Screening of Diabetic Nephropathy Progression-Related Genes Based on Weighted Gene Co-expression Network Analysis.

The purpose of this study is to explore the progression-related genes of diabetic nephropathy (DN) through weighted gene co-expression network analysis (WGCNA). The gene expression dataset GSE14202 was downloaded from the GEO database for differential expression analysis. WGCNA v1.69 was used to perform co-expression analysis on differentially expressed genes. 25 modular genes were selected through WGCNA. The motif enrichment analysis was performed on 25 genes, and 34 motifs were obtained, of which 8 transcription factors (TFs) were differentially expressed. GENIE3 was applied to analyze the expression correlation of 8 differentially expressed TFs and 25 genes. Combined with the predicted TF-target gene relationship, 69 interactions between 8 TFs and 18 genes were obtained. The functional enrichment analysis of 18 genes showed that 7 key genes were obviously enriched in adaptive immune response and were clearly up-regulated in advanced DN patients. The expression of C1S, LAIR1, CD84, SIT1, SASH3, and CD180 in glomerular samples from DN patients was significantly up-regulated in compared with normal samples, and the expression of these genes was negatively correlated with GFR. We observed that in the in vitro cell model of DN, the relative expression levels of 5 key genes (except SASH3) were obviously elevated in the high-glucose group. Five key genes were identified to be related to the progression of DN. The findings of this study may provide new ideas and therapeutic targets for exploring the pathogenesis of DN.

Carbon dots conjugated to SN38 for improved colorectal anticancer therapy.

Irinotecan (CTP-11) is one of the standard therapies for colorectal cancer (CRC). CTP-11 is enzymatically converted to the hydrophobic 7-ethyl-10-hydroxycamptothecin (SN38), a one hundred-fold more active metabolite. Conjugation of hydrophobic anticancer drugs to nanomaterials is a strategy to improve their solubility, efficacy, and selectivity. Carbon dots (CDs) have garnered interest for their small sizes (<10 â€‹nm), low toxicity, high water solubility, and bright fluorescence. This paper describes the use of CDs to improve drug vehiculation, stability, and chemotherapeutic efficiency of SN38 through a direct intracellular uptake in CRC. The covalent conjugation of SN38 to CDs via a carbamate bond provides a CD-SN38 hybrid material for slow, sustained, and pH-responsive drug release. CD-SN38 successfully penetrates the CRC cells with a release in the nucleus affecting first the cell cycle and then the cytoskeleton. Moreover, CD-SN38 leads to a deregulation of the extracellular matrix (ECM), one of the major components of the cancer niche considered a possible target therapy for reducing the cancer progression. This work shows the combined therapeutic and imaging potential of CD-based hybrid materials for the treatment of CRC. Future efforts for targeted therapy of chronic diseases characterized by altered ECM deposition, such as chronic kidney disease and chronic allograft nephropathy in kidney transplant patients are envisaged.

Protective Effects of Combination of Radix Astragali and Radix Salviae Miltiorrhizae on Kidney of Spontaneously Hypertensive Rats and Renal Intrinsic Cells.

OBJECTIVE: To evaluate the effects of combination of Radix Astragali (RA) and Radix Salviae Miltiorrhizae (RS) on kidney of spontaneously hypertensive rats (SHRs) and renal intrinsic cells. METHODS: SHRs were intragastrically administrated with RA (5.09 g/kg) and RS (2.55 g/kg) either alone or with combination for 4 weeks; valsartan (13.35 mg/kg) was used as a positive control. Blood pressure and renal ultrasonography were monitored periodically. The biomarkers [microalbumin (mALB), cystatin ^C, angiotensin II (Ang II), interleukin-1 beta (IL-1ß), and ß2-microglobulin (ß2-Mg), etc.] in serum and urine were measured by enzyme-linked immunosorbent assay (ELISA). The protein expressions [phosphorylated adenosine 5'-monophosphate-activated protein kinase-α1 (p-AMPKα1), sestrin-ß, calcium/calmodulin-dependent protein kinase kinase-ß (CaMKK-ß), phosphoinositide 3-kinases (PI3K), serine-threonine protein kinase 1 (AKT1), and vascular endothelial growth factor receptor 2 (VEGFR2)] in renal cortex were determined by Western blot. In vitro, the hypertensive cellular model was established by applying 2×10-6 mol/L Ang ^II. The primary human podocytes, human glomerular endothelial cells (HRGECs), and human proximal tubular epithelial cells (HK-2s) were pre-incubated with sulfotanshinone sodium (Tan, 10 µg/mL) and/or calycosin-7-O-ß-D-glucoside (Cal, 5 µg/mL). The cellular viability and apoptosis were assayed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and Annexin V/PI staining, respectively. The level of endothelial nitric oxide synthase (eNOS) in culture supernatant was determined by ELISA. RESULTS: RA+RS signifificantly decreased the diastolic blood pressure, renal vascular resistance index, and parenchymal thickness, increased 24 h urinary volume as well as lowered the levels of urine mALB and serum cystatin ^C, IL-1ß and ß2-Mg of SHRs (P <0.05 vs. SHRs). The decreased protein levels of p-AMPKα1, sestrinß and CaMKK-ß and the increased protein levels of PI3K, AKT1 and VEGFR2 in renal cortex of SHRs were normalized after RA+RS treatment (P <0.05). In vitro, Tan and Cal attenuated the Ang II-induced abnormal proliferation and increased the apoptosis of HRGECs and HK-2s and improved the level of eNOS in culture supernatant. Whereas, neither of them showed powerful effect on podocyte. CONCLUSION: The combination of RA and RS had potential effects on alleviating the renal damages of SHRs and the renoprotection was independent of blood pressure level.

Toll-Like Receptor 3 Signaling Contributes to Regional Neutrophil Recruitment in Cultured Human Glomerular Endothelial Cells.

BACKGROUND: Given the importance of neutrophil recruitment in the pathogenesis of glomerulonephritis (GN), the representative neutrophil chemoattractant C-X-C motif chemokine 1 (CXCL1)/GROα and the adhesion molecule E-selectin in glomerular endothelial cells (GECs) play a pivotal role in the development of GN. Endothelial Toll-like receptor 3 (TLR3) is thought to be involved in the inflammatory response via innate immunity. However, the role of endothelial TLR3 signaling in the expression of neutrophil chemoattractants and adhesion molecules remains to be elucidated. Thus, we aimed to examine this issue. METHODS: We treated normal human GECs with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA, and analyzed the expressions of CXCL1 and E-selectin using quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and enzyme-linked immunosorbent assay. To further elucidate the poly IC-induced signaling pathway, we subjected the cells to RNA interference against TLR3, interferon (IFN)-ß, nuclear factor (NF)-κB p65, and IFN regulatory factor (IRF) 3. We also used immunofluorescence to examine the endothelial expression of CXCL1 in biopsy specimens from patients with crescentic and non-crescentic purpura nephritis (PN). RESULTS: We found that the activation of TLR3 induced the endothelial expression of CXCL1 and E-selectin, and that this involved TLR3, -NF-κB, IRF3, and IFN-ß. Intense endothelial CXCL1 expression was observed in biopsy specimens from patients with crescentic PN. CONCLUSION: These findings support a role for glomerular antiviral innate immunity in the pathogenesis of GN. Intervention of glomerular TLR3 signaling may therefore be a suitable therapeutic strategy for treating GN in the future.

Angiotensin 1-7 attenuates angiotensin Ⅱ-induced human glomerular en-dothelial cell injury via Mas receptor / 中国病理生理杂志

AIM:To investigate the effect of angiotensin 1-7(Ang1-7)on the human glomerular endothelial cells(HGECs)injury induced by angiotensin Ⅱ(Ang Ⅱ)and its possible mechanism.METHODS: Cultured HGECs were divided into 6 groups randomly: control group, Ang Ⅱ group, Ang1-7 group, Ang Ⅱ +Ang1-7 group, Ang Ⅱ +Ang1-7+A779(an inhibitor of Mas receptor)group and A779 group.The apoptotic rate and reactive oxygen species (ROS)of HGECs were analyzed by flow cytometry and photographed by fluorescence microscopy.The levels of lactate de-hydrogenase(LDH),nitric oxide(NO),endothelin-1(ET-1),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α), transforming growth factor-β(TGF-β),monocyte chemoattractant protein-1(MCP-1)and intercellular adhesion molecule-1(ICAM-1)in the supernatant of cell cultures were measured.RESULTS:Compared with the control group,the apoptot-ic rate and the average fluorescence intensity of ROS were increased in the Ang Ⅱ group,IL-6,TNF-α,TGF-β,ICAM-1 and MCP-1 in cell supernatants were also increased in the Ang Ⅱ group(P<0.05).Compared with the Ang Ⅱ group,the apoptotic rate,ROS level, and the above inflammatory factors were decreased in Ang Ⅱ +Ang1-7 group(P<0.05). Compared with the Ang Ⅱ +Ang1-7 group,adding A779 increased the cell apoptotic rate,ROS production and the releases of the above inflammatory factors in cell supernatants(P<0.05).Compared with the Ang Ⅱ group,adding Ang1-7 inhibi-ted the LDH leakage, ET-1 secretion and promoted the release of NO in a dose-dependent manner(P<0.05).CON-CLUSION:Ang1-7 attenuates the HGECs injury induced by Ang Ⅱ by inhibiting the Mas receptor.

Matrix metalloproteinase 9 induces endothelial-mesenchymal transition via Notch activation in human kidney glomerular endothelial cells.

BACKGROUND: Endothelial-mesenchymal transition (EndoMT) is a major source of myofibroblast formation in kidney fibrosis. Our previous study showed a profibrotic role for matrix metalloproteinase 9 (MMP-9) in kidney fibrosis via induction of epithelial-mesenchymal transition (EMT). Inhibition of MMP-9 activity reduced kidney fibrosis in murine unilateral ureteral obstruction. This study investigated whether MMP-9 also plays a role in EndoMT in human glomerular endothelial cells. RESULTS: TGF-ß1 (10 or 20 ng/ml) induced EndoMT in HKGECs as shown by morphological changes. In addition, VE-cadherin and CD31 were significantly downregulated, whereas α-SMA, vimentin, and N-cadherin were upregulated. RT-PCR revealed that Snail, a known inducer of EMT, was upregulated. The MMP inhibitor GM6001 abrogated TGF-ß1-induced EndoMT. Zymography indicated that MMP-9 was also upregulated in TGF-ß1-treated HKGECs. Recombinant MMP-9 (2 µg/ml) induced EndoMT in HKGECs via Notch signaling, as evidenced by increased formation of the Notch intracellular domain (NICD) and decreased Notch 1. Inhibition of MMP-9 activity by its inhibitor showed a dose-dependent response in preventing TGF-ß1-induced α-SMA and NICD in HKGECs, whereas inhibition of Notch signaling by γ-secretase inhibitor (GSI) blocked rMMP-9-induced EndoMT. CONCLUSIONS: Taken together, our results demonstrate that MMP-9 plays an important role in TGF-ß1-induced EndoMT via upregulation of Notch signaling in HKGECs.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells.

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo.

Effect of TGF-β/Smad signal pathway mediated by integrin linked kinase in the progress of renal fibrosis / 重庆医学

Objective To observe the effect of TGF‐β1 in the endothelial‐mesenchymal transition EndM T of glomeruli ,and to explore the effect of TGF‐β/Smad signaling pathway mediated by integrin linked kinase(ILK ) in the progress of renal fibrosis . Methods Human glomerular endothelial cells (HGEnC) incubated in vitro were divided into blank control group ,TGF‐β1 (12 .5 , 25 .0 ,50 .0 ng/mL) group .TGF‐β1 50 .0 ng/mL receptor type one antagonist (LY364749)5 .0 μmol/L group ,ILK (QLT‐0267)5 .0μmol/L antagonist group .The mRNA level of P‐Smad 2/3 and ILK was determined by RT‐PCR ,and the protein level of P‐Smad 2/3 ,ILK ,E‐cadherin ,CD31 ,α‐SMA and FSP‐1 was determined by Western blot after 48 h and 72 h after incubation in each group un‐der the above‐mentioned condition .Results (1)TGF‐β1 could significantly increased the mRNA level of P‐Smad2/3 and ILK (P0 .05) .Conclusion TGF‐β1 as the effector molecule in downstream can promote endothelial‐mesenchymal transition of HGEnC ,and TGF‐β/Smad signaling pathway mediated integrin linked kinase participate in this process ,which probably play important role in the progress of renal fibrosis .

Culture of the Human Glomerular Endothelial Cells / 대한신장학회잡지

Whereas mesangial and epithelial cells from glomeruli are commonly grown in vitro, there has been major difficulties in developing homogenous cultures of human glomerular endothelial cells. This study defines the conditions for the reproducible isolation and growth of homogenous monolayers of human glomerular endothelial cells based on the method of Green DF et al published in 1992. Using the selective media and the sieving method, fibronectin was required as a surface matrix after adequate collagenase treatment, and endothelial cell growth factor and heparin was needed for the continuous growth of endothelial cells. The endothelial cell growth factor was isolated from the bovine hypothalamic extracts. Glomerular capillary endothelial cells exhibited a cobblestone morphology at confluence and stained homogenously with von Willebrand factor(factor VIII). The cytokeratin and the actin were not stained. This study might be helpful for in vitro study to know the biological characteristics of human glomerular endothelial cells under the predetermined condition.