Cardioprotection Conferred by Sitagliptin Is Associated with Reduced Cardiac Angiotensin II/Angiotensin-(1-7) Balance in Experimental Chronic Kidney Disease

Juliana Isa Beraldo, Acaris Benetti, Flávio Araújo Borges-Júnior, Daniel F Arruda-Junior, Flavia Letícia Martins, Leonardo Jensen, Rafael Dariolli, Maria Heloisa Shimizu, Antonio C Seguro, Weverton M Luchi, Adriana C C Girardi, Juliana Isa Beraldo, Acaris Benetti, Flávio Araújo Borges-Júnior, Daniel F Arruda-Junior, Flavia Letícia Martins, Leonardo Jensen, Rafael Dariolli, Maria Heloisa Shimizu, Antonio C Seguro, Weverton M Luchi, Adriana C C Girardi

Abstract

Dipeptidyl peptidase IV (DPPIV) inhibitors are antidiabetic agents that exert renoprotective actions independently of glucose lowering. Cardiac dysfunction is one of the main outcomes of chronic kidney disease (CKD); however, the effects of DPPIV inhibition on cardiac impairment during CKD progression remain elusive. This study investigated whether DPPIV inhibition mitigates cardiac dysfunction and remodeling in rats with a 5/6 renal ablation and evaluated if these effects are associated with changes in the cardiac renin-angiotensin system (RAS). To this end, male Wistar rats underwent a 5/6 nephrectomy (Nx) or sham operation, followed by an 8-week treatment period with the DPPIV inhibitor sitagliptin (IDPPIV) or vehicle. Nx rats had lower glomerular filtration rate, overt albuminuria and higher blood pressure compared to sham rats, whereas CKD progression was attenuated in Nx + IDPPIV rats. Additionally, Nx rats exhibited cardiac hypertrophy and fibrosis, which were associated with higher cardiac DPPIV activity and expression. The sitagliptin treatment prevented cardiac fibrosis and mitigated cardiac hypertrophy. The isovolumic relaxation time (IRVT) was higher in Nx than in sham rats, which was suggestive of CKD-associated-diastolic dysfunction. Sitagliptin significantly attenuated the increase in IRVT. Levels of angiotensin II (Ang II) in the heart tissue from Nx rats were higher while those of angiotensin-(1-7) Ang-(1-7) were lower than that in sham rats. This cardiac hormonal imbalance was completely prevented by sitagliptin. Collectively, these results suggest that DPPIV inhibition may delay the onset of cardiovascular impairment in CKD. Furthermore, these findings strengthen the hypothesis that a crosstalk between DPPIV and the renin-angiotensin system plays a role in the pathophysiology of cardiorenal syndromes.

Keywords: 5/6 renal ablation; cardiorenal syndromes; dipeptidyl peptidase IV; renin-angiotensin system.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cardiac dipeptidyl peptidase IV (DPPIV) activity and expression are upregulated in chronic kidney disease (CKD) rats. (A) DPPIV activity was measured using colorimetry in the heart tissue from sham and CKD rats (Nx) treated with vehicle or sitagliptin (IDPPIV); (B,C) the DPPIV protein level in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats was evaluated by immunoblotting; (B) representative immunoblot; (C) graphical representation of the relative levels of DPPIV protein in the rat. The GAPDH protein level was used as an internal control for normalization. n = 6 rats/group; (D) graphical representation of the relative gene expression of DPPIV in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats. The levels of mRNA of DPPIV were measured using quantitative PCR, and cyclophilin was used as an internal control. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05).
Figure 2
Figure 2
Treatment with sitagliptin attenuates cardiac remodeling in CKD rats. (A) The ratio between the heart weight (mg) and body weight (g) was determined in sham and CKD rats treated with vehicle or sitagliptin; (B,C) cardiac hypertrophy was assessed in sham, sham + IDPPIV, Nx and Nx + IDPPIV rats by measuring the cardiomyocyte nuclear volumes in heart sections stained with hematoxylin-eosin (400× magnification); (D,E) cardiac interstitial fibrosis was evaluated in heart sections stained with Picrosirius red (200× magnification); (F,G) the Na+/H+ exchanger isoform 1 (NHE1) protein level in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats was evaluated by immunoblotting; (F) representative immunoblot; (G) graphical representation of the relative levels of NHE1 protein in the rat. The GAPDH protein level was used as an internal control for normalization. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05).
Figure 3
Figure 3
The effect of sitagliptin on the cardiac function and B-type natriuretic peptide (BNP) levels in CKD rats. (A,B) Doppler echocardiography was performed in sham and CKD rats treated with vehicle or sitagliptin; (A) left ventricle (LV) ejection fraction, as a marker of LV systolic function; (B) isovolumic relaxation time (IVRT), as a marker of LV diastolic function; (C,D) the levels of systemic and cardiac BNP were assessed in the four groups of rats; (C) serum BNP levels were determined by ELISA in sham, sham + IDPPIV, Nx and Nx + IDPPIV rats; (D) graphical representation of the relative gene expression of BNP in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats. The levels of BNP mRNA were measured using quantitative PCR, and cyclophilin was used as an internal control. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05).
Figure 4
Figure 4
The effect of sitagliptin on the expression of cardiac renin-angiotensin system components in CKD rats. Quantitative PCR was used to determine the mRNA expression of the main components of the local renin-angiotensin system (RAS) in the heart tissue from sham rats treated with vehicle (n = 12) or sitagliptin (sham + IDPPIV, n = 9) and CKD rats treated with vehicle (Nx, n = 10) or sitagliptin (Nx + IDPPIV, n = 10). (A) Angiotensinogen (AGT); (B) angiotensin-converting enzyme (ACE); (C) angiotensin-converting enzyme-2 (ACE2); (D) angiotensin II type 1 receptor (AT1R); (E) angiotensin II type 2 receptor (AT2R) and (F) mas receptor expression (MasR). Cyclophilin was used as an internal control. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05).
Figure 5
Figure 5
The effect of sitagliptin on the expression of ACE and ACE2 and on the concentrations of angiotensin II (Ang II) and angiotensin-(1-7) Ang-(1-7) in the heart of CKD rats. (A–D) The levels of ACE and ACE2 protein were measured in the heart tissue from sham rats and CKD rats treated with vehicle or sitagliptin (IDPPIV) by immunoblotting. (A) Representative immunoblot; (B) graphical representation of the relative levels of ACE protein in the rat. The GAPDH protein level was used as an internal control for normalization; (C) graphical representation of the relative levels of ACE2 protein in the rat; (D) the ratio of ACE to ACE2 protein expression; (E–G) the levels of Ang II and Ang-(1-7) were measured in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats using ELISA. The heart peptide content was normalized to protein concentration and reported as a pg/mg protein; (E) Ang II concentration; (F) Ang-(1-7) concentration; (G) the ratio of Ang II to Ang-(1-7) concentration. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05); (H,I) correlations between the heart DPPIV activity and (H) levels of Ang II in the heart tissue; (I) levels of Ang II in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats. The correlation coefficients and p values were obtained using the Pearson correlation test, and the lines represent linear regression plotting.
Figure 6
Figure 6
The serum concentrations of Ang II and Ang-(1-7) of sham and CKD rats are not significantly affected by sitagliptin. The levels of Ang II and angiotensin 1-7 Ang-(1-7) were measured in the serum from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats using ELISA. (A) The serum Ang II concentration; (B) serum Ang-(1-7) concentration; (C) the ratio of Ang II to Ang-(1-7) concentration in the serum. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM.
Figure 7
Figure 7
Treatment with sitagliptin exerts antioxidant and anti-inflammatory effects in the heart of CKD rats. (A) The levels of nitrotyrosine were measured by ELISA in the heart tissue from sham and CKD rats treated with vehicle or sitagliptin (IDPPIV); (B–F) quantitative PCR was used to determine the mRNA expression of inflammatory cytokines in the heart tissue from sham, sham + IDPPIV, Nx and Nx + IDPPIV rats. Graphical representation of the relative gene expression of (B) MCP-1, (C) TNF-α, (D) IL-6, (E) IL-1β and (F) IL-10. Cyclophilin was used as an internal control. The number of rats per experimental group is indicated in the bars. The data represent the mean ± SEM. Bars with different lowercase letters are significantly different (p < 0.05).

References

    1. Scirica B.M., Bhatt D.L., Braunwald E., Steg P.G., Davidson J., Hirshberg B., Ohman P., Frederich R., Wiviott S.D., Hoffman E.B., et al. Saxagliptin and cardiovascular outcomes in patients with type 2 diabetes mellitus. N. Engl. J. Med. 2013;369:1317–1326. doi: 10.1056/NEJMoa1307684.
    1. Udell J.A., Bhatt D.L., Braunwald E., Cavender M.A., Mosenzon O., Steg P.G., Davidson J.A., Nicolau J.C., Corbalan R., Hirshberg B., et al. Saxagliptin and cardiovascular outcomes in patients with type 2 diabetes and moderate or severe renal impairment: Observations from the SAVOR-TIMI 53 Trial. Diabetes Care. 2015;38:696–705. doi: 10.2337/dc14-1850.
    1. Joo K.W., Kim S., Ahn S.Y., Chin H.J., Chae D.W., Lee J., Han J.S., Na K.Y. Dipeptidyl peptidase IV inhibitor attenuates kidney injury in rat remnant kidney. BMC Nephrol. 2013;14:98. doi: 10.1186/1471-2369-14-98.
    1. Tsuprykov O., Ando R., Reichetzeder C., von Websky K., Antonenko V., Sharkovska Y., Chaykovska L., Rahnenführer J., Hasan A.A., Tammen H., et al. The dipeptidyl peptidase inhibitor linagliptin and the angiotensin II receptor blocker telmisartan show renal benefit by different pathways in rats with 5/6 nephrectomy. Kidney Int. 2016;89:1049–1061. doi: 10.1016/j.kint.2016.01.016.
    1. Eun Lee J., Kim J.E., Lee M.H., Song H.K., Ghee J.Y., Kang Y.S., Min H.S., Kim H.W., Cha J.J., Han J.Y., et al. DA-1229, a dipeptidyl peptidase IV inhibitor, protects against renal injury by preventing podocyte damage in an animal model of progressive renal injury. Lab. Investig. 2016;96:547–560. doi: 10.1038/labinvest.2016.34.
    1. Chaykovska L., von Websky K., Rahnenführer J., Alter M., Heiden S., Fuchs H., Runge F., Klein T., Hocher B. Effects of DPP-4 inhibitors on the heart in a rat model of uremic cardiomyopathy. PLoS ONE. 2011;6:e27861. doi: 10.1371/journal.pone.0027861.
    1. Dos Santos L., Salles T.A., Arruda-Junior D.F., Campos L.C.G., Pereira A.C., Barreto A.L.T., Antonio E.L., Mansur A.J., Tucci P.J.F., Krieger J.E., et al. Circulating dipeptidyl peptidase IV activity correlates with cardiac dysfunction in human and experimental heart failure. Circul. Heart Fail. 2013;6:1029–1038. doi: 10.1161/CIRCHEARTFAILURE.112.000057.
    1. Arruda-Junior D.F., Martins F.L., Dariolli R., Jensen L., Antonio E.L., Dos Santos L., Tucci P.J., Girardi A.C. Dipeptidyl Peptidase IV Inhibition Exerts Renoprotective Effects in Rats with Established Heart Failure. Front. Physiol. 2016;7:293. doi: 10.3389/fphys.2016.00293.
    1. Gomez N., Touihri K., Matheeussen V., Mendes Da Costa A., Mahmoudabady M., Mathieu M., Baerts L., Peace A., Lybaert P., Scharpé S., et al. Dipeptidyl peptidase IV inhibition improves cardiorenal function in overpacing-induced heart failure. Eur. J. Heart Fail. 2012;14:14–21. doi: 10.1093/eurjhf/hfr146.
    1. Salles T.A., dos Santos L., Barauna V.G., Girardi A.C.C. Potential role of dipeptidyl peptidase IV in the pathophysiology of heart failure. Int. J. Mol. Sci. 2015;16:4226–4249. doi: 10.3390/ijms16024226.
    1. Takashima S., Fujita H., Fujishima H., Shimizu T., Sato T., Morii T., Tsukiyama K., Narita T., Takahashi T., Drucker D.J., et al. Stromal cell-derived factor-1 is upregulated by dipeptidyl peptidase-4 inhibition and has protective roles in progressive diabetic nephropathy. Kidney Int. 2016;90:783–796. doi: 10.1016/j.kint.2016.06.012.
    1. Farah L.X., Valentini V., Pessoa T.D., Malnic G., McDonough A.A., Girardi A.C. The physiological role of glucagon-like peptide-1 in the regulation of renal function. Am. J. Physiol. Renal Physiol. 2016;310:F123–F127. doi: 10.1152/ajprenal.00394.2015.
    1. Holst J.J., Deacon C.F. Glucagon-like peptide-1 mediates the therapeutic actions of DPP-IV inhibitors. Diabetologia. 2005;48:612–615. doi: 10.1007/s00125-005-1705-7.
    1. Siragy H.M., Carey R.M. Role of the intrarenal renin-angiotensin-aldosterone system in chronic kidney disease. Am. J. Nephrol. 2010;31:541–550. doi: 10.1159/000313363.
    1. Remuzzi G., Perico N., Macia M., Ruggenenti P. The role of renin-angiotensin-aldosterone system in the progression of chronic kidney disease. Kidney Int. Suppl. 2005:S57–S65. doi: 10.1111/j.1523-1755.2005.09911.x.
    1. Ma T.K., Kam K.K., Yan B.P., Lam Y.Y. Renin-angiotensin-aldosterone system blockade for cardiovascular diseases: Current status. Br. J. Pharmacol. 2010;160:1273–1292. doi: 10.1111/j.1476-5381.2010.00750.x.
    1. Aroor A., Zuberek M., Duta C., Meuth A., Sowers J.R., Whaley-Connell A., Nistala R. Angiotensin II Stimulation of DPP4 Activity Regulates Megalin in the Proximal Tubules. Int. J. Mol. Sci. 2016;17:780. doi: 10.3390/ijms17050780.
    1. Kawase H., Bando Y.K., Nishimura K., Aoyama M., Monji A., Murohara T. A dipeptidyl peptidase-4 inhibitor ameliorates hypertensive cardiac remodeling via angiotensin-II/sodium-proton pump exchanger-1 axis. J. Mol. Cell Cardiol. 2016;98:37–47. doi: 10.1016/j.yjmcc.2016.06.066.
    1. Karmazyn M., Kilić A., Javadov S. The role of NHE-1 in myocardial hypertrophy and remodelling. J. Mol. Cell Cardiol. 2008;44:647–653. doi: 10.1016/j.yjmcc.2008.01.005.
    1. Filippatos G., Farmakis D., Parissis J. Renal dysfunction and heart failure: Things are seldom what they seem. Eur. Heart J. 2014;35:416–418. doi: 10.1093/eurheartj/eht515.
    1. Segall L., Nistor I., Covic A. Heart failure in patients with chronic kidney disease: A systematic integrative review. Biomed. Res. Int. 2014;2014:937398. doi: 10.1155/2014/937398.
    1. Sharkovska Y., Reichetzeder C., Alter M., Tsuprykov O., Bachmann S., Secher T., Klein T., Hocher B. Blood pressure and glucose independent renoprotective effects of dipeptidyl peptidase-4 inhibition in a mouse model of type-2 diabetic nephropathy. J. Hypertens. 2014;32:2211–2223. doi: 10.1097/HJH.0000000000000328.
    1. Pacheco B.P.M., Crajoinas R.O., Couto G.K., Davel A.P.C., Lessa L.M., Rossoni L.V., Girardi A.C.C. Dipeptidyl peptidase IV inhibition attenuates blood pressure rising in young spontaneously hypertensive rats. J. Hypertens. 2011;29:520–528. doi: 10.1097/HJH.0b013e328341939d.
    1. Lim S.W., Jin L., Piao S.G., Chung B.H., Yang C.W. Inhibition of dipeptidyl peptidase IV protects tacrolimus-induced kidney injury. Lab. Investig. 2015;95:1174–1185. doi: 10.1038/labinvest.2015.93.
    1. Shigeta T., Aoyama M., Bando Y.K., Monji A., Mitsui T., Takatsu M., Cheng X.W., Okumura T., Hirashiki A., Nagata K., et al. Dipeptidyl peptidase-4 modulates left ventricular dysfunction in chronic heart failure via angiogenesis-dependent and -independent actions. Circulation. 2012;126:1838–1851. doi: 10.1161/CIRCULATIONAHA.112.096479.
    1. Lin C.H., Lin C.C. Sitagliptin attenuates inflammatory responses in lipopolysaccharide-stimulated cardiomyocytes via nuclear factor-κB pathway inhibition. Exp. Ther. Med. 2016;11:2609–2615. doi: 10.3892/etm.2016.3255.
    1. Picatoste B., Ramírez E., Caro-Vadillo A., Iborra C., Ares-Carrasco S., Egido J., Tuñón J., Lorenzo O. Sitagliptin reduces cardiac apoptosis, hypertrophy and fibrosis primarily by insulin-dependent mechanisms in experimental type-II diabetes. Potential roles of GLP-1 isoforms. PLoS ONE. 2013;8:e78330. doi: 10.1371/annotation/df98874f-c1bd-4a13-b474-942fbb956287.
    1. Zhang H., Xiong Z., Wang J., Zhang S., Lei L., Yang L., Zhang Z. Glucagon-like peptide-1 protects cardiomyocytes from advanced oxidation protein product-induced apoptosis via the PI3K/Akt/Bad signaling pathway. Mol. Med. Rep. 2016;13:1593–1601. doi: 10.3892/mmr.2015.4724.
    1. Balteau M., Van Steenbergen A., Timmermans A.D., Dessy C., Behets-Wydemans G., Tajeddine N., Castanares-Zapatero D., Gilon P., Vanoverschelde J.L., Horman S., et al. AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes. Am. J. Physiol. Heart Circ. Physiol. 2014;307:H1120–H1133. doi: 10.1152/ajpheart.00210.2014.
    1. Kanasaki K., Shi S., Kanasaki M., He J., Nagai T., Nakamura Y., Ishigaki Y., Kitada M., Srivastava S.P., Koya D. Linagliptin-mediated DPP-4 inhibition ameliorates kidney fibrosis in streptozotocin-induced diabetic mice by inhibiting endothelial-to-mesenchymal transition in a therapeutic regimen. Diabetes. 2014;63:2120–2131. doi: 10.2337/db13-1029.
    1. Connelly K.A., Bowskill B.B., Advani S.L., Thai K., Chen L.H., Kabir M.G., Gilbert R.E., Advani A. Dipeptidyl peptidase-4 inhibition improves left ventricular function in chronic kidney disease. Clin. Investig. Med. 2014;37:E172. doi: 10.25011/cim.v37i3.21384.
    1. Yamada H., Tanaka A., Kusunose K., Amano R., Matsuhisa M., Daida H., Ito M., Tsutsui H., Nanasato M., Kamiya H., et al. Effect of sitagliptin on the echocardiographic parameters of left ventricular diastolic function in patients with type 2 diabetes: A subgroup analysis of the PROLOGUE study. Cardiovasc. Diabetol. 2017;16:63. doi: 10.1186/s12933-017-0546-2.
    1. Ferrario C.M. Cardiac remodelling and RAS inhibition. Ther. Adv. Cardiovasc. Dis. 2016;10:162–171. doi: 10.1177/1753944716642677.
    1. Zhang L.H., Pang X.F., Bai F., Wang N.P., Shah A.I., McKallip R.J., Li X.W., Wang X., Zhao Z.Q. Preservation of Glucagon-Like Peptide-1 Level Attenuates Angiotensin II-Induced Tissue Fibrosis by Altering AT1/AT 2 Receptor Expression and Angiotensin-Converting Enzyme 2 Activity in Rat Heart. Cardiovasc. Drugs Ther. 2015;29:243–255. doi: 10.1007/s10557-015-6592-7.
    1. Matsui H., Barry W.H., Livsey C., Spitzer K.W. Angiotensin II stimulates sodium-hydrogen exchange in adult rabbit ventricular myocytes. Cardiovasc. Res. 1995;29:215–221. doi: 10.1016/S0008-6363(96)88573-7.
    1. Girardi A.C.C., Knauf F., Demuth H.U., Aronson P.S. Role of dipeptidyl peptidase IV in regulating activity of Na +/H+ exchanger isoform NHE3 in proximal tubule cells. Am. J. Physiol. Cell Physiol. 2004;287:C1238–C1245. doi: 10.1152/ajpcell.00186.2004.
    1. Hori M., Nishida K. Oxidative stress and left ventricular remodelling after myocardial infarction. Cardiovasc. Res. 2009;81:457–464. doi: 10.1093/cvr/cvn335.
    1. Choi S.Y., Ryu H.M., Oh E.J., Choi J.Y., Cho J.H., Kim C.D., Kim Y.L., Park S.H. Dipeptidyl peptidase-4 inhibitor gemigliptin protects against vascular calcification in an experimental chronic kidney disease and vascular smooth muscle cells. PLoS ONE. 2017;12:e0180393. doi: 10.1371/journal.pone.0180393.
    1. Choi S.H., Park S., Oh C.J., Leem J., Park K.G., Lee I.K. Dipeptidyl peptidase-4 inhibition by gemigliptin prevents abnormal vascular remodeling via NF-E2-related factor 2 activation. Vascul. Pharmacol. 2015;73:11–19. doi: 10.1016/j.vph.2015.07.005.
    1. Koibuchi N., Hasegawa Y., Katayama T., Toyama K., Uekawa K., Sueta D., Kusaka H., Ma M., Nakagawa T., Lin B., et al. DPP-4 inhibitor linagliptin ameliorates cardiovascular injury in salt-sensitive hypertensive rats independently of blood glucose and blood pressure. Cardiovasc. Diabetol. 2014;13:157. doi: 10.1186/s12933-014-0157-0.
    1. Sauvé M., Ban K., Momen M.A., Zhou Y.Q., Henkelman R.M., Husain M., Drucker D.J. Genetic deletion or pharmacological inhibition of dipeptidyl peptidase-4 improves cardiovascular outcomes after myocardial infarction in mice. Diabetes. 2010;59:1063–1073. doi: 10.2337/db09-0955.
    1. Suda M., Shimizu I., Yoshida Y., Hayashi Y., Ikegami R., Katsuumi G., Wakasugi T., Okuda S., Soga T., Minamino T. Inhibition of dipeptidyl peptidase-4 ameliorates cardiac ischemia and systolic dysfunction by up-regulating the FGF-2/EGR-1 pathway. PLoS ONE. 2017;12:e0182422. doi: 10.1371/journal.pone.0182422.
    1. Shimizu M.H., Coimbra T.M., de Araujo M., Menezes L.F., Seguro A.C. N-acetylcysteine attenuates the progression of chronic renal failure. Kidney Int. 2005;68:2208–2217. doi: 10.1111/j.1523-1755.2005.00677.x.
    1. Inoue B.H., Arruda-Junior D.F., Campos L.C.G., Barreto A.L.T., Rodrigues M.V., Krieger J.E., Girardi A.C.C. Progression of microalbuminuria in SHR is associated with lower expression of critical components of the apical endocytic machinery in the renal proximal tubule. Am. J. Physiol. Renal Physiol. 2013;305:F216–F226. doi: 10.1152/ajprenal.00255.2012.
    1. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265–275.
    1. Ariyoshi M., Mizuno M., Morisue Y., Shimada M., Fujita S., Nasu J., Okada H., Shimomura H., Yamamoto K., Tsuji T. Identification of a target antigen recognized by a mouse monoclonal antibody to the bile canalicular surface of rat hepatocytes with a random phage display library. Acta Med. Okayama. 2002;56:187–191. doi: 10.18926/AMO/31684.
    1. Rutherford P.A., Pizzonia J.H., Biemesderfer D., Abu-Alfa A., Reilly R., Aronson P.S. Expression of Na(+)-H+ exchanger isoforms NHE1 and NHE3 in kidney and blood cells of rabbit and rat. Exp. Nephrol. 1997;5:490–497.

Source: PubMed

Sponsorer og samarbejdspartnere

Medicinske tilstande

Narkotikainterventioner

3
Abonner