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URINARY CYTOKINES AS MARKERS OF REFLUX NEPHROPATHY
0022-5347/99/1625-1739/0 THEJOURNAL OF UROLOGY Copyright 0 1999 by AMERICAN UROLOCICAL ASSOCIATION, INC
Vol. 162,1739-1742,November 1999 Printed in U.S.A
URINARY CYTOKINES AS MARKERS OF REFLUX NEPHROPATHY G. K. NINAN," R. SINGH JUTLEY
AND
0. EREMIN
From the Department of Paediatric Surgery and Paediatric Urology, Royal Aberdeen Children's Hospital and Department of Surgery, University of Aberdeen Medical School, Foresterhill, Aberdeen, United Kingdom
ABSTRACT
Purpose: We established whether the urinary cytokines interleukin-6 (IL-61, tumor necrosis factor (TNFI-a and soluble TNF receptor-1 have a role as noninvasive markers of renal damage in children with vesicoureteral reflux. Materials and Methods: We performed a n observational study in a surgical and urological unit a t a pediatric teaching hospital. Urine cytokine levels of IL-6, TNF-a and soluble TNF receptor-1 were measured using a standard enzyme-linked immunosorbent assay technique in children stratified into group 1-11 with vesicoureteral reflux and reflux nephropathy, group 2-6 with vesicoureteral reflux only and no associated nephropathy, and group 3-15 age and sex matched controls. Results: Urinary levels of the cytokines IL-6 and soluble TNF receptor-1 were significantly elevated in group 1versus group 3 (0.048 to 13.25 pg./Fmol. creatinine, mean 3.658 versus 0.027 to 0.677, mean 0.247 and 102.89 to 4,502.9 pg./pmol. creatinine, mean 1,395.3 versus 13.06 to 569.6, mean 145.357, respectively). Neither cytokine in group 2 (0.074 to 10.96 pg./pmol. creatinine, mean 2.94 and 51.52 to 1,115.48, mean 413.137, respectively) was elevated compared to that in group 3. TNF-a was not elevated in group 1or 2 compared to that in group 3 (0.104 to 2.518 pg./pmol. creatinine, mean 0.56,0.094 to 1.278, mean 0.334 and 0.065 to 0.694, mean 0.241, respectively). Conclusions: Measuring the urinary levels of the cytokines IL-6 and soluble TNF receptor-1 may be useful as a noninvasive marker of reflux associated renal damage. Further studies with larger patient groups are necessary to locate the source of production of the elevated urinary cytokines measured in our study. KEY WORDS: vesicoureteral reflux, kidney, cytokines, tumor markers Vesicoureteral reflux is the most common anomaly of the urinary tract in children who present with urinary tract infection. Reflux nephropathy secondary to vesicoureteral reflux is a significant cause of morbidity in children with 30% of refluxing kidneys sustaining scars during the initial episode of sepsis.' Detecting renal scarring currently depends on imaging modalities with the associated implicit problems of exposure to radiation, invasiveness and high expense. To our knowledge attempts to identify reliable noninvasive markers of reflux nephropathy have not been successful and the compelling need for such a method for the initial early detection of reflux nephropathy in children remains extant. We evaluated urinary levels of the cytokines interleukin-6 (IL-61, tumor necrosis factor (TNF)-a and soluble TNF receptor-1 in children with vesicoureteral reflux and reflux nephropathy to assess their value as simple, noninvasive and reproducible indicators of renal injury. Cytokines are a group of small peptides that regulate the humoral and cellular components of the immune system, and modulate the inflammatory response in vivo. Each process has been implicated as having a central role in reflux associated renal damage.2-4 1L-6 is a key inducer of B and T-cell activation and differentiation during i n f l a r n m a t i ~ n .More~.~ over, with TNF-a it acts as a co-stimulant to increase the efficacy of antigen presentation to T cells, augmenting the immune response.7 Thus, these cytokines may have a role in the perpetuation and progression of renal damage in reflux nephropathy.
TNF-a is produced by macrophages in vivo. It induces phagocytosis and the respiratory burst in neutrophils with the concomitant release of highly reactive free radicals.7 These processes have been demonstrated to be central in the pathogenesis of renal scarring in pyelonephritis.3 This cytokine also activates vascular endothelium during inflammatory processes. The prolonged and enhanced release of TNF-a in the host is deleterious, resulting in tissue damage and inflammation.8 Soluble TNF receptor-1 represents the truncated form of the extracellular domain of membrane bound TNF-a receptor-Xi.9 It is possible that the soluble receptor serves as a mechanism for desensitizing cells to the effects of TNF-a. Moreover, because soluble TNF receptor-1 binds to TNF-a, it may form a stable complex that represents a readily available reservoir of biologically active cytokine.10 Preliminary results of the attributes of these 3 cytokines in the serum of children with vesicoureteral reflux and associated reflux nephropathy versus sex and age matched controls indicate significantly higher levels of all 3 in those with reflux nephropathy." In light of the serum cytokine profiles we hypothesized that the urinary measurement of IL-6, TNF-a and soluble TNF receptor-1 may be useful as a noninvasive marker of reflux associated renal parenchymal damage. MATERIALS AND METHODS
Patients. We evaluated 3 groups of children, representing sequential patients recruited during hospitalization for elective surgery from October 1994 to March 1995. Patients in groups 1and 2 were hospitalized for routine evaluation of the urinary tract after documented urinary tract infections. In
Accepted for publication May 28, 1999. * Current address: Department of Paediatric Surgery and Paediatric Urology, Children's Hospital, Leicester Royal Infirmary, Leicester LE1 5WW, United Kingdom. 1739
1740
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY
all cases informed consent was obtained before study recruit- 1as well as age and reflux grade. All ChikIren also underwent cystoscopy and voiding cystourethrogaphy as part of the invesment. Group 1 consisted of 2 girls and 9 boys 7 months to 13.33 tigative protocol. Sample collection and analysis. Urine was co~~ected from years old with vesicoureteral reflux and established reflux nephropathy. Reflux was unilateral in 8 children and bilat- children in groups 1 and 2 using aseptic technique during era1 in 3, and ranged from unilateral grade 2 to bilateral cystoscopy. In group 3 a mid stream ckan-catch specimen grade 5 on radiographic study. One boy had a congenital was obtained from those with adequate bhdder control. In refluxing solitary kidney. Group 2 consisted of 3 girls and 3 immobilized children and those with inadequate bladder conboys 5 months to 11.83 years old with radiographic grades 1 trol urine specimen bags were used. All urine specimens were to 4 vesicoureteral reflux only and no associated nephropa- stored at -2OC within 3 hours of collection. Stored urine thy. Group 3 comprised 5 girls and 10 boys matched to all samples were thawed to room temperature before cytokine group 1 and 2 patients by sex and age within 6 months, analysis. Samples were centrifuged at 800 X gravity for 10 excluding 1in each of groups 1 and 2. These controls had no minutes to remove cellular and particulate matter. The suurological abnormalities and no history of urinary tract in- pernatant was used for all subsequent analysis. The urinary profiles of TNF-a, IL-6 and soluble TNF receptor-1 were fection. Group 3 patients were selected from children hospitalized determined using the quantitative sandwich enzyme-linked for minor surgical procedures, including tooth extraction in 4, immunosorbent assay (ELISA) specific for each cytokine. removal of minor skin lesions such as skin tags and benign Assay technique. Commercially available sandwich ELISA naevi in 4, elective insertion of grommets in 3, elective or- involves a monoclonal cytokine antibody coated to microtiter chiopexy in 2 and elective repair of a supraumbilical hernia wells. Precise aliquots of the urine sample (200 11.1. each of in 1. None of the children had urological abnormalities or TNF-a, IL-6 and soluble TNF receptor-1 urine samples) were previous documentation of urinary sepsis. All children se- added to each microtiter well coated with the appropriate lected for study were free of urinary tract infection at evalu- cytokine antibody. The plate was covered with a n adhesive ation, as determined by normal white cell count, serum strip to prevent sample evaporation. The apparatus was then C-reactive protein and urine microscopy. incubated at room temperature for 2 hours. Each well was Znvestigative protocol. Patients in groups 1 and 2 were aspirated and washed after incubation for a total of 3 washes admitted to our hospital to evaluate the urinary tract after using an automated plate washer and wash buffer provided presenting with a documented urinary tract infection. In all in the kit. Another horseradish peroxidase conjugated polycases hospitalization was elective and investigations were clonal antibody (200 ~ 1 .directed ) against the cytokine was performed after the urine was rendered sterile by antibiotics added and the assay was incubated for 1hour at room ternadministered elsewhere. Interval from the most recent uri- perature. The aspiration-wash cycle was then repeated. nary tract infection to evaluation was 8 to 12 weeks. Color developing reagent (200 pl.) was added and the assay Renal ultrasonography was performed initially to determine was incubated for 20 minutes at room temperature. A stop kidney size and outline, and indicate any abnormalities second- solution was added and the optical density of each well was arY to vesicou~eteralr e f l u , hPdYsPlasia O r dYsPlasia. AnY determined immediately on a dual wavelength ELISA reader reflux nePhroPathY changes were documented by r d h u c l i d e at 450 and 570 nm., respectively, which corrects OD,,, readimaging using the 9 9 ~ C h n e t i u mducoheptonate (99mTc-GH) ing by subtracting OD,,,. Each cytokine result was obtained given intravenously. This radionuclide is rapidly cleared from from optical density values using a standard ELISA curve, the circulation by g l o m e d w filtration. It is also Partially re- All measurements were performed in duplicate. In addition, absorbed by the renal tubules and retained in the renal co~-&x, cytokine levels were compared to reflux grade in group 1 where it binds to the proximal convoluted tubules. For 20 min- patients with established nephropathy. In cases of bilateral Utes *r injection Psterior, and right and left oblique POSte- reflux a cumulative score was determined for comparison. n o r images were obtained using 20-second dynamic image To allow for the dilutional effect of varying urine output frames. A renogram was also constmctedforeach P a t i e n t u s k urinary levels of cytokines were expressed as the ratio of a gamma interfaced to a data Processing cytokine-to-urinary creatinine (pg./pmol. creatinine). Normal minutes &r radionuclide injection the contribution of each Serum laboratory values for creatininewere 30 to 80 p m o l . ~ , kidney to total renal function was determined by the Assay values were compared using the Mann-Whitney U sum accumulated ineach 99"Tc-GH scan was consid- test. Results were calculated with the aid of a computerized abnormalwhen Or more areas Of decreased up- statistical software package with p <0.05 considered signiftake were noted with or without preservation of the cortical icant. outline. All frames were evaluated visually and interpreted while blinded to cytokine results. Scans were examined by the same interpreter in all cases. The "Tc-GH study was also RESULTS considered abnormal when relative function in each kidney at 2 A comparison of the urinary levels of IL-6 in group 1 and minutes on the computer generated renogram differed by more than 6%. The table shows relative function in children in group control group 3 (0.048 to 13.25 pg./pmol. creatinine, mean 3.658 and 0.027 to 0.677, mean 0.247, respectively) showed a significant increase in group 1 with documented vesicoureteral reflux and associated reflux nephropathy (p = RtJLt. 0.003). No difference in urinary IL-6 was documented in Pt.No. - Age group 2 children (0.074 t o 10.96 pg./pmol. creatinine, mean Reflux Grade % Renal Function 2.94) compared to that in the group 3 controls (fig. 1). This 1 - 10 Mos. 515 22/78 relationship was also observed in serum levels of soluble TNF 2 - 10 Mos. 410 27/73 receptor-1 in groups 1 to 3 (102.89 to 4,502.9 pg./pmol. cre3 - 13.3 Yrs. 515 Nonelpoor uptake 4 - 13.3Yrs. 510 30170 atinine, mean 1,395.3, 51.52 t o 1,115.48, mean 413.137 and 5 - 6.75 Yrs. 2/3 81/19 13.06 to 569.6, mean 145.357, respectively, p = 0.001, fig. 2). 6 - 8 Mos. 2/0 Nonelnormal There were no significant changes in urinary TNF-a in 7 - 3.5 Yrs. 4/0 15/85 40 30/70 groups 1t o 3 (0.104 to 2.518 pg./pmol. creatinine, mean 0.56, 8 - 3.5 Yrs. 9 - 1.17 Yrs. 4 Decrease&0.094 t o 1.278, mean 0.334 and 0.065 to 0.694, mean 0.241, 10 - 6.08 Yrs. 4/0 30170 fig. 3). Similarly analysis of reflux grade and cytokine level 11 - 3.83 Yrs. 0/4 69/31 _____. did not show any statistical correlation (figs. 4 to 6 ) .
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY
1741
'P = 0.003
-
ns
-
E
--.3 0 0 Q C
Y
y .E *
da
2: Q .-cL
rn
3 NEPHROPATHY CHANGES
REFLUX ONLY
CONTROLS
FIG.1. Scattergram shows trends in urinary IL-6 in 3 patient categories. Horizontal lines represent median values. NEPHROPATHY CHANGES
*P = 0.001
5000
:g U
5 0
0
X
\
Y
&v
4500 4000 3500 3000 2500 2000
I
5
1000
?'
500
.-:
o
.m a
:
rn
1
1
I
NEPHROPATHY CHANGES
rn -DREFLUX ONLY
-aCONTROLS
FIG.2. Scattergram demonstrates trends in urinary soluble TNF receptor-1 in 3 patient categories. Horizontal lines represent median values.
CONTROLS
FIG. 3. Scattergram reveals trends in urinary TNF-a in 3 patient categories. Horizontal lines represent median values. ns, not significant.
I
-
& 1500
2
REFLUX ONLY
-
:
0
0
10
E l *
grade of reflux (cumulative if bilateral)
FIG. 4. Relationship between urinary IL-6 and reflux grade in group 1 patients.
DISCUSSION
The mechanism of release of the elevated urinary cytokines IL-6 and soluble TNF receptor-1 in association with reflux nephropathy is unclear. Proteinuria is a hallmark of reflux nephropathy due to focal and segmental glomerulosclerosis in association with the disease.'* Preliminary studies documenting serum profiles of IL-6 and soluble TNF receptor-1 indicated significantly elevated levels in children with established reflux nephropathy.11 It is possible that the elevated urinary levels of IL-6 and soluble TNF receptor-1 in our current study are the result of serum cytokines filtering through the diseased glomeruli into the urinary tract. TNF-a does not appear to have a role in established reflux nephropathy since urinary levels were not significantly elevated in this study. It is possible that any molecules excreted in the urinary tract may bind to inhibitors in the urine, preventing detection by ELISA. TNF receptor-1 is a physiological inhibitor of TNF-a, and so any TNF-a molecules excreted in the urine may bind to the soluble receptor and escape detection. Immune cells are key producers of cytokines in vivo. The histological feature associated with reflux nephropathy includes a chronic inflammatory cell infiltrate.12 These immune cells may be responsible for the elevated levels of IL-6 and soluble TNF receptor-1 documented in this study. It is possible that a self-perpetuating sterile immune response is mounted against certain antigens deposited at the time of
12
. .
. .
~-
0 0
2
4
6
8
10
72
grade of reflux (cumulative if bilateral)
FIG.5. Relationship between urinary ThT-a and reflux grade in group 1 patients.
initial damage. Fliger et a1 reported that the glycoprotein tubular basement membrane induces inflammation in the kidneys.I3 This antigen may have been deposited in the interstitium as a result of the urodynamic pressure of refluxing urine. Tamm-Horsfall protein or uromodulin may also produce a host immune response similar to that of tubular
1742
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY nity for earlier surgical intervention, minimizing reflux associated renal scarring. Currently there are little established data o n the role of cytokines in vesicoureteral reflux a n d reflux nephropathy. To our knowledge o u r report is the first to document urinary cytokine profiles i n children w i t h vesicoureteral reflux a n d reflux nephropathy. In view of our findings w e believe that further detailed studies with larger patient groups a n d a wider range of cytokines are of merit to investigate fully the potential of urinary cytokines as simple and noninvasive markers of reflux associated renal damage.
=I
REFERENCES
1. Rollerston, G . L., Maling, T. M. J . and Hodson, C. J.: Intra renal i reflux and the scarred kidney. Arch. Dis. Child., 49 531,1974. 0 1 4 6 a 10 12 2. Torres, V. E., Kramer, S. A. and Holley, K. E.: Effect of bacterial grade of reflux (cumulatlve if bilateral) immunization on experimental reflux nephropathy. J . Urol., 131:722, 1984. FIG.6. Relationship between urinary soluble TNF receptor-1 and 3. Roberts, J . A.: Vesicoureteric reflux and pyelonephritis in the reflux grade in group 1 patients. monkey: a review. J. Urol., 148:1721, 1992. 4. Shimamura, T.: Mechanisms of renal tissue destruction in experimental acute pyelonephritis. Exp. Molec. Path., 3 4 34, basement membrane. Hodson et a1 reported that serum an1981. 5. Hirano, T., Teranishi, T. and Lin, B.: Human helper T-cell factibodies t o Tamm-Horsfall protein a n d cellular infiltrates tods). IV.Demonstration of a human late-acting an cell difsurrounding Tamm-Horsfall protein h a v e been noted in referentiation factor acting on Staphylococcus Aureus Cowan flux nephropathy, suggesting that i t may h a v e a role i n the I-stimulated B-cells. J. Immunol., 133 798, 1984. perpetuation of renal damage even after the initial damage 6. Lotz, M., Jirik, F. and Kabouridis, P.: B-cell stimulating factor has subsided.14 Angell et a1 showed that bacterial antigens 2/IL-6 is a co-stimulant for human thymocytes and also persist i n renal scars long after urine has been rendered T-lymphocytes. J . Exp. Med., 161: 1253, 1988. ~ t e r i 1 e . ITherefore, ~ further investigations documenting the 7. Rubin-Kelly, V. E. and Jevnikar, A. M.: Antigen presentation by source a n d site of cytokine production would h a v e merit. renal tubular epithelial cells. J . Amer. SOC.Nephrol., 2: 13, There i s considerable overlap i n cytokine levels i n patients 1991. with established reflux nephropathy a n d those with vesi8. Tracey, K. J., Beutler, B. and Lowry, S. F.: Shock and tissue injury induced by recombinant human cachectin. Science, 234 coureteral reflux changes only. It i s possible that renal scar470, 1986. ring is developing in patients w i t h reflux only who h a v e a 9. Engelmann, H., Novick, D. and Wallach, D.: 2 TNF-BPs purified high cytokine level, comparable t o those i n group 1 with from human urine. Evidence for immunological crossestablished reflux nephropathy. Similarly the low cytokine reactivity with cell surface TNF receptors. J. Biol. Chem., 265 level in patients with established reflux nephropathy m a y be 1531, 1990. explained by immunological reactions that are i n fact non- 10. Aderka, D., Engelmann, H. and Maor, Y.: Stabilization of the progressive. There is merit in the serial cytokine measbioactivity of TNF by its soluble receptors. J. Exp. Med., 175: urement of IL-6 a n d soluble TNF receptor-1 as well as in 323,1992. repeat 99mTc-GH scanning i n the former group to discover 11. Singh Jutley, R., Eremin, O . , Youngson, G. G. and Ninan, G. K.: Serum cytokine profile in reflux nephropathy. Read at Interwhether there i s progression t o renal scarring. national Symposium on Paediatric Surgical Research, Royal Urinary markers in re5ux nephropathy are an intensive area of College of Surgeons, Dublin, Ireland, July 24-25, 1995. research but currently there is little c o n s e m among research group. Anderson16 and Kunin17 et al examined the role of the 12. Becker, G. J . and Kincaid-Smith, P.: Reflux nephropathy: the glomerular lesion and progression of renal failure. Ped. Nephrenal tubular enzymes N-acetyl-PD-glucosaminidase and rol., 7: 365, 1993. g a m m a - g l u t a m y l t r a s e . In addition, Cam et al reported that 13. Fliger, F. D., Wieslander, J., Brentjens, J . R., Andres. G. A. and N-acety~/%D-glkxminidaseis also grade dependent.18 In our Butkowski, R. J.: Identification of a target antigen in human study there was no correlation between re5ux grade and level of anti-tubular basement membrane nephritis. Kidney Int., 31: urinary cytokine. N-acetyl-/%D-glucosaminidaseand gamma800, 1987. glutamyltransferase represent tubular enzymes that are sensitive 14. Hodson, J., Maling, T. M. J., McManamon, P. J . and Lewis, M. G.: Reflux nephropathy. Kidney Int., 8: S50, 1975. to umdynamic pressure in vesicomteral re5ux due to their ana15. Angell, M. E., Relman, A. S. and Robbins, S. L.: Active chronic tomical pasition in the renal system. Sincethe immune response in pyelonephritis without evidence of bacterial infection. New reflux nephropathy is interstitial, cytokine levels would be exEngl. J. Med., 278 1303, 1968. pected to be independent of ongoing reflux. 16. Anderson, D. N., Driver, C. P., Whiting, P. H. and Youngson, G. G.: Reflux-inducedrenal parenchymal injury: evaluation by CONCLUSIONS urinary enzyme management. Ped. Surg. Int., 10: 108, 1995. Our findings support measurement of the urinary cyto- 17. Kunin, C. M., Chesney, R. W., Craig, W. A., England, A. G. and De Angelis, C.: Enzymuria as a marker of renal injury and kines IL-6 a n d soluble TNF receptor-1 as simple a n d nonindisease: studies of N-acetyl-B-D-glucosaminidase in the genvasive indicators of ongoing renal parenchymal damage i n eral population and in patients with renal disease. Pediatrics, association with vesicoureteral reflux. Serial measurement of 6 2 751, 1978. these urinary cytokines may help i n t h e early identification 18. Carr, M. C., Peters, C. A., Retik, A. B. and Mandell, J.: Urinary of children at risk for reflux nephropathy before the developlevels of renal tubular enzyme N-acetyl-P-D-glucosaminidase ment of renal scarring. This m a r k e r optimizes the opportuin relation to grade of reflux. J . Urol., 146 654, 1991.
Vol. 162,1739-1742,November 1999 Printed in U.S.A
URINARY CYTOKINES AS MARKERS OF REFLUX NEPHROPATHY G. K. NINAN," R. SINGH JUTLEY
AND
0. EREMIN
From the Department of Paediatric Surgery and Paediatric Urology, Royal Aberdeen Children's Hospital and Department of Surgery, University of Aberdeen Medical School, Foresterhill, Aberdeen, United Kingdom
ABSTRACT
Purpose: We established whether the urinary cytokines interleukin-6 (IL-61, tumor necrosis factor (TNFI-a and soluble TNF receptor-1 have a role as noninvasive markers of renal damage in children with vesicoureteral reflux. Materials and Methods: We performed a n observational study in a surgical and urological unit a t a pediatric teaching hospital. Urine cytokine levels of IL-6, TNF-a and soluble TNF receptor-1 were measured using a standard enzyme-linked immunosorbent assay technique in children stratified into group 1-11 with vesicoureteral reflux and reflux nephropathy, group 2-6 with vesicoureteral reflux only and no associated nephropathy, and group 3-15 age and sex matched controls. Results: Urinary levels of the cytokines IL-6 and soluble TNF receptor-1 were significantly elevated in group 1versus group 3 (0.048 to 13.25 pg./Fmol. creatinine, mean 3.658 versus 0.027 to 0.677, mean 0.247 and 102.89 to 4,502.9 pg./pmol. creatinine, mean 1,395.3 versus 13.06 to 569.6, mean 145.357, respectively). Neither cytokine in group 2 (0.074 to 10.96 pg./pmol. creatinine, mean 2.94 and 51.52 to 1,115.48, mean 413.137, respectively) was elevated compared to that in group 3. TNF-a was not elevated in group 1or 2 compared to that in group 3 (0.104 to 2.518 pg./pmol. creatinine, mean 0.56,0.094 to 1.278, mean 0.334 and 0.065 to 0.694, mean 0.241, respectively). Conclusions: Measuring the urinary levels of the cytokines IL-6 and soluble TNF receptor-1 may be useful as a noninvasive marker of reflux associated renal damage. Further studies with larger patient groups are necessary to locate the source of production of the elevated urinary cytokines measured in our study. KEY WORDS: vesicoureteral reflux, kidney, cytokines, tumor markers Vesicoureteral reflux is the most common anomaly of the urinary tract in children who present with urinary tract infection. Reflux nephropathy secondary to vesicoureteral reflux is a significant cause of morbidity in children with 30% of refluxing kidneys sustaining scars during the initial episode of sepsis.' Detecting renal scarring currently depends on imaging modalities with the associated implicit problems of exposure to radiation, invasiveness and high expense. To our knowledge attempts to identify reliable noninvasive markers of reflux nephropathy have not been successful and the compelling need for such a method for the initial early detection of reflux nephropathy in children remains extant. We evaluated urinary levels of the cytokines interleukin-6 (IL-61, tumor necrosis factor (TNF)-a and soluble TNF receptor-1 in children with vesicoureteral reflux and reflux nephropathy to assess their value as simple, noninvasive and reproducible indicators of renal injury. Cytokines are a group of small peptides that regulate the humoral and cellular components of the immune system, and modulate the inflammatory response in vivo. Each process has been implicated as having a central role in reflux associated renal damage.2-4 1L-6 is a key inducer of B and T-cell activation and differentiation during i n f l a r n m a t i ~ n .More~.~ over, with TNF-a it acts as a co-stimulant to increase the efficacy of antigen presentation to T cells, augmenting the immune response.7 Thus, these cytokines may have a role in the perpetuation and progression of renal damage in reflux nephropathy.
TNF-a is produced by macrophages in vivo. It induces phagocytosis and the respiratory burst in neutrophils with the concomitant release of highly reactive free radicals.7 These processes have been demonstrated to be central in the pathogenesis of renal scarring in pyelonephritis.3 This cytokine also activates vascular endothelium during inflammatory processes. The prolonged and enhanced release of TNF-a in the host is deleterious, resulting in tissue damage and inflammation.8 Soluble TNF receptor-1 represents the truncated form of the extracellular domain of membrane bound TNF-a receptor-Xi.9 It is possible that the soluble receptor serves as a mechanism for desensitizing cells to the effects of TNF-a. Moreover, because soluble TNF receptor-1 binds to TNF-a, it may form a stable complex that represents a readily available reservoir of biologically active cytokine.10 Preliminary results of the attributes of these 3 cytokines in the serum of children with vesicoureteral reflux and associated reflux nephropathy versus sex and age matched controls indicate significantly higher levels of all 3 in those with reflux nephropathy." In light of the serum cytokine profiles we hypothesized that the urinary measurement of IL-6, TNF-a and soluble TNF receptor-1 may be useful as a noninvasive marker of reflux associated renal parenchymal damage. MATERIALS AND METHODS
Patients. We evaluated 3 groups of children, representing sequential patients recruited during hospitalization for elective surgery from October 1994 to March 1995. Patients in groups 1and 2 were hospitalized for routine evaluation of the urinary tract after documented urinary tract infections. In
Accepted for publication May 28, 1999. * Current address: Department of Paediatric Surgery and Paediatric Urology, Children's Hospital, Leicester Royal Infirmary, Leicester LE1 5WW, United Kingdom. 1739
1740
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY
all cases informed consent was obtained before study recruit- 1as well as age and reflux grade. All ChikIren also underwent cystoscopy and voiding cystourethrogaphy as part of the invesment. Group 1 consisted of 2 girls and 9 boys 7 months to 13.33 tigative protocol. Sample collection and analysis. Urine was co~~ected from years old with vesicoureteral reflux and established reflux nephropathy. Reflux was unilateral in 8 children and bilat- children in groups 1 and 2 using aseptic technique during era1 in 3, and ranged from unilateral grade 2 to bilateral cystoscopy. In group 3 a mid stream ckan-catch specimen grade 5 on radiographic study. One boy had a congenital was obtained from those with adequate bhdder control. In refluxing solitary kidney. Group 2 consisted of 3 girls and 3 immobilized children and those with inadequate bladder conboys 5 months to 11.83 years old with radiographic grades 1 trol urine specimen bags were used. All urine specimens were to 4 vesicoureteral reflux only and no associated nephropa- stored at -2OC within 3 hours of collection. Stored urine thy. Group 3 comprised 5 girls and 10 boys matched to all samples were thawed to room temperature before cytokine group 1 and 2 patients by sex and age within 6 months, analysis. Samples were centrifuged at 800 X gravity for 10 excluding 1in each of groups 1 and 2. These controls had no minutes to remove cellular and particulate matter. The suurological abnormalities and no history of urinary tract in- pernatant was used for all subsequent analysis. The urinary profiles of TNF-a, IL-6 and soluble TNF receptor-1 were fection. Group 3 patients were selected from children hospitalized determined using the quantitative sandwich enzyme-linked for minor surgical procedures, including tooth extraction in 4, immunosorbent assay (ELISA) specific for each cytokine. removal of minor skin lesions such as skin tags and benign Assay technique. Commercially available sandwich ELISA naevi in 4, elective insertion of grommets in 3, elective or- involves a monoclonal cytokine antibody coated to microtiter chiopexy in 2 and elective repair of a supraumbilical hernia wells. Precise aliquots of the urine sample (200 11.1. each of in 1. None of the children had urological abnormalities or TNF-a, IL-6 and soluble TNF receptor-1 urine samples) were previous documentation of urinary sepsis. All children se- added to each microtiter well coated with the appropriate lected for study were free of urinary tract infection at evalu- cytokine antibody. The plate was covered with a n adhesive ation, as determined by normal white cell count, serum strip to prevent sample evaporation. The apparatus was then C-reactive protein and urine microscopy. incubated at room temperature for 2 hours. Each well was Znvestigative protocol. Patients in groups 1 and 2 were aspirated and washed after incubation for a total of 3 washes admitted to our hospital to evaluate the urinary tract after using an automated plate washer and wash buffer provided presenting with a documented urinary tract infection. In all in the kit. Another horseradish peroxidase conjugated polycases hospitalization was elective and investigations were clonal antibody (200 ~ 1 .directed ) against the cytokine was performed after the urine was rendered sterile by antibiotics added and the assay was incubated for 1hour at room ternadministered elsewhere. Interval from the most recent uri- perature. The aspiration-wash cycle was then repeated. nary tract infection to evaluation was 8 to 12 weeks. Color developing reagent (200 pl.) was added and the assay Renal ultrasonography was performed initially to determine was incubated for 20 minutes at room temperature. A stop kidney size and outline, and indicate any abnormalities second- solution was added and the optical density of each well was arY to vesicou~eteralr e f l u , hPdYsPlasia O r dYsPlasia. AnY determined immediately on a dual wavelength ELISA reader reflux nePhroPathY changes were documented by r d h u c l i d e at 450 and 570 nm., respectively, which corrects OD,,, readimaging using the 9 9 ~ C h n e t i u mducoheptonate (99mTc-GH) ing by subtracting OD,,,. Each cytokine result was obtained given intravenously. This radionuclide is rapidly cleared from from optical density values using a standard ELISA curve, the circulation by g l o m e d w filtration. It is also Partially re- All measurements were performed in duplicate. In addition, absorbed by the renal tubules and retained in the renal co~-&x, cytokine levels were compared to reflux grade in group 1 where it binds to the proximal convoluted tubules. For 20 min- patients with established nephropathy. In cases of bilateral Utes *r injection Psterior, and right and left oblique POSte- reflux a cumulative score was determined for comparison. n o r images were obtained using 20-second dynamic image To allow for the dilutional effect of varying urine output frames. A renogram was also constmctedforeach P a t i e n t u s k urinary levels of cytokines were expressed as the ratio of a gamma interfaced to a data Processing cytokine-to-urinary creatinine (pg./pmol. creatinine). Normal minutes &r radionuclide injection the contribution of each Serum laboratory values for creatininewere 30 to 80 p m o l . ~ , kidney to total renal function was determined by the Assay values were compared using the Mann-Whitney U sum accumulated ineach 99"Tc-GH scan was consid- test. Results were calculated with the aid of a computerized abnormalwhen Or more areas Of decreased up- statistical software package with p <0.05 considered signiftake were noted with or without preservation of the cortical icant. outline. All frames were evaluated visually and interpreted while blinded to cytokine results. Scans were examined by the same interpreter in all cases. The "Tc-GH study was also RESULTS considered abnormal when relative function in each kidney at 2 A comparison of the urinary levels of IL-6 in group 1 and minutes on the computer generated renogram differed by more than 6%. The table shows relative function in children in group control group 3 (0.048 to 13.25 pg./pmol. creatinine, mean 3.658 and 0.027 to 0.677, mean 0.247, respectively) showed a significant increase in group 1 with documented vesicoureteral reflux and associated reflux nephropathy (p = RtJLt. 0.003). No difference in urinary IL-6 was documented in Pt.No. - Age group 2 children (0.074 t o 10.96 pg./pmol. creatinine, mean Reflux Grade % Renal Function 2.94) compared to that in the group 3 controls (fig. 1). This 1 - 10 Mos. 515 22/78 relationship was also observed in serum levels of soluble TNF 2 - 10 Mos. 410 27/73 receptor-1 in groups 1 to 3 (102.89 to 4,502.9 pg./pmol. cre3 - 13.3 Yrs. 515 Nonelpoor uptake 4 - 13.3Yrs. 510 30170 atinine, mean 1,395.3, 51.52 t o 1,115.48, mean 413.137 and 5 - 6.75 Yrs. 2/3 81/19 13.06 to 569.6, mean 145.357, respectively, p = 0.001, fig. 2). 6 - 8 Mos. 2/0 Nonelnormal There were no significant changes in urinary TNF-a in 7 - 3.5 Yrs. 4/0 15/85 40 30/70 groups 1t o 3 (0.104 to 2.518 pg./pmol. creatinine, mean 0.56, 8 - 3.5 Yrs. 9 - 1.17 Yrs. 4 Decrease&0.094 t o 1.278, mean 0.334 and 0.065 to 0.694, mean 0.241, 10 - 6.08 Yrs. 4/0 30170 fig. 3). Similarly analysis of reflux grade and cytokine level 11 - 3.83 Yrs. 0/4 69/31 _____. did not show any statistical correlation (figs. 4 to 6 ) .
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY
1741
'P = 0.003
-
ns
-
E
--.3 0 0 Q C
Y
y .E *
da
2: Q .-cL
rn
3 NEPHROPATHY CHANGES
REFLUX ONLY
CONTROLS
FIG.1. Scattergram shows trends in urinary IL-6 in 3 patient categories. Horizontal lines represent median values. NEPHROPATHY CHANGES
*P = 0.001
5000
:g U
5 0
0
X
\
Y
&v
4500 4000 3500 3000 2500 2000
I
5
1000
?'
500
.-:
o
.m a
:
rn
1
1
I
NEPHROPATHY CHANGES
rn -DREFLUX ONLY
-aCONTROLS
FIG.2. Scattergram demonstrates trends in urinary soluble TNF receptor-1 in 3 patient categories. Horizontal lines represent median values.
CONTROLS
FIG. 3. Scattergram reveals trends in urinary TNF-a in 3 patient categories. Horizontal lines represent median values. ns, not significant.
I
-
& 1500
2
REFLUX ONLY
-
:
0
0
10
E l *
grade of reflux (cumulative if bilateral)
FIG. 4. Relationship between urinary IL-6 and reflux grade in group 1 patients.
DISCUSSION
The mechanism of release of the elevated urinary cytokines IL-6 and soluble TNF receptor-1 in association with reflux nephropathy is unclear. Proteinuria is a hallmark of reflux nephropathy due to focal and segmental glomerulosclerosis in association with the disease.'* Preliminary studies documenting serum profiles of IL-6 and soluble TNF receptor-1 indicated significantly elevated levels in children with established reflux nephropathy.11 It is possible that the elevated urinary levels of IL-6 and soluble TNF receptor-1 in our current study are the result of serum cytokines filtering through the diseased glomeruli into the urinary tract. TNF-a does not appear to have a role in established reflux nephropathy since urinary levels were not significantly elevated in this study. It is possible that any molecules excreted in the urinary tract may bind to inhibitors in the urine, preventing detection by ELISA. TNF receptor-1 is a physiological inhibitor of TNF-a, and so any TNF-a molecules excreted in the urine may bind to the soluble receptor and escape detection. Immune cells are key producers of cytokines in vivo. The histological feature associated with reflux nephropathy includes a chronic inflammatory cell infiltrate.12 These immune cells may be responsible for the elevated levels of IL-6 and soluble TNF receptor-1 documented in this study. It is possible that a self-perpetuating sterile immune response is mounted against certain antigens deposited at the time of
12
. .
. .
~-
0 0
2
4
6
8
10
72
grade of reflux (cumulative if bilateral)
FIG.5. Relationship between urinary ThT-a and reflux grade in group 1 patients.
initial damage. Fliger et a1 reported that the glycoprotein tubular basement membrane induces inflammation in the kidneys.I3 This antigen may have been deposited in the interstitium as a result of the urodynamic pressure of refluxing urine. Tamm-Horsfall protein or uromodulin may also produce a host immune response similar to that of tubular
1742
URINARY CYTOKINES AS MARKERS OF REFLUX NEUROPATHY nity for earlier surgical intervention, minimizing reflux associated renal scarring. Currently there are little established data o n the role of cytokines in vesicoureteral reflux a n d reflux nephropathy. To our knowledge o u r report is the first to document urinary cytokine profiles i n children w i t h vesicoureteral reflux a n d reflux nephropathy. In view of our findings w e believe that further detailed studies with larger patient groups a n d a wider range of cytokines are of merit to investigate fully the potential of urinary cytokines as simple and noninvasive markers of reflux associated renal damage.
=I
REFERENCES
1. Rollerston, G . L., Maling, T. M. J . and Hodson, C. J.: Intra renal i reflux and the scarred kidney. Arch. Dis. Child., 49 531,1974. 0 1 4 6 a 10 12 2. Torres, V. E., Kramer, S. A. and Holley, K. E.: Effect of bacterial grade of reflux (cumulatlve if bilateral) immunization on experimental reflux nephropathy. J . Urol., 131:722, 1984. FIG.6. Relationship between urinary soluble TNF receptor-1 and 3. Roberts, J . A.: Vesicoureteric reflux and pyelonephritis in the reflux grade in group 1 patients. monkey: a review. J. Urol., 148:1721, 1992. 4. Shimamura, T.: Mechanisms of renal tissue destruction in experimental acute pyelonephritis. Exp. Molec. Path., 3 4 34, basement membrane. Hodson et a1 reported that serum an1981. 5. Hirano, T., Teranishi, T. and Lin, B.: Human helper T-cell factibodies t o Tamm-Horsfall protein a n d cellular infiltrates tods). IV.Demonstration of a human late-acting an cell difsurrounding Tamm-Horsfall protein h a v e been noted in referentiation factor acting on Staphylococcus Aureus Cowan flux nephropathy, suggesting that i t may h a v e a role i n the I-stimulated B-cells. J. Immunol., 133 798, 1984. perpetuation of renal damage even after the initial damage 6. Lotz, M., Jirik, F. and Kabouridis, P.: B-cell stimulating factor has subsided.14 Angell et a1 showed that bacterial antigens 2/IL-6 is a co-stimulant for human thymocytes and also persist i n renal scars long after urine has been rendered T-lymphocytes. J . Exp. Med., 161: 1253, 1988. ~ t e r i 1 e . ITherefore, ~ further investigations documenting the 7. Rubin-Kelly, V. E. and Jevnikar, A. M.: Antigen presentation by source a n d site of cytokine production would h a v e merit. renal tubular epithelial cells. J . Amer. SOC.Nephrol., 2: 13, There i s considerable overlap i n cytokine levels i n patients 1991. with established reflux nephropathy a n d those with vesi8. Tracey, K. J., Beutler, B. and Lowry, S. F.: Shock and tissue injury induced by recombinant human cachectin. Science, 234 coureteral reflux changes only. It i s possible that renal scar470, 1986. ring is developing in patients w i t h reflux only who h a v e a 9. Engelmann, H., Novick, D. and Wallach, D.: 2 TNF-BPs purified high cytokine level, comparable t o those i n group 1 with from human urine. Evidence for immunological crossestablished reflux nephropathy. Similarly the low cytokine reactivity with cell surface TNF receptors. J. Biol. Chem., 265 level in patients with established reflux nephropathy m a y be 1531, 1990. explained by immunological reactions that are i n fact non- 10. Aderka, D., Engelmann, H. and Maor, Y.: Stabilization of the progressive. There is merit in the serial cytokine measbioactivity of TNF by its soluble receptors. J. Exp. Med., 175: urement of IL-6 a n d soluble TNF receptor-1 as well as in 323,1992. repeat 99mTc-GH scanning i n the former group to discover 11. Singh Jutley, R., Eremin, O . , Youngson, G. G. and Ninan, G. K.: Serum cytokine profile in reflux nephropathy. Read at Interwhether there i s progression t o renal scarring. national Symposium on Paediatric Surgical Research, Royal Urinary markers in re5ux nephropathy are an intensive area of College of Surgeons, Dublin, Ireland, July 24-25, 1995. research but currently there is little c o n s e m among research group. Anderson16 and Kunin17 et al examined the role of the 12. Becker, G. J . and Kincaid-Smith, P.: Reflux nephropathy: the glomerular lesion and progression of renal failure. Ped. Nephrenal tubular enzymes N-acetyl-PD-glucosaminidase and rol., 7: 365, 1993. g a m m a - g l u t a m y l t r a s e . In addition, Cam et al reported that 13. Fliger, F. D., Wieslander, J., Brentjens, J . R., Andres. G. A. and N-acety~/%D-glkxminidaseis also grade dependent.18 In our Butkowski, R. J.: Identification of a target antigen in human study there was no correlation between re5ux grade and level of anti-tubular basement membrane nephritis. Kidney Int., 31: urinary cytokine. N-acetyl-/%D-glucosaminidaseand gamma800, 1987. glutamyltransferase represent tubular enzymes that are sensitive 14. Hodson, J., Maling, T. M. J., McManamon, P. J . and Lewis, M. G.: Reflux nephropathy. Kidney Int., 8: S50, 1975. to umdynamic pressure in vesicomteral re5ux due to their ana15. Angell, M. E., Relman, A. S. and Robbins, S. L.: Active chronic tomical pasition in the renal system. Sincethe immune response in pyelonephritis without evidence of bacterial infection. New reflux nephropathy is interstitial, cytokine levels would be exEngl. J. Med., 278 1303, 1968. pected to be independent of ongoing reflux. 16. Anderson, D. N., Driver, C. P., Whiting, P. H. and Youngson, G. G.: Reflux-inducedrenal parenchymal injury: evaluation by CONCLUSIONS urinary enzyme management. Ped. Surg. Int., 10: 108, 1995. Our findings support measurement of the urinary cyto- 17. Kunin, C. M., Chesney, R. W., Craig, W. A., England, A. G. and De Angelis, C.: Enzymuria as a marker of renal injury and kines IL-6 a n d soluble TNF receptor-1 as simple a n d nonindisease: studies of N-acetyl-B-D-glucosaminidase in the genvasive indicators of ongoing renal parenchymal damage i n eral population and in patients with renal disease. Pediatrics, association with vesicoureteral reflux. Serial measurement of 6 2 751, 1978. these urinary cytokines may help i n t h e early identification 18. Carr, M. C., Peters, C. A., Retik, A. B. and Mandell, J.: Urinary of children at risk for reflux nephropathy before the developlevels of renal tubular enzyme N-acetyl-P-D-glucosaminidase ment of renal scarring. This m a r k e r optimizes the opportuin relation to grade of reflux. J . Urol., 146 654, 1991.