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Functional role of atrial natriuretic peptide in acute renal transplant rejection
ImmunopharmacoN ELSEVIER
lmmunopharmacology33 (1996) 161-163
Functional role of atrial natriuretic peptide in acute renal transplant rejection Sarala Naicker a,*, Derseree Moodley b Anandh Nadar c, Prem Gathiram c "~Departments of Medicine, Universi~" of Durban-Westville, Durban, South Africa b Clinical and Experimental Pharmacology, University ofDurban-Westville, Durban, South Africa c University of Natal Medical School and Department 6~fPhysiology, University ofDurban-Westville, Durban, South Africa
Keywords: Atrial natriureticpeptide; Renal transplant; Renal failure; Acute rejection
1. Introduction
Atrial natriuretic peptides (ANP) are a family of peptides, secreted by cardiac atria, brain and kidney (Vollmar, 1990; Al-Haidary et al., 1990; Snyman et al., 1994). Secretion of ANP by cultured neonatal cells has been reported by Ritter et al. (1991). ANP exerts its effects on several targets including blood vessels, adrenals, brain and kidneys. These effects combine to reduce central blood volume and therefore, inhibit ANP secretion via a feedback mechanism. Plasma ANP is elevated in a number of diseases, including both human and experimental forms of acute renal failure (ARF) (Kanfer et al., 1989; Zuber et al., 1993). In animal studies, ANP administration has been shown to be beneficial in preserving glomerular filtration rate in both acute (Shaw et al., 1987) and established phase of ischaemic renal injury. In a human double-blind randomised study, however, it had no significant effect in improving post-ischaemic renal allograft function (Ratcliffe et al., 1991). We have examined the levels of ANP in
* Correspondingauthor. Departmentof Experimentaland Clinical Pharmacology,Natal Medical School, P.O. Box 17039, Congella, 4013, South Africa.
the blood and urine of renal transplant patients undergoing acute rejection.
2. Materials and methods
Blood was collected from 18 renal transplant patients (on the day of acute rejection) and 14 normal subjects in chilled sodium citrate tubes. The samples were centrifuged at 3200 rpm for 20 rain and 1 ml plasma aliquoted into Eppendorfs containing 100 txl kinin cocktail (60 mM EDTA, 6 mM 1,10-phenanthraline, 40 m g / m l aprotinin, 4 m g / m l soya bean trypsin inhibitor, 10 txM Captopril and 10 IxM phosphoramidon) and stored at - 2 0 ° C until the assay. Urine samples were collected in chilled tubes containing 100 txl thiomersal and 100 jxl SBTI 4 mg/ml. After being centrifuged, 1 ml supernatant was put into eppendorfs containing 100 txl, 0.2 M Tris buffer (pH 8.2). ANP was extracted from plasma and urine samples using Sep-Pak C 18 cartridges. The cartridges were activated with 1 ml 60% acetonitrile in 0.1% trifluoroacetic acid (TFA) followed by 1 ml 0.1% TFA (3 times). The plasma sample was acidified with 0.1% TFA and loaded onto the Sep-Pak cartridge. After washing the column, ANP was eluted
0162-3109/96/$15.00 © 1996 ElsevierScienceB.V. All rights reserved PII SO 162-3109(96)00033- 1
162
S. Naicker et al./lmmunopharmacology 33 (1996) 161 163
Table 1 Plasma and urinary ANP levels during acute renal transplant rejection Plasma ANP (pg/ml)
Urine ANP (g/ml)
Control
mean 24 range 11.7-42.5 SEM 2.8 n= 14
mean 2.7 range 2.3-3 SEM 0.14 n=4
Serum creatinine < 250 ~mol/1
mean 39 range 13-75 SEM 8.4 n=8
mean 21 range 2.5-52.5 SEM 6.3 n=8
Serum creatinine > 250 p~mol/l
mean 54 range 21-80 SEM 6.2 n = 10
mean 36 range 17-74 SEM 8.4 n = 10
SEM, standard error of mean; n, number of patients. with 60% acetonitrile in 0.1% TFA. The eluant was evaporated to dryness under a nitrogen stream at 37°C. The residues were reconstituted with RIA buffer and subjected to RIA using a kit (Peninsula Laboratories, Belmont, California). Acute rejection was confirmed by renal histology in all patients using haemotoxylin and eosin and periodic acid Schiff stains according to the Banff classification of renal transplant pathology (Solez et al., 1993).
3. Results The mean ANP values in patients undergoing acute rejection with concomitant severe renal failure (serum creatinine > 250 ~ m o l / 1 ) were greater than those in patients with acute rejection and associated milder renal failure (serum creatinine < 250 ~xmol/1) but significantly more so ( p = 0 . 0 1 6 7 , Wilcoxon-Mann-Whitney test) when compared to volunteer subjects (Table 1). All 10 patients with acute rejection and severe renal failure showed marked oedema and weight gain, whereas only 25% of rejecting patients with milder renal failure were oedematous.
4. Discussion Acute renal transplant rejection is an immunological process within which the pathological changes
may be mediated by circulating or locally formed vasoactive peptides. The precise nature and role of these mediator peptides has not been well defined in the existing literature. The significance of these elevated plasma and urinary ANP levels has to be further elucidated, taking into consideration that ANP is cleared from the body by neutral endopeptidases and by proteases in the brush border of renal tubules (Olins et al., 1987). We postulate that immune stimulation during the acute rejection process results in the release of ANP from distal convoluted tubular cells and collecting ducts (Figueroa et al., 1990), suggesting that ANP has an intra-renal paracrine function. Preliminary work by our group (D. Moodley, C. Snyman, S. Naicker, R. Ramsaroop and K.D. Bhoola, unpublished results) showing reduction of A N P immunostaining in distal tubular cells and collecting ducts in renal tissue during acute rejection appears to support this hypothesis.
References A1-HaidaryAD, Mackay IG, Bhoola KD, Millar ND, Watson ML and Mackenzie JC. Electrolyte and humoral responses of renal transplant patients to head-out water immersion. Nephrol Dial Transplant 1990; 5: 535-541. Figueroa CD, Lewis HM, MacIver AG, Mackenzie JC and Bhoola KD. Cellular localisation of atrial natriuretic factor in the human kidney. Nephrol Dial Transplant 1990; 5: 25-31.
S. Naicker et a l . / lmmunopharmacology 33 (1996) 161 163
Kanfer A, Dussaule JC, Czekalski S, Rondeau E, Sraer JD, Ardaillou R. Physiological significance of increased levels of endogenous atrial natriuretic factor in human acute renal failure. Clin Nephrol 1989; 32: 51-56. Olins GM, Spear KL, Siegel NR, Zurcher-Neely HA. Inactivation of atrial natriuretic factor by the renal brush border. Biochim Biophys Acta 1987; 901: 97-100. Ratcliffe PJ, Richardson AJ, Kirby JE, Moyses C, Shelton JR, Morris PJ. Effect of intravenous infusion of atriopeptin 3 on immediate renal allograft function. Kidney Int. 1991; 39: 164-168. Ritter D, Needleman P, Greenwald JE. Synthesis and secretion of an atriopeptin - like protein in rat kidney cell culture. J Clin Invest 1991; 87:208 212.
163
Shaw SG, Weidemann P, Hodler, Zimmermann A, Paternostro A. Atrial natriuretic peptide protects against ischaemic renal failure in the rat. J Clin Invest. 1987; 80:1232 -1237. Solez K, Axelsen RA, Benediktsson H, Burdick JP, Cohen AH et al. International standardisation of criteria for the histological diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int. 1993; 44:411 -422. Vollmar AM. Atrial natriuretic peptide in peripheral organs other than the heart. Klin Wochenchr 1990; 68: 699-708. Zuber M, Kiowski W, Huser B, Erne P, Buhler FR, Thiel G. Atrial natriuretic peptide release and volume regulation following kidney transplantation. Clin Nephrol 1993; 40: 91-95.
lmmunopharmacology33 (1996) 161-163
Functional role of atrial natriuretic peptide in acute renal transplant rejection Sarala Naicker a,*, Derseree Moodley b Anandh Nadar c, Prem Gathiram c "~Departments of Medicine, Universi~" of Durban-Westville, Durban, South Africa b Clinical and Experimental Pharmacology, University ofDurban-Westville, Durban, South Africa c University of Natal Medical School and Department 6~fPhysiology, University ofDurban-Westville, Durban, South Africa
Keywords: Atrial natriureticpeptide; Renal transplant; Renal failure; Acute rejection
1. Introduction
Atrial natriuretic peptides (ANP) are a family of peptides, secreted by cardiac atria, brain and kidney (Vollmar, 1990; Al-Haidary et al., 1990; Snyman et al., 1994). Secretion of ANP by cultured neonatal cells has been reported by Ritter et al. (1991). ANP exerts its effects on several targets including blood vessels, adrenals, brain and kidneys. These effects combine to reduce central blood volume and therefore, inhibit ANP secretion via a feedback mechanism. Plasma ANP is elevated in a number of diseases, including both human and experimental forms of acute renal failure (ARF) (Kanfer et al., 1989; Zuber et al., 1993). In animal studies, ANP administration has been shown to be beneficial in preserving glomerular filtration rate in both acute (Shaw et al., 1987) and established phase of ischaemic renal injury. In a human double-blind randomised study, however, it had no significant effect in improving post-ischaemic renal allograft function (Ratcliffe et al., 1991). We have examined the levels of ANP in
* Correspondingauthor. Departmentof Experimentaland Clinical Pharmacology,Natal Medical School, P.O. Box 17039, Congella, 4013, South Africa.
the blood and urine of renal transplant patients undergoing acute rejection.
2. Materials and methods
Blood was collected from 18 renal transplant patients (on the day of acute rejection) and 14 normal subjects in chilled sodium citrate tubes. The samples were centrifuged at 3200 rpm for 20 rain and 1 ml plasma aliquoted into Eppendorfs containing 100 txl kinin cocktail (60 mM EDTA, 6 mM 1,10-phenanthraline, 40 m g / m l aprotinin, 4 m g / m l soya bean trypsin inhibitor, 10 txM Captopril and 10 IxM phosphoramidon) and stored at - 2 0 ° C until the assay. Urine samples were collected in chilled tubes containing 100 txl thiomersal and 100 jxl SBTI 4 mg/ml. After being centrifuged, 1 ml supernatant was put into eppendorfs containing 100 txl, 0.2 M Tris buffer (pH 8.2). ANP was extracted from plasma and urine samples using Sep-Pak C 18 cartridges. The cartridges were activated with 1 ml 60% acetonitrile in 0.1% trifluoroacetic acid (TFA) followed by 1 ml 0.1% TFA (3 times). The plasma sample was acidified with 0.1% TFA and loaded onto the Sep-Pak cartridge. After washing the column, ANP was eluted
0162-3109/96/$15.00 © 1996 ElsevierScienceB.V. All rights reserved PII SO 162-3109(96)00033- 1
162
S. Naicker et al./lmmunopharmacology 33 (1996) 161 163
Table 1 Plasma and urinary ANP levels during acute renal transplant rejection Plasma ANP (pg/ml)
Urine ANP (g/ml)
Control
mean 24 range 11.7-42.5 SEM 2.8 n= 14
mean 2.7 range 2.3-3 SEM 0.14 n=4
Serum creatinine < 250 ~mol/1
mean 39 range 13-75 SEM 8.4 n=8
mean 21 range 2.5-52.5 SEM 6.3 n=8
Serum creatinine > 250 p~mol/l
mean 54 range 21-80 SEM 6.2 n = 10
mean 36 range 17-74 SEM 8.4 n = 10
SEM, standard error of mean; n, number of patients. with 60% acetonitrile in 0.1% TFA. The eluant was evaporated to dryness under a nitrogen stream at 37°C. The residues were reconstituted with RIA buffer and subjected to RIA using a kit (Peninsula Laboratories, Belmont, California). Acute rejection was confirmed by renal histology in all patients using haemotoxylin and eosin and periodic acid Schiff stains according to the Banff classification of renal transplant pathology (Solez et al., 1993).
3. Results The mean ANP values in patients undergoing acute rejection with concomitant severe renal failure (serum creatinine > 250 ~ m o l / 1 ) were greater than those in patients with acute rejection and associated milder renal failure (serum creatinine < 250 ~xmol/1) but significantly more so ( p = 0 . 0 1 6 7 , Wilcoxon-Mann-Whitney test) when compared to volunteer subjects (Table 1). All 10 patients with acute rejection and severe renal failure showed marked oedema and weight gain, whereas only 25% of rejecting patients with milder renal failure were oedematous.
4. Discussion Acute renal transplant rejection is an immunological process within which the pathological changes
may be mediated by circulating or locally formed vasoactive peptides. The precise nature and role of these mediator peptides has not been well defined in the existing literature. The significance of these elevated plasma and urinary ANP levels has to be further elucidated, taking into consideration that ANP is cleared from the body by neutral endopeptidases and by proteases in the brush border of renal tubules (Olins et al., 1987). We postulate that immune stimulation during the acute rejection process results in the release of ANP from distal convoluted tubular cells and collecting ducts (Figueroa et al., 1990), suggesting that ANP has an intra-renal paracrine function. Preliminary work by our group (D. Moodley, C. Snyman, S. Naicker, R. Ramsaroop and K.D. Bhoola, unpublished results) showing reduction of A N P immunostaining in distal tubular cells and collecting ducts in renal tissue during acute rejection appears to support this hypothesis.
References A1-HaidaryAD, Mackay IG, Bhoola KD, Millar ND, Watson ML and Mackenzie JC. Electrolyte and humoral responses of renal transplant patients to head-out water immersion. Nephrol Dial Transplant 1990; 5: 535-541. Figueroa CD, Lewis HM, MacIver AG, Mackenzie JC and Bhoola KD. Cellular localisation of atrial natriuretic factor in the human kidney. Nephrol Dial Transplant 1990; 5: 25-31.
S. Naicker et a l . / lmmunopharmacology 33 (1996) 161 163
Kanfer A, Dussaule JC, Czekalski S, Rondeau E, Sraer JD, Ardaillou R. Physiological significance of increased levels of endogenous atrial natriuretic factor in human acute renal failure. Clin Nephrol 1989; 32: 51-56. Olins GM, Spear KL, Siegel NR, Zurcher-Neely HA. Inactivation of atrial natriuretic factor by the renal brush border. Biochim Biophys Acta 1987; 901: 97-100. Ratcliffe PJ, Richardson AJ, Kirby JE, Moyses C, Shelton JR, Morris PJ. Effect of intravenous infusion of atriopeptin 3 on immediate renal allograft function. Kidney Int. 1991; 39: 164-168. Ritter D, Needleman P, Greenwald JE. Synthesis and secretion of an atriopeptin - like protein in rat kidney cell culture. J Clin Invest 1991; 87:208 212.
163
Shaw SG, Weidemann P, Hodler, Zimmermann A, Paternostro A. Atrial natriuretic peptide protects against ischaemic renal failure in the rat. J Clin Invest. 1987; 80:1232 -1237. Solez K, Axelsen RA, Benediktsson H, Burdick JP, Cohen AH et al. International standardisation of criteria for the histological diagnosis of renal allograft rejection: The Banff working classification of kidney transplant pathology. Kidney Int. 1993; 44:411 -422. Vollmar AM. Atrial natriuretic peptide in peripheral organs other than the heart. Klin Wochenchr 1990; 68: 699-708. Zuber M, Kiowski W, Huser B, Erne P, Buhler FR, Thiel G. Atrial natriuretic peptide release and volume regulation following kidney transplantation. Clin Nephrol 1993; 40: 91-95.