Potent Therapy and Transcriptional Profile of Combined Erythropoietin-Derived Peptide Cyclic Helix B Surface Peptide and Caspase-3 siRNA against Kidney Ischemia/Reperfusion Injury in Mice.

Cause-specific treatment and timely diagnosis are still not available for acute kidney injury (AKI) apart from supportive therapy and serum creatinine measurement. A novel erythropoietin-derived cyclic helix B surface peptide (CHBP) protects kidneys against AKI with different causes, but the underlying mechanism is not fully defined. Herein, we investigated the transcriptional profile of renoprotection induced by CHBP and its potential synergistic effects with siRNA targeting caspase-3, an executing enzyme of apoptosis and inflammation (CASP3siRNA), on ischemia/reperfusion (IR)-induced AKI. Utilizing a mouse model with 30-minute renal bilateral ischemia and 48-hour reperfusion, the renoprotection of CHBP or CASP3siRNA was demonstrated in renal function and structure, active caspase-3 and HMGB1 expression. Combined treatment of CHBP and CASP3siRNA further preserved kidney structure and reduced active caspase-3 and HMGB1. Furthermore, differentially expressed genes (DEGs) were identified with fold change >1.414 and P < 0.05. In IR kidneys, 281 DEGs induced by CHBP were mainly involved in promoting cell division and improving cellular function and metabolism (upregulated signal transducer and activator of transcription 5B and solute carrier family 22 member 7). The additional administration of CASP3siRNA caused 504 and 418 DEGs in IR + CHBP kidneys with or without negative control small-interfering RNA, with 37 genes in common. These DEGs were associated with modulated apoptosis and inflammation (upregulated BCL6, SLPI, and SERPINA3M) as well as immunity, injury, and microvascular homeostasis (upregulated complement factor H and GREM1 and downregulated ANGPTL2). This proof-of-effect study indicated the potent renoprotection of CASP3siRNA upon CHBP at the early stage of IR-induced AKI. Underlying genes, BCL6, SLPI, SERPINA3M, GREM1, and ANGPTL2, might be potential new biomarkers for clinical applications. SIGNIFICANCE STATEMENT: It is imperative to explore new strategies of cause-specific treatment and timely diagnosis for acute kidney injury (AKI). CHBP and CASP3siRNA synergistically protected kidney structure after 48-hour ischemia/reperfusion-induced AKI with reduced injury mediators CASP3 and high mobility group box 1. CHBP upregulated cell division-, function-, and metabolism-related genes, whereas CASP3siRNA further regulated immune response- and tissue homeostasis-associated genes. Combined CHBP and CASP3siRNA might be a potent and specific treatment for AKI, and certain dysregulated genes secretory leukocyte peptidase inhibitor and SERPINA3M could facilitate timely diagnosis.

Protection of CTGF Antibody Against Diabetic Nephropathy in Mice Via Reducing Glomerular ß-Catenin Expression and Podocyte Epithelial-Mesenchymal Transition.

Despite substantial progress in medical care, the morbidity rate of diabetic nephropathy (DN) remains high in patients with diabetes. Evidence suggests that connective tissue growth factor (CTGF) induced podocyte injury may contribute to DN and CTGF inhibition could reduce albuminuria. However, to date the mechanisms involved in the effect of CTGF on podocyte injury have not been fully understood. The aim of this study is to investigate the effects of therapeutic CTGF antibody on glomerular ß-catenin expression and podocyte epithelial-mesenchymal transition (EMT) in diabetic mice. C57BL/6J mice were randomly divided into three groups as the following: the control, DN, and DN treated by CTGF antibody group. DN was induced by a single intraperitoneal injection of streptozotocin and then CTGF antibody was administrated three times per week for 8 weeks. Urinary albumin excretion, mesangial proliferation and matrix deposition, and ß-catenin expression in glomeruli at mRNA and protein level were all increased in DN mice compared to that in the control. Besides, the development of EMT in podocytes from diabetic mice, demonstrated by the downregulation of nephrin and upregulation of desmin in glomeruli, was detected. Furthermore, blocking CTGF by specific antibody reduced albuminuria, prevented the overexpression of CTGF, as well as ß-catenin, in glomeruli and subsequently ameliorated podocyte EMT in DN mice. In summary, this study suggested that CTGF antibody protected podocytes against injury in DN mice by reducing ß-catenin overexpression and preventing podocyte EMT, which might provide new insight into the mechanism of CTGF inhibition in the treatment of DN. J. Cell. Biochem. 118: 3706-3712, 2017. © 2017 Wiley Periodicals, Inc.

Diagnostic and prognostic value of galectin-3, serum creatinine, and cystatin C in chronic kidney diseases.

OBJECTIVES: This study was undertaken to determine the diagnostic and prognostic values of galectin-3 (Gal-3) in patients with chronic kidney disease (CKD). METHODS: Patients with CKD (n=150) were enrolled as the CKD group, which was divided into six groups according to glomerular filtration rates (GFR) indexes. At the same time, 50 healthy adults were chosen for the control group (NC). Measured data included the levels of serum Gal-3, serum creatinine (SCr), ß2 -microglobulin (ß2 -MG), 24-hour urinary protein, cystatin C (CysC), serum albumin (Alb) and other related indicators. RESULTS: There was no significant difference between CKD and NC group in age, gender and the level of Alb. CKD group had lower estimated glomerular filtration rate (eGFR) but higher Gal-3, CysC, SCr, ß2 MG and 24-hour urinary protein excretion than control group (P<.001). Moreover, the receiver operating characteristic (ROC) analysis of Gal-3, CysC and SCr revealed that the corresponding areas under the curve (AUC) were 0.89, 0.83 and 0.85, respectively, and the AUC value of joint ROC curve of Gal-3, CysC and SCr was 0.96. In addition, the 6-year kidney survival rates of low Gal-3 group and high Gal-3 group were 47.3% and 22.8% respectively (HR=2.65; P<.01). CONCLUSIONS: Our study verified Gal-3, CysC and SCr were negatively related to eGFR. Besides, it is suggested that Gal-3 can be used as an indicating factor in the diagnosis of CKD; the joint analysis of Gal-3, CysC and SCr for CKD may distinctly improve diagnostic accuracy.

[The Role of HMGN2 in the Development of Periodontitis Dental Plaque].

OBJECTIVES: To determine the role of high mobility group chromosomal protein N2 (HMGN2) in the development of periodontitis plaque biofilm. METHODS: Saliva samples were collected before clinical interventions in patients with periodontitis (25 Mild periodontitis, 25 Moderate periodontitis and 25 Severe periodontitis) and healthy controls ( n=25).Following an estimation of dental plaque index, the level of HMGN2 in saliva was determined by enzyme-linked immunosorbent assay (ELISA).The recombinant human HMGN2 protein was purified and tested, its inhibitive effects on Porphyromonas gingivalis (P.g)growth and formation of P.g bioflim were detected by K-B antibacterial annulus and crystal violet staining, respectively. RESULTS: The healthy controls had lower levels of HMGN2 in saliva than those with periodontitis, especially those with severe periodontitis ( P<0.01).The level of HMGN2 was negatively correlated with dental plaque index ( r=-0.363, P<0.05). HMGN2 inhibited the development of main periodontal bacteria biofilm ( P<0.05). CONCLUSIONS: HMGN2 is involved in the development of periodontitis plaque biofilm and plays an important role in the development of periodontitis.

[Inhibition of Cigarettes Smoke-induced Epithelial to Mesenchymal Transition by the SMO Inhibitor PF-5274857 in Beas-2b Epithelial Cells].

OBJECTIVES: To explore the effects of the Smoothened (Smo) inhibitor PF-5274857 on cigarette smoke (CS)-induced epithelial-mesenchymal transition (EMT) and apply a new idea fororal squamous cell carcinoma (OSCC) treatment. METHODS: Beas-2b cells were used to investigate the CS-induced EMT. Both pretreat and post-treat were performed in the study. In pretreat group, after being pretreated with 3 µmol/L PF-5274857 for 2 h, cells were cultured with CS for 8 d; In post-treat group, cells were treated by 3 µmol/L PF-5274857 for 4 d after CS cultrue. Western blot and immunofluorescence were applied to examine the EMT markers E-cadherin, vimentin, and smooth muscle actin α (α-SMA) in Beas-2b epithelial cells. The transwell culture system was used in migration assays. RESULTS: Pretreat Beas-2b cells with PF-5274857 for 2 h can prevent the CS-induced EMT for epithelial and mesenchymal markers, as well as migration capacity. Up regulated E-cadherin and down regulated vimentin and α-SMA were observed by Western blot. Furthermore Beas-2b cells induced by CS that underwent EMT showed increased E-cadherin and decreased vimentin and α-SMA after treatment with PF-5274857 for 4 d. Importantly, the elevated migration capacity level was also decreased. CONCLUSIONS: The Smo inhibitor PF-5274857 has both preventive effect and therapeutic potential against CS-induced EMT.

Changes of adiponectin and its receptors in rats following chronic renal failure.

OBJECTIVE: To investigate changes of adiponectin and its receptors (Adipo R) in rats following chronic renal failure. METHODS: Male SD rats were randomly divided into two groups: control group and chronic renal failure (CRF) group. The CRF group were gavaged with adenine (300 mg/kg/d) for 4 weeks and the control group with drinking water. All rats were anesthetized at 2nd or 4th week and blood and urine samples were collected for detection of renal function, 24 h urine protein and adiponectin concentration. Renal tissues were also collected for HE staining, immunohistochemistry and RT-PCR screening. RESULTS: Compared with control group, the serum concentrations of urea and creatinine, 24 h urine protein excretion in the CRF group were significantly higher at 2nd week and further increased at 4th week (p < 0.05). The adiponectin levels in serum and urine in the CRF group were significantly higher than those of control group (p < 0.01). The renal expressions of Adipo R1 and Adipo R2 in CRF group were also significantly increased compared to control group (p < 0.01). The increased expressions of Adipo R1 and Adipo R2 were positively related to the adiponectin levels in serum, urine, and 24 h urine protein. CONCLUSION: The significant changes in expression of adiponectin and its receptors in rat CRF model could be an adaptive response that may provide the basis to understand pathological changes in chronic kidney disease.

In situ forming hydrogels via catalyst-free and bioorthogonal "tetrazole-alkene" photo-click chemistry.

In situ forming hydrogels were developed from 4-arm poly(ethylene glycol)-methacrylate (PEG-4-MA) and -tetrazole (PEG-4-Tet) derivatives through catalyst-free and bioorthogonal "tetrazole-alkene" photo-click chemistry. PEG-4-MA and PEG-4-Tet (Mn = 10 kg/mol) were soluble at 37 °C in phosphate buffer (PB, pH 7.4, 10 mM) at total polymer concentrations ranging from 20 to 60 wt % but formed fluorescent hydrogels upon 365 nm UV irradiation at an intensity of 20.6, 30.7, or 60 mW/cm(2). The gelation times ranged from ca. 50 s to 5 min, and storage moduli varied from 0.65 to 25.2 kPa depending on polymer concentrations and degrees of Tet substitution in PEG-4-Tet conjugates. The cell experiments via an indirect contact assay demonstrated that these "tetrazole-alkene" photo-click PEG hydrogels were noncytotoxic. The high specificity of photo-click reaction renders thus obtained PEG hydrogels particularly interesting for controlled protein release. Notably, in vitro release studies showed that cytochrome c (CC), γ-globulins (Ig), and recombinant human interleukin-2 (rhIL-2) all were released from PEG hydrogels in a sustained and quantitative manner over a period of 14-20 days. Importantly, released CC and rhIL-2 exhibited comparable biological activities to native CC and rhIL-2, respectively. These results confirm that "tetrazole-alkene" photo-click reaction is highly compatible with these loaded proteins. This photo-controlled, specific, efficient, and catalyst-free click chemistry provides a new and versatile strategy to in situ forming hydrogels that hold tremendous potentials for protein delivery and tissue engineering.

Protective effects of HBSP on ischemia reperfusion and cyclosporine a induced renal injury.

Ischemia reperfusion (IR) and cyclosporine A (CsA) injuries are unavoidable in kidney transplantation and are associated with allograft dysfunction. Herein, the effect and mechanism of a novel tissue protective peptide, helix B surface peptide (HBSP) derived from erythropoietin, were investigated in a rat model. The right kidney was subjected to 45 min ischemia, followed by left nephrectomy and 2-week reperfusion, with or without daily treatment of CsA 25 mg/kg and/or HBSP 8 nmol/kg. Blood urea nitrogen was increased by CsA but decreased by HBSP at 1 week and 2 weeks, while the same changes were revealed in urinary protein/creatinine only at 2 weeks. HBSP also significantly ameliorated tubulointerstitial damage and interstitial fibrosis, which were gradually increased by IR and CsA. In addition, apoptotic cells, infiltrated inflammatory cells, and active caspase-3+ cells were greatly reduced by HBSP in the both IR and IR + CsA groups. The 17 kD active caspase-3 protein was decreased by HBSP in the IR and IR + CsA kidneys, with decreased mRNA only in the IR + CsA kidneys. Taken together, it has been demonstrated, for the first time, that HBSP effectively improved renal function and tissue damage caused by IR and/or CsA, which might be through reducing caspase-3 activation and synthesis, apoptosis, and inflammation.

Lanthanum carbonate for the treatment of hyperphosphatemia in CKD 5D: multicenter, double blind, randomized, controlled trial in mainland China.

BACKGROUND: Serum phosphorus control is critical for chronic kidney disease (CKD) 5D patients. Currently, clinical profile for an oral phosphorus binder in the mainland Chinese population is not available. OBJECTIVE: To establish the efficacy, safety, and tolerability of lanthanum carbonate in CKD 5D patients. DESIGN: Multicenter, randomized, double blind, placebo-controlled study. A central randomization center used computer generated tables to allocate treatments. SETTING: Twelve tertiary teaching hospitals and medical university affiliated hospitals in mainland China. PARTICIPANTS: Overall, 258 hemodialysis or continuous ambulatory peritoneal dialysis (CAPD) adult patients were enrolled. INTERVENTION: After a 0-3-week washout period and a 4-week lanthanum carbonate dose-titration period, 230 patients were randomized 1:1 to receive lanthanum carbonate (1500 mg-3000 mg) or placebo for a further 4-week maintenance phase. MAIN OUTCOME MEASURES: Efficacy and safety of lanthanum carbonate to achieve and maintain target serum phosphorus concentrations were assessed. RESULTS: In the titration phase, serum phosphorus concentrations of all patients decreased significantly. About three-fifths achieved target levels without significantly disturbing serum calcium levels. At the end of the maintenance period, the mean difference in serum phosphorus was significantly different between the lanthanum carbonate and placebo-treated groups (0.63±0.62 mmol/L vs. 0.15±0.52 mmol/L, P < 0.001). The drug-related adverse effects were mild and mostly gastrointestinal in nature. CONCLUSION: Lanthanum carbonate is an efficacious and well-tolerated oral phosphate binder with a mild AE profile in hemodialysis and CAPD patients. This agent may provide an alternative for the treatment of hyperphosphatemia in CKD 5D patients in mainland China. TRIAL REGISTRATION: No. ChiCTR-TRC-10000817.

Multiple nocardial abscesses secondary to anti­neutrophil cytoplasmic antibody­associated vasculitis in an elderly patient: A case report and literature review.

A nocardial abscess is a relatively rare opportunistic infection that typically occurs after immunosuppressive treatment and is a clinical challenge. In the present study, the case of a 69-year-old patient with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and lung and kidney involvement, was reported. The patient received systemic glucocorticoid and cyclophosphamide treatment for 6 months, after which a large encapsulated abscess appeared on magnetic resonance imaging and CT in the subcutaneous tissue of the left hip and lung, respectively, and the pus culture showed Nocardia. Orthopedic abscess incision and ultrasound-guided thoracic puncture drainage were performed, and the lesion was completely absorbed after 1 month of treatment with linezolid and compound sulfamethoxazole. Tests for ANCA were negative, and renal function and urine tests were completely normal after 1 year of follow-up. Furthermore, a literature review performed for the present study retrieved a few reports of successful treatment of multiple nocardial abscesses secondary to ANCA-associated vasculitis in elderly patients in a short period of time. Therefore, the present case report and literature review have been reported to improve awareness of this rare disease, so as to facilitate its early diagnosis and treatment.

Platelet glycoprotein IIb HPA-3 a/b polymorphism is associated with native arteriovenous fistula thrombosis in chronic hemodialysis patients.

OBJECTIVE: The aim of this study was to investigate the association of Glycoprotein IIb (GPIIb) human platelet antigen-3 (HPA-3) a/b polymorphism with end-stage renal disease (ESRD) on hemodialysis (HD) and native Arteriovenous fistula (AVF) thrombosis. METHODS: The polymorphism in the GPIIb subunit of the receptor HPA-3 (a and b alleles) was identified by polymerase chain reaction with sequence-specific primers (PCR-SSP) in 145 HD patients and 120 healthy controls from a Chinese Han population. The HD patients were classified into two groups: G1 and G2. G1 included 65 HD patients presented at least one AVF thrombosis episode and G2 included 80 HD patients without any episode of AVF thrombosis. RESULTS: There were no significant differences in either HPA-3 a/b genotypes (aa, ab, and bb) frequency distribution (p = 0.396) or allele (a and b) frequency distribution (p = 0.146) between HD patients and control groups. However, there were significant differences in both HPA-3 a/b genotypes (aa, ab, and bb) distribution (χ(2) = 6.127, p = 0.047) and allele (a and b) frequency distribution (χ(2) = 5.954, p = 0.015) between G1 and G2. The relative risk of native AVF dysfunction in ab + bb patients compared with that of aa patients was 2.31 (95% confidence interval: 1.18-4.52). CONCLUSIONS: These findings suggest an association between AVF thrombosis and the HPA-3b allele, and it is likely that HPA-3 a/b polymorphisms could be useful markers for potential risk of native AVF thrombosis in HD patients.

XCL1 Aggravates Diabetic Nephropathy-Mediated Renal Glomerular Endothelial Cell Apoptosis and Inflammatory Response via Regulating p53/Nuclear Factor-Kappa B Pathway.

BACKGROUND: Glomerular endothelial cell damage plays an important role in the occurrence and development of diabetic nephropathy (DN). OBJECTIVES: This study aimed to clarify the role of XCL1 in DN-mediated glomerular endothelial cell apoptosis and whether the function was related to the activation of the p53/nuclear factor-kappa B (NF-κB) signaling pathway. METHODS: Candidate biomarkers were identified by least absolute shrinkage and selection operator (LASSO) regression model analysis. The area under the receiver operating characteristic curve value was calculated and used to evaluate the discriminating ability. Cell viability, apoptosis, and interleukin-1ß and tumor necrosis factor-α expression at messenger RNA and protein levels were detected by using the Cell Counting Kit-8, flow cytometry, ELISA, real-time polymerase chain reaction, and Western blotting assays. In vivo studies were conducted in the DN mice. RESULTS: The LASSO regression model displayed good discriminating performance, with a C-index of 0.803 and good calibration, and high XCL1 expression was identified as the predicting factor for DN in diabetes mellitus patients. XCL1 expression was upregulated in glomeruli of db/db mice, which was closely related to the expression of its receptor (XCR1). XCL1 overexpression played an important role in the apoptosis and inflammatory response of high glucose (HG)-treated human renal glomerular endothelial cells. Meanwhile, the expression of p53 and the levels of inflammatory cytokines were upregulated upon XCL1 overexpression. p53 silencing with its inhibitor blocked the apoptotic response and inflammatory response in XCL1-overexpressed cells exposed to HG. Besides, the XCL1 overexpression-induced downregulation of NF-κB was reversed by pifithrin-α pretreatment. CONCLUSIONS: Our findings in this work provided the mechanistic insights into the effects of XCL1 on the modulation of DN development, illustrating that XCL1 might serve as an essential prognostic indicator and therapeutic target for DN progression.

Suppression of the PI3K-Akt pathway is involved in the decreased adhesion and migration of bone marrow-derived mesenchymal stem cells from non-obese diabetic mice.

T1DM (type 1 diabetes mellitus) is an autoimmune disease characterized by T-cell-mediated damage of islet ß-cells. The pathology of NOD (non-obese diabetic) mouse involves the insulitis induced by infiltration of T-cells, a similar pathogenic mechanism in T1DM patient. BM-MSCs (bone marrow mesenchymal stem cells) are multipotent progenitor cells that can be isolated from a number of sources. Recent studies have shown that transplantation of MSCs to the NOD mice could prevent the process and have the therapeutic effects on T1DM. In our studies, we have found that migration and adhesion of BM-MSCs from NOD mice were suppressed compared with the BM-MSCs from ICR (imprinting control region) mice, accompanying with the abnormal distribution of FAK (focal adhesion kinase) and F-actin (filamentous actin). Further, we have found that the activation of PI3K (phosphoinositide 3-kinase)-Akt pathway was suppressed in BM-MSCs from NOD mice. When the PI3K-Akt pathway was inhibited by LY294002, the adhesion and migration of BM-MSCs from ICR mice were suppressed as well. These results indicated that the suppression of PI3K-Akt pathway is involved in the decreased adhesion and migration of BM-MSCs from NOD mice.

Analyzing clinical characteristics of patients with different cumulative hemodialysis durations: a cross-sectional study.

BACKGROUND: The objective of this study was to examine the clinical characteristics of patients with different cumulative hemodialysis (HD) durations, so as to improve their survival rate. METHODS: In this cross-sectional study, we extracted background information and relevant clinical data from 145 patients who were undergoing maintenance HD three times a week at the Affiliated Hospital of Nantong University between January 1998 and January 2019. The study subjects were divided into four groups according to the duration of their HD: <5 years, 5-10 years, 10-15 years, and >15 years of HD. We collected the medical history and relevant clinical parameters for each subject, and measured the urea reduction ratio (URR), hemoglobin (Hb), serum calcium, phosphorus, parathyroid hormone (iPTH), and serum albumin (ALB) levels for each group. RESULTS: The average patient age was 52.06 ±  11.93 years old. The average patient age in the 10-15 years and >15 years groups was significantly lower than in the <5 years and 5-10 years groups (P = 0.002, P < 0.001, P = 0.012, and P = 0.0025, respectively). The most common cause of end-stage renal disease (ESRD) was chronic glomerulonephritis. We found no significant differences in URR, Hb, serum calcium, serum phosphorus, iPTH, and ALB levels. CONCLUSION: A prolonged HD duration was related to a younger mean age at the start of HD treatment. The leading cause of ESRD was chronic glomerulonephritis. We predominantly found diabetic nephropathy in the group with a duration of <5 years cumulative HD. Most of the indexes related to hemodialysis almost satisfied the recommended values in these patients.

Long-Term Protection of CHBP Against Combinational Renal Injury Induced by Both Ischemia-Reperfusion and Cyclosporine A in Mice.

Renal ischemia-reperfusion (IR) injury and cyclosporine A (CsA) nephrotoxicity affect allograft function and survival. The prolonged effects and underlying mechanisms of erythropoietin derived cyclic helix B peptide (CHBP) and/or caspase-3 small interfering RNA (CASP-3siRNA) were investigated in mouse kidneys, as well as kidney epithelial cells (TCMK-1), subjected to transplant-related injuries. Bilateral renal pedicles were clamped for 30 min followed by reperfusion for 2 and 8 weeks, with/without 35 mg/kg CsA gavage daily and/or 24 nmol/kg CHBP intraperitoneal injection every 3 days. The ratio of urinary albumin to creatinine was raised by IR injury, further increased by CsA and lowered by CHBP at 2, 4, 6 and 8 weeks, whereas the level of SCr was not significantly affected. Similar change trends were revealed in tubulointerstitial damage and fibrosis, HMGB1 and active CASP-3 protein. Increased apoptotic cells in IR kidneys were decreased by CsA and CHBP at 2 and/or 8 weeks. p70 S6 kinase and mTOR were reduced by CsA with/without CHBP at 2 weeks, so were S6 ribosomal protein and GSK-3ß at 8 weeks, with reduced CASP-3 at both time points. CASP-3 was further decreased by CHBP in IR or IR + CsA kidneys at 2 or 8 weeks. Furthermore, in TCMK-1 cells CsA induced apoptosis was decreased by CHBP and/or CASP-3siRNA treatment. Taken together, CHBP predominantly protects kidneys against IR injury at 2 weeks and/or CsA nephrotoxicity at 8 weeks, with different underlying mechanisms. Urinary albumin/creatinine is a good biomarker in monitoring the progression of transplant-related injuries. CsA divergently affects apoptosis in kidneys and cultured kidney epithelial cells, in which CHBP and/or CASP-3siRNA reduces inflammation and apoptosis.

Erythropoietin Derived Peptide Improved Endoplasmic Reticulum Stress and Ischemia-Reperfusion Related Cellular and Renal Injury.

Ischemia-reperfusion (IR) injury often affects transplant and native kidneys alike. IR injury is one of the main causes of acute kidney injury (AKI) and further associated with the progression of chronic kidney disease. Our previous study revealed the renoprotection of erythropoietin derived cyclic helix-B surface peptide (CHBP) against IR injury. However, the precise role and underlying mechanism of endoplasmic reticulum stress (ERS) in the injury and the renoprotection induced by IR or CHBP, respectively, have not been fully defined. This study using mouse kidney epithelial cells (TCMK-1) revealed that the level of CHOP (a key marker of ERS), PERK, and JNK (regulators of CHOP) was gradually increased by the prolonged time of hydrogen peroxide (H2O2) stimulation. In addition, CHOP mRNA and protein were significantly reduced by small interfering RNA (siRNA) target CHOP, as were apoptotic and inflammatory mediator caspase-3 and HMGB-1, and early apoptosis. Furthermore, CHOP mRNA was correlated positively with PERK protein, active caspase-3, HMGB-1 and apoptosis, but negatively with cell viability in vitro, while CHOP protein was also correlated positively with the level of tubulointerstitial damage and active caspase-3 protein in vivo. Finally, CHBP improved the viability of TCMK-1 cells subjected to H2O2 stimulation time-dependently, with reduced level of CHOP mRNA. CHBP also inhibited the increase of CHOP protein, not only in TCMK-1 cells, but also in the IR injury kidneys at 2 weeks. Moreover, CHBP reduced the expression of PERK mRNA and protein, JNK and HMGB-1 protein, as well as early and later apoptosis. In addition, raised CHOP at 12 h post IR injury might be an early time window for intervention. In conclusion, the differential role of ERS and CHBP in IR-related injury was proved in mouse TCMK-1 cells and kidneys, in which the mechanistic signaling pathway was associated with CHOP/PERK/JNK, HMGB-1/caspase-3, and apoptosis. CHOP might be a potential biomarker and CHBP might be therapeutic drug for IR-induced AKI.

Streptococcosis in farmed Litopenaeus vannamei: a new emerging bacterial disease of penaeid shrimp.

Presumptive systemic streptococcal infections were detected histologically in farmed Litopenaeus vannamei juveniles submitted from a Latin American country and the bacteria isolated. Characterization work demonstrated that the Gram-positive cocci form chains, grow aerobically and anaerobically, are oxidase- and catalase-negative, non-hemolytic, non-motile, Lancefield Group B positive and PCR positive when amplified with a universal streptococcal primer set. Differing Streptococcus identifications were obtained using API 20 Strep and Biolog systems, the former identifying the isolate as S. uberis and the latter as S. parauberis. Injection of specific pathogen-free (SPF) L. vannamei with the bacteria resulted in 100% mortality by 3 d post-injection with successful recovery of the agent from moribund test shrimp hemolymph samples. The recovered isolate was used in per os and waterborne exposure studies of SPF L. vannamei with mortalities ranging from 40 to 100% and 80 to 100%, respectively. Histologic analysis of 5 to 8 moribund shrimp from each exposure method demonstrated that all contained a severe bacteremia characterized by numerous free cocci within the hemolymph and aggregates of vacuolated hemocytes with notable intravacuolar cocci. This unique lesion type was most pronounced within the lymphoid organ and considered pathodiagnostic for this disease. Experimentally induced lesions were identical to those in naturally infected farmed shrimp and the Streptococcus sp. responsible was re-isolated, fulfilling Koch's postulates. Five freeze/thaw cycles of 10 experimentally infected shrimp were performed over a 2 mo period and the bacteria successfully cultured from all shrimp at each interval. These collective findings describe the first reported case of streptococcosis in marine penaeid shrimp in the Western Hemisphere and indicate that the agent may be disseminated via live or frozen infected shrimp.

Fibroblasts rescue oral squamous cancer cell from metformin-induced apoptosis via alleviating metabolic disbalance and inhibiting AMPK pathway.

Metformin is an antidiabetic drug widely used for the treatment of type 2 diabetes. Growing evidence suggests that it may exert antitumor effects in vivo and in vitro. However, even with the promising potency on defeating cancer cells, the pre-clinical and epidemiological studies of metformin on various kinds of cancers are not satisfactory, and the reasons and underlying mechanisms remain unknown. Since cancer is a complex system, dependent on a promoting microenvironment, we hypothesize that the interactions between cancer cells and their neighborhood fibroblasts are essential for metformin resistance. To test this, we used a cell co-culture model closely mimicking the in vivo interactions and metabolic exchanges between normal stromal cells (NOFs) and oral squamous cancer cells (OSCC). Here we show that while metformin can significantly inhibit cell growth and induce apoptosis of OSCC cultured alone in a dose-dependent manner through activating p-AMPKT172 and modulating Bcl-2, Bax, and cleaved PARP. However, when OSCC are co-cultured with NOFs the metformin effects on OSCC cells are annihilated. NOFs are rescuing OSCC from metformin - induced apoptosis, at least partially, through inhibiting the activity of AMPK and PARP, maintaining mitochondrial membrane potential and increasing the oxidative stress. Our results indicate that metformin effects on oral cancer cells are modulated by the microenvironment and that this has to be taken into consideration in the context of developing a new combination of drugs for oral cancer treatment.

[Role of protein kinase D1 in regulating the growth, apoptosis and drug sensitivity of oral squamous carcinoma cells].

OBJECTIVE: This study aimed to investigate the role of protein kinase D (PKD)1 in regulating the growth, apop-tosis, and drug sensitivity of the squamous carcinoma cell line SCC-25. METHODS: The SCC-25 cell line was transfected with either the control-shRNA or PKD1-shRNA plasmids. The stable transfected cells were selected, and the efficiency of PKD1 knockdown was detected by Western blot. The growth and apoptosis of SCC-25 were analyzed with a cell counting kit-8 (CCK8) and flow cytometry. The 50% inhibitory concentrations (IC50) of paclitaxel in the control and PKD1 knockdown cell lines were detected by CCK-8. The expression levels of Bax, Bcl-2, and P-gp were detected by Western blot. RESULTS: PKD1 was constitutively expressed and phosphorylated in various cancer cell lines. Inhibiting the expression of PKD1 in SCC-25 cells by RNA interference could inhibit the growth and promote the apoptosis of SCC-25 cells via downregulating Bcl-2 expression. Additionally, inhibiting PKD1 expression could downregulate the expression of P-gp, thereby decreasing both the IC50 and resistance index of paclitaxel. CONCLUSIONS: PKD1 plays an important role in regulating the biobehavior of SCC-25. It is a potential therapeutic target for oral squamous carcinoma.

Salivary protease spectrum biomarkers of oral cancer.

Proteases are important molecules that are involved in many physiological and pathological processes of the human body, such as growth, apoptosis and metastasis cancer cells. They are potential targets in cancer diagnosis and biotherapy. In this study, we analyzed the salivary protease spectrum of patients with oral squamous cell carcinoma (OSCC), oral benign masses and chronic periodontitis, as well as that of health, using human protease array kits, enzyme-linked immunosorbent assay, western blot and immunofluorescence. The salivary protease spectrum was found to be associated with oral diseases. For example, the saliva of patients with OSCC contained increased numbers of proteases than those of other oral diseases and health. The levels of matrix metalloproteinase (MMP)-1, MMP-2, MMP-10, MMP-12, A disintegrin and metalloprotease (ADAM)9, A disintegrin and metalloprotease with thrombospondin type 13 motifs (ADAMST13), cathepsin V and kallikrein 5 in the saliva of patients with OSCC were significantly increased compared with those of other groups. Taking MMP-1, cathepsin V, kallikrein 5 and ADAM9 as biomarkers of OSCC, cutoff values were199, 11.34, 9.29 and 202.55 pg·mL-1, respectively. From the area under the curve, sensitivity and specificity, the combination of cathepsin V/kallikrein5/ADAM9 was an optimal biomarker for diagnosing OSCC. Thus, analysis of the salivary protease spectrum may be an innovative and cost-efficient approach to evaluating the health status of the oral cavity. Specifically, increases in cathepsin V, kallikrein 5 and ADAM9 may be useful biomarkers in the screening and diagnosis of OSCC.