Journal: Stem cells (Dayton, Ohio)

Article Title: Characterization of renal progenitors committed toward tubular lineage and their regenerative potential in renal tubular injury.

doi: 10.1002/stem.1130

Figure Lengend Snippet: Figure 1. CD133þCD24þ tubular cells are distinguished from CD133þCD24þ cells of Bowman’s capsule by CD106 expression and localize in specific segments of the tubule. Fluorescence-activated cell sorting analysis for the contemporaneous expression of CD133 and CD24 (A) or CD133, CD106, and PDX (B) in freshly isolated total renal cells after CD45 depletion. Isotype controls are shown in Supporting Information Figure 2. (C): Pie-graph representing the percentage of CD133þCD106þPDXþ cells (red), CD133þCD106þPDX cells (green), and CD133þCD106PDX cells (blue) in freshly isolated total renal cells analyzed in (A) and (B). Results represent mean values 6 SEM (three sepa- rate experiments). (D): Triple-label immunofluorescence for CD106 (red), CD133 (green), and CD13 (blue) in human kidney. G ¼ glomerulus. (E, F): Triple-label immunofluorescence for CD133 (green), CD13 (red), cytokeratin 7, and vimentin (blue). (G): Double-label immunofluorescence for CD133 (green) and aquaporin-1 (red). (H): Double-label immunofluorescence for CD133 (green) and CLC-KA (red). (I): Double-label immunofluo- rescence for CD133 (green) and THP (red). (J): Double-label immunofluorescence for CD133 (green) and NCCT (red). (K): Double-label immuno- fluorescence for CD133 (green) and AQP2 (red) in the medulla of adult human kidney. Topro-3 was used to counterstain nuclei. Bars 20 lm. Details are shown in (D0, E0, F0). Abbreviations: AQP2, aquaporin-2; NCCT, Na/Cl cotransporter; THP, Tamm-Horsfall glycoprotein.

Article Snippet: The following antibodies were used: mAb anti-CD24 (clone SN3), mAb anti-Vimentin (V9), pAb antinephrin (C17), pAb anti-aquaporin-2 (AQP2) (C-17), pAb anti-aquaporin-1 (AQP1) (H-55), pAb antimegalin, pAb anti-chloride channel-A (CLC-KA, clone K-16), pAb anti-thiazide-sensitive Na/Cl cotransporter (NCCT, clone N-19) (all from Santa Cruz Biotechnology, Santa Cruz, CA, www.scbt.com), mAb anti-CD133/2 (clone 293C3, Miltenyi Biotec GmbH, Bergish Gladbach, Germany, www. miltenyibiotec.com), mAb anti-CD106 (clone 1.4C1, SigmaAldrich, St. Louis, MO, www.sigmaaldrich.com), mAb anti-podocalyxin (PDX) (clone 222328) (R&D Systems, Minneapolis, MN, www.rndsystems.com), mAb anti-cytokeratin 7 (CK7) (clone OVTL 12/30, Dako, Carpinteria, CA, www.dako.com), mAb antiCD13 (22A5, Abcam, Cambridge, U.K., www.abcam.com), mAb anti-Histone-H3 (phospho S10) (Abcam), mAb antisynaptopodin (G1D4, Progen, Heidelberg, Germany, www.progen.de), mAb anti-epithelial membrane antigen (EMA) (clone E29, Dako), pAb anti-Tamm-Horsfall glycoprotein (THP) (MP Biomedicals, Verona, Italy, www.mpbio.com), phycoerythrin (PE)-conjugated antiCD106 (IE10, R&D Systems), unlabeled anti-CD106 (1.G1B1, Southern Biotech, Birmingham, AL, www.southernbiotech.com), allophycocyanin (APC)-conjugated anti-CD133/2 (clone 293C3), anti-CD45 Microbeads (mouse IgG2a), and anti-mouse IgG1 microbeads (all purchased from Miltenyi).

Techniques: Expressing, Fluorescence, FACS, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Immunohistochemical analysis of ASIC1a expression in renal tissue. Immunostaining for ASIC1a, AQP1, THP and SYN in renal tissue. ASIC1a was colocalized with AQP and SYN, but not THP. ASIC1a, acid sensing ion channel 1a; AQP1, aquaporin 1, proximal tubular cells marker; THP, Tamm‐Horsfall protein, thick ascending limb and distal tubular cells marker; SYN, Synaptopodin, podocyte marker; DAPI, 4,6‐diamidino‐2‐phenylindole, nuclear

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Immunohistochemical staining, Expressing, Immunostaining, Marker

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Inhibiting ASIC1a by PcTx1 attenuate I/R induced kidney injury. Different doses of PcTx1 (0.2, 2, 4, 10 nmol L −1 kg −1 body weight) or vehicle were injected by tail vein, 30 min before I/R operation. Twenty‐four hours after reperfusion, the expression of ASIC1a in the kidney of sham and I/R operating mouse was measured by Western blotting and PCR; functional and histological changes were assessed by serum creatinine, Urea and PAS staining; apoptosis of renal tubular epithelia cells was assessed by TUNEL staining. A, I/R increase protein and RNA level of ASIC1a in the kidney, however, administration of PcTx1 had no effect on expression of ASIC1a. B‐C, injection of PcTx1 reduced serum creatinine and urea in a dose dependent manner; D, Typical visual field of PAS staining and pathological score calculated from PAS staining. PcTx1 (4 nmol L −1 kg −1 body weight) significantly attenuated I/R induced renal injury. * P < 0.05, ** P < 0.01, *** P < 0.001, compared with sham; # P < 0.05, ### P < 0.001, compared with vehicle+I/R, n = 6

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Injection, Expressing, Western Blot, Functional Assay, Staining, TUNEL Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Inhibiting ASIC1a by PcTx1 attenuate I/R induced apoptosis of renal tubule, in vivo. Typical image of TUNEL staining in renal coronal section from sham, vehicle+I/R and PcTx1+ I/R animal. *** P < 0.001, compared with sham; # P < 0.05, ### P < 0.001, compared with vehicle+I/R, n = 6

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: In Vivo, TUNEL Assay, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Inhibiting ASIC1a by PcTx1 reduced H/R induced apoptosis of HK‐2 cells. A, H/R increase protein level of ASIC1a in the HK‐2 cells. * P < 0.05, n = 6. B, HK‐2 cells were pre‐treated with different doses of PcTx1 (5, 25, 100, and 500 ng/mL) or vehicle before H/R treatment. Apoptosis of HK‐2 cells was measured by Annexin‐V/PI staining and evaluated by flow cytometry. C, the group data from B. PcTx1 attenuated H/R induced apoptosis especially early apoptosis dose dependently. *** P < 0.001, compared with Normoxia; # P < 0.05, ## P < 0.01, compared with vehicle+H/R, n = 6. E, Cell variability was measured by MTT assay. Treatment of PcTx1 for 6 h had no effect on variability of HK‐2 cells

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Staining, Flow Cytometry, MTT Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Inhibiting ASIC1a by PcTx1 reduced H/R induced intracellular calcium overload and loss of mitochondrial membrane potential. HK‐2 cells were administrated with different doses of PcTx1 as Figure . A, Intracellular calcium concentration were measured by fluo4 and evaluated by flow cytometry. PcTx1 attenuated H/R induced intracellular calcium overload dose dependently. B, Intracellular calcium concentration was measured by fluo4 immunofluorescence staining. As expected, PcTx1 attenuated H/R induced intracellular calcium overload. C, mitochondrial membrane potential were monitored by JC‐1 dye and evaluated by flow cytometry. *** P < 0.001, compared with Normoxia; # P < 0.05, ## P < 0.01, compared with vehicle+H/R, n = 6

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Concentration Assay, Flow Cytometry, Immunofluorescence, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Inhibiting ASIC1a by PcTx1 reduced expression of cleaved‐caspase3 in renal tubule. A: PcTx1 (4 nmol L −1 kg −1 body weight) or vehicle were injected by tail vein, 30 min before I/R operation. Twenty‐four hours after reperfusion, apoptosis of renal tubular epithelia cells was assessed by immunohistochemical reaction of cleaved‐caspase3. Typical image of cleaved‐caspase3 immunohistochemical staining in renal coronal section from sham, vehicle + I/R and PcTx1+ I/R animal; B, detailed information of immunohistochemical reaction of cleaved‐caspase3 under high‐power lens; ** P < 0.01, compared with sham; ## P < 0.001, compared with vehicle + I/R, n = 6. C, HK‐2 cells were pre‐treated with PcTx1 (100 ng/mL) before H/R treatment, cleaved‐caspase3 was measured by Western blotting; ** P < 0.01, compared with Normoxia; ## P < 0.01, compared with vehicle+H/R, n = 6

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Expressing, Injection, Immunohistochemical staining, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Acid‐sensing ion channel 1a is involved in ischaemia/reperfusion induced kidney injury by increasing renal epithelia cell apoptosis

doi: 10.1111/jcmm.14238

Figure Lengend Snippet: Schema of I/R induced the local microenvironment acidification and the activation of ASIC1a on renal injury and the involved underlying mechanism. Ischaemia caused accumulation of extracellular protons (H + ). ASIC1a are activated by extracellular H + and induce influx of Ca 2+ , which induced loss of mitochondrial membrane potential. The damage of mitochondrial increased cleaved‐caspase3, which resulted in apoptosis of renal tubular epithelia cells. Administration of the specific inhibitor of ASIC1a, PcTx1 ameliorated ischaemic renal injury by inhibiting ASIC1a, which implied a potential therapeutic choice for AKI

Article Snippet: After incubated in 0.3% BSA, the slices was incubated in the mixed primary antibodies: Guinea pig anti‐ASIC1a (1:50 alomone lab, Israel)+Rabbit anti‐aquaporin 1 (AQP1, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐ Tamm‐Horsfall protein (THP, 1:100, Abcam, UAS), Guinea pig anti‐ASIC1a (1:50)+Rabbit anti‐Synaptopodin (SYN, 1:100, Abcam, USA), Guinea pig anti‐ASIC2a (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC2a (1:50)+Rabbit anti‐SYN (1:100), Guinea pig anti‐ASIC3 (1:50 alomone lab, Israel)+Rabbit anti‐AQP1(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐THP(1:100), Guinea pig anti‐ASIC3 (1:50)+Rabbit anti‐ SYN (1:100).

Techniques: Activation Assay