; Insulin ; Ketosis ; Kidney Tubules, Proximal ; Lower Urinary Tract Symptoms ; Medical Records ; Pharmacovigilance* ; Risk Factors ; Seoul

PURPOSE: Diabetic nephropathy (DN) is a prevalent chronic microvascular complication of diabetes mellitus involving disturbances in electrolytes and the acid-base balance caused by a disorder of glucose metabolism. NHE1 is a Na+/H+ exchanger responsible for keeping intracellular pH (pHi) balance and cell growth. Our study aimed to investigate roles of NHE1 in high glucose (HG)-induced apoptosis in renal tubular epithelial cells. MATERIALS AND METHODS: Renal epithelial tubular cell line HK-2 was cultured in medium containing 5 mM or 30 mM glucose. Then, cell apoptosis, oxidative stress, NHE1 expression, and pHi were evaluated. NHE1 siRNA and inhibitor were used to evaluate its role in cell apoptosis. RESULTS: HG significantly increased cell apoptosis and the production of reactive oxygen species (ROS) and 8-OHdG (p<0.05). Meanwhile, we found that HG induced the expression of NHE1 and increased the pHi from 7.0 to 7.6 after 48 h of incubation. However, inhibiting NHE1 using its specific siRNA or antagonist DMA markedly reduced cell apoptosis stimulated by HG. In addition, suppressing cellular oxidative stress using antioxidants, such as glutathione and N-acetyl cysteine, significantly reduced the production of ROS, accompanied by a decrease in NHE1. We also found that activated cyclic GMP-Dependent Protein Kinase Type I (PKG) signaling promoted the production of ROS, which contributed to the regulation of NHE1 functions. CONCLUSION: Our study indicated that HG activates PKG signaling and elevates the production of ROS, which was responsible for the induction of NHE1 expression and dysfunction, as well as subsequent cell apoptosis, in renal tubular epithelial cells.
Antioxidants/metabolism ; Apoptosis/*drug effects ; Cation Transport Proteins/*metabolism ; Cell Cycle/drug effects ; Cell Line ; Dose-Response Relationship, Drug ; Epithelial Cells/*cytology/drug effects/*metabolism ; Glucose/*pharmacology ; Glutathione/metabolism ; Humans ; Kidney Tubules/*cytology ; Oxidative Stress/*drug effects ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects ; Sodium-Hydrogen Antiporter/*metabolism

OBJECTIVETo investigate the effect of arctiin on advanced oxidation protein product (AOPP)-induced epithelial-to-mesenchymal transition (EMT) in tubular cells and explore the mechanisms underlying this effect.

METHODSHuman proximal tubular cells (HK-2 cells) were treated with bovine serum albumin (BSA) or AOPPs in the presence or absence of arctiin. The expressions of E-cadherin, vimentin, and GRP78 at the protein and mRNA levels in the cells were examined using Western blotting and quantitative real-time PCR. The level of reactive oxygen species (ROS) was measured by flow cytometry with DCFH-DA as the fluorescent probe.

RESULTSCompared with BSA-treated cells, the cells treated with AOPPs showed decreased expression of epithelial cell marker E-cadherin and overexpression of mesenchymal marker vimentin and endoplasmic reticulum stress marker GRP78 with an increased ROS level. These changes induced by AOPPs were partly inhibited by arctiin.

CONCLUSIONArctiin can ameliorate AOPP-induced EMT in tubular cells by inhibiting endoplasmic reticulum stress, and oxidative stress response may participate in this process.

Advanced Oxidation Protein Products ; adverse effects ; Cadherins ; metabolism ; Cell Line ; Endoplasmic Reticulum Stress ; Epithelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; Furans ; pharmacology ; Glucosides ; pharmacology ; Heat-Shock Proteins ; metabolism ; Humans ; Kidney Tubules ; cytology ; drug effects ; Oxidative Stress ; Reactive Oxygen Species ; metabolism ; Vimentin ; metabolism

OBJECTIVETo study the clinical and histopathologic features of post-transplant kidney biopsy tissues from pediatric C-III donors.

METHODSThe clinical and pathologic features of 20 cases (22 case-times) of renal transplant biopsies from pediatric cadaveric donors were analyzed by light microscopy and immunohistochemistry according to the Banff system of working classification of renal allograft pathology. Biopsies were compared to those from adult C-III donors and adult cadaveric donors.

RESULTSSixteen cases (72.7%) showed renal allograft drug toxicity damage by Tacrolimus, seven cases (31.8%) showed degeneration and necrosis of renal tubular epithelial cells, four cases (18.2%) showed T cell-mediated acute rejection and six cases (27.3%) showed renal interstitial inflammation. There were two cases (9.1%) of renal dysplasia and one case (4.5%) of renal infarction. There was insufficient evidence for diagnosis of renal allograft nephropathy. Compared to post-transplant kidney from adult C-III donors, the proportion of drug toxicity damage was higher (P<0.05). Compared to post-transplant kidney from adult cadavers, the proportions of drug toxicity damage, degeneration and necrosis of renal tubular epithelial cells were higher (P<0.05) while the proportion of acute rejection was lower (P<0.05).

CONCLUSIONSThe pathologic changes in the post-transplant kidneys from pediatric donors are different from those from adult donors. Optimal long-term outcome can be accomplished by effective treatment based on timely or procedural biopsy.

Adult ; Age Factors ; Biopsy ; Cadaver ; Child ; Graft Rejection ; pathology ; Humans ; Immunohistochemistry ; Immunosuppressive Agents ; adverse effects ; Infarction ; pathology ; Kidney ; blood supply ; drug effects ; pathology ; Kidney Transplantation ; Kidney Tubules ; drug effects ; pathology ; Necrosis ; Tacrolimus ; adverse effects ; Transplantation, Homologous ; Treatment Outcome