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Splenectomy protects the kidneys against ischemic reperfusion injury in the rat
Transplant Immunology 27 (2012) 8–11
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Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Splenectomy protects the kidneys against ischemic reperfusion injury in the rat Toshiya Hiroyoshi ⁎, Masahiro Tsuchida, Koichi Uchiyama, Koki Fujikawa, Takahiro Komatsu, Yoshihiro Kanaoka, Hideyasu Matsuyama Department of Urology, Graduate School of Medicine, Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 26 March 2012 Accepted 27 March 2012 Keywords: Renal ischemic reperfusion Renal injury Kidney transplantation Splenectomy
a b s t r a c t Background: Ischemic reperfusion (I/R) injury of the kidney is closely associated with delayed graft function, increased acute rejection, and late allograft dysfunction. Splenectomy reduced hepatic I/R injury by inhibiting leukocyte infiltration in the liver, release of TNF-α, cell apoptosis, and expression of caspase-3. Thus, we investigated the effects of splenectomy on renal I/R injury in the rat. Methods: Male Wistar rats were assigned to four groups: sham operation (sham group), sham operation + splenectomy (sham + SPLN group), right nephrectomy followed by clamping the left renal pedicle for 30 min (I/R 30 group), and I/R 30 + splenectomy (I/R 30 + SPLN group). Renal function was determined by measuring the concentration of blood urea nitrogen (BUN) and serum creatinine (S-Cr). The serum level of tumor necrosis factor-α (TNF-α) was measured as the marker for inflammation. Left kidneys were obtained 24 h after reperfusion. TUNEL assay was assessed for cell apoptosis. Spleens were obtained immediately (0-h group) and 3 h after reperfusion (3-h group). The removed spleens were histologically evaluated. Results: The BUN and S-Cr levels were significantly lower in the I/R 30 + SPLN group than in the I/R 30 group (p b 0.05 for both). Apoptotic cells were significantly lower in the I/R 30 + SPLN group than in the I/R 30 group. The serum level of TNF-α, which was increased after I/R, was significantly lower in the I/R 30 + SPLN group than in the I/R 30 group (p b 0.05). Spleen weights were significantly lower in the 3-h group than in the 0-h group (p b 0.05). Conclusion: These results suggest that splenectomy reduces renal I/R injury, and this effect may occur by an anti-inflammatory pathway and inhibition of cell apoptosis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Objective
Renal ischemic reperfusion (I/R) injury is a clinically significant problem in renal transplantation. Renal I/R injury in the early transplant period has been associated with acute rejection, delayed graft function, and late allograft failure [1–6]. I/R injury is characterized by decreased local oxygen, cellular metabolism impairments with decreased levels of ATP metabolic substrate and glucose, inflammation, free radical production, apoptosis, and necrosis, all of which lead to deterioration of tubular cells [7–13]. Several agents, such as heparin [14,15], erythropoietin [16–19], mycophenolate mofetil [7,20–23], and cyclosporine [2,23,24], have been reported to prevent renal I/R injury. These agents protect renal I/R injury by antiinflammatory activities. Likewise, splenectomy has been reported to reduce hepatic I/R injury, probably by anti-inflammatory and antiapoptotic mechanisms [25–27]. The definite role of splenectomy in renal I/R injury has not yet been elucidated.
We investigated whether splenectomy can reduce renal I/R injury using a rat experimental model and attempted to identify the proof of principle of splenectomy in the clinical setting.
Abbreviations: I/R, ischemic reperfusion; BUN, blood urea nitrogen; TNF-α, tumor necrosis factor-α; TUNEL, in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling; AI, apoptotic index. ⁎ Corresponding author. Tel.: + 81 836 22 2275; fax: + 81 836 22 2276. E-mail address: [email protected] (T. Hiroyoshi). 0966-3274/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2012.03.005
3. Materials and methods 3.1. Animals and experimental design Animal experiments were strictly performed according to standards for care and keeping of laboratory animals. Adult male Wistar rats (Kyudo, Saga, Japan) weighing 242 to 296 g were housed in individual cages in a temperature- and light-controlled environment. Food and water were available ad libitum. Rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg). A midline skin incision was made after the skin was shaved and sterilized with 10% povidone-iodine. Renal I/R injury was induced by clamping the left renal pedicle for 30 min. The right kidney was removed immediately after unclamping the renal pedicle. Splenectomy was performed 0 and 3 h after the right nephrectomy. Animals were assigned to four groups: sham operation (sham group), sham operation + splenectomy (sham + SPLN group), right
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
nephrectomy followed by clamping the left renal pedicle for 30 min (I/R 30 group), and I/R 30 + splenectomy (I/R 30 + SPLN group). 3.2. Biochemical analysis Blood samples were obtained 24 h after reperfusion, centrifuged (2500×g for 10 min) to separate the serum and plasma supernatants, and stored at − 80 °C until measurement. Blood urea nitrogen (BUN) and creatinine (S-Cr) were measured with a 7180 Clinical Analyzer (Hitachi, Tokyo, Japan). 3.3. Tumor necrosis factor-α assays
9
Left kidneys 24 h after reperfusion were used for TUNEL staining. An apoptosis in situ detection kit (Wako Pure Chemicals, Osaka, Japan) was used for the TUNEL assay. The apoptotic index (AI) was defined as number of apoptotic cells× 100 / total number of nucleated cells. 3.7. Statistical analysis The data were expressed as mean ± standard deviation. The oneway ANOVA t-test was used to compare differences between the I/R group and the I/R + SPLN group in terms of BUN, S-Cr, TNF-α, and AI. A p-value of b0.05 was considered to be statistically significant. 4. Results
Serum samples were stored at −80 °C until assays were performed. Serum levels of tumor necrosis factor-α (TNF-α) were measured with a TNF-α ELISA kit according to the manufacturer's instructions (Invitrogen Corporation, CA, US). 3.4. Spleen weight The weights of spleens obtained immediately after reperfusion (0-h group) and 3 h after right nephrectomy with or without I/R injury (3-h group and sham group, respectively) were measured.
4.1. Renal function The BUN and S-Cr levels in the I/R 30 group were significantly higher than those in the sham group (50.9±14.1 vs. 27.2±3.1 mg/dl, pb 0.01 and 0.59±0.17 vs. 0.30 ±0.04 mg/dl, p b 0.01, respectively). The BUN and S-Cr levels in the I/R 30+ SPLN group were significantly lower than those in the I/R 30 group (27.7±3.6 vs. 50.9 ±14.1 mg/dl, pb 0.01 and 0.34± 0.09 vs. 0.59± 0.17 mg/dl, pb 0.05, respectively) (Fig. 1).
A
3.5. Histology Left kidneys were obtained 24 h after reperfusion. Tissue specimens were fixed in 10% formalin and embedded in paraffin. Sections were cut 5-μm thick and stained with hematoxylin–eosin for light microscope examination. 3.6. In situ detection of apoptotic cells
Serum creatinine (mg/dL)
Blood urea nitrogen (mg/dL)
In situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining was performed. Serial sections of 5-μm thickness were prepared.
B
* 70 60 50 40 30 20 10 0 sham
sham SPLN
I/R30
I/R30 SPLN
*
C
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 sham
sham SPLN
I/R30
I/R30 SPLN
Fig. 1. BUN and S-Cr levels. BUN and S-Cr levels were 27.1 ± 3.15 and 0.30 ± 0.04 mg/dl in the sham group, 26.1 ± 4.25 and 0.34 ± 0.04 mg/dl in the sham + SPLN group, 50.9 ± 14.1 and 0.59 ± 0.17 mg/dl in the I/R 30 group, and 27.7 ± 3.6 and 0.34 ± 0.09 mg/dl in the I/R 30 + SPLN group, respectively. *p b 0.05.
Fig. 2. Histological examination.(A) Sham group. (B) I/R 30 group. (C) I/R 30 + SPLN group. Original magnification: ×400. In the I/R 30 + SPLN group, the degree of tubular damage and interstitial edema and number of inflammatory cells were less than those in the I/R 30 group.
10
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
C
A
25
B
*
D
Apoptosis index
20 15 10 5 0 sham
sham SPLN
I/R30
I/R30 SPLN
Fig. 3. TUNEL analysis of apoptotic cells.(A) Sham group. (B) I/R 30 group. (C) I/R 30 + SPLN group. Original magnification: ×400. (D) The apoptotic index (AI). The AI was 0 in the sham group, 1.4 ± 2.6 in the sham + SPLN group, 15.2 ± 3.9 in the I/R 30 group, and 2.4 ± 0.9 in the I/R 30 + SPLN group. *p b 0.05.
4.2. Histological examination 4.4. Plasma TNF-α activities In the I/R 30 group, tubular damage mainly occurred in the outer stripe of the outer medulla. Tubular cell swelling, tubular dilatation, and tubular epithelial cell necrosis were observed. Luminal hyaline casts, interstitial edema, and infiltration of inflammatory cells were also seen. In the I/R 30 + SPLN group, the degree of tubular damage and interstitial edema and number of inflammatory cells were less prominent compared with those in the I/R 30 group (Fig. 2).
The TNF-α levels in the sham group were similar to those in the sham + SPLN group. In the I/R 30 group, the TNF-α levels were significantly increased compared with those in the sham group (21.6 ± 3.4 vs. 5.0 ± 7.7 pg/ml, respectively; p b 0.05). In the I/R 30 + SPLN group, the TNF-α levels were significantly lower compared with those in the I/R 30 group (8.8 ± 10.9 vs. 21.6 ± 3.4 pg/ml, respectively; p b 0.05) (Fig. 4).
4.3. TUNEL staining and AI TUNEL-positive cells were mainly present in the tubules in the outer stripe of the outer medulla. The AI was similar in the sham group and sham + SPLN group. In the I/R 30 group, the AI was significantly increased compared with that in the sham group (15.2 ± 3.9 vs. 0, respectively; p b 0.001). In the I/R 30 + SPLN group, the AI was significantly lower compared with that in the I/R 30 group (2.4 ± 0.89 vs. 15.2 ± 3.9, respectively; p b 0.01) (Fig. 3).
4.5. Spleen weight There was no statistically significant difference in spleen weights between the 0-h and sham groups (p = 0.92). In the 3-h group, the spleen weight was significantly lower compared with that in the 0-h and sham groups (3-h vs. 0-h groups: 0.61 ± 0.09 vs. 0.84 ± 0.022 g, respectively; p b 0.05 and 3-h vs. sham groups: 0.61 ± 0.09 vs. 0.88 ± 0.19 g, respectively; p b 0.05) (Fig. 5).
* 30
* *
1.2
20
Spleen weight (g)
TNF-α α (pg/dl)
25
15 10 5
1 0.8 0.6 0.4 0.2
0 sham
sham SPLN
I/R30
I/R30 SPLN
0 0h
Fig. 4. Plasma TNF-α activities.TNF-α levels were 5.04 ± 7.66 pg/ml in the sham group, 5.54 ± 9.98 pg/dl in the sham + SPLN group, 21.6 ± 3.38 pg/ml in the I/R 30 group, and 8.78 ± 10.9 pg/ml in the I/R 30 + SPLN group. *p b 0.05.
sham
3h
Fig. 5. Spleen weight.The spleen weights were 0.84 ± 0.022 g in the 0-h group, 0.88 ± 0.19 g in the sham group, and 0.61 ± 0.09 g in the 3-h group. *p b 0.05.
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
5. Discussion The results of our study show that ischemic reperfusion combined with splenectomy improved renal function, improved renal histopathological degeneration, and decreased the level of TNF-α, leading to the reduction of renal injury caused by ischemic reperfusion. Many agents have been reported to prevent renal I/R injury. Heparin can protect against renal I/R injury by acting on the complement system to prevent inflammatory activity and inhibiting the adhesion and activation of neutrophils mediated by P-selectin [14,15]. Erythropoietin protected against renal I/R injury by reducing polymorphonuclease leukocyte accumulation in rat kidneys and up-regulating heat shock protein 70, anti-apoptotic protein B-cell lymphoma-2, and hypoxia inducible factor-1α expression [16–19]. Mycophenolate mofetil protected against renal I/R injury by decreasing the infiltration of lymphocytes and macrophages and reduced the expression of adhesion molecule intercellular adhesion molecule-1 [7,20–23]. Cyclosporine A reduced renal I/R injury by decreasing levels of inflammatory cytokines (interleukin-1 and TNF-α) and interstitial leukocyte accumulation [2,23,24]. All of these agents protect against renal I/R injury by anti-inflammatory activities. Because the spleen is the largest single reservoir of lymphocytes, it is suggested that splenectomy can reduce I/R injury by decreasing the number of lymphocytes that excrete inflammatory cytokines. Kirstan reported that TNF-α is an important mediator in renal I/R injury [28]. TNF-α reduced glomerular blood flow and the glomerular filtration rate by stimulating mesangial cells to produce a variety of vasoconstrictive mediators (platelet activating factor, endothelin-1, and prostaglandins) and recruit neutrophils and monocytes to the kidney [28]. TNF-α itself induced renal epithelial cell apoptosis, the major cell death pattern associated with renal I/R injury. Our study demonstrates that splenectomy reduces TNF-α production, which may be a major mechanism by which splenectomy reduces renal I/R injury. Our results are consistent with those of other reports showing that splenectomy reduced hepatic I/R injury [25–27]. Okuaki [25] reported that in hepatic I/R injury, splenectomy reduced the number of polymorphonuclear cells in the liver and TNF-α production; thus, the spleen may be a source of inflammatory mediators and play an important role in TNF-α production. Inflammatory cells were suggested to be mobilized from the spleen in previous research related to hepatic I/R injury [25]. The study showed that the weight of the spleen was significantly reduced after reperfusion, which may be explained by mobilization of a large amount of inflammatory cells from the spleen. The mechanisms of renal I/R injury have been considered to be abnormalities in regional blood flow, endothelial and epithelial cell dysfunction, inflammation and caspase activation, free radical production, apoptosis, and necrosis, and all of them are interdependent and act as distinct entities [29,30]. However, the precise mechanisms of I/R injury are complex. Although the definite mechanism of escaping renal I/R injury by splenectomy has not been elucidated, splenectomy is likely to decrease the damage of renal I/R injury by inhibiting TNF-α production. Our findings may serve as theoretical proof of the successful use of splenectomy for better renal function of the transplanted kidney in the clinical setting. Further experimental and clinical studies are needed to clarify the definite mechanism of renal I/R injury and the role of splenectomy. References [1] Requiao-Moura LR, Durao Mde S, Tonato EJ, et al. Effects of ischemia and reperfusion injury on long-term graft function. Transplant Proc 43:70–3.
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[2] Yang CW, Ahn HJ, Han HJ, et al. Pharmacological preconditioning with low-dose cyclosporine or FK506 reduces subsequent ischemia/reperfusion injury in rat kidney. Transplantation 2001;72:1753–9. [3] van Es A, Hermans J, van Bockel JH, Persijn GG, van Hooff JP, de Graeff J. Effect of warm ischemia time and HLA (A and B) matching on renal cadaveric graft survival and rejection episodes. Transplantation 1983;36:255–8. [4] Sanfilippo F, Vaughn WK, Spees EK, Lucas BA. The detrimental effects of delayed graft function in cadaver donor renal transplantation. Transplantation 1984;38: 643–8. [5] Goes N, Urmson J, Ramassar V, Halloran PF. Ischemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-gamma, transforming growth factor-beta 1, granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-10. Transplantation 1995;59:565–72. [6] Cole E, Naimark D, Aprile M, et al. An analysis of predictors of long-term cadaveric renal allograft survival. Clin Transplant 1995;9:282–8. [7] Chavez-Velasquez M, Pons H, Medina M, Quiroz Y, Parra G, Herrera J. Effects of mycophenolate mofetil in ischemic acute renal failure in rats. Nefrologia 2007;27:448–58. [8] Kosieradzki M, Rowinski W. Ischemia/reperfusion injury in kidney transplantation: mechanisms and prevention. Transplant Proc 2008;40:3279–88. [9] Bonventre JV. Mechanisms of ischemic acute renal failure. Kidney Int 1993;43: 1160–78. [10] Eickelberg O, Seebach F, Riordan M, et al. Functional activation of heat shock factor and hypoxia-inducible factor in the kidney. J Am Soc Nephrol 2002;13: 2094–101. [11] Gobe G, Willgoss D, Hogg N, Schoch E, Endre Z. Cell survival or death in renal tubular epithelium after ischemia–reperfusion injury. Kidney Int 1999;56: 1299–304. [12] Morimoto RI. Regulation of the heat shock transcriptional response: cross talk between a family of heat shock factors, molecular chaperones, and negative regulators. Genes Dev 1998;12:3788–96. [13] Safirstein R. Gene expression in nephrotoxic and ischemic acute renal failure. J Am Soc Nephrol 1994;4:1387–95. [14] Zhou T, Chen JL, Song W, et al. Effect of N-desulfated heparin on hepatic/renal ischemia reperfusion injury in rats. World J Gastroenterol 2002;8:897–900. [15] Yagmurdur MC, Colak T, Emiroglu R, et al. Antiinflammatory action of heparin via the complement system in renal ischemia–reperfusion. Transplant Proc 2003;35: 2566–70. [16] Sharples EJ, Patel N, Brown P, et al. Erythropoietin protects the kidney against the injury and dysfunction caused by ischemia–reperfusion. J Am Soc Nephrol 2004;15:2115–24. [17] Patel NS, Sharples EJ, Cuzzocrea S, et al. Pretreatment with EPO reduces the injury and dysfunction caused by ischemia/reperfusion in the mouse kidney in vivo. Kidney Int 2004;66:983–9. [18] Ates E, Yalcin AU, Yilmaz S, Koken T, Tokyol C. Protective effect of erythropoietin on renal ischemia and reperfusion injury. ANZ J Surg 2005;75:1100–5. [19] Imamura R, Moriyama T, Isaka Y, et al. Erythropoietin protects the kidneys against ischemia reperfusion injury by activating hypoxia inducible factor-1alpha. Transplantation 2007;83:1371–9. [20] Muller V, Hamar P, Szabo A, Knust E, Vogelsang M, Heemann U. Effect of mycophenolate mofetil on the in vivo infiltration of lymphocytes in the rat remnant kidney. Transplant Proc 1998;30:982. [21] Seujang Y, Kittikowit W, Eiam-Ong S. Lipid peroxidation and renal injury in renal ischemic reperfusion: effect of angiotensin inhibition. J Med Assoc Thai 2006;89: 1686–93. [22] Ventura CG, Coimbra TM, de Campos SB, de Castro I, Yu L, Seguro AC. Mycophenolate mofetil attenuates renal ischemia/reperfusion injury. J Am Soc Nephrol 2002;13:2524–33. [23] Bloudickova S, Rajnoch J, Lodererova A, Honsova E, Viklicky O. Mycophenolate mofetil ameliorates accelerated progressive nephropathy in rat. Kidney Blood Press Res 2006;29:60–6. [24] Ysebaert DK, De Greef KE, Vercauteren SR, et al. Effect of immunosuppression on damage, leukocyte infiltration, and regeneration after severe warm ischemia/reperfusion renal injury. Kidney Int 2003;64:864–73. [25] Okuaki Y, Miyazaki H, Zeniya M, et al. Splenectomy-reduced hepatic injury induced by ischemia/reperfusion in the rat. Liver 1996;16:188–94. [26] Ito K, Ozasa H, Yoneya R, Horikawa S. Splenectomy ameliorates hepatic ischemia and reperfusion injury mediated by heme oxygenase-1 induction in the rat. Liver 2002;22:467–73. [27] Jiang H, Meng F, Li W, Tong L, Qiao H, Sun X. Splenectomy ameliorates acute multiple organ damage induced by liver warm ischemia reperfusion in rats. Surgery 2007;141:32–40. [28] Donnahoo KK, Shames BD, Harken AH, Meldrum DR. Review article: the role of tumor necrosis factor in renal ischemia–reperfusion injury. J Urol 1999;162: 196–203. [29] Burne-Taney MJ, Kofler J, Yokota N, Weisfeldt M, Traystman RJ, Rabb H. Acute renal failure after whole body ischemia is characterized by inflammation and T cell-mediated injury. Am J Physiol Renal Physiol 2003;285:F87–94. [30] Yang B, Jain S, Pawluczyk IZ, et al. Inflammation and caspase activation in long-term renal ischemia/reperfusion injury and immunosuppression in rats. Kidney Int 2005;68:2050–67.
Contents lists available at SciVerse ScienceDirect
Transplant Immunology journal homepage: www.elsevier.com/locate/trim
Splenectomy protects the kidneys against ischemic reperfusion injury in the rat Toshiya Hiroyoshi ⁎, Masahiro Tsuchida, Koichi Uchiyama, Koki Fujikawa, Takahiro Komatsu, Yoshihiro Kanaoka, Hideyasu Matsuyama Department of Urology, Graduate School of Medicine, Yamaguchi University, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan
a r t i c l e
i n f o
Article history: Received 21 January 2012 Received in revised form 26 March 2012 Accepted 27 March 2012 Keywords: Renal ischemic reperfusion Renal injury Kidney transplantation Splenectomy
a b s t r a c t Background: Ischemic reperfusion (I/R) injury of the kidney is closely associated with delayed graft function, increased acute rejection, and late allograft dysfunction. Splenectomy reduced hepatic I/R injury by inhibiting leukocyte infiltration in the liver, release of TNF-α, cell apoptosis, and expression of caspase-3. Thus, we investigated the effects of splenectomy on renal I/R injury in the rat. Methods: Male Wistar rats were assigned to four groups: sham operation (sham group), sham operation + splenectomy (sham + SPLN group), right nephrectomy followed by clamping the left renal pedicle for 30 min (I/R 30 group), and I/R 30 + splenectomy (I/R 30 + SPLN group). Renal function was determined by measuring the concentration of blood urea nitrogen (BUN) and serum creatinine (S-Cr). The serum level of tumor necrosis factor-α (TNF-α) was measured as the marker for inflammation. Left kidneys were obtained 24 h after reperfusion. TUNEL assay was assessed for cell apoptosis. Spleens were obtained immediately (0-h group) and 3 h after reperfusion (3-h group). The removed spleens were histologically evaluated. Results: The BUN and S-Cr levels were significantly lower in the I/R 30 + SPLN group than in the I/R 30 group (p b 0.05 for both). Apoptotic cells were significantly lower in the I/R 30 + SPLN group than in the I/R 30 group. The serum level of TNF-α, which was increased after I/R, was significantly lower in the I/R 30 + SPLN group than in the I/R 30 group (p b 0.05). Spleen weights were significantly lower in the 3-h group than in the 0-h group (p b 0.05). Conclusion: These results suggest that splenectomy reduces renal I/R injury, and this effect may occur by an anti-inflammatory pathway and inhibition of cell apoptosis. © 2012 Elsevier B.V. All rights reserved.
1. Introduction
2. Objective
Renal ischemic reperfusion (I/R) injury is a clinically significant problem in renal transplantation. Renal I/R injury in the early transplant period has been associated with acute rejection, delayed graft function, and late allograft failure [1–6]. I/R injury is characterized by decreased local oxygen, cellular metabolism impairments with decreased levels of ATP metabolic substrate and glucose, inflammation, free radical production, apoptosis, and necrosis, all of which lead to deterioration of tubular cells [7–13]. Several agents, such as heparin [14,15], erythropoietin [16–19], mycophenolate mofetil [7,20–23], and cyclosporine [2,23,24], have been reported to prevent renal I/R injury. These agents protect renal I/R injury by antiinflammatory activities. Likewise, splenectomy has been reported to reduce hepatic I/R injury, probably by anti-inflammatory and antiapoptotic mechanisms [25–27]. The definite role of splenectomy in renal I/R injury has not yet been elucidated.
We investigated whether splenectomy can reduce renal I/R injury using a rat experimental model and attempted to identify the proof of principle of splenectomy in the clinical setting.
Abbreviations: I/R, ischemic reperfusion; BUN, blood urea nitrogen; TNF-α, tumor necrosis factor-α; TUNEL, in situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling; AI, apoptotic index. ⁎ Corresponding author. Tel.: + 81 836 22 2275; fax: + 81 836 22 2276. E-mail address: [email protected] (T. Hiroyoshi). 0966-3274/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2012.03.005
3. Materials and methods 3.1. Animals and experimental design Animal experiments were strictly performed according to standards for care and keeping of laboratory animals. Adult male Wistar rats (Kyudo, Saga, Japan) weighing 242 to 296 g were housed in individual cages in a temperature- and light-controlled environment. Food and water were available ad libitum. Rats were anesthetized with intraperitoneal pentobarbital (50 mg/kg). A midline skin incision was made after the skin was shaved and sterilized with 10% povidone-iodine. Renal I/R injury was induced by clamping the left renal pedicle for 30 min. The right kidney was removed immediately after unclamping the renal pedicle. Splenectomy was performed 0 and 3 h after the right nephrectomy. Animals were assigned to four groups: sham operation (sham group), sham operation + splenectomy (sham + SPLN group), right
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
nephrectomy followed by clamping the left renal pedicle for 30 min (I/R 30 group), and I/R 30 + splenectomy (I/R 30 + SPLN group). 3.2. Biochemical analysis Blood samples were obtained 24 h after reperfusion, centrifuged (2500×g for 10 min) to separate the serum and plasma supernatants, and stored at − 80 °C until measurement. Blood urea nitrogen (BUN) and creatinine (S-Cr) were measured with a 7180 Clinical Analyzer (Hitachi, Tokyo, Japan). 3.3. Tumor necrosis factor-α assays
9
Left kidneys 24 h after reperfusion were used for TUNEL staining. An apoptosis in situ detection kit (Wako Pure Chemicals, Osaka, Japan) was used for the TUNEL assay. The apoptotic index (AI) was defined as number of apoptotic cells× 100 / total number of nucleated cells. 3.7. Statistical analysis The data were expressed as mean ± standard deviation. The oneway ANOVA t-test was used to compare differences between the I/R group and the I/R + SPLN group in terms of BUN, S-Cr, TNF-α, and AI. A p-value of b0.05 was considered to be statistically significant. 4. Results
Serum samples were stored at −80 °C until assays were performed. Serum levels of tumor necrosis factor-α (TNF-α) were measured with a TNF-α ELISA kit according to the manufacturer's instructions (Invitrogen Corporation, CA, US). 3.4. Spleen weight The weights of spleens obtained immediately after reperfusion (0-h group) and 3 h after right nephrectomy with or without I/R injury (3-h group and sham group, respectively) were measured.
4.1. Renal function The BUN and S-Cr levels in the I/R 30 group were significantly higher than those in the sham group (50.9±14.1 vs. 27.2±3.1 mg/dl, pb 0.01 and 0.59±0.17 vs. 0.30 ±0.04 mg/dl, p b 0.01, respectively). The BUN and S-Cr levels in the I/R 30+ SPLN group were significantly lower than those in the I/R 30 group (27.7±3.6 vs. 50.9 ±14.1 mg/dl, pb 0.01 and 0.34± 0.09 vs. 0.59± 0.17 mg/dl, pb 0.05, respectively) (Fig. 1).
A
3.5. Histology Left kidneys were obtained 24 h after reperfusion. Tissue specimens were fixed in 10% formalin and embedded in paraffin. Sections were cut 5-μm thick and stained with hematoxylin–eosin for light microscope examination. 3.6. In situ detection of apoptotic cells
Serum creatinine (mg/dL)
Blood urea nitrogen (mg/dL)
In situ terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end-labeling (TUNEL) staining was performed. Serial sections of 5-μm thickness were prepared.
B
* 70 60 50 40 30 20 10 0 sham
sham SPLN
I/R30
I/R30 SPLN
*
C
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 sham
sham SPLN
I/R30
I/R30 SPLN
Fig. 1. BUN and S-Cr levels. BUN and S-Cr levels were 27.1 ± 3.15 and 0.30 ± 0.04 mg/dl in the sham group, 26.1 ± 4.25 and 0.34 ± 0.04 mg/dl in the sham + SPLN group, 50.9 ± 14.1 and 0.59 ± 0.17 mg/dl in the I/R 30 group, and 27.7 ± 3.6 and 0.34 ± 0.09 mg/dl in the I/R 30 + SPLN group, respectively. *p b 0.05.
Fig. 2. Histological examination.(A) Sham group. (B) I/R 30 group. (C) I/R 30 + SPLN group. Original magnification: ×400. In the I/R 30 + SPLN group, the degree of tubular damage and interstitial edema and number of inflammatory cells were less than those in the I/R 30 group.
10
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
C
A
25
B
*
D
Apoptosis index
20 15 10 5 0 sham
sham SPLN
I/R30
I/R30 SPLN
Fig. 3. TUNEL analysis of apoptotic cells.(A) Sham group. (B) I/R 30 group. (C) I/R 30 + SPLN group. Original magnification: ×400. (D) The apoptotic index (AI). The AI was 0 in the sham group, 1.4 ± 2.6 in the sham + SPLN group, 15.2 ± 3.9 in the I/R 30 group, and 2.4 ± 0.9 in the I/R 30 + SPLN group. *p b 0.05.
4.2. Histological examination 4.4. Plasma TNF-α activities In the I/R 30 group, tubular damage mainly occurred in the outer stripe of the outer medulla. Tubular cell swelling, tubular dilatation, and tubular epithelial cell necrosis were observed. Luminal hyaline casts, interstitial edema, and infiltration of inflammatory cells were also seen. In the I/R 30 + SPLN group, the degree of tubular damage and interstitial edema and number of inflammatory cells were less prominent compared with those in the I/R 30 group (Fig. 2).
The TNF-α levels in the sham group were similar to those in the sham + SPLN group. In the I/R 30 group, the TNF-α levels were significantly increased compared with those in the sham group (21.6 ± 3.4 vs. 5.0 ± 7.7 pg/ml, respectively; p b 0.05). In the I/R 30 + SPLN group, the TNF-α levels were significantly lower compared with those in the I/R 30 group (8.8 ± 10.9 vs. 21.6 ± 3.4 pg/ml, respectively; p b 0.05) (Fig. 4).
4.3. TUNEL staining and AI TUNEL-positive cells were mainly present in the tubules in the outer stripe of the outer medulla. The AI was similar in the sham group and sham + SPLN group. In the I/R 30 group, the AI was significantly increased compared with that in the sham group (15.2 ± 3.9 vs. 0, respectively; p b 0.001). In the I/R 30 + SPLN group, the AI was significantly lower compared with that in the I/R 30 group (2.4 ± 0.89 vs. 15.2 ± 3.9, respectively; p b 0.01) (Fig. 3).
4.5. Spleen weight There was no statistically significant difference in spleen weights between the 0-h and sham groups (p = 0.92). In the 3-h group, the spleen weight was significantly lower compared with that in the 0-h and sham groups (3-h vs. 0-h groups: 0.61 ± 0.09 vs. 0.84 ± 0.022 g, respectively; p b 0.05 and 3-h vs. sham groups: 0.61 ± 0.09 vs. 0.88 ± 0.19 g, respectively; p b 0.05) (Fig. 5).
* 30
* *
1.2
20
Spleen weight (g)
TNF-α α (pg/dl)
25
15 10 5
1 0.8 0.6 0.4 0.2
0 sham
sham SPLN
I/R30
I/R30 SPLN
0 0h
Fig. 4. Plasma TNF-α activities.TNF-α levels were 5.04 ± 7.66 pg/ml in the sham group, 5.54 ± 9.98 pg/dl in the sham + SPLN group, 21.6 ± 3.38 pg/ml in the I/R 30 group, and 8.78 ± 10.9 pg/ml in the I/R 30 + SPLN group. *p b 0.05.
sham
3h
Fig. 5. Spleen weight.The spleen weights were 0.84 ± 0.022 g in the 0-h group, 0.88 ± 0.19 g in the sham group, and 0.61 ± 0.09 g in the 3-h group. *p b 0.05.
T. Hiroyoshi et al. / Transplant Immunology 27 (2012) 8–11
5. Discussion The results of our study show that ischemic reperfusion combined with splenectomy improved renal function, improved renal histopathological degeneration, and decreased the level of TNF-α, leading to the reduction of renal injury caused by ischemic reperfusion. Many agents have been reported to prevent renal I/R injury. Heparin can protect against renal I/R injury by acting on the complement system to prevent inflammatory activity and inhibiting the adhesion and activation of neutrophils mediated by P-selectin [14,15]. Erythropoietin protected against renal I/R injury by reducing polymorphonuclease leukocyte accumulation in rat kidneys and up-regulating heat shock protein 70, anti-apoptotic protein B-cell lymphoma-2, and hypoxia inducible factor-1α expression [16–19]. Mycophenolate mofetil protected against renal I/R injury by decreasing the infiltration of lymphocytes and macrophages and reduced the expression of adhesion molecule intercellular adhesion molecule-1 [7,20–23]. Cyclosporine A reduced renal I/R injury by decreasing levels of inflammatory cytokines (interleukin-1 and TNF-α) and interstitial leukocyte accumulation [2,23,24]. All of these agents protect against renal I/R injury by anti-inflammatory activities. Because the spleen is the largest single reservoir of lymphocytes, it is suggested that splenectomy can reduce I/R injury by decreasing the number of lymphocytes that excrete inflammatory cytokines. Kirstan reported that TNF-α is an important mediator in renal I/R injury [28]. TNF-α reduced glomerular blood flow and the glomerular filtration rate by stimulating mesangial cells to produce a variety of vasoconstrictive mediators (platelet activating factor, endothelin-1, and prostaglandins) and recruit neutrophils and monocytes to the kidney [28]. TNF-α itself induced renal epithelial cell apoptosis, the major cell death pattern associated with renal I/R injury. Our study demonstrates that splenectomy reduces TNF-α production, which may be a major mechanism by which splenectomy reduces renal I/R injury. Our results are consistent with those of other reports showing that splenectomy reduced hepatic I/R injury [25–27]. Okuaki [25] reported that in hepatic I/R injury, splenectomy reduced the number of polymorphonuclear cells in the liver and TNF-α production; thus, the spleen may be a source of inflammatory mediators and play an important role in TNF-α production. Inflammatory cells were suggested to be mobilized from the spleen in previous research related to hepatic I/R injury [25]. The study showed that the weight of the spleen was significantly reduced after reperfusion, which may be explained by mobilization of a large amount of inflammatory cells from the spleen. The mechanisms of renal I/R injury have been considered to be abnormalities in regional blood flow, endothelial and epithelial cell dysfunction, inflammation and caspase activation, free radical production, apoptosis, and necrosis, and all of them are interdependent and act as distinct entities [29,30]. However, the precise mechanisms of I/R injury are complex. Although the definite mechanism of escaping renal I/R injury by splenectomy has not been elucidated, splenectomy is likely to decrease the damage of renal I/R injury by inhibiting TNF-α production. Our findings may serve as theoretical proof of the successful use of splenectomy for better renal function of the transplanted kidney in the clinical setting. Further experimental and clinical studies are needed to clarify the definite mechanism of renal I/R injury and the role of splenectomy. References [1] Requiao-Moura LR, Durao Mde S, Tonato EJ, et al. Effects of ischemia and reperfusion injury on long-term graft function. Transplant Proc 43:70–3.
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