- Email: [email protected]
Splenectomy Attenuates the Course of Kidney Ischemia-Reperfusion Injury in Rats
Splenectomy Attenuates the Course of Kidney Ischemia-Reperfusion Injury in Rats W. Wystrychowskia,*, L. Filipczykb, L. Cierpkaa, E. Obuchowiczc, A. Więcekd, and A. Wystrychowskid a Department of General, Vascular and Transplant Surgery, Medical University of Silesia, Katowice, Poland; bDepartment of Nephrology, Municipal Hospital, Bytom, Poland; cDepartment of Pharmacology, Medical University of Silesia, Katowice, Poland; and dDepartment of Nephrology, Endocrinology and Metabolic Disorders; Medical University of Silesia, Katowice, Poland
ABSTRACT Introduction. Renal ischemia-reperfusion injury (IRI) initiates inflammatory response with synthesis of free oxygen radicals, chemokines, and cytokines which attract neutrophils and monocytes, which then differentiate into macrophages and dendritic cells, activating adaptive immune response. The spleen is the main source of both monocytes and lymphocytes. The aim of this study was to assess whether splenectomy performed before or upon IRI affects post-ischemic and long-term renal function. Methods. Two weeks after right nephrectomy, the left kidney pedicle was clamped for 45 minutes in 24 rats. After the clip insertion, the spleen was removed in 12 animals and the remaining 12 rats underwent sham splenectomy. In the second experiment, splenectomy (n ¼ 9) or sham procedure (n ¼ 9) was performed simultaneously with right nephrectomy, 2 weeks before left kidney ischemia. The excretory function of the kidney was evaluated 48 hours and 7 days after ischemia. In the experimental model of chronic renal failure, 14 days before right nephrectomy, the prolonged 90-minute ischemia was induced in 32 rats with simultaneous splenectomy (n ¼ 16) or sham procedure (n ¼ 16). In long-term observation, the renal function and mortality rate was evaluated. Results. Kidney function preservation was superior in rats that underwent splenectomy together with renal ischemia when compared to controls. This was further expressed with a 2 times lower mortality rate in splenectomized animals in 6 months observation after prolonged renal ischemia. Renoprotective effect was not observed when splenectomy was performed 2 weeks before IRI. Conclusions. The results suggest a detrimental influence of the spleen on the development of renal IRI.
R
ENAL ISCHEMIA-REPERFUSION INJURY (IRI) causes glomerular capillary endothelial dysfunction and tubular epithelial necrosis leading to ischemic acute kidney injury (AKI) with activation of inflammatory reaction mediated by increased cytokine and adhesion molecule expression, synthesis of reactive oxygen species and chemoattractants, leading to neutrophil, monocyte, and lymphocyte activation and infiltration [1]. As the part of innate immune system, macrophages play a crucial role in the inflammatory reaction and development of AKI through synthesis of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, IL-12, tumor necrosis factor alpha, and complement components [2]. Furthermore, macrophage-derived transforming
growth factor beta promotes interstitial fibrosis leading ultimately to chronic renal failure [3]. Some of the infiltrating monocytes differentiate into antigen-presenting dendritic cells (DCs) [4]. Activation of DC expressed Toll-like receptors leads to stimulation of an adaptive immune response responsible for chronic renal failure and which, in the setting of a kidney allograft, is co-responsible for its chronic rejection.
*Address correspondence to Wojciech Wystrychowski, MD, PhD, Department of General, Vascular and Transplant Surgery, Medical University of Silesia, ul. Francuska 20-24, 40-027 Katowice, Poland. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.056
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2558
Transplantation Proceedings, 46, 2558e2561 (2014)
SPLENECTOMY
2559
Heat shock proteins released during IRI, as well as components of necrotic cells act as endogenous ligands for Toll-like receptors, leading to DC activation with increased major histocompatibility complex II expression. Antigen-presenting cells migrating to the secondary lymphatic organs activate naive T lymphocytes with their differentiation into cytotoxic, as well as type 1 and 2 T helper cells [5,6]. These dependencies have been confirmed by observing kidney infiltration with CD4þ and CD8þ T cells 6 weeks after experimental 60minute renal ischemia, as well as improved post-ischemic renal function in CD4þ-deficient rats [7]. This phenomenon of interplay between innate and adaptive immune responses mediated, inter alia, by infiltrating monocytes and lymphocytes, may explain significant influences of IRI and the course of AKI on long-term renal function. The spleen is the main unit of the mononuclear phagocytic system (MPS) comprising monocytes, macrophages, and DCs. Furthermore, as the largest single lymphoid organ, it is the main reservoir and source of lymphocytes, and the predominant site of T cells activation. The aim of this study was to assess the influence of splenectomy (Splx) performed 2 weeks before or upon kidney ischemia on the course of ischemic AKI and long-term renal function in the rat.
kidney ischemia. In the third experiment, 90-minute ischemia of the left kidney was performed, with simultaneous splenectomy (group 5, n ¼ 16) or sham operation (group 6, control, n ¼ 16). The right nephrectomy was performed 2 weeks later. Forty-five-minute warm ischemia is a standard model of experimental AKI, but not applicable for analysis of post-ischemic chronic renal failure. Concurrently, 90 minutes of solitary kidney warm ischemia induces exceedingly severe IRI leading to almost 100% of mortality in an acute post-ischemic phase. Thus, the model of 90-minute renal ischemia with delayed healthy kidney nephrectomy was applied as the experimental model of chronic renal failure. The surgical procedures of in situ left kidney vascular pedicle clamping followed by splenectomy and subsequent clamp release were performed under ether anesthesia. During the ischemia period rats were awaken. Twenty-four hours and 6 days after 45-minute ischemia, the animals were placed in metabolic cages for 24-hour urine collection and subsequently underwent blood sample collection from the retroorbital plexus. Diuresis (D; mL/24 hours), creatinine clearance (ClCr; mL/min), proteinuria to creatinine clearance ratio (UProt/ ClCr; mg/mL) and fractional excretion of sodium (FENa, %), as well as kidney mass (mg/100 mg body weight) were estimated 48 hours and 7 days after ischemia. In the third study, long-term renal function and mortality rate were assessed 6 months after 90-minute IRI. The study protocol was approved by the local bioethical committee for experiments on animals.
METHODS
RESULTS
Seventy-four male Sprague-Dawley rats (9-weeks old, weighing 250 to 300 g) were obtained from the Experimental Medicine Centre, Medical University of Silesia, Katowice, Poland. Animals received humane care in compliance with the “Principals of Laboratory Animal Care.” All rats were provided with standard laboratory chow and water ad libitum in a temperature-controlled environment (21 C) with a 12-hour light-dark cycle. In the first experiment, 2 weeks after right nephrectomy, the left kidney pedicle was clamped for 45 minutes in 24 rats. After the clip insertion, the spleen was removed in 12 randomly chosen animals (group 1). The remaining 12 rats underwent sham splenectomy (group 2, control). In the second experiment including 18 rats, splenectomy (group 3, n ¼ 9) or sham procedure (group 4, control, n ¼ 9) was performed simultaneously with right nephrectomy, 2 weeks before solitary
As shown in Table 1, splenectomy performed simultaneously with 45-minute solitary kidney ischemia alleviated ischemiainduced kidney function impairment in a 48-hourlong observation. Seven days after IRI induction the renal function has normalized in all animals, however, significantly higher kidney mass expressed more intense inflammatory edema in the control group. Long-term observation confirmed development of post-ischemic chronic renal failure in asplenic (group 5, N ¼ 8), as well as sham group (group 6, N ¼ 4) reflected with increased proteinuria (UProt/ClCr: 0.034 0.0.02 and 0.035 0.01 mg/mL, respectively); however, these results were much influenced by high mortality rate in the latter. Six-month observation revealed a twice higher survival
Table 1. Results at 48 Hours and 7 Days After 45-minute Solitary Kidney Ischemia*,† Group 1
Group 2
Group 3
Group 4
Splx
Sham
Splx
Sham
Upon 45-min Renal Ischemia N
48 h
7d
12
D ClCr FENa UProt/ClCr D ClCr FENa UProt/ClCr kidney mass
22.08 1.32 0.76 0.009 11.79 3.15 0.07 0.001 0.63
4.53 0.51 0.93 0.007 3.42 0.49 0.04 0.0004 0.06
2 Weeks Before 45-min Renal Ischemia 12
28.29 0.50 3.88 0.029 20.2 2.77 0.08 0.0013 0.81
9
8.72 0.33‡ 5.11§ 0.016‡ 10.14¶ 0.55 0.05 0.0004 0.25‡
27.28 0.74 2.10 0.018 16.56 2.81 0.15 0.001 0.67
9
4.89 0.37 1.26 0.015 6.92 0.72 0.14 0.001 0.15
24.44 0.81 2.31 0.018 15.56 2.58 0.13 0.002 0.69
8.42 0.30 2.54 0.012 5.75 0.67 0.11 0.001 0.1
Abbreviations: Splx, splenectomy; D, diuresis; ClCr, creatinine clearance; FENa, fractional excretion of sodium; UProt/ClCr, proteinuria to creatinine clearance ratio; N, number of animals. *Splenectomy (groups 1 and 3) or sham splenectomy (groups 2 and 4) performed 2 weeks before (groups 3 and 4) or upon ischemia (groups 1 and 2). † Means SD, Mann-Whitney U test: ‡ < 0.001 versus group 1; § < 0.005 versus group 1, ¶ < 0.05 versus group 1.
2560
rate in splenectomized animals (50%) in comparison to the sham splenectomy group (25%; Fig 1). DISCUSSION
An inflammatory reaction with activation of the MPS and subsequent stimulation of adaptive immune response are the key constituents of IRI. The remote effect of splenectomy on IRI has been a subject of few studies so far. It has been stated that spleen removal attenuates multi-organ failure after experimental intestinal IRI [8]. Other observations revealed beneficial effects of Splx on the course of spinal cord and cerebral ischemia [9]. Swirski et al proved ischemia-induced mobilization of the splenic undifferentiated monocytes reservoir with their migration and accumulation in ischemia-injured myocardium [10]. Hiroyoshi et al, in an experimental model of renal IRI in the rat, confirmed better kidney function when 30-minute ischemia was followed by spleen removal. However, the post-ischemic observation was limited to just 24 hours [11]. This outcome is consistent with clinical observation of shortening of the delay in renal graft function in splenectomized recipients of cadaveric donor kidneys [12]. On the other hand, splenectomy has been proven to increase systemic inflammatory response in course of AKI through decreased synthesis of anti-inflammatory IL-10 [13]. Multiple recent observations indicate IRI as the major factor beside antigen compatibility influencing renal graft early function and long-term survival [14]. Influence of the splenectomy on the immune reaction after organ transplantation has been studied for years. First clinical trials showed a beneficial effect of Splx on renal graft survival with decreased frequency of acute graft rejection. These positive results were best explained with higher azathioprine
Fig 1. Survival analysis 6 months after prolonged 90-minute renal ischemia accompanied by splenectomy (Splx, gr 5) or sham splenectomy (sham, gr 6) e Kaplan-Meier cumulative survival (Cox F test P ¼ .07).
WYSTRYCHOWSKI, FILIPCZYK, CIERPKA ET AL
tolerance as the consequence of post-splenectomy leukocytosis, and transient impairment of humoral immunity with mitigation of the spleen-derived antibody response [15]. However, potential benefits of splenectomy in organ recipients were outweighed by possible complications related to the additional surgical exposure and increased long-term risk of infection in asplenic patients. Furthermore, potential application has been limited with introduction of new immunosuppressive agents and therapy protocols. Nevertheless, splenectomy is still proposed as the rescue procedure in case of acute antibody-mediated renal graft rejection refractory to other treatment modalities [16]. It is also part of current kidney transplantation protocols in case of an AB0incompatible living donor [17]. The long-term lymphocytosis with increased levels of B and T cells is commonly observed in splenectomized patients, and the phenomenon of immunosuppressive influence of splenectomy remains still unclear. Revealed impaired distribution of lymphocyte subsets with long-term decrease in naive (CD45RA) CD4þ T cells and immunoglobulin M memory B cells is one of possible mechanisms [18,19]. However, Ashimine et al revealed lack of inhibitory effect of pretransplantation splenectomy on HLA antibody synthesis in renal graft recipients [20]. Our results revive the rationale of such practice, showing another potential mechanism of beneficial effect of Splx on long-term renal function after IRI through alleviation of the course of AKI; however, only when splenectomy accompanies ischemia-reperfusion. It may explain early observations of improved renal graft survival in splenectomized recipients only in cases of cadaveric and not living donor transplants, which might have been related to longer ischemia and concurrently greater degree of IRI and MPS activation in the former [12]. The observed lack of beneficial influence when Splx was performed 2 weeks before ischemia
SPLENECTOMY
can be explained with compensation of its role by other MPS units, mainly liver, as well as increased level of circulating monocytes when spleen reservoir function is lost. CONCLUSIONS
Our results suggest a detrimental influence of the spleen on the development of ischemia-reperfusion kidney injury and long-term renal function. This calls for devising methods of inhibition of mononuclear release from the spleen in clinical conditions involving ischemia-reperfusion, such as the peritransplantation period. REFERENCES [1] Kinsey GR, Li L, Okusa MD. Inflammation in acute kidney injury. Nephron Exp Nephrol 2008;109:e102. [2] Jang HR, Rabb H. The innate immune response in ischemic acute kidney injury. Clin Immunol 2009;130:41. [3] Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and repair. J Clin Invest 2008;118:3522. [4] Li Li, Okusa M. Macrophages, dendritic cells and kidney ischemia-reperfusion injury. Semin Nephrol 2010;30:268. [5] Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol 2004;5:971. [6] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987. [7] Ascon M, Ascon DB, Liu M, et al. Renal ischemiareperfusion leads to long term infiltration of activated and effector-memory T lymphocytes. Kidney Int 2009;75:526. [8] Savas C, Ozogul C, Karaöz E, et al. Splenectomy reduces remote organ damage after intestinal ischaemia-reperfusion injury. Acta Chir Belg 2003;103:315.
2561 [9] Temiz C, Solmaz I, Tehli O, et al. The effects of splenectomy on lipid peroxidation and neuronal loss in experimental spinal cord ischemia/reperfusion injury. Turk Neurosurg 2013;23:67. [10] Swirski FK, Nahrendorf M, Etzrodt M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 2009;325:612. [11] Hiroyoshi T, Tsuchida M, Uchiyama K, et al. Splenectomy protects the kidneys against ischemic reperfusion injury in the rat. Transpl Immunol 2012;27:8. [12] Okiye S, Zincke H, Engen D, et al. Splenectomy in high-risk primary renal transplant recipients. Am J Surg 1983;146:594. [13] Andrés-Hernando A, Altmann C, Ahuja N, et al. Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice. Am J Physiol Renal Physiol 2011;301:F907. [14] Salahudeen AK, Haider N, May W. Cold ischemia and the reduced long-term survival of cadaveric renal allografts. Kidney Int 2004;65:713. [15] Stuart FP, Reckard CR, Ketel BL, et al. Effect of splenectomy on first cadaver kidney transplants. Ann Surg 1980;192:553. [16] Kaplan B, Gangemi A, Thielke J. Successful rescue of refractory, severe antibody mediated rejection with splenectomy. Transplantation 2007;83:99. [17] Magee C. Transplantation across previously incompatible immunological barriers. Transpl Int 2006;19:87. [18] Wolf HM, Eibl MM, Georgi E, et al. Long-term decrease of CD4þCD45RAþ T cells and impaired primary immune response after post-traumatic splenectomy. Br J Haematol 1999;107:55. [19] Cameron PU, Jones P, Gorniak M, et al. Splenectomy associated changes in IgM memory B cells in an adult spleen registry cohort. PLoS One 2011;6:e23164. [20] Ashimine S, Watarai Y, Yamamoto T, et al. Neither pretransplant rituximab nor splenectomy affects de novo HLA antibody production after renal transplantation. Kidney Int 2014;85: 425.
ABSTRACT Introduction. Renal ischemia-reperfusion injury (IRI) initiates inflammatory response with synthesis of free oxygen radicals, chemokines, and cytokines which attract neutrophils and monocytes, which then differentiate into macrophages and dendritic cells, activating adaptive immune response. The spleen is the main source of both monocytes and lymphocytes. The aim of this study was to assess whether splenectomy performed before or upon IRI affects post-ischemic and long-term renal function. Methods. Two weeks after right nephrectomy, the left kidney pedicle was clamped for 45 minutes in 24 rats. After the clip insertion, the spleen was removed in 12 animals and the remaining 12 rats underwent sham splenectomy. In the second experiment, splenectomy (n ¼ 9) or sham procedure (n ¼ 9) was performed simultaneously with right nephrectomy, 2 weeks before left kidney ischemia. The excretory function of the kidney was evaluated 48 hours and 7 days after ischemia. In the experimental model of chronic renal failure, 14 days before right nephrectomy, the prolonged 90-minute ischemia was induced in 32 rats with simultaneous splenectomy (n ¼ 16) or sham procedure (n ¼ 16). In long-term observation, the renal function and mortality rate was evaluated. Results. Kidney function preservation was superior in rats that underwent splenectomy together with renal ischemia when compared to controls. This was further expressed with a 2 times lower mortality rate in splenectomized animals in 6 months observation after prolonged renal ischemia. Renoprotective effect was not observed when splenectomy was performed 2 weeks before IRI. Conclusions. The results suggest a detrimental influence of the spleen on the development of renal IRI.
R
ENAL ISCHEMIA-REPERFUSION INJURY (IRI) causes glomerular capillary endothelial dysfunction and tubular epithelial necrosis leading to ischemic acute kidney injury (AKI) with activation of inflammatory reaction mediated by increased cytokine and adhesion molecule expression, synthesis of reactive oxygen species and chemoattractants, leading to neutrophil, monocyte, and lymphocyte activation and infiltration [1]. As the part of innate immune system, macrophages play a crucial role in the inflammatory reaction and development of AKI through synthesis of proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, IL-12, tumor necrosis factor alpha, and complement components [2]. Furthermore, macrophage-derived transforming
growth factor beta promotes interstitial fibrosis leading ultimately to chronic renal failure [3]. Some of the infiltrating monocytes differentiate into antigen-presenting dendritic cells (DCs) [4]. Activation of DC expressed Toll-like receptors leads to stimulation of an adaptive immune response responsible for chronic renal failure and which, in the setting of a kidney allograft, is co-responsible for its chronic rejection.
*Address correspondence to Wojciech Wystrychowski, MD, PhD, Department of General, Vascular and Transplant Surgery, Medical University of Silesia, ul. Francuska 20-24, 40-027 Katowice, Poland. E-mail: [email protected]
0041-1345/14 http://dx.doi.org/10.1016/j.transproceed.2014.09.056
ª 2014 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
2558
Transplantation Proceedings, 46, 2558e2561 (2014)
SPLENECTOMY
2559
Heat shock proteins released during IRI, as well as components of necrotic cells act as endogenous ligands for Toll-like receptors, leading to DC activation with increased major histocompatibility complex II expression. Antigen-presenting cells migrating to the secondary lymphatic organs activate naive T lymphocytes with their differentiation into cytotoxic, as well as type 1 and 2 T helper cells [5,6]. These dependencies have been confirmed by observing kidney infiltration with CD4þ and CD8þ T cells 6 weeks after experimental 60minute renal ischemia, as well as improved post-ischemic renal function in CD4þ-deficient rats [7]. This phenomenon of interplay between innate and adaptive immune responses mediated, inter alia, by infiltrating monocytes and lymphocytes, may explain significant influences of IRI and the course of AKI on long-term renal function. The spleen is the main unit of the mononuclear phagocytic system (MPS) comprising monocytes, macrophages, and DCs. Furthermore, as the largest single lymphoid organ, it is the main reservoir and source of lymphocytes, and the predominant site of T cells activation. The aim of this study was to assess the influence of splenectomy (Splx) performed 2 weeks before or upon kidney ischemia on the course of ischemic AKI and long-term renal function in the rat.
kidney ischemia. In the third experiment, 90-minute ischemia of the left kidney was performed, with simultaneous splenectomy (group 5, n ¼ 16) or sham operation (group 6, control, n ¼ 16). The right nephrectomy was performed 2 weeks later. Forty-five-minute warm ischemia is a standard model of experimental AKI, but not applicable for analysis of post-ischemic chronic renal failure. Concurrently, 90 minutes of solitary kidney warm ischemia induces exceedingly severe IRI leading to almost 100% of mortality in an acute post-ischemic phase. Thus, the model of 90-minute renal ischemia with delayed healthy kidney nephrectomy was applied as the experimental model of chronic renal failure. The surgical procedures of in situ left kidney vascular pedicle clamping followed by splenectomy and subsequent clamp release were performed under ether anesthesia. During the ischemia period rats were awaken. Twenty-four hours and 6 days after 45-minute ischemia, the animals were placed in metabolic cages for 24-hour urine collection and subsequently underwent blood sample collection from the retroorbital plexus. Diuresis (D; mL/24 hours), creatinine clearance (ClCr; mL/min), proteinuria to creatinine clearance ratio (UProt/ ClCr; mg/mL) and fractional excretion of sodium (FENa, %), as well as kidney mass (mg/100 mg body weight) were estimated 48 hours and 7 days after ischemia. In the third study, long-term renal function and mortality rate were assessed 6 months after 90-minute IRI. The study protocol was approved by the local bioethical committee for experiments on animals.
METHODS
RESULTS
Seventy-four male Sprague-Dawley rats (9-weeks old, weighing 250 to 300 g) were obtained from the Experimental Medicine Centre, Medical University of Silesia, Katowice, Poland. Animals received humane care in compliance with the “Principals of Laboratory Animal Care.” All rats were provided with standard laboratory chow and water ad libitum in a temperature-controlled environment (21 C) with a 12-hour light-dark cycle. In the first experiment, 2 weeks after right nephrectomy, the left kidney pedicle was clamped for 45 minutes in 24 rats. After the clip insertion, the spleen was removed in 12 randomly chosen animals (group 1). The remaining 12 rats underwent sham splenectomy (group 2, control). In the second experiment including 18 rats, splenectomy (group 3, n ¼ 9) or sham procedure (group 4, control, n ¼ 9) was performed simultaneously with right nephrectomy, 2 weeks before solitary
As shown in Table 1, splenectomy performed simultaneously with 45-minute solitary kidney ischemia alleviated ischemiainduced kidney function impairment in a 48-hourlong observation. Seven days after IRI induction the renal function has normalized in all animals, however, significantly higher kidney mass expressed more intense inflammatory edema in the control group. Long-term observation confirmed development of post-ischemic chronic renal failure in asplenic (group 5, N ¼ 8), as well as sham group (group 6, N ¼ 4) reflected with increased proteinuria (UProt/ClCr: 0.034 0.0.02 and 0.035 0.01 mg/mL, respectively); however, these results were much influenced by high mortality rate in the latter. Six-month observation revealed a twice higher survival
Table 1. Results at 48 Hours and 7 Days After 45-minute Solitary Kidney Ischemia*,† Group 1
Group 2
Group 3
Group 4
Splx
Sham
Splx
Sham
Upon 45-min Renal Ischemia N
48 h
7d
12
D ClCr FENa UProt/ClCr D ClCr FENa UProt/ClCr kidney mass
22.08 1.32 0.76 0.009 11.79 3.15 0.07 0.001 0.63
4.53 0.51 0.93 0.007 3.42 0.49 0.04 0.0004 0.06
2 Weeks Before 45-min Renal Ischemia 12
28.29 0.50 3.88 0.029 20.2 2.77 0.08 0.0013 0.81
9
8.72 0.33‡ 5.11§ 0.016‡ 10.14¶ 0.55 0.05 0.0004 0.25‡
27.28 0.74 2.10 0.018 16.56 2.81 0.15 0.001 0.67
9
4.89 0.37 1.26 0.015 6.92 0.72 0.14 0.001 0.15
24.44 0.81 2.31 0.018 15.56 2.58 0.13 0.002 0.69
8.42 0.30 2.54 0.012 5.75 0.67 0.11 0.001 0.1
Abbreviations: Splx, splenectomy; D, diuresis; ClCr, creatinine clearance; FENa, fractional excretion of sodium; UProt/ClCr, proteinuria to creatinine clearance ratio; N, number of animals. *Splenectomy (groups 1 and 3) or sham splenectomy (groups 2 and 4) performed 2 weeks before (groups 3 and 4) or upon ischemia (groups 1 and 2). † Means SD, Mann-Whitney U test: ‡ < 0.001 versus group 1; § < 0.005 versus group 1, ¶ < 0.05 versus group 1.
2560
rate in splenectomized animals (50%) in comparison to the sham splenectomy group (25%; Fig 1). DISCUSSION
An inflammatory reaction with activation of the MPS and subsequent stimulation of adaptive immune response are the key constituents of IRI. The remote effect of splenectomy on IRI has been a subject of few studies so far. It has been stated that spleen removal attenuates multi-organ failure after experimental intestinal IRI [8]. Other observations revealed beneficial effects of Splx on the course of spinal cord and cerebral ischemia [9]. Swirski et al proved ischemia-induced mobilization of the splenic undifferentiated monocytes reservoir with their migration and accumulation in ischemia-injured myocardium [10]. Hiroyoshi et al, in an experimental model of renal IRI in the rat, confirmed better kidney function when 30-minute ischemia was followed by spleen removal. However, the post-ischemic observation was limited to just 24 hours [11]. This outcome is consistent with clinical observation of shortening of the delay in renal graft function in splenectomized recipients of cadaveric donor kidneys [12]. On the other hand, splenectomy has been proven to increase systemic inflammatory response in course of AKI through decreased synthesis of anti-inflammatory IL-10 [13]. Multiple recent observations indicate IRI as the major factor beside antigen compatibility influencing renal graft early function and long-term survival [14]. Influence of the splenectomy on the immune reaction after organ transplantation has been studied for years. First clinical trials showed a beneficial effect of Splx on renal graft survival with decreased frequency of acute graft rejection. These positive results were best explained with higher azathioprine
Fig 1. Survival analysis 6 months after prolonged 90-minute renal ischemia accompanied by splenectomy (Splx, gr 5) or sham splenectomy (sham, gr 6) e Kaplan-Meier cumulative survival (Cox F test P ¼ .07).
WYSTRYCHOWSKI, FILIPCZYK, CIERPKA ET AL
tolerance as the consequence of post-splenectomy leukocytosis, and transient impairment of humoral immunity with mitigation of the spleen-derived antibody response [15]. However, potential benefits of splenectomy in organ recipients were outweighed by possible complications related to the additional surgical exposure and increased long-term risk of infection in asplenic patients. Furthermore, potential application has been limited with introduction of new immunosuppressive agents and therapy protocols. Nevertheless, splenectomy is still proposed as the rescue procedure in case of acute antibody-mediated renal graft rejection refractory to other treatment modalities [16]. It is also part of current kidney transplantation protocols in case of an AB0incompatible living donor [17]. The long-term lymphocytosis with increased levels of B and T cells is commonly observed in splenectomized patients, and the phenomenon of immunosuppressive influence of splenectomy remains still unclear. Revealed impaired distribution of lymphocyte subsets with long-term decrease in naive (CD45RA) CD4þ T cells and immunoglobulin M memory B cells is one of possible mechanisms [18,19]. However, Ashimine et al revealed lack of inhibitory effect of pretransplantation splenectomy on HLA antibody synthesis in renal graft recipients [20]. Our results revive the rationale of such practice, showing another potential mechanism of beneficial effect of Splx on long-term renal function after IRI through alleviation of the course of AKI; however, only when splenectomy accompanies ischemia-reperfusion. It may explain early observations of improved renal graft survival in splenectomized recipients only in cases of cadaveric and not living donor transplants, which might have been related to longer ischemia and concurrently greater degree of IRI and MPS activation in the former [12]. The observed lack of beneficial influence when Splx was performed 2 weeks before ischemia
SPLENECTOMY
can be explained with compensation of its role by other MPS units, mainly liver, as well as increased level of circulating monocytes when spleen reservoir function is lost. CONCLUSIONS
Our results suggest a detrimental influence of the spleen on the development of ischemia-reperfusion kidney injury and long-term renal function. This calls for devising methods of inhibition of mononuclear release from the spleen in clinical conditions involving ischemia-reperfusion, such as the peritransplantation period. REFERENCES [1] Kinsey GR, Li L, Okusa MD. Inflammation in acute kidney injury. Nephron Exp Nephrol 2008;109:e102. [2] Jang HR, Rabb H. The innate immune response in ischemic acute kidney injury. Clin Immunol 2009;130:41. [3] Ricardo SD, van Goor H, Eddy AA. Macrophage diversity in renal injury and repair. J Clin Invest 2008;118:3522. [4] Li Li, Okusa M. Macrophages, dendritic cells and kidney ischemia-reperfusion injury. Semin Nephrol 2010;30:268. [5] Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol 2004;5:971. [6] Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004;5:987. [7] Ascon M, Ascon DB, Liu M, et al. Renal ischemiareperfusion leads to long term infiltration of activated and effector-memory T lymphocytes. Kidney Int 2009;75:526. [8] Savas C, Ozogul C, Karaöz E, et al. Splenectomy reduces remote organ damage after intestinal ischaemia-reperfusion injury. Acta Chir Belg 2003;103:315.
2561 [9] Temiz C, Solmaz I, Tehli O, et al. The effects of splenectomy on lipid peroxidation and neuronal loss in experimental spinal cord ischemia/reperfusion injury. Turk Neurosurg 2013;23:67. [10] Swirski FK, Nahrendorf M, Etzrodt M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 2009;325:612. [11] Hiroyoshi T, Tsuchida M, Uchiyama K, et al. Splenectomy protects the kidneys against ischemic reperfusion injury in the rat. Transpl Immunol 2012;27:8. [12] Okiye S, Zincke H, Engen D, et al. Splenectomy in high-risk primary renal transplant recipients. Am J Surg 1983;146:594. [13] Andrés-Hernando A, Altmann C, Ahuja N, et al. Splenectomy exacerbates lung injury after ischemic acute kidney injury in mice. Am J Physiol Renal Physiol 2011;301:F907. [14] Salahudeen AK, Haider N, May W. Cold ischemia and the reduced long-term survival of cadaveric renal allografts. Kidney Int 2004;65:713. [15] Stuart FP, Reckard CR, Ketel BL, et al. Effect of splenectomy on first cadaver kidney transplants. Ann Surg 1980;192:553. [16] Kaplan B, Gangemi A, Thielke J. Successful rescue of refractory, severe antibody mediated rejection with splenectomy. Transplantation 2007;83:99. [17] Magee C. Transplantation across previously incompatible immunological barriers. Transpl Int 2006;19:87. [18] Wolf HM, Eibl MM, Georgi E, et al. Long-term decrease of CD4þCD45RAþ T cells and impaired primary immune response after post-traumatic splenectomy. Br J Haematol 1999;107:55. [19] Cameron PU, Jones P, Gorniak M, et al. Splenectomy associated changes in IgM memory B cells in an adult spleen registry cohort. PLoS One 2011;6:e23164. [20] Ashimine S, Watarai Y, Yamamoto T, et al. Neither pretransplant rituximab nor splenectomy affects de novo HLA antibody production after renal transplantation. Kidney Int 2014;85: 425.