Antithrombin III/SerpinC1 insufficiency exacerbates renal ischemia/reperfusion injury

Feng Wang, Guangyuan Zhang, Zeyuan Lu, Aron M Geurts, Kristie Usa, Howard J Jacob, Allen W Cowley, Niansong Wang, Mingyu Liang, Feng Wang, Guangyuan Zhang, Zeyuan Lu, Aron M Geurts, Kristie Usa, Howard J Jacob, Allen W Cowley, Niansong Wang, Mingyu Liang

Abstract

Antithrombin III, encoded by SerpinC1, is a major anti-coagulation molecule in vivo and has anti-inflammatory effects. We found that patients with low antithrombin III activities presented a higher risk of developing acute kidney injury after cardiac surgery. To study this further, we generated SerpinC1 heterozygous knockout rats and followed the development of acute kidney injury in a model of modest renal ischemia/reperfusion injury. Renal injury, assessed by serum creatinine and renal tubular injury scores after 24 h of reperfusion, was significantly exacerbated in SerpinC1(+/-) rats compared to wild-type littermates. Concomitantly, renal oxidative stress, tubular apoptosis, and macrophage infiltration following this injury were significantly aggravated in SerpinC1(+/-) rats. However, significant thrombosis was not found in the kidneys of any group of rats. Antithrombin III is reported to stimulate the production of prostaglandin I2, a known regulator of renal cortical blood flow, in addition to having anti-inflammatory effects and to protect against renal failure. Prostaglandin F1α, an assayable metabolite of prostaglandin I2, was increased in the kidneys of the wild-type rats at 3 h after reperfusion. The increase of prostaglandin F1α was significantly blunted in SerpinC1(+/-) rats, which preceded increased tubular injury and oxidative stress. Thus, our study found a novel role of SerpinC1 insufficiency in increasing the severity of renal ischemia/reperfusion injury.

Figures

Figure 1
Figure 1
Renal function following ischemia/reperfusion injury (IRI) was worsened in rats with SerpinC1 insufficiency. Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Blood and tissues were collected 24 h after reperfusion. (a) Serum creatinine. (b) Blood urea nitrogen. (c) Plasma levels of ATIII. (d) SerpinC1 mRNA abundance in liver. (e) ATIII protein abundance in liver. (f) ATIII protein abundance in renal cortex. (g) SerpinC1 mRNA abundance in renal cortex. N=6. ATIII, antithrombin III; Het, SerpinC1+/− rat; IR, ischemia/reperfusion; SD, Sprague–Dawley; Sham, sham operated; WT, wild-type littermate.
Figure 2
Figure 2
SerpinC1 insufficiency exacerbated renal histological injury in ischemia/reperfusion injury (IRI). Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Kidneys were harvested 24 h after reperfusion. (a) Representative images of periodic acid–Schiff (PAS) staining (× 200). (b) Tubule injury scores. N=6. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.
Figure 3
Figure 3
SerpinC1 insufficiency did not result in renal thrombosis. Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Unflushed kidneys were harvested 24 h after reperfusion. (a) Several representative images of Masson trichrome staining are shown for a broad region of the kidney (left, × 100) and glomerular capillary, arteriole, and small veins (right, × 400). Red blood cells were observed in the blood vessels, consistent with the kidneys not being flushed before harvesting. Thrombi were not observed. (b) Plasma levels of fibrinogen. (c) Plasma levels of fibrinogen degradation products (FDPs). N=6. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.
Figure 4
Figure 4
SerpinC1 insufficiency increased renal cortical malondialdehyde (MDA) levels in rats with ischemia/reperfusion injury (IRI). Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Kidneys were harvested 24 h after reperfusion. N=6. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.
Figure 5
Figure 5
SerpinC1 insufficiency increased renal tubular apoptosis in rats with ischemia/reperfusion injury (IRI). Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Kidneys were harvested 24 h after reperfusion. (a) Representative images of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) staining (× 400, left) and overlays with 4',6-diamidino-2-phenylindole (DAPI) staining (right). (b) Quantatitive analysis. N=6. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.
Figure 6
Figure 6
SerpinC1 insufficiency increased renal macrophage infiltration in rats with ischemia/reperfusion injury (IRI). Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Kidneys were harvested 24 h after reperfusion. (a) Representative images (× 200) of immunohistochemistry analysis using an anti-F4/80 antibody. (b) Quantatitive analysis. N=6. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.
Figure 7
Figure 7
SerpinC1 insufficiency blunted the increase in renal prostaglandin (PGI2) following ischemia/reperfusion injury (IRI) before significantly exacerbating tubular injury. Rats were subjected to uninephrectomy and 30 min of warm ischemia of the remaining kidney. Kidneys were harvested 3 h after reperfusion. (a) Renal cortical levels of prostaglandin F1α (PGF1α). (b) Tubule injury score. (c) Renal cortical levels of malondialdehyde (MDA). N=4–5. Het, SerpinC1+/− rat; IR, ischemia/reperfusion; Sham, sham operated; WT, wild-type littermate.

References

    1. 1Rosner MH, Okusa MD. Acute kidney injury associated with cardiac surgery. Clin J Am Soc Nephrol 2006; 1: 19–32.
    1. 2Waikar SS, Liu KD, Chertow GM. Diagnosis, epidemiology and outcomes of acute kidney injury. Clin J Am Soc Nephrol 2008; 3: 844–861.
    1. 3Wald R, Quinn RR, Luo J et al. Chronic dialysis and death among survivors of acute kidney injury requiring dialysis. JAMA 2009; 302: 1179–1185.
    1. 4Rosenberg RD. Biochemistry of heparin antithrombin interactions, and the physiologic role of this natural anticoagulant mechanism. Am J Med 1989; 87: 2S–9S.
    1. 5Horie S, Ishii H, Kazama M. Heparin-like glycosaminoglycan is a receptor for antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells. Thromb Res 1990; 59: 895–904.
    1. 6Harada N, Okajima K, Kushimoto S et al. Antithrombin reduces ischemia/reperfusion injury of rat liver by increasing the hepatic level of prostacyclin. Blood 1999; 93: 157–164.
    1. 7Harada N, Okajima K, Uchiba M et al. Antithrombin reduces ischemia/reperfusion-induced liver injury in rats by activation of cyclooxygenase-1. Thromb Haemost 2004; 92: 550–558.
    1. 8Ozden A, Sarioglu A, Demirkan NC et al. Antithrombin III reduces renal ischemia-reperfusion injury in rats. Res Exp Med (Berl) 2001; 200: 195–203.
    1. 9Fourrier F. Therapeutic applications of antithrombin concentrates in systemic inflammatory disorders. Blood Coagul Fibrinolysis 1998; 9(Suppl 2): S39–S45.
    1. 10Ostermann H. Antithrombin III in sepsis. New evidences and open questions. Minerva Anestesiol 2002; 68: 445–448.
    1. 11Roemisch J, Gray E, Hoffmann JN et al. Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 2002; 13: 657–670.
    1. 12Opal SM. Interactions between coagulation and inflammation. Scand J Infect Dis 2003; 35: 545–554.
    1. 13Souter PJ, Thomas S, Hubbard AR et al. Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood. Crit Care Med 2001; 29: 134–139.
    1. 14Dunzendorfer S, Kaneider N, Rabensteiner A et al. Cell-surface heparan sulfate proteoglycan-mediated regulation of human neutrophil migration by the serpin antithrombin III. Blood 2001; 97: 1079–1085.
    1. 15Sahsivar MO, Narin C, Kiyici A et al. The effect of iloprost on renal dysfunction after renal I/R using cystatin C and beta2-microglobulin monitoring. Shock 2009; 32: 498–502.
    1. 16Canacankatan N, Sucu N, Aytacoglu B et al. Affirmative effects of iloprost on apoptosis during ischemia-reperfusion injury in kidney as a distant organ. Ren Fail 2012; 34: 111–118.
    1. 17Lelcuk S, Alexander F, Kobzik L et al. Prostacyclin and thromboxane A2 moderate postischemic renal failure. Surgery 1985; 98: 207–212.
    1. 18Johannes T, Ince C, Klingel K et al. Iloprost preserves renal oxygenation and restores kidney function in endotoxemia-related acute renal failure in the rat. Crit Care Med 2009; 37: 1423–1432.
    1. 19Geurts AM, Cost GJ, Freyvert Y et al. Knockout rats via embryo microinjection of zinc-finger nucleases. Science 2009; 325: 433.
    1. 20Hirsh J, Piovella F, Pini M. Congenital antithrombin III deficiency. Incidence and clinical features. Am J Med 1989; 87: 34S–38S.
    1. 21Palevsky PM, Liu KD, Brophy PD et al. KDOQI US commentary on the 2012 KDIGO clinical practice guideline for acute kidney injury. Am J Kidney Dis 2013; 61: 649–672.
    1. 22Hunt SA, Abraham WT, Chin MH et al. ACC/AHA 2005 Guideline Update for the Diagnosis and Management of Chronic Heart Failure in the Adult: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines (Writing Committee to Update the 2001 Guidelines for the Evaluation and Management of Heart Failure): developed in collaboration with the American College of Chest Physicians and the International Society for Heart and Lung Transplantation: endorsed by the Heart Rhythm Society. Circulation 2005 112: e154–e235.
    1. 23Feng D, Yang C, Geurts AM et al. Increased expression of NAD(P)H oxidase subunit p67(phox) in the renal medulla contributes to excess oxidative stress and salt-sensitive hypertension. Cell Metab 2012; 15: 201–208.
    1. 24Schumer M, Colombel MC, Sawczuk IS et al. Morphologic, biochemical, and molecular evidence of apoptosis during the reperfusion phase after brief periods of renal ischemia. Am J Pathol 1992; 140: 831–838.
    1. 25Liang M, Pietrusz JL. Thiol-related genes in diabetic complications: a novel protective role for endogenous thioredoxin 2. Arterioscler Thromb Vasc Biol 2007; 27: 77–83.
    1. 26Tian Z, Greene AS, Pietrusz JL et al. MicroRNA-target pairs in the rat kidney identified by microRNA microarray, proteomic, and bioinformatic analysis. Genome Res 2008; 18: 404–411.
    1. 27Mori T, Polichnowski A, Glocka P et al. High perfusion pressure accelerates renal injury in salt-sensitive hypertension. J Am Soc Nephrol 2008; 19: 1472–1482.
    1. 28Xu X, Kriegel AJ, Liu Y et al. Delayed ischemic preconditioning contributes to renal protection by upregulation of miR-21. Kidney Int 2012; 82: 1167–1175.
    1. 29Melnikov VY, Faubel S, Siegmund B et al. Neutrophil-independent mechanisms of caspase-1- and IL-18-mediated ischemic acute tubular necrosis in mice. J Clin Invest 2002; 110: 1083–1091.
    1. 30Liu Y, Taylor NE, Lu L et al. Renal medullary microRNAs in Dahl salt-sensitive rats: miR-29b regulates several collagens and related genes. Hypertension 2010; 55: 974–982.
    1. 31Mladinov D, Liu Y, Mattson DL et al. MicroRNAs contribute to the maintenance of cell-type-specific physiological characteristics: miR-192 targets Na+/K+-ATPase beta1. Nucleic Acids Res 2013; 41: 1273–1283.

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