Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Expression levels of miR-145-5p are decreased in HG podocytes. Podocytes were incubated with 5 mM (NG group) and 25 mM (HG group) glucose for different time periods (12, 24, 48 and 72 h). The expression levels of miR-145-5p were measured by reverse transcription-quantitative PCR. Data are presented as the mean ± SD, and as the fold change relative to the control group (n=3). **P<0.01 vs. NG. miR-145-5p, microRNA-145-5p; HG, high glucose; NG, normal glucose.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Expressing, Incubation, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Effect of miR-145-5p on HG-induced podocyte apoptosis. (A) Podocyte apoptosis was detected by flow cytometry following HG (25 mM) stimulation for 48 h. Podocytes treated with NG (5 mM) were used as the negative control group. (B) Quantification of the percentage of apoptotic cells. (C) TUNEL staining of apoptotic podocytes. Magnification, ×200. (D) Percentages of apoptotic cells identified by TUNEL staining. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and as the fold change relative to the control group (n=3). *P<0.05 and **P<0.01 vs. the scrambled control. HG, high glucose; miR-145-5p, microRNA-145-5p; NG, normal glucose.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Flow Cytometry, Negative Control, TUNEL Assay, Staining, Comparison, Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: miR-145-5p is a direct target of Notch1 in podocytes. (A) Venn diagram showing the 267 genes identified as potential targets of miR-145-5p using three prediction tools (B) Predicted binding sequences of miR-145-5p with the 3′-UTR of Notch1. (C) Relative luciferase activity was analyzed after WT or mut 3′-UTR reporter plasmids were co-transfected with miR-145-5p mimic or miR-145-5p inhibitor in podocytes. (D) Reverse transcription-quantitative PCR detection of Notch1 mRNA expression in podocytes transfected with miR-145-5p mimic or miR-145-5p inhibitor. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group (n=3). *P<0.05 and **P<0.01 vs. the respective NC or scrambled control. HG, high glucose; miR-145-5p, microRNA-145-5p; NC, negative control; MUT, mutant; NG, normal glucose; UTR, untranslated region; WT, wild-type.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Comparison, Control, Negative Control, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: Overexpression of miR-145-5p suppresses the HG-induced activation of the Notch signaling pathway. (A-C) RT-qPCR detection of (A) NICD, (B) Hes1 and (C) Hey1 mRNA expression in control (NG) and transfected podocytes under HG conditions. (D) Western blotting detection of NICD, Hes1 and Hey1 protein expression in podocytes. (E) Semi-quantification of the protein levels of NICD, Hes1 and Hey1. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the scrambled control. miR-145-5p, microRNA-145-5p; HG, high glucose; NG, normal glucose; RT-qPCR, reverse transcription-quantitative PCR; NICD, intracellular domain of Notch; Hes1, hes family bHLH transcription factor 1; Hey1, hes related family bHLH transcription factor with YRPW motif 1.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Over Expression, Activation Assay, Quantitative RT-PCR, Expressing, Control, Transfection, Western Blot, Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-145-5p attenuates high glucose-induced apoptosis by targeting the Notch signaling pathway in podocytes

doi: 10.3892/etm.2020.8427

Figure Lengend Snippet: mRNA and protein expression levels of caspase-3, Bcl-2 and Bax in podocytes. (A-C) RT-qPCR detection of (A) caspase-3, (B) Bcl-2 and (C) Bax mRNA expression in podocytes. (D) Western blotting detection of cleaved caspase-3, Bcl-2 and Bax protein levels in podocytes transfected with miR-145-5p mimics and inhibitors. (E) Semi-quantification of protein levels of cleaved caspase-3, Bcl-2 and Bax. Statistical analysis was performed using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Data are presented as the mean ± SD, and shown as the fold change relative to the control group. GAPDH was used as an internal control (n=3). *P<0.05, **P<0.01 and ***P<0.001 vs. the scrambled control. miR-145-5p, microRNA-145-5p; NG, normal glucose; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Mouse podocytes (cat. no. M1710) and all cell culture media, FBS and other supplements were purchased from ScienCell Research Laboratories, Inc.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Comparison, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of ECM on the differentiation of kidney organoids in a static culture condition. A Fluorescent images of immunostained kidney organoids cultured with various conditions of ECMs for 6 days. The cells were immunostained by NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue). B Quantitative analysis of kidney antibody-specific markers from kidney organoids (* indicates p <0.05 for experimental group vs. control group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Cell Culture

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of the shear stress on the differentiation of kidney organoids in a kidney organoid-on-a-chip. A Fluorescent images showing hPSCs-derived kidney organoid-related cell differentiation under a shear stress condition. Kidney organoids were cultured within kidney organoid-on-a-chip for 6 days. After culturing for 6 days, the cells were stained for NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue) to identify the kidney-related cells, respectively. B Quantitative analysis of the kidney-related markers from kidney organoids. (* indicates p < 0.05 for LTL expression of Matrigel-coated group vs. Matrigel/VEGF-coated group, *** indicates p < 0.001 for PECAM expression of Matrigel-coated group vs. Matrigel/VEGF-coated group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Derivative Assay, Cell Differentiation, Cell Culture, Staining, Expressing

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis

doi: 10.1681/ASN.2014111136

Figure Lengend Snippet: IL-17 receptor A (IL-17RA), podocyte, and tubular injury marker expression in kidney and urine. (A) Immunohistochemical localization of IL-17RA and KIM-1 in control and diabetic kidney. IL-17RA expression is mostly seen in proximal tubular epithelial cells. Diabetes increased staining intensity, but the pattern is not changed. IL-17A administration did not alter IL-17A expression. KIM-1 expression is absent in control kidney and expression of KIM-1 is increased in diabetic mice kidney. IL-17A treatment suppressed KIM-1 expression in diabetic kidney. Scale bar=100 µM. (B) Western blot analysis of KIM-1 and sema3A excretion in urine. (C) Quantification of podocyte injury marker nephrin excretion in urine. STZ diabetic mice (12 weeks), Ins2Akita mice (30 weeks) and db/db mice (20 weeks) treated with vehicle or IL-17A and excretion of nephrin was quantified by ELISA as described in the Concise Methods. IL-17A administration significantly reduced the excretion of nephrin in urine. *P<0.001 versus other groups. #P<0.001 versus vehicle-treated diabetic mice. (D and E) Quantification of podocytes in glomerular section that are immunostained with Wilms tumor-1 antibody (D) and positive cells in 20 glomeruli in each kidney was counted then averaged (E). Scale bar=100 µM. *P<0.05 versus other group. n=4–6.

Article Snippet: Protein extracted from kidneys, TKPTS cells, human podocytes, 47 and mouse podocytes (CLS cell lines, Germany) were used for Western blot analysis as described previously.

Techniques: Marker, Expressing, Immunohistochemical staining, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Wilms Tumor Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis

doi: 10.1681/ASN.2014111136

Figure Lengend Snippet: Regulation of AMWAP and IL-10 by IL-17A in different cells. (A) Administration of IL-17A induced a large increase in AMWAP expression in Ins2Akita kidney analyzed by real-time PCR. *P<0.001 versus WT control; #P<0.001 versus vehicle treated Ins2Akita mice. (B) RT-PCR analysis of AMWAP and IL-10 expression in mouse podocyte. *P<0.01 versus vehicle treated control. (C) RT-PCR analysis of AMWAP expression in TKPTS (mouse proximal tubular epithelial cells). *P<0.01 versus 0 hour. (D) AMPK inhibitor suppressed IL-17A–induced AMWAP expression in TKPTS cells. **P<0.001 versus other groups. (E) IL-17A administration enhanced IL-10 excretion in urine of Ins2Akita mice. *P<0.001 versus WT control; #P<0.001 versus vehicle-treated Ins2Akita mice. (F) IL-17A administration enhanced IL-10 excretion in diabetic mouse urine at 12 weeks after STZ administration. *P<0.01 versus vehicle treated nondiabetic control; #P<0.001 versus vehicle-treated diabetic control. IL-17A (50 ng/ml) (G) and IL-17F (50 ng/ml) (H) treatment induced AMWAP expression in macrophages within hours, whereas IL-10 expression takes 72 hours. *P<0.001 versus 0 hour. (I) Recombinant AMWAP treatment induced IL-10 and arginase-1 expression in macrophages. *P<0.001 versus vehicle-treated. (J) Recombinant AMWAP treatment induced IL-10 protein expression in macrophages. IL-10 protein in culture supernatant was quantified by ELISA. *P<0.01 versus vehicle-treated. (K) AMWAP suppressed LPS-induced IL-1β expression in macrophages. **P<0.001 versus other groups. (L) Recombinant AMWAP treatment induced IL-10 expression in mouse podocyte. *P<0.001 versus vehicle treated. n=6–10.

Article Snippet: Protein extracted from kidneys, TKPTS cells, human podocytes, 47 and mouse podocytes (CLS cell lines, Germany) were used for Western blot analysis as described previously.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: Low-Dose IL-17 Therapy Prevents and Reverses Diabetic Nephropathy, Metabolic Syndrome, and Associated Organ Fibrosis

doi: 10.1681/ASN.2014111136

Figure Lengend Snippet: Epithelial cell–specific overexpression of IL-17A is sufficient to suppress diabetic nephropathy. Data from IL-17A transgenic Line 2. (A) IL-17A transgenic mice were crossed with nephropathy prone strain DBA/2J. Six-week-old WT and IL-17A–positive F1 mice were given a single dose of STZ (150 mg/kg body wt). Mice were euthanized 8 weeks after STZ administration and albuminuria was quantified. (B) Blood glucose level at 8 weeks of diabetes. (C) Kidney hypertrophy was calculated as ratio of kidney weight and body weight (KW/BW). (D) Albumin excretion rate (AER) expressed as μg/24-hour urine. (E) AER expressed as μg/mg of creatinine. (F) Serum IL-17A levels. (G) Urine IL-17A levels. (H) Mesangial index. (I) Glomerular area. (J–M) PAS-hematoxylin–stained kidney section. Scale bar=100 µM. (J) WT control. (K) IL-17A transgenic control. (K) WT diabetic mouse kidney. (L) IL-17A transgenic diabetic mouse kidney. (N–Q) Electron microscope images. Original magnification, ×15,000. WT control (N) and IL-17A transgenic control (O) kidney show normal podocyte foot process and basement membrane. Diabetes-induced podocyte foot process effacement (red arrowhead) and basement membrane thickening (red arrow) in WT (P), which was suppressed in IL-17A transgenic animals (Q and R) AMWAP mRNA expression in the kidney. (S) Mannose receptor mRNA in the kidney. (T) IL-10 mRNA expression in the kidney. (U) Quantification of TBARS for oxidative stress. *P<0.001 versus other groups; #P<0.001 versus WT diabetic. n=10–12.

Article Snippet: Protein extracted from kidneys, TKPTS cells, human podocytes, 47 and mouse podocytes (CLS cell lines, Germany) were used for Western blot analysis as described previously.

Techniques: Over Expression, Transgenic Assay, Staining, Microscopy, Expressing