- Email: [email protected]
Pentosidine and Its Deposition in Renal Tissue in Renal Transplantation
Pentosidine and Its Deposition in Renal Tissue in Renal Transplantation K. Yoshida, T. Yoneda, K. Fujimoto, Y. Hirao, and N. Konishi ABSTRACT Background. Advanced glycation end products (AGEs) accumulate in lesions of arteriosclerosis, Alzheimer’s disease, rheumatoid arthritis, diabetic retinopathy, and diabetic nephropathy. Among AGEs, chemical quantification and immunohistologic methods for pentosidine have been established. Free pentosidine— eliminated by renal excretion— is mainly affected by renal function. In this study, we measured concentrations of plasma free and total pentosidine and immunohistologically investigated kidney graft biopsy specimens in patients after renal transplantation to investigate the renal function, plasma free and total pentosidine, and its relationship with deposition in the renal tissue. Patients and methods. In 28 patients who underwent renal transplantation from 1996 to 2003, we measured the time course of plasma concentrations of free pentosidine, total pentosidine, and serum creatinine starting right after renal transplantation. Thirty-four graft biopsy specimens were immunohistologically investigated using antipentosidine antibody. Plasma free and total pentosidine, and serum creatinine were measured at the same time. Results. Plasma free and total pentosidine were positively correlated with serum creatinine. Plasma free pentosidine and serum creatinine reached nadir values on day 34.2 ⫾ 14.2, when the blood concentrations were 5.1 ⫾ 1.6 pmol/mL and 1.7 ⫾ 0.7 mg/dL, respectively. Plasma total pentosidine reached a nadir on day 116.5 ⫾ 39.7 when the plasma concentration was 4.0 ⫾ 1.5 pmol/mg. We correlated the time required to reach the nadir of plasma free and total pentosidine concentrations. However, neither the concentration of plasma free nor plasma total pentosidine at nadir correlated with serum creatinine. The intensity of immunostaining with anti-pentosidine antibody in proximal tubular cells was graded as weakly positive, positive, or strongly positive. Significant differences were obtained among plasma free pentosidine values between the weakly positive and strongly positive groups. Conclusions. Renal transplantation improves renal function and decreases renal excretion of free pentosidine. Accordingly, total pentosidine also decreases. However, the concentrations of plasma free and total pentosidine at nadir varied among individuals; the blood concentrations were not determined by renal function alone. It was suggested that deposition of pentosidine in proximal tubular cells was more severe among patients with higher plasma free pentosidine and serum creatinine values.
A
DVANCED GLYCATION end products (AGEs) are produced by the Maillard reaction, a nonenzymatic reaction of saccharides and proteins.1 AGEs are detected in the blood of healthy individuals; their concentrations are high in renal failure patients.2– 4 AGEs have been demonstrated in vivo in recent years to accumulate in lesions of diabetic nephropathy, diabetic retinopathy, dialysis amyloidosis, arteriosclerosis, Alzheimer’s disease, and rheumatoid arthritis.5–9 A chemical method has been established to quantify pentosidine using high-performance liquid chro-
matography.10 Immunohistologic investigations have also been performed on renal tissues using anti-pentosidine antibody.11,12 Among AGEs, those with relatively low and From the Department of Urology (K.Y., T.Y., K.F., Y.H.), and the Second Department of Pathology (N.K.), Nara Medical University, Nara, Japan. Address reprint requests to K. Yoshida, Department of Urology, Nara Medical University, Shijyo-cho 840, Kashihara-shi, Nara-ken 634-8522, Japan. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.11.018
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
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high molecular weights are called AGE peptides and AGE proteins, respectively. Pentosidine is an AGE peptide with a relatively low molecular weight accounting for a small amount of AGEs.13 However, its presence in vivo closely reflects the degrees of carbonylation and oxidation.2,14 About 95% of blood pentosidine is bound to blood protein; the remaining molecules are present as the free form.2– 4 It has been reported that renal excretion decreases free pentosidine concentration in blood, which is filtered through the glomeruli and excreted in urine while being resorbed from the proximal renal tubule, taking several tens of hours. Pentosidine deposition in the proximal renal tubule is assumed to be due to resorption.10,11,15 In this study, we measured the concentrations of plasma free and total pentosidine and immunohistologically investigated biopsy specimens of the transplanted kidney in patients after renal transplantation to investigate relations with serum creatinine, pentosidine, and the degree of deposition of pentosidine in the proximal renal tubule. PATIENTS AND METHODS Patients From 1996 to 2003, 28 patients received a renal transplant in our hospital (19 men, 9 women). Renal transplantation was performed without complication. Immunosuppressive therapy consisted of cyclosporine or tacrolimus, mizoribine or azathioprine, and prednisolone. In addition for cadaveric renal transplantation, horse antilymphocyte immunoglobulin was administered for 1 week after transplantation. All patients who underwent living renal transplantation left dialysis immediately after surgery. In contrast, all patients who received cadaveric renal transplantation underwent dialysis 9.5 ⫾ 5.9 times and left dialysis 13.7 ⫾ 7.5 days after surgery. Informed consent was obtained from each patient. In 28 patients, plasma free and total pentosidine and serum creatinine were measured in 578 blood samples. To investigate correlations between plasma free pentosidine and serum creatinine and between plasma total pentosidine and serum creatinine, simple regression analysis was performed, and graphs prepared (Fig 1). In 28 patients, plasma free and total pentosidine and serum creatinine were measured serially, starting immediately after transplantation. The patients were divided into four groups: patients who underwent living renal transplantation and developed no acute rejection for 120 days (group A); those who underwent living renal transplantation and developed acute rejection within 120 days (group B); those who underwent cadaveric renal transplantation and developed no acute rejection reaction for 120 days (group C); and those who underwent cadaveric renal transplantation and developed acute rejection within 120 days (group D) (Table 1). For rejection, steroid pulse therapy was performed in all patients.
Fig 1. (A) Relationship between serum creatinine and plasma free pentosidine. There was a positive correlation between serum creatinine and plasma free pentosidine (y ⫽ 2.518 ⫹ 2.348x; r ⫽ 0.681; P ⬍.01). (b) Relationship between serum creatinine and plasma total pentosidine. There was a positive correlation between serum creatinine and plasma total pentosidine (y ⫽ 6.341 ⫹ 1.589x; r ⫽ 0.544; P ⬍.01).
Gusperimus hydrochloride was additionally used for four patients in whom the effect of steroid pulse therapy was insufficient; all patients overcame acute rejection reactions. Blood sampling was performed on the day of and 5, 10, 15, 20, 25, 30, 60, and 120 days after living renal transplantation. Patients who received cadaveric renal transplantation underwent dialysis after transplantation and the dialyzed kidney graft began functioning. Blood sampling was performed on the day of discontinuation of dialysis and 5, 10, 15, 20, 25, 30, 60, and 120 days there after among patients who underwent cadaveric renal transplantation. The results of the time course of blood sampling in the four groups are presented (Fig 2). The stable concentration of plasma free pentosidine after it reached the nadir owing to functional improvement of the grafted kidney in the 28 patients was defined as the free pentosidine nadir
Table 1. Patient Demographics and Clinical Characteristics Group
Acute Rejection
n
Age (y)
Donor Age (y)
Duration of Dialysis (mo)
A living B living C cadaver D cadaver All patients
⫺ ⫹ ⫺ ⫹
7 5 10 6 28
38.0 ⫾ 11.6 35.0 ⫾ 11.2 39.4 ⫾ 9.3 45.7 ⫾ 4.0 39.5 ⫾ 9.5
53.6 ⫾ 10.7 58.6 ⫾ 4.0 41.3 ⫾ 17.5 52.6 ⫾ 17.1 51.5 ⫾ 15.1
45.4 ⫾ 30.8 43.2 ⫾ 39.9 62.4 ⫾ 26.9 107.0 ⫾ 73.7 64.5 ⫾ 53.8
The differences in the average age, donor age, and duration of dialysis between each group were not significant.
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YOSHIDA, YONEDA, FUJIMOTO ET AL
Fig 2. Results of time course blood sampling in the four groups are shown. Plasma total pentosidine (mmol/mg)(); plasma free pentosidine (mmol/mL)( ); serum creatinine (mg/dL)().
‘
concentration. Serum creatinine measured as the time nadir was reached was presented as the number of days taken to reach nadir (Table 2). Similarly, the stable concentration of plasma total pentosidine after it reached the initial lowest level owing to functional improvement of the grafted kidney was defined as the total pentosidine nadir concentration. The plasma free pentosidine and serum creatinine levels at the time of reaching nadir and the number of days taken to reach nadir are shown (Table 3). In patients who received a cadaveric kidney graft, days were counted from the day of discontinuation of dialysis, not from the transplantation day. Correlation between the number of days taken for plasma free and total pentosidine to reach nadir was tabulated (Fig 3). The correlation was investigated between plasma free pentosidine and serum creatinine at the time of reaching nadir, between plasma total pentosidine and serum creatinine at the time of reaching nadir, and between plasma total and free pentosidine at the time of reaching nadir (see Fig 3). Of 28 patients, 18 underwent graft biopsy (13 men, 5 women), and the 34 graft biopsy specimens were immunohistologically investigated using anti-pentosidine antibody. Blood was collected during graft biopsy, and plasma free and total pentosidine were measured. Biopsy was performed in all patients 1 hour after
transplantation, but these specimens were not used. The intensity and distribution of immunostaining were investigated. The intensity of immunostaining with anti-pentosidine antibody in proximal tubular cells was graded as weakly positive, positive, and strongly positive compare (Fig 4) to plasma free and total pentosidine, serum creatinine, time between transplantation and allograft biopsy, and age (Table 4).
Measurement Method Measurements of Concentrations of Plasma Free and Total Pentosidine. The concentrations were measured by HPLC using patient plasma. Plasma free pentosidine (pmol/mL) was measured after pretreatment with 10% trichloroacetic acid (TCA) for deproteination. For pretreatment of plasma total pentosidine, 12N hydrochloric acid was added to hydrolyze the sample for 16 hours. Because 95% or more of total pentosidine was bound to blood proteins, the total pentosidine level was presented as the amount per 1 mg of blood protein in pmol/mg. These procedures were performed according to the method reported by Miyata et al.13 Most of the total pentosidine was bound to proteins and the free pentosidine level was within the allowable margin of error, giving
Table 2. Data at the Time of Free Pentosidine Nadir Group
A
B
C
D
All Patients
Plasma free pentosidine (pmol/mL) Serum creatinine (mg/dL) Time after transplantation (d)
5.0 ⫾ 1.5 1.3 ⫾ 0.4 27.0 ⫾ 4.4
4.3 ⫾ 1.2 2.0 ⫾ 0.3 37.4 ⫾ 22.7
4.8 ⫾ 1.5 1.2 ⫾ 0.5 25.3 ⫾ 4.8
6.3 ⫾ 1.7 2.2 ⫾ 0.8 47.0 ⫾ 13.0
5.1 ⫾ 1.6 1.7 ⫾ 0.7 34.2 ⫾ 14.2
The differences in the average plasma free pentosidine, serum creatinine and time after transplantation between each group were not significant.
PENTOSIDINE IN RENAL TISSUE
4269 Table 3. Data at the Time of Total Pentosidine Nadir
Group
A
B
C
D
All Patients
Plasma total pentosidine (pmol/mg) Plasma free pentosidine (pmol/mL) Serum creatinine (mg/dL) Time after transplantation (d)
4.5 ⫾ 1.4 4.8 ⫾ 0.9 1.4 ⫾ 0.4 107.8 ⫾ 22.8
4.1 ⫾ 2.9 4.3 ⫾ 1.5 2.0 ⫾ 0.1 140.3 ⫾ 36.6
2.9 ⫾ 1.2 4.5 ⫾ 2.2 1.2 ⫾ 0.3 100.3 ⫾ 42.1
4.3 ⫾ 3.2 6.0 ⫾ 1.2 2.1 ⫾ 0.8 117.4 ⫾ 60.6
4.0 ⫾ 1.5 4.9 ⫾ 1.7 1.7 ⫾ 0.6 116.5 ⫾ 39.7
The differences in the average plasma total pentosidine, plasma free pentosidine, serum creatinine, and time after transplantation between each group were not significant.
no influence on the result. Thus, total pentosidine was selected versus free pentosidine. Serum Creatinine. Serum creatinine was measured using sera from blood samples. The unit is mg/dL. Immunohistologic Investigation. Immunohistologic investigation was performed using anti-pentosidine rabbit polyclonal IgG provided by Tokai University.11,12 Kidney biopsy specimens were fixed in 10% formalin, embedded in paraffin, and sectioned at 3 m. The sections were mounted on 3-aminopropyltriethoxy silane-coated glass slides and deparaffinized. After endogenous peroxidase was removed by treatment with 3% hydrogen peroxide-supplemented methanol for 30 minutes, the slides were treated with 10 mmol of sodium citrate buffer (pH 6.0) and autoclaved at 120°C for 5 minutes. The slides were then subjected to blocking treatment using a kit (Histofine SAB-PO, Nichirei, Tokyo, Japan). The slides
were incubated with anti-pentosidine rabbit polyclonal IgG (10 mg/mL) at room temperature for 2 hours then with the biotinlabeled secondary antibody and peroxidase-conjugated treptavidin. Finally, the slides were counterstained with hematoxylin. Statistical Analysis. The measured results were presented as mean values ⫾ standard deviations (SD). For analysis between two groups, the Mann-Whitney U test was used. Correlation was analyzed using simple regression analysis. A P level less than 1% was regarded as significant.
RESULTS
Plasma free pentosidine and serum creatinine were positively correlated (y ⫽ 2.518 ⫹ 2.348x, r ⫽ .681, P ⬍ .01) (see Fig 1). Plasma total pentosidine was also positively corre-
Fig 3. (A) Relationship between plasma total pentosidine nadir duration and plasma free pentosidine nadir duration. There was a positive correlation between plasma total pentosidine nadir duration and plasma free pentosidine nadir duration (y ⫽ 9.672 ⫹ 0.229x; r ⫽ 0.679; P ⬍ .01). (B–D) There was no correlation between plasma free pentosidine at nadir and serum creatinine, between plasma total pentosidine at nadir and serum creatinine, and between plasma free pentosidine at nadir and plasma free pentosidine at nadir.
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The course after renal transplantation and changes in plasma free and total pentosidine and serum creatinine are shown. In all groups, the pentosidine levels decreased with functional improvement of the kidney graft. Serum creatinine also decreased with improvement of renal function. Plasma free pentosidine began to decrease with serum creatinine at almost the same time, and plasma total pentosidine decreased (see Figs 2a– d). Plasma free pentosidine reached a nadir on day 34.2 ⫾ 14.2, and its concentration and the serum creatinine level were 5.1 ⫾ 1.6 pmol/mL and 1.7 ⫾ 0.7 mg/dL, respectively (Table 2). Plasma total pentosidine reached nadir on day 116.5 ⫾ 39.7, and serum creatinine, plasma free and total pentosidine at nadir were 1.7 ⫾ 0.6 mg/dL, 4.9 ⫾ 1.7 pmol/mL, and 4.0 ⫾ 1.5 pmol/mg, respectively (Table 3). A positive correlation was observed between the days required to reach the nadir of plasma free and total pentosidine (y ⫽ 2.518 ⫹ 2.348x; r ⫽ 0.681; P ⬍ .01); Fig. 3a). The concentration of plasma free pentosidine at nadir was not correlated with serum creatinine (Fig. 3b). The concentration of plasma total pentosidine at nadir was correlated neither with serum creatinine nor plasma free pentosidine (Figs. 3c,d). As reported, anti-pentosidine antibody stained proximal tubular cells. When the staining intensity was graded; the intensity was weakly positive in 5, positive in 9, and strongly positive in 12 specimens (Fig 4). Significant differences were detected in plasma free pentosidine between the weakly positive and strongly positive groups (P ⬍ .001; Table 4). However, there were no significant differences in age, serum creatinine, time after transplantation, or plasma total pentosidine. DISCUSSION
Fig 4. The nuclei and cytoplasm of proximal tubular cells were stained. The patients were divided into three groups by the staining intensity: weakly positive (A), positive (B), and strongly positive groups (C). Because hematoxylin was used for counterstaining, hematoxylin color was intermingled (⫻100).
lated with serum creatinine, although the degree was less than that of plasma free pentosidine (y ⫽ 6.341 ⫹ 1.589x, r ⫽ .544, P ⬍ .01) (see Fig 1b).
As reported, the blood pentosidine concentration was affected by renal function.3 The positive correlation between plasma pentosidine and serum creatinine suggests that free pentosidine in blood decreases as renal function improves. It has been reported that about 80% of intravenously injected pentosidine was excreted in urine within 24 hours in rats with normal renal function. Plasma total pentosidine decreases with decreased free pentosidine, which may have resulted in correlation with serum creatinine. Creatinine constantly and sensitively reflected renal function in all patients. Unlike creatinine, reflection of renal function by free pentosidine varied. This variation may have been due to large individual differences in production and tissue pooling of pentosidine in vivo.16 Variation of plasma total pentosidine was larger than that of plasma free pentosidine, but it similarly exhibited a positive correlation with serum creatinine. Washing out of protein-bound pentosidine takes time after improvement of renal function. It may have resulted in greater variation of renal function when evaluated by total pentosidine than free pentosidine. Time courses of serum creatinine and plasma free and total pentosidine as renal function improved by renal transplantation exhibited smooth recovery without hemodi-
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Table 4. Parameters Among the Groups Divided by Intensity of Immunostaining in Proximal Tubular Cells
Age (yr) Time after transplantation (mos) Serum creatinine (mg/dL) Plasma free pentosidine (pmol/mL) Plasma total pentosidine (pmol/mg)
Weakly Positive (n ⫽ 6)
Positive (n ⫽ 16)
Strongly Positive (n ⫽ 12)
P
37.9 ⫾ 10.5 29.9 ⫾ 11.4 2.4 ⫾ 0.9 3.6 ⫾ 0.7 6.6 ⫾ 4.3
38.8 ⫾ 10.1 30.4 ⫾ 12.1 3.8 ⫾ 3.5 5.7 ⫾ 2.5 6.7 ⫾ 4.1
38.7 ⫾ 14.8 28.5 ⫾ 20.3 4.8 ⫾ 3.7 15.0 ⫾ 9.1 12.3 ⫾ 8.9
NS NS NS ⬍.001 NS
The difference in the average plasma free pentosidine between weekly positive and strongly positive groups was significant. The differences in the average age, plasma total pentosidine, serum creatinine, and time after transplantation between weekly positive and strongly positive groups were not significant.
alysis in patients who underwent living renal transplantation. Group A may have exhibited a typical course with the least bias. Serum creatinine and plasma free pentosidine simultaneously reached a nadir on day 27.0 on average. Then plasma total pentosidine reached a nadir on day 107.8 on average, showing a mean delay of 80.8 days. This delay was the time required to wash out protein-bound pentosidine. The washout period varied among the patients. Similar results for the time course of plasma free and total pentosidine after transplantation were also obtained in previous reports.4 The total pentosidine washout period after renal transplantation may have been more prolonged in patients with a higher total pentosidine concentration at the time of transplantation (living kidney-grafted patients) or discontinuation of dialysis (cadaver-donor kidneygrafted patients). When we investigated the period in 28 patients in all groups, no correlation was obtained. Neither age nor dialysis history was correlated. This study could not identify the factor determining the wash out period. Because more rejection occurred in group B, nadirs of serum creatinine and plasma free pentosidine were delayed compared to those in group A, and a nadir of plasma total pentosidine was also displayed. Because the period between discontinuation of dialysis and nadir was regarded as the time required to reach nadir in group C, it may have been slightly shorter than those in groups A and B. In group D, rejection reaction persisted in more patients, resulting in the longest delays to nadir. There was a positive correlation between the numbers of days required to reach the nadir of plasma total and free pentosidine. This finding was natural because renal excretion of free pentosidine gradually cleared total pentosidine. The concentration of plasma free pentosidine at nadir was not correlated with serum creatinine. Similarly, the concentration of plasma total pentosidine at nadir was not correlated with serum creatinine. These findings indicated that the pentosidine level varied among individuals regardless of renal function when renal function was stable. As described, the variation of blood pentosidine concentrations may have been determined by not only renal function but also various factors such as difference in the amount of production; further analysis is necessary. The regions stained with anti-pentosidine antibody were proximal tubular cells, glomerular loop, mesangium, and glomerular vascular endothelium. Only some glomerular
regions were stained and the staining factor was not clear in this study. Because a relationship between pentosidine deposition in the glomeruli and pathologic aggravation of diabetic nephropathy has been reported, further investigation is necessary.6 The varied staining intensity in proximal tubular cells was easily graded by three steps. Free pentosidine easily passes through glomerular vascular endothelium and is resorbed from the proximal urinary tubule. An in vitro experiment demonstrated that proximal tubular cells incorporate pentosidine. It has also been reported that intravenously injected pentosidine was deposited in urinary tubular cells in a rat experiment.10,15 In this study, the plasma free pentosidine level was significantly higher as the staining intensity in the proximal urinary tubule increased, suggesting that as the concentration of free pentosidine filtered through the proximal tubular glomeruli increased, resorption from proximal tubular cells increased and the cells were more strongly stained. In conclusion, improved renal function by renal transplantation decreased blood free pentosidine, and total blood pentosidine. However, the concentration varied among individuals even though renal excretion decreased blood pentosidine, and the blood pentosidine concentration was not determined by renal clearance alone. Deposition of pentosidine in proximal tubular cells may increase in patients with higher free pentosidine and serum creatinine values. ACKNOWLEDGMENT We acknowledge the doctors and staff in Tokai University for instruction in the measurement and immunohistologic investigation of pentosidine and providing us with anti-pentosidine antibody.
REFERENCES 1. Baynes JW, Watkins NG, Fisher CI, et al: The Amadori product on protein: structure and reactions. Prog Clin Biol Res 304:43, 1989 2. Miyata T, Fu MX, Kurokawa K, et al: Autoxidation products of both carbohydrates and lipids are increased in uremic plasma. Evidence for a generalized increase in oxidative stress in uremia? Kidney Int 54:1290, 1998 3. Miyata T, Ueda Y, Shinzato T, et al: Accumulation of albumin-linked and free-form pentosidine in the circulation of uremic patients with end-stage renal failure: renal implications in the pathophysiology of pentosidine. J Am Soc Nephrol 7:1198, 1996
4272 4. Miyata T, Ueda Y, Yoshida A, et al: Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy. Kidney Int 51:880, 1997 5. Miyata T, Ueda Y, Yoshida A, et al: Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy. Kidney Int 51:880, 1997 6. Horie K, Miyata T, Maeda K, et al: Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy. J Clin Invest 100:2995, 1997 7. Makita Z, Bucala R, Rayfield EJ, et al: Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure. Lancet 343:1519, 1994 8. Miyata T, Wada Y, Cai Z, et al: Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 51:1170, 1997 9. Asano M, Fujita Y, Ueda Y, et al: Renal proximal tubular metabolism of protein-linked pentosidine, an advanced glycation end product. Nephron 91:688, 2002 10. Kume S, Takeya M, Mori T, et al: Immunohistochemical and ultrastructural detection of advanced glycation end products in
YOSHIDA, YONEDA, FUJIMOTO ET AL atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am J Pathol 147:654, 1995 11. Yamagishi S, Yamamoto Y, Harada S, et al: Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with their receptors. FEBS Lett 384:103, 1996 12. Smith MA, Taneda S, Richey PL, et al: Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc Natl Acad Sci U S A 91:5710, 1994 13. Miyata T, Ueda Y, Yamada Y, et al: Accumulation of carbonyls accelerates the formation of pentosidine, an advanced glycation end product: carbonyl stress in uremia. J Am Soc Nephrol 9:2349, 1998 14. Miyata T, Ishiguro N, Yasuda Y, et al: Increased pentosidine, an advanced glycation end product, in plasma and synovial fluid from patients with rheumatoid arthritis and its relation with inflammatory markers. Bioch Biophys Res Comm 244:45, 1998 15. Miyata T, Ueda Y, Horie K, et al: Renal catabolism of advanced glycation end products: the fate of pentosidine. Kidney Int 53:416, 1998 16. Bucala R, Makita Z, Vega G, et al: Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc Natl Acad Sci U S A 91:9441, 1994
A
DVANCED GLYCATION end products (AGEs) are produced by the Maillard reaction, a nonenzymatic reaction of saccharides and proteins.1 AGEs are detected in the blood of healthy individuals; their concentrations are high in renal failure patients.2– 4 AGEs have been demonstrated in vivo in recent years to accumulate in lesions of diabetic nephropathy, diabetic retinopathy, dialysis amyloidosis, arteriosclerosis, Alzheimer’s disease, and rheumatoid arthritis.5–9 A chemical method has been established to quantify pentosidine using high-performance liquid chro-
matography.10 Immunohistologic investigations have also been performed on renal tissues using anti-pentosidine antibody.11,12 Among AGEs, those with relatively low and From the Department of Urology (K.Y., T.Y., K.F., Y.H.), and the Second Department of Pathology (N.K.), Nara Medical University, Nara, Japan. Address reprint requests to K. Yoshida, Department of Urology, Nara Medical University, Shijyo-cho 840, Kashihara-shi, Nara-ken 634-8522, Japan. E-mail: [email protected]
0041-1345/05/$–see front matter doi:10.1016/j.transproceed.2005.11.018
© 2005 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
4266
Transplantation Proceedings, 37, 4266 – 4272 (2005)
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high molecular weights are called AGE peptides and AGE proteins, respectively. Pentosidine is an AGE peptide with a relatively low molecular weight accounting for a small amount of AGEs.13 However, its presence in vivo closely reflects the degrees of carbonylation and oxidation.2,14 About 95% of blood pentosidine is bound to blood protein; the remaining molecules are present as the free form.2– 4 It has been reported that renal excretion decreases free pentosidine concentration in blood, which is filtered through the glomeruli and excreted in urine while being resorbed from the proximal renal tubule, taking several tens of hours. Pentosidine deposition in the proximal renal tubule is assumed to be due to resorption.10,11,15 In this study, we measured the concentrations of plasma free and total pentosidine and immunohistologically investigated biopsy specimens of the transplanted kidney in patients after renal transplantation to investigate relations with serum creatinine, pentosidine, and the degree of deposition of pentosidine in the proximal renal tubule. PATIENTS AND METHODS Patients From 1996 to 2003, 28 patients received a renal transplant in our hospital (19 men, 9 women). Renal transplantation was performed without complication. Immunosuppressive therapy consisted of cyclosporine or tacrolimus, mizoribine or azathioprine, and prednisolone. In addition for cadaveric renal transplantation, horse antilymphocyte immunoglobulin was administered for 1 week after transplantation. All patients who underwent living renal transplantation left dialysis immediately after surgery. In contrast, all patients who received cadaveric renal transplantation underwent dialysis 9.5 ⫾ 5.9 times and left dialysis 13.7 ⫾ 7.5 days after surgery. Informed consent was obtained from each patient. In 28 patients, plasma free and total pentosidine and serum creatinine were measured in 578 blood samples. To investigate correlations between plasma free pentosidine and serum creatinine and between plasma total pentosidine and serum creatinine, simple regression analysis was performed, and graphs prepared (Fig 1). In 28 patients, plasma free and total pentosidine and serum creatinine were measured serially, starting immediately after transplantation. The patients were divided into four groups: patients who underwent living renal transplantation and developed no acute rejection for 120 days (group A); those who underwent living renal transplantation and developed acute rejection within 120 days (group B); those who underwent cadaveric renal transplantation and developed no acute rejection reaction for 120 days (group C); and those who underwent cadaveric renal transplantation and developed acute rejection within 120 days (group D) (Table 1). For rejection, steroid pulse therapy was performed in all patients.
Fig 1. (A) Relationship between serum creatinine and plasma free pentosidine. There was a positive correlation between serum creatinine and plasma free pentosidine (y ⫽ 2.518 ⫹ 2.348x; r ⫽ 0.681; P ⬍.01). (b) Relationship between serum creatinine and plasma total pentosidine. There was a positive correlation between serum creatinine and plasma total pentosidine (y ⫽ 6.341 ⫹ 1.589x; r ⫽ 0.544; P ⬍.01).
Gusperimus hydrochloride was additionally used for four patients in whom the effect of steroid pulse therapy was insufficient; all patients overcame acute rejection reactions. Blood sampling was performed on the day of and 5, 10, 15, 20, 25, 30, 60, and 120 days after living renal transplantation. Patients who received cadaveric renal transplantation underwent dialysis after transplantation and the dialyzed kidney graft began functioning. Blood sampling was performed on the day of discontinuation of dialysis and 5, 10, 15, 20, 25, 30, 60, and 120 days there after among patients who underwent cadaveric renal transplantation. The results of the time course of blood sampling in the four groups are presented (Fig 2). The stable concentration of plasma free pentosidine after it reached the nadir owing to functional improvement of the grafted kidney in the 28 patients was defined as the free pentosidine nadir
Table 1. Patient Demographics and Clinical Characteristics Group
Acute Rejection
n
Age (y)
Donor Age (y)
Duration of Dialysis (mo)
A living B living C cadaver D cadaver All patients
⫺ ⫹ ⫺ ⫹
7 5 10 6 28
38.0 ⫾ 11.6 35.0 ⫾ 11.2 39.4 ⫾ 9.3 45.7 ⫾ 4.0 39.5 ⫾ 9.5
53.6 ⫾ 10.7 58.6 ⫾ 4.0 41.3 ⫾ 17.5 52.6 ⫾ 17.1 51.5 ⫾ 15.1
45.4 ⫾ 30.8 43.2 ⫾ 39.9 62.4 ⫾ 26.9 107.0 ⫾ 73.7 64.5 ⫾ 53.8
The differences in the average age, donor age, and duration of dialysis between each group were not significant.
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YOSHIDA, YONEDA, FUJIMOTO ET AL
Fig 2. Results of time course blood sampling in the four groups are shown. Plasma total pentosidine (mmol/mg)(); plasma free pentosidine (mmol/mL)( ); serum creatinine (mg/dL)().
‘
concentration. Serum creatinine measured as the time nadir was reached was presented as the number of days taken to reach nadir (Table 2). Similarly, the stable concentration of plasma total pentosidine after it reached the initial lowest level owing to functional improvement of the grafted kidney was defined as the total pentosidine nadir concentration. The plasma free pentosidine and serum creatinine levels at the time of reaching nadir and the number of days taken to reach nadir are shown (Table 3). In patients who received a cadaveric kidney graft, days were counted from the day of discontinuation of dialysis, not from the transplantation day. Correlation between the number of days taken for plasma free and total pentosidine to reach nadir was tabulated (Fig 3). The correlation was investigated between plasma free pentosidine and serum creatinine at the time of reaching nadir, between plasma total pentosidine and serum creatinine at the time of reaching nadir, and between plasma total and free pentosidine at the time of reaching nadir (see Fig 3). Of 28 patients, 18 underwent graft biopsy (13 men, 5 women), and the 34 graft biopsy specimens were immunohistologically investigated using anti-pentosidine antibody. Blood was collected during graft biopsy, and plasma free and total pentosidine were measured. Biopsy was performed in all patients 1 hour after
transplantation, but these specimens were not used. The intensity and distribution of immunostaining were investigated. The intensity of immunostaining with anti-pentosidine antibody in proximal tubular cells was graded as weakly positive, positive, and strongly positive compare (Fig 4) to plasma free and total pentosidine, serum creatinine, time between transplantation and allograft biopsy, and age (Table 4).
Measurement Method Measurements of Concentrations of Plasma Free and Total Pentosidine. The concentrations were measured by HPLC using patient plasma. Plasma free pentosidine (pmol/mL) was measured after pretreatment with 10% trichloroacetic acid (TCA) for deproteination. For pretreatment of plasma total pentosidine, 12N hydrochloric acid was added to hydrolyze the sample for 16 hours. Because 95% or more of total pentosidine was bound to blood proteins, the total pentosidine level was presented as the amount per 1 mg of blood protein in pmol/mg. These procedures were performed according to the method reported by Miyata et al.13 Most of the total pentosidine was bound to proteins and the free pentosidine level was within the allowable margin of error, giving
Table 2. Data at the Time of Free Pentosidine Nadir Group
A
B
C
D
All Patients
Plasma free pentosidine (pmol/mL) Serum creatinine (mg/dL) Time after transplantation (d)
5.0 ⫾ 1.5 1.3 ⫾ 0.4 27.0 ⫾ 4.4
4.3 ⫾ 1.2 2.0 ⫾ 0.3 37.4 ⫾ 22.7
4.8 ⫾ 1.5 1.2 ⫾ 0.5 25.3 ⫾ 4.8
6.3 ⫾ 1.7 2.2 ⫾ 0.8 47.0 ⫾ 13.0
5.1 ⫾ 1.6 1.7 ⫾ 0.7 34.2 ⫾ 14.2
The differences in the average plasma free pentosidine, serum creatinine and time after transplantation between each group were not significant.
PENTOSIDINE IN RENAL TISSUE
4269 Table 3. Data at the Time of Total Pentosidine Nadir
Group
A
B
C
D
All Patients
Plasma total pentosidine (pmol/mg) Plasma free pentosidine (pmol/mL) Serum creatinine (mg/dL) Time after transplantation (d)
4.5 ⫾ 1.4 4.8 ⫾ 0.9 1.4 ⫾ 0.4 107.8 ⫾ 22.8
4.1 ⫾ 2.9 4.3 ⫾ 1.5 2.0 ⫾ 0.1 140.3 ⫾ 36.6
2.9 ⫾ 1.2 4.5 ⫾ 2.2 1.2 ⫾ 0.3 100.3 ⫾ 42.1
4.3 ⫾ 3.2 6.0 ⫾ 1.2 2.1 ⫾ 0.8 117.4 ⫾ 60.6
4.0 ⫾ 1.5 4.9 ⫾ 1.7 1.7 ⫾ 0.6 116.5 ⫾ 39.7
The differences in the average plasma total pentosidine, plasma free pentosidine, serum creatinine, and time after transplantation between each group were not significant.
no influence on the result. Thus, total pentosidine was selected versus free pentosidine. Serum Creatinine. Serum creatinine was measured using sera from blood samples. The unit is mg/dL. Immunohistologic Investigation. Immunohistologic investigation was performed using anti-pentosidine rabbit polyclonal IgG provided by Tokai University.11,12 Kidney biopsy specimens were fixed in 10% formalin, embedded in paraffin, and sectioned at 3 m. The sections were mounted on 3-aminopropyltriethoxy silane-coated glass slides and deparaffinized. After endogenous peroxidase was removed by treatment with 3% hydrogen peroxide-supplemented methanol for 30 minutes, the slides were treated with 10 mmol of sodium citrate buffer (pH 6.0) and autoclaved at 120°C for 5 minutes. The slides were then subjected to blocking treatment using a kit (Histofine SAB-PO, Nichirei, Tokyo, Japan). The slides
were incubated with anti-pentosidine rabbit polyclonal IgG (10 mg/mL) at room temperature for 2 hours then with the biotinlabeled secondary antibody and peroxidase-conjugated treptavidin. Finally, the slides were counterstained with hematoxylin. Statistical Analysis. The measured results were presented as mean values ⫾ standard deviations (SD). For analysis between two groups, the Mann-Whitney U test was used. Correlation was analyzed using simple regression analysis. A P level less than 1% was regarded as significant.
RESULTS
Plasma free pentosidine and serum creatinine were positively correlated (y ⫽ 2.518 ⫹ 2.348x, r ⫽ .681, P ⬍ .01) (see Fig 1). Plasma total pentosidine was also positively corre-
Fig 3. (A) Relationship between plasma total pentosidine nadir duration and plasma free pentosidine nadir duration. There was a positive correlation between plasma total pentosidine nadir duration and plasma free pentosidine nadir duration (y ⫽ 9.672 ⫹ 0.229x; r ⫽ 0.679; P ⬍ .01). (B–D) There was no correlation between plasma free pentosidine at nadir and serum creatinine, between plasma total pentosidine at nadir and serum creatinine, and between plasma free pentosidine at nadir and plasma free pentosidine at nadir.
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YOSHIDA, YONEDA, FUJIMOTO ET AL
The course after renal transplantation and changes in plasma free and total pentosidine and serum creatinine are shown. In all groups, the pentosidine levels decreased with functional improvement of the kidney graft. Serum creatinine also decreased with improvement of renal function. Plasma free pentosidine began to decrease with serum creatinine at almost the same time, and plasma total pentosidine decreased (see Figs 2a– d). Plasma free pentosidine reached a nadir on day 34.2 ⫾ 14.2, and its concentration and the serum creatinine level were 5.1 ⫾ 1.6 pmol/mL and 1.7 ⫾ 0.7 mg/dL, respectively (Table 2). Plasma total pentosidine reached nadir on day 116.5 ⫾ 39.7, and serum creatinine, plasma free and total pentosidine at nadir were 1.7 ⫾ 0.6 mg/dL, 4.9 ⫾ 1.7 pmol/mL, and 4.0 ⫾ 1.5 pmol/mg, respectively (Table 3). A positive correlation was observed between the days required to reach the nadir of plasma free and total pentosidine (y ⫽ 2.518 ⫹ 2.348x; r ⫽ 0.681; P ⬍ .01); Fig. 3a). The concentration of plasma free pentosidine at nadir was not correlated with serum creatinine (Fig. 3b). The concentration of plasma total pentosidine at nadir was correlated neither with serum creatinine nor plasma free pentosidine (Figs. 3c,d). As reported, anti-pentosidine antibody stained proximal tubular cells. When the staining intensity was graded; the intensity was weakly positive in 5, positive in 9, and strongly positive in 12 specimens (Fig 4). Significant differences were detected in plasma free pentosidine between the weakly positive and strongly positive groups (P ⬍ .001; Table 4). However, there were no significant differences in age, serum creatinine, time after transplantation, or plasma total pentosidine. DISCUSSION
Fig 4. The nuclei and cytoplasm of proximal tubular cells were stained. The patients were divided into three groups by the staining intensity: weakly positive (A), positive (B), and strongly positive groups (C). Because hematoxylin was used for counterstaining, hematoxylin color was intermingled (⫻100).
lated with serum creatinine, although the degree was less than that of plasma free pentosidine (y ⫽ 6.341 ⫹ 1.589x, r ⫽ .544, P ⬍ .01) (see Fig 1b).
As reported, the blood pentosidine concentration was affected by renal function.3 The positive correlation between plasma pentosidine and serum creatinine suggests that free pentosidine in blood decreases as renal function improves. It has been reported that about 80% of intravenously injected pentosidine was excreted in urine within 24 hours in rats with normal renal function. Plasma total pentosidine decreases with decreased free pentosidine, which may have resulted in correlation with serum creatinine. Creatinine constantly and sensitively reflected renal function in all patients. Unlike creatinine, reflection of renal function by free pentosidine varied. This variation may have been due to large individual differences in production and tissue pooling of pentosidine in vivo.16 Variation of plasma total pentosidine was larger than that of plasma free pentosidine, but it similarly exhibited a positive correlation with serum creatinine. Washing out of protein-bound pentosidine takes time after improvement of renal function. It may have resulted in greater variation of renal function when evaluated by total pentosidine than free pentosidine. Time courses of serum creatinine and plasma free and total pentosidine as renal function improved by renal transplantation exhibited smooth recovery without hemodi-
PENTOSIDINE IN RENAL TISSUE
4271
Table 4. Parameters Among the Groups Divided by Intensity of Immunostaining in Proximal Tubular Cells
Age (yr) Time after transplantation (mos) Serum creatinine (mg/dL) Plasma free pentosidine (pmol/mL) Plasma total pentosidine (pmol/mg)
Weakly Positive (n ⫽ 6)
Positive (n ⫽ 16)
Strongly Positive (n ⫽ 12)
P
37.9 ⫾ 10.5 29.9 ⫾ 11.4 2.4 ⫾ 0.9 3.6 ⫾ 0.7 6.6 ⫾ 4.3
38.8 ⫾ 10.1 30.4 ⫾ 12.1 3.8 ⫾ 3.5 5.7 ⫾ 2.5 6.7 ⫾ 4.1
38.7 ⫾ 14.8 28.5 ⫾ 20.3 4.8 ⫾ 3.7 15.0 ⫾ 9.1 12.3 ⫾ 8.9
NS NS NS ⬍.001 NS
The difference in the average plasma free pentosidine between weekly positive and strongly positive groups was significant. The differences in the average age, plasma total pentosidine, serum creatinine, and time after transplantation between weekly positive and strongly positive groups were not significant.
alysis in patients who underwent living renal transplantation. Group A may have exhibited a typical course with the least bias. Serum creatinine and plasma free pentosidine simultaneously reached a nadir on day 27.0 on average. Then plasma total pentosidine reached a nadir on day 107.8 on average, showing a mean delay of 80.8 days. This delay was the time required to wash out protein-bound pentosidine. The washout period varied among the patients. Similar results for the time course of plasma free and total pentosidine after transplantation were also obtained in previous reports.4 The total pentosidine washout period after renal transplantation may have been more prolonged in patients with a higher total pentosidine concentration at the time of transplantation (living kidney-grafted patients) or discontinuation of dialysis (cadaver-donor kidneygrafted patients). When we investigated the period in 28 patients in all groups, no correlation was obtained. Neither age nor dialysis history was correlated. This study could not identify the factor determining the wash out period. Because more rejection occurred in group B, nadirs of serum creatinine and plasma free pentosidine were delayed compared to those in group A, and a nadir of plasma total pentosidine was also displayed. Because the period between discontinuation of dialysis and nadir was regarded as the time required to reach nadir in group C, it may have been slightly shorter than those in groups A and B. In group D, rejection reaction persisted in more patients, resulting in the longest delays to nadir. There was a positive correlation between the numbers of days required to reach the nadir of plasma total and free pentosidine. This finding was natural because renal excretion of free pentosidine gradually cleared total pentosidine. The concentration of plasma free pentosidine at nadir was not correlated with serum creatinine. Similarly, the concentration of plasma total pentosidine at nadir was not correlated with serum creatinine. These findings indicated that the pentosidine level varied among individuals regardless of renal function when renal function was stable. As described, the variation of blood pentosidine concentrations may have been determined by not only renal function but also various factors such as difference in the amount of production; further analysis is necessary. The regions stained with anti-pentosidine antibody were proximal tubular cells, glomerular loop, mesangium, and glomerular vascular endothelium. Only some glomerular
regions were stained and the staining factor was not clear in this study. Because a relationship between pentosidine deposition in the glomeruli and pathologic aggravation of diabetic nephropathy has been reported, further investigation is necessary.6 The varied staining intensity in proximal tubular cells was easily graded by three steps. Free pentosidine easily passes through glomerular vascular endothelium and is resorbed from the proximal urinary tubule. An in vitro experiment demonstrated that proximal tubular cells incorporate pentosidine. It has also been reported that intravenously injected pentosidine was deposited in urinary tubular cells in a rat experiment.10,15 In this study, the plasma free pentosidine level was significantly higher as the staining intensity in the proximal urinary tubule increased, suggesting that as the concentration of free pentosidine filtered through the proximal tubular glomeruli increased, resorption from proximal tubular cells increased and the cells were more strongly stained. In conclusion, improved renal function by renal transplantation decreased blood free pentosidine, and total blood pentosidine. However, the concentration varied among individuals even though renal excretion decreased blood pentosidine, and the blood pentosidine concentration was not determined by renal clearance alone. Deposition of pentosidine in proximal tubular cells may increase in patients with higher free pentosidine and serum creatinine values. ACKNOWLEDGMENT We acknowledge the doctors and staff in Tokai University for instruction in the measurement and immunohistologic investigation of pentosidine and providing us with anti-pentosidine antibody.
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4272 4. Miyata T, Ueda Y, Yoshida A, et al: Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy. Kidney Int 51:880, 1997 5. Miyata T, Ueda Y, Yoshida A, et al: Clearance of pentosidine, an advanced glycation end product, by different modalities of renal replacement therapy. Kidney Int 51:880, 1997 6. Horie K, Miyata T, Maeda K, et al: Immunohistochemical colocalization of glycoxidation products and lipid peroxidation products in diabetic renal glomerular lesions. Implication for glycoxidative stress in the pathogenesis of diabetic nephropathy. J Clin Invest 100:2995, 1997 7. Makita Z, Bucala R, Rayfield EJ, et al: Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure. Lancet 343:1519, 1994 8. Miyata T, Wada Y, Cai Z, et al: Implication of an increased oxidative stress in the formation of advanced glycation end products in patients with end-stage renal failure. Kidney Int 51:1170, 1997 9. Asano M, Fujita Y, Ueda Y, et al: Renal proximal tubular metabolism of protein-linked pentosidine, an advanced glycation end product. Nephron 91:688, 2002 10. Kume S, Takeya M, Mori T, et al: Immunohistochemical and ultrastructural detection of advanced glycation end products in
YOSHIDA, YONEDA, FUJIMOTO ET AL atherosclerotic lesions of human aorta with a novel specific monoclonal antibody. Am J Pathol 147:654, 1995 11. Yamagishi S, Yamamoto Y, Harada S, et al: Advanced glycosylation end products stimulate the growth but inhibit the prostacyclin-producing ability of endothelial cells through interactions with their receptors. FEBS Lett 384:103, 1996 12. Smith MA, Taneda S, Richey PL, et al: Advanced Maillard reaction end products are associated with Alzheimer disease pathology. Proc Natl Acad Sci U S A 91:5710, 1994 13. Miyata T, Ueda Y, Yamada Y, et al: Accumulation of carbonyls accelerates the formation of pentosidine, an advanced glycation end product: carbonyl stress in uremia. J Am Soc Nephrol 9:2349, 1998 14. Miyata T, Ishiguro N, Yasuda Y, et al: Increased pentosidine, an advanced glycation end product, in plasma and synovial fluid from patients with rheumatoid arthritis and its relation with inflammatory markers. Bioch Biophys Res Comm 244:45, 1998 15. Miyata T, Ueda Y, Horie K, et al: Renal catabolism of advanced glycation end products: the fate of pentosidine. Kidney Int 53:416, 1998 16. Bucala R, Makita Z, Vega G, et al: Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency. Proc Natl Acad Sci U S A 91:9441, 1994