Q J Med 2003; 96:363–367 doi:10.1093/qjmed/hcg052

Urinary retinol binding protein in Indo-Asian patients with idiopathic interstitial nephritis S.T. BALL, M. LAPSLEY 1 , A.G.W. NORDEN 2 , T.D.H. CAIRNS, A.B.D. PALMER and D.H. TAUBE From the Renal Unit, St Mary’s Hospital, London, 1Department of Chemical Pathology, St Helier Hospital, Carshalton, and 2Department of Clinical Biochemistry, Addenbrookes Hospital, Cambridge, UK Received 24 June 2002 and in revised form 29 January 2003

Summary Background: Idiopathic interstitial nephritis (IIN) is common in the UK Indo-Asian population. Lack of systemic involvement and unremarkable urinalysis on stick testing suggest that it may underlie some cases of end-stage renal failure of undetermined cause. If IIN is diagnosed early, prompt initiation of treatment can improve long-term outcome. Aims: To investigate whether urinary retinol binding protein (RBP) is elevated more commonly than urinary albumin in IIN, and might be useful in the early detection of renal disease in Indo-Asian patients. Design: Preliminary observational study Methods: We measured urinary RBP and urinary albumin in 19 Indo-Asian patients in whom a renal biopsy had shown IIN, 10 of whom had already been treated with corticosteroids at the time of specimen collection. A further 28 Indo-Asian patients

with glomerular disease, and six with normal light-microscopic renal biopsy, were assessed in parallel. Results: Urinary RBP/creatinine ratio (RCR) was elevated in all 19 cases of IIN, compared to 12/19 in whom the albumin/creatinine ratio (ACR) was elevated. Elevated urinary RBP was thus significantly more common than albuminuria in this group (p-0.01). Twelve of the 19 cases also satisfied the criteria for tubular proteinuria. RCR was elevated to )30 times the upper limit of normal in 7/9 who had not previously received corticosteroids, of whom four had normal ACR; none had ACR )5 times the upper limit of normal. Discussion: These data suggest that measurement of urinary RBP should be explored as an adjunct to albuminuria, if screening for renal disease in the Indo-Asian population is contemplated.

Introduction There is a high incidence of end-stage renal failure (ESRF) in the UK Indo-Asian population.1,2 In the Indo-Asian population from which our cases are drawn, the incidence is estimated to be ) 300/106/year, )3 times that in the Caucasian population; when adjusted for age, the relative risk for ESRF is )5 times that for the Caucasian population.3 The most important underlying causes are diabetes mellitus4 and late presentation with ESRF

of undetermined cause.2,3,5,6 The latter group typically present with an unremarkable stick urinalysis and smooth small kidneys. These cases constitute an opportunity for therapeutic intervention, since established forms of treatment may be beneficial if instigated in good time. For example, although idiopathic interstitial nephritis (IIN) (which is common in Indo-Asian patients and typically presents with chronic renal failure, an unremarkable

Address correspondence to Dr S.T. Ball, Renal Unit, University Hospitals Birmingham, Edgbaston, Birmingham B15 2TH. e-mail: [email protected] ß Association of Physicians 2003

364

S.T. Ball et al.

urinary sediment and smooth small kidneys3,7) may progress to ESRF, progression may be delayed by early treatment with a regimen incorporating corticosteroids.8 As the incidence of IIN in this population may be as high as 38/106/year, this represents an important opportunity for treatment. Urinalysis for haematuria and albuminuria is frequently unremarkable in these cases,7 and therefore we hypothesized that a urinary marker of tubular injury such as RBP might be elevated more commonly than albumin in cases of IIN. If so, this marker might be used to improve early detection of renal disease in this group.3 RBP is a low-molecular-weight (21 kDa) protein that is filtered at the glomerulus and reabsorbed and catabolized in the proximal tubule after binding to megalin.9 Elevated levels of urinary RBP reflect proximal tubular dysfunction10,11 and in conjunction with urinary albumin, it has been used to define tubular proteinuria.10 RBP is easily measured12 and is stable at room temperature for )12 h, a property of considerable value in any screening strategy for renal disease.13 We therefore undertook this pilot study of 19 Indo-Asian patients with IIN (10 already corticosteroid-treated); 28 Indo-Asians attending the same out-patient clinic with documented glomerular disease and six with normal light microscopic renal histology are also reported for illustrative purposes.

Methods The indications for renal biopsy were haematuria, proteinuria )0.5 g/day or undiagnosed renal impairment. All biopsies were examined in multiple sections at four levels and stained with haematoxylin and eosin, periodic acid-Schiff and elastic van Gieson. Immunoperoxidase staining was carried out in paraffin-embedded formalin-fixed material, using antibodies to IgA, IgG, IgM, C1q, C3 and C4. Electron microscopy was performed in all cases. A mid-stream urine specimen was obtained at a routine morning consultation. The specimen was frozen at 80 8C without preservative, within 6 h of collection, and thawed to room temperature at the time of assay. The urinary concentration of albumin was determined by immunoturbidimetry on an Array analyser (Beckman-Coulter), RBP by ELISA,12 and creatinine by a Jaffe´ method. In our laboratory, a normal albumin/creatinine ratio (ACR) is -2.5 mg/mmol, microalbuminuria corresponds to ACR -25 mg/mmol but )2.5 mg/mmol, and albuminuria, ACR )25 mg/mmol. The upper limit of normal for urinary RBP/creatinine ratio (RCR) is

0.017mg/mmol.12 Stick urinalysis was performed using Multistix (Bayer Diagnostics), according to the manufacturer’s instructions. Glomerular filtration rate was estimated using the Cockcroft-Gault equation (eGFR).14

Results There were six individuals with a normal renal biopsy or an electron-microscopic diagnosis of thin basement membrane disease. The ACR and RCR were normal in all six cases. Figure 1 shows ACR and RCR for the other 47 cases. The 19 cases of IIN included 7 males and 12 females, mean (range) age 51.0 (31.5–69.2) years. Specimens of urine were obtained prior to treatment with corticosteroids in nine (4 males, 5 females, mean age 53.9 years). In the other 10, specimens were obtained at routine follow-up, a mean of 4.2 (1.0–8.1) years following renal biopsy. Renal impairment was present in all cases at the time of specimen collection; the mean eGFR was 35.3 ml/min (range 18.6–63.7 ml/min). On stick urinalysis, no patient with IIN had microscopic haematuria, but four had proteinuria. Twelve of the 19 had an elevated ACR (8 in the microalbuminuric range). Of the untreated patients, 5/9 had an elevated ACR (all in the microalbuminuric range). Figure 1 shows ACR on the abscissa. The RCR was elevated in all 19/19 cases of IIN, significantly more frequently than the ACR (p = 0.008, Fisher’s exact test). In 13/19, the RCR was )10 times the upper limit of normal, and this was so in 7/9 not treated with corticosteroids. In 12/19 (8 of the 9 not treated with corticosteroids), the criterion for tubular proteinuria10 was satisfied. Figure 1 shows RCR on the ordinate. Of the nine patients not treated with corticosteroids, seven had an RCR )30 times the upper limit of normal, of whom two had no elevation of ACR, and five had a maximum 5-fold elevation over the upper limit of normal. There were two more in whom a normal ACR was associated with a lesser elevation in the RCR. Urinary RBP correlated reciprocally with eGFR, but with only borderline significance (Pearson correlation coefficient 0.513, 95%CI 0.78 to 0.08). There was no correlation between albuminuria and eGFR, nor between albuminuria and urinary RBP. Figure 1 also shows the urinary RCR and ACR in 28 Indo-Asian patients with predominant glomerular pathology attending the same clinic as the patients with IIN (IgA nephropathy (n = 10), focal segmental glomerulosclerosis (n = 9), membranous

RBP and idiopathic interstitial nephritis

365

Figure 1. Retinol binding protein (RBP)/creatinine and albumin/creatinine ratios in 10 patients with corticosteroid-treated idiopathic interstitial nephritis (IIN) (m), nine with corticosteroid-untreated IIN (j) and 28 with glomerular disease (n). The solid line denotes the function ACR = (RBP/creatinine310)q2, which defines pure tubular proteinuria, and the horizontal broken line the upper limit of normal for RBP/creatinine. The vertical broken lines denote the upper limit of normoalbuminuria at 2.5 mg/mmol and of microalbuminuria at 25 mg/mmol. The figures adjacent to cases of IIN represent the estimated glomerular filtration rate at the time of urine collection.

nephropathy (n = 4), SLE (n = 3) and diabetic nephropathy (n = 2)). These patients had a wide range of eGFR (20.3–123.1 ml/min) and of albuminuria. The distribution of urinary RCR and ACR in this group was similar to that of non-Indo-Asian patients with glomerular disease.10

Discussion In the UK, the demand for renal replacement therapy is increasing. This is particularly apparent in the Indo-Asian community, because renal disease is so common and a currently young population is ageing (ESRF increases with age).2,15 The incidence of ESRF in the Indo-Asian population is at least three times that in the Caucasian population, and is likely to become considerably higher.1–3 Early identification of renal disease may help prevent this expansion by permitting the timely instigation of aetiologyspecific and non-specific treatment.16,17 This

depends on both detection and referral;18 for example, Kissmeyer and colleagues, studying a mixed population of hypertensives and diabetics aged between 50 and 75, estimated that 11% had renal insufficiency but only a quarter had been referred for specialist review. At our own centre, late-presenting ESRF of undetermined cause accounts for 34.7% of Indo-Asian patients starting renal replacement therapy, who themselves account for 41.4% of ‘dialysis starters’.3 This occurs despite a high level of local awareness and specialist provision, illustrated by the high rate of investigation by renal biopsy in this ethnic group (460/106/year overall and 1520/106/year in the over50 age group attending our centre).3 We believe that such a high incidence may justify screening for renal disease in this ethnic group, and this is a subject of ongoing debate in the UK.19 The methods used to screen must however take account of the high levels of tubulo-interstitial pathology in Indo-Asian patients,3 in which there is little or no albuminuria, as is apparent in the cases of IIN reported above.

366

S.T. Ball et al.

The measurement of urinary albumin excretion by stick urinalysis, spot urinary ACR or timed urine collection is a sensitive means to detect glomerular pathology.20 These methods are in widespread use in the UK, and are likely to underpin any proposed screening strategy in the Indo-Asian population, but as Figure 1 shows, a normal urinary ACR may be present in patients with significant chronic renal impairment caused by IIN. It is the third most frequent diagnosis arising from renal biopsy amongst Indo-Asians investigated at our centre, and may underlie some of the many cases of ESRF of undetermined cause.6,7 As IIN is an important, potentially treatable pathology,8 we believe that urinary RBP measurement should be evaluated if a screening strategy for renal disease is considered in this ethnic group,19 as it is very likely to increase the sensitivity of detection of renal disease over urinary ACR alone. Although a number of low-molecularweight proteins and tubular enzymes have been used as urinary markers of tubulo-interstitial damage,11,21–24 RBP has the benefit of being both stable and easily measured. Our series is somewhat heterogeneous in the cases of IIN analysed, some being studied prior to, and others following, treatment with corticosteroids, but all had significant renal impairment and all had histologically demonstrated tubulo-interstitial atrophy and fibrosis. Of the corticosteroid-untreated group, all but one satisfied the criterion for tubular proteinuria. All the cases had elevated urinary RCR, and this was often of much greater degree than the corresponding rise in ACR. This is despite the fact that some albumin is filtered at the glomerulus and reabsorbed in the proximal tubule,25 perhaps because there are differences in the handling of these proteins.10,26 It is of further interest that in a megalin knock-out mouse, abnormal vitamin D metabolism27 and TGFb1 target gene up-regulation have been reported28 and that the urine in Dent’s disease has been shown to contain high concentrations of bioactive hormones.25 Thus putative mechanisms of renal disease progression may arise from deficient megalin-mediated proximal tubular endocytosis, which also causes elevated urinary RBP. This may be pertinent to the cases described above. We believe that the data presented offer preliminary support to our hypothesis, suggesting that the measurement of urinary RBP could be developed to enhance the detection of renal disease in Indo-Asians, specifically those with IIN and possibly those with other forms of predominant tubulointerstitial damage (data not shown). It is a candidate for further investigation in developing strategies for

the early diagnosis of renal disease in this ethnic group.19

References 1. Roderick PJ, Jones I, Raleigh VS, McGeown M, Mallick N. Population need for renal replacement therapy in Thames regions: ethnic dimension. Br Med J 1994; 309:1111–14. 2. Roderick PJ, Raleigh VS, Hallam L, Mallick NP. The need and demand for renal replacement therapy in ethnic minorities in England. J Epidemiol Community Health 1996; 50:334–9. 3. Ball S, Lloyd J, Cattell V, Cook T, Palmer A, Cairns T, et al. Why is there so much end stage renal failure of undetermined cause in the Indo-Asian population? Q J Med 2001; 94:187–93. 4. Burden AC, McNally P, Feehally J, Walls J. Increased incidence of end stage renal failure secondary to diabetes mellitus in Asian ethnic groups in the United Kingdom. Diabet Med 1992; 9:641–5. 5. Pazianas M, Eastwood JB, MacRae KD, Phillips ME. Racial origin and primary renal diagnosis in 771 patients with end- stage renal disease. Nephrol Dial Transplant 1991; 6:931–5. 6. Lightstone L, Rees AJ, Tomson C, Walls J, Winearls CG, Feehally J. High incidence of end-stage renal disease in Indo-Asians in the UK wsee commentsx. Q J Med 1995; 88:191–5. 7. Ball S, Cook T, Hulme B, Palmer A, Taube D. The diagnosis and racial origin of 394 patients undergoing renal biopsy: an association between Indian race and interstitial nephritis. Nephrol Dial Transplant 1997; 12:71–7. 8. Ball S, Tagore R, Wahba M, Cairns T, Palmer A, Taube D. Corticosteroid therapy improves outcomes in Indo-Asians with idiopathic interstitial nephritis. Renal Association Autumn Meeting 2000, Cambridge, 2000. 9. Christensen EI, Moskaug JO, Vorum H, Jacobsen C, Gundersen TE, Nykjaer A, et al. Evidence for an essential role of megalin in transepithelial transport of retinol. J Am Soc Nephrol 1999; 10:685–95. 10. Norden AGW, Scheinman SJ, Deschodt-Lanckman MM, Lapsley M, Nortier JL, Thakker RV, et al. Tubular proteinuria defined by a study of Dent’s (CLCN5 mutation) and other tubular diseases. Kidney International 2000; 57:1240–49. 11. Kabanda A, Jadoul M, Lauwerys M, Bernard A, van Ypersele de Strihou C. Low molecular weight proteinuria in Chinese herbs nephropathy. Kidney International 1995; 48:1571–6. 12. Lapsley M, Akers K, Norden AG. Sensitive assays for urinary retinol-binding protein and beta-2- glycoprotein-1 based on commercially available standards. Ann Clin Biochem 1998; 35:115–19. 13. Donaldson MDC, Chambers RE, Woolridge MW, Whicher JT. Stability of a1-microglobulin, b2-microglobulin and retinol binding protein in urine. Clin Chim Acta 1989; 179:73–8. 14. Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976; 16:31–41. 15. Roderick P, Clements S, Diamond I, Storkey M, Raleigh VS. Estimating the demand for renal replacement therapy in Greater London: the impact of demographc trends in ethnic minority populations. Health Trends 1998; 30:46–50.

RBP and idiopathic interstitial nephritis

367

16. Levin A. Consequences of late referral on patient outcomes. Nephrol Dial Transplant 2000; 15(Suppl. 3):8–13.

0. due to diabetic nephropathy and primary glomerulonephritis. Kidney Int Suppl 1994; 47:S101–4.

17. Jungers P. Screening for renal insufficiency: is it worth while? is it feasible? Nephrol Dial Transplant 1999; 14:2082–4.

24. Guy JM, Brammah TB, Holt L, Bernstein RM, McMurray JR, Tieszen K, et al. Urinary excretion of albumin and retinol binding protein in systemic lupus erythematosus. Ann Clin Biochem 1997; 34:668–74.

18. Kissmeyer L, Kong C, Cohen J, Unwin RJ, Woolfson RG, Neild GH. Community nephrology: audit of screening for renal insufficiency in a high risk population. Nephrol Dial Transplant 1999; 14:2150–5. 19. Lightstone E. Preventing kidney disease: the ethnic challenge. Peterborough, National Kidney Research Fund, 2001. 20. Ballantyne FC, Gibbons J, O’Reilly DS. Urine albumin should replace total protein for the assessment of glomerular proteinuria. Ann Clin Biochem 1993; 30:101–3. 21. Bernard A, Buchet JP, Roels H, Masson P, Lauwerys R. Renal excretion of proteins and enzymes in workers exposed to cadmium. Eur J Clin Invest 1979; 9:11–22. 22. Holm J, Hemmingsen L, Nielsen NV. Low molecular mass proteinuria as a marker of proximal renal tubular dysfunction in normo- and microalbuminuric non-insulin dependent diabetic subjects. Clin Chem 1993; 9:517–19. 23. Yaqoob M, McClelland P, Patrick AW, Stevenson A, Mason H, Bell GM. Tubulopathy with macroalbuminuria

25. Norden AG, Lapsley M, Lee PJ, Pusey CD, Scheinman SJ, Tam FW, et al. Glomerular protein sieving and implications for renal failure in Fanconi syndrome. Kidney Int 2001; 60:1885–92. 26. Verroust PJ, Birn H, Nielsen R, Kozyraki R, Christensen EI. The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology. Kidney Int 2002; 62:745–56. 27. Nykjaer A, Dragun D, Walther D, Vorum H, Jacobsen C, Herz J, et al. An endocytic pathway essential for renal uptake and activation of the steroid 25-( OH) vitamin D3. Cell 1999; 96:507–15. 28. Hilpert J, Wogensen L, Thykjaer T, Wellner M, Schlichting U, Orntoft TF, et al. Expression profiling confirms the role of endocytic receptor megalin in renal vitamin D3 metabolism. Kidney Int 2002; 62:1672–81.