1. Volume 75, Issue 2, Pages 227-234 (January 2009)
Inhibition of JAK2 protects renal endothelial and epithelial cells from oxidative stress and cyclosporin A toxicity 
Fernando Neria, Maria A. Castilla, Ruth Fernandez Sanchez, Francisco R. Gonzalez Pacheco, Juan J.P. Deudero, Olalla Calabia, Alberto Tejedor, Felix Manzarbeitia, Alberto Ortiz, Carlos Caramelo 
Kidney International 
Volume 75, Issue 2, Pages (January 2009)
DOI: /ki
Copyright © 2009 International Society of Nephrology Terms and Conditions

2. Figure 1
JAK2/STAT pathway inhibition with AG490 protects from H2O2-induced cell death. Cells were treated with 50 μmol/l AG490 or vehicle (0.1% DMSO) before exposure to H2O2 for 24 h. (a) AG490 protects bovine aorta endothelial cells (BAECs) from H2O2-induced cell death (n=6, *P<0.05 with respect to vehicle-treated cells; **P<0.001 with respect to vehicle-treated cells). (b) AG490 protects human umbilical vein endothelial cells (HUVECs) from H2O2 (n=4, *P<0.05 with respect to vehicle-treated cells). (c) AG490 protects proximal tubular epithelial cells (PTECs) from H2O2 (n=5, *P<0.05 with respect to vehicle-treated cells). Cell death was assayed by an LDH release assay.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

3. Figure 2
JAK2/STAT pathway inhibition with AG490 protects from superoxide-induced cell death and a dominant-negative form of JAK2 (JAK2-DN) protects from oxidative stress-induced cell death. (a) AG490 protects BAEC from XO/HX-induced death (n=3; *P<0.05 with respect to vehicle-treated cells; **P<0.001 with respect to vehicle-treated cells). (b) AG490 also protects PTEC from XO/HX (n=3; *P<0.05 with respect to vehicle-treated cells). (c) JAK2-DN protects BAEC from cell death induced by 250 μmol/l H2O2 (n=3; *P<0.05 with respect to the baseline). (d) JAK2-DN protects PTEC from cell death induced by 1 mmol/l H2O2 (n=3; *P<0.02 with respect to the baseline). Cell death was assayed by an LDH release assay.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

4. Figure 3
AG490 preserves Bcl2 expression and prevents caspase-3 activation induced by H2O2 in BAEC. (a) Western blot. Cells were incubated for 1 h with vehicle or 50 μmol/l AG490, in the absence or presence of 250 μmol/l H2O2. α-Tubulin was used as loading control. The Bcl2/Bax ratio was determined by band intensity quantitation (*P<0.01 with respect to the other conditions, n=4). (b) Caspase 3 activity was measured as processing of the fluorogenic substrate Ac-DEVD-AMC. Cells were incubated for 24 h with vehicle or 50 μmol/l AG490, in the absence or presence of 250 μmol/l H2O2. Results are expressed as % change over vehicle control in the absence of H2O2 (n=3, *P<0.01 with respect to all the other conditions).
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

5. Figure 4
AG490 protects BAEC and PTEC from cyclosporin A (CsA)-induced death. (a) AG490 protects BAEC from death induced by 10 μmol/l CsA, as assayed by LDH release at 24 h (n=4, *P<0.05 with respect to vehicle-treated cells; **P<0.01 with respect to vehicle-treated cells). (b) Cytoprotective effect of 50 μmol/l AG490 against 25 μmol/l CsA in PTEC, as assayed by LDH release at 24 h (n=3, *P<0.01 vs vehicle-treated cells). (c) Western blot analysis of JAK2 and STAT3 phosphorylation profile in BAEC activated by H2O2 or CsA. (d) Western blot analysis of JAK2 and STAT3 phosphorylation profile in PTEC activated by H2O2 or CsA. Total JAK2 and STAT3 were used as loading controls. Values represent phospho-JAK2 and phospho-STAT3 relative band intensity with respect to total JAK2 and STAT3, expressed as fold-change over time=0 min. Blots on the right show inhibition of JAK2 and STAT3 phosphorylation by AG490, assayed a t=30 min. All the experiments were performed with the corresponding time control; no changes in phosphorylation were observed in any condition. The experiments shown are representative of 3 with identical results.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

6. Figure 5
AG490 or dominant-negative JAK2 (JAK2-DN) reduce oxidative stress in response to H2O2 or CsA. Confocal microphotographs of rhodamine 123 fluorescence: (a) confluent BAEC grown on glass cover slips were treated with 100 μmol/l H2O2 or 10 μmol/l CsA for 1 h in the presence of AG490 or vehicle and incubated for the last 15 min with dihydrorhodamine 123. *P<0.01 vs its respective vehicle-treated control. (b) Similar results were obtained in PTEC treated with 100 μmol/l H2O2 or 10 μmol/l CsA for 1 h. *P<0.01 vs its respective vehicle-treated control. (c) JAK2-DN overexpression decreased the rhodamine signal in transfected BAEC exposed to H2O2. Images were taken with identical exposure conditions. Photographs are representative of three different experiments yielding similar results. *P<0.05 vs empty vector; **P<0.05 vs H2O2-treated cells. Relative red fluorescence was quantified and expressed as fold-change±s.d. over cells treated with vehicle or empty vector (n=4).
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

7. Figure 6
AG490 decreases superoxide anion production in response to H2O2 or CsA. Microphotographs of ethidium fluorescence. (a) Confluent BAEC grown on glass cover slips were treated with 100 μmol/l H2O2 or 10 μmol/l CsA for 1 h in the presence of AG490 or vehicle and incubated for the last 15 min with dihydroethidium. (b) Similar results were obtained in PTEC treated with 100 μmol/l H2O2 or 10 μmol/l CsA for 1 h. Images were taken with identical exposure conditions and gray-scaled. Photographs are representative of three different experiments yielding similar results. Numbers indicate relative red fluorescent quantification, expressed as mean fold-change±s.d. over cells treated with vehicle (n=3). *P<0.05 vs its respective vehicle-treated control.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

8. Figure 7
AG490 protects from CsA-induced renal damage in mice. Representative images of groups of six mice treated with AG490 (a, d, g, j), CsA (b, e, h, k), or CsA+AG490 (c, f, i, l) stained with hematoxylin/eosin (a–f), mannose-binding lectin (g–i), and anti-malondialdehyde (MDA) antibodies (j–l). Black arrows on (b) and (e) indicate damaged tubules in CsA-treated kidneys. White arrow on (c) indicates well-preserved tubules in CsA+AG490-treated kidneys. White arrowheads on (f) point to well-preserved brush borders despite some degree of vacuolization in some tubules in CsA+AG490, as opposed to more severe injury in CsA alone, that included increased numbers of pyknotic nuclei, a paradigm of apoptotic cells (e, black arrowheads). Lectin binding to peritubular capillaries as a marker of injury was increased in CsA-treated animals (h) and this was prevented by AG490 (i). Oxidative stress in vivo was detected by anti-MDA antibodies (j–l). Increased MDA staining was observed in CsA-treated kidneys but not in CsA+AG490-treated kidneys. Numbers indicate mean relative staining intensity±s.d. from six different kidneys for each condition. *P<0.05 vs other groups.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions

9. Figure 8
Effect of AG490 on I/R: renal-induced damage in mice. Representative images of groups of four mice sham-operated (a, d, g, j), treated with DMSO 1 h before of I/R (b, e, h, k) or treated with AG490 1 h before of I/R (c, f, i, l) stained with hematoxylin/eosin (a–f), mannose-binding lectin (g–i), and antimalondialdehyde (MDA) antibodies (j–l). Black arrows on (b) and (e) indicate damaged tubules filled with debris in DMSO kidneys. Black arrowheads on (e) indicate leukocyte infiltration. Lectin binding to peritubular capillaries as a marker of endothelial injury was increased in DMSO kidneys (h) and this was prevented by AG490 (i). Oxidative stress in vivo was detected by anti-MDA antibodies (j–l). Increased MDA staining was observed in DMSO+I/R-treated kidneys but not in AG490+I/R-treated kidneys. Numbers indicate mean relative staining intensity±s.d. from four different kidneys for each condition. *P<0.05 vs other groups.
Kidney International  , DOI: ( /ki )
Copyright © 2009 International Society of Nephrology Terms and Conditions