Gut 1997; 40: 761-766

Renal tubular dysfunction in patients withinflammatory bowel disease treated withaminosalicylate

S Schreiber, J Hamling, E Zehnter, S Howaldt, W Daerr, A Raedler, W Kruis

Humboldt University,Charite UniversityHospital, IVthDepartent ofMedicine(Gastroenterology)S Schreiber

University ofHamburg,Department ofMedicine,GastroenterologySection,Hamburg, GermanyS SchreiberJ HamlingS HowaldtW DaerrA Raedler

EvangelischesKrankenhaus Kalk,Department ofMedicine,Cologne, GermanyE ZehnterW Kruis

Correspondence to:Dr Stefan Schreiber,Charite der HumboldtUniversitaet,IVth Department ofMedicine,Schumannstrasse20/2110117,Berlin, Germany.Accepted for publication23 January 1997

AbstractBackground-An increasing number ofcase reports indicate potential nephro-toxicity of 5-aminosalicylic acid (5-ASA),which shares similarities with thechemical structures of both phenacetinand acetylsalicylic acid.Aim-In a point prevalence study theoccurrence of sensitive indices indicativeof early kidney malfunction was assessedin outpatients with inflammatory boweldisease.Methods-Routine indices of kidneyfunction (creatinine clearance, urinaryprotein content, pH, electrolytes, andmicroscopy) were investigated in 223patients with inflammatory bowel diseaseas well as sensitive markers of glomerularor tubular dysfunction (microproteinuriaby SDS polyacrylamide gel electrophor-esis (SDS-PAGE), urinary concentra-tions of N-acetyl-1-D-glucosamidase,aol-microglobulin, -y-glutamyltransferase(GGT), alkaline phosphatase (AP), andalbumin). Histories of exposure to 5-ASAwere assessed by questionnaire.Results-Patients receiving high amountsof 5-ASA, both actual as well as on alifetime basis, showed an increasedprevalence oftubular proteinuria by SDS-PAGE. Raised values for urinary AP andGGT indicate proximal tubular epithelialcells as the source. All other kidneyfunction tests were normal. Analysis ofcovariates indicated strong associationsbetween disease activity and size of5-ASAdoses as well as alterations in kidneytubular function.Conclusion-The possibility exists thathigh doses of 5-ASA may be associatedwith proximal tubular proteinuria. Thispoint prevalence study cannot dissect thepossible impact of chronic inflammationfrom high dose 5-ASA treatment andfiurther prospective studies are war-ranted.(Gut 1997; 40: 761-766)

Keywords: 5-aminosalicylic acid, 4-aminosalicylic acid,sulphasalazine, inflammatory bowel disease,nephrotoxicity.

Aminosalicylate compounds are widely used inboth acute phase and remission maintenancetreatment of inflammatory bowel disease.Considerations of long term toxicity seemimportant as lifetime maintenance treatiment is

usually recommended for ulcerative colitis.'`Both acetylsalicylic acid and phenacetin, whichhave been implicated in the occurrence of non-steroidal anti-inflammatory drug (NSAID)induced nephropathy, share structural simi-larities with 5-ASA.4 In addition, one studyhas indicated that 5-ASA may cause lesions tokidney tubular epithelial cells in both rats anddogs when fed in high doses.7 Studies byMahmud et al, which were conducted inparallel with the work presented here, indicatethat patients with inflammatory bowel diseasetreated with 5-ASA show an increased renalleakage of albumin, as detected by sensitivetechniques.8 9 These studies also suggestedthat microalbuminuria in patients with inflam-matory bowel disease may be activity relatedbecause a correlation with increased serumtumour necrosis factor-at (TNF-ct) concentra-tions was seen.9 No clinically relevant longterm nephrotoxicity has so far been reporteddue to chronic sulphasalazine (SASP) ormesalazine treatment.'0 The introduction ofmesalazine (5-aminosalicylic acid, 5-ASA) indifferent pharmaceutical formulations into thetreatment of patients with inflammatory boweldisease has offered the opportunity of increas-ing dosages much further than had beenpossible with sulphonamide carrier com-pounds such as sulphasalazine." 1'4 However,systemic absorption and plasma concentra-tions of 5-ASA delivered from SASP werefound to be lower than from equivalent dosesof delayed or slow release mesalazine formula-tions. "-'7 The potential for dose relatedchronic nephrotoxicity caused by long term useof mesalazines may therefore be furtherenhanced. We have conducted a point preva-lence study using sensitive indices of renaltubular and glomerular function to assess theoccurrence of altered kidney function inaminosalicylate (SASP or mesalazine) treatedpatients with inflammatory bowel disease.

MethodsA point prevalence study was performed withindividuals selected from consecutive patientsattending the Universities of Hamburg andCologne inflammatory bowel disease out-patient clinics.

ENTRY CRITERIAPatients with a confirmed diagnosis of Crohn'sdisease or ulcerative colitis of at least sixmonths duration were included. Salicylate

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Schreiber, Himling, Zehnter, Howaldt, Daerr, Raedler, Kruis

treatment had to be unchanged in the past twomonths preceding the study. Only patientsreceiving a delayed release mesalazine formu-lation, SASP, or no treatment were selected.Including patients on slow release mesalazineor olsalazine would have generated subgroupswith small patient numbers. Concomitanttreatment with corticosteroids was allowed.The maximum age was set at 60 years.Childbirth or lactation less than six monthspreviously were exclusion criteria as well asabdominal or other major operations less thansix months earlier, pre-existing renal disease(any history of renal disease dating back furtherthan the diagnosis of inflammatory boweldisease), hypertension, diabetes mellitus, or ahistory of chronic ingestion of analgesics. Allpatients having received cytotoxic drugs(methotrexate, azathioprine), cyclosporine A,or any experimental treatment for inflam-matory bowel disease within the six monthspreceding selection were excluded as well.Patients were also excluded who had signs ofurinary tract infection by microscopy whichcould be confirmed by culture.

PATIENTS

A total of 223 patients out of 497 screenedwere entered into the study (93 with ulcerativecolitis, 130 with Crohn's disease). Reasons forexclusion of 252 patients were previous useof immunosuppressive drugs or immuno-mudulators (61), previous experimentaltreatment (49), slow release mesalazine orolsalazine treatment (43), non-compliance incollecting urine or giving plasma samples (26),unwillingness to participate in the study (19),major operations (19), age >60 (15), signs ofurinary tract infections (17), history of arterialhypertension (10), chronic use of NSAIDanalgesics (9), diabetes mellitus (2), preg-nancy/childbirth (2), preexisting renal disease(2). Mean duration of disease was 58.3 (range12-132) months with a mean of 3-3 (range1-17) acute relapses. Patients were groupedinto those receiving no 5-ASA, those receivingup to 1-5 g/day, more than 1-5 but less than3 0 g/day, and those receiving 3 0 g/day ormore. In patients treated with SASP theamount of 5-ASA contained in the moleculewas calculated for evaluation. No differences indisease characteristics, disease activity, or sexwere seen between patients younger than than30 and those aged 30 or older. Moreover,differences in disease characteristics (location,Crohn's disease v ulcerative colitis, presence ofextra intestinal manifestations) betweendifferent treatment groups did not reachsignificance.

PATIENT DATA COTLLECTIONInformation on previous exposure to 5-ASAcontaining drugs, other drugs used for thetreatment of inflammatory bowel disease, andnon-steroidal analgesic drugs was obtained bya questionnaire, which was administered by thephysician to the patient. To improve theaccuracy of these data, the patients' medical

history (prescription records) was checkedwhenever possible. Disease activity scores werecalculated from diary cards which were filled inby the patients during the week preceding andthe week after analysis of urine. Patientsrecorded the occurrence of specific symptomsused to calculate the Crohn's disease activityindex'8 '9 or the clinical colitis activity index.20The Crohn's disease activity index'8 9 providesa calculated composite score which in-corporates eight items: number of liquid orvery soft stools, general wellbeing, abdominalpain, abdominal mass, relative packed redblood cell volume, body weight, extraintestinalmanifestations, use of opiate receptorstimulating drugs for the control of diarrhoea.Higher scores indicate more disease activity.Patients with a score below 150 are consideredto be in clinical remission. Scores above 450reflect severely active Crohn's disease. Theclinical colitis activity index20 incorporatesseven clinical items: number of stools, blood instools, investigator's global assessment of sym-ptomatic state, body temperature due to colitis,abdominal pain, extraintestinal manifestations,and erythrocyte sedimentation rate. Patientswith a clinical colitis activity index <4 areconsidered to be in remission.20

ANALYTICAL TECHNIQUESAll chemicals were obtained from Sigma(Munich, Germany) if not stated otherwise.Morning urine was collected and an aliquotimmediately frozen. In addition, most patientscollected 24 hour urine during the next day. Analiquot was taken and frozen for determinationof enzyme activity. The morning urine wassubjected to protein analysis by sodiumdodecyl sulphate polyacrylamide gel electro-phoresis (SDS-PAGE) as described by Pesce eta12' and Schiwara et al.22 The analysis ofmicroproteinuria by SDS-PAGE allows thedifferentiation between glomerular and tubularproteinuria by protein size and pattern. Thedetection of significant amounts of proteinwith a tubular pattern, which represents pro-teins originating from the tubular epithelium,was considered a pathological finding.2' 22 Thelower sensitivity limit for urinary proteindetection and analysis by SDS-PAGE was 5mg/l, the reference range was between 7 and 56mg/24 h (90% confidence interval). xtl-Micro-globulin (cdl-MG) and albumin concentrationswere determined by nephelometry and theactivities of N-acetyl-,B-D-glucosaminidase(NAG), -y-glutamyl-transferase (GGT), andalkaline phosphatase (AP) by their respectiveenzyme activities in 24 hour urine (BoehringerMannheim, Mannheim, Germany). To elimi-nate errors in 24 hour urine sampling agradient between enzyme activity andcreatinine concentration was calculated.23 24Normal values in healthy volunteers have beenobtained by Scherberich et al.25

STATISTICS

The Mann-Whitney U test was carried out toevaluate differences between groups.26 Analysis

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Tubular proteinuria in inflammatory bowel disease

of variance (ANOVA) or multiple cross tableanalysis was used to test for significancebetween 5-ASA exposure and prevalence oftubular proteinuria. All values are given asmean (SEM) if not indicated otherwise. Signi-ficance of differences was assumed at p<005.

Results

TREATMENTSThe Table shows 5-ASA doses taken andpopulation characteristics of the patients withinflammatory bowel disease. Forty two percent of patients received .3O0 g 5-ASA/day, 8%between 1-5 g/day and 3 0 g/day, and 32-6%a maximum of 1-5 g/day. The remaining (17%)patients received no 5-ASA during the twomonths preceding the study. Active disease wasrelated to higher 5-ASA dosages (p<0-0001,Table). Average duration of disease was 58-3(range 12-132) months with an average of3-3 (range 1-17) acute relapses. Meanduration of 5-ASA treatment was found to be32 months.

PREVALENCE OF TUBUIAR PROTEINURIA ANDRAISED URINE ALKALINE PHOSPHATASE

CONCENTRATIONSProteinuria of tubular origin detected by SDS-PAGE is a pathological finding. In our patientpopulation an increase in prevalence of tubularproteinuria was seen with increasing daily doseof oral 5-ASA (Fig 1, p=0 0086). The parallelincreased prevalence of raised urinary APconcentrations (>0.9 U/mmol creatinine,23 24p=0001), which coincided highly significantlywith the detection of a tubular protein pattern(p<O 000 1), indicated that the origin ofurinaryproteins was most likely from the proximaltubular epithelial cells (Fig 1). Eleven per centof patients receiving no 5-ASA treatmentshowed a tubular proteinuria by SDS-PAGEand 5% had raised urinary AP concentrations.Disease activity, defined by the Crohn's diseaseactivity index or the colitis activity index, wasexplored as an alternative variable of eitherprimary importance or as a confounder ofaltered renal function indices. The detection oftubular proteinuria by SDS-PAGE was moreoften seen in active disease (39 of 51 patientswith a positive SDS-PAGE had active disease,p<00001) as well as a raised urinary APexcretion (38 active out of 44 patients,p<0-0001). In active patients with inflamma-tory bowel disease 43% (51/91) showed a

Population characteristics

>1lSgand5-ASA treatment None 15 glday <30 glday 3-0 glday Total

Patients/group 38 72 18 95 223UC/CD 7/31 46/26 8/10 32/63 93/130Active patients* 3/38 8/72 8/18 72/95 91/223Male/Female 21/17 30/42 10/8 44/51 105/118Weight (kg) (mean (SD)) 66 (10-4) 64 (9 2) 63 (7-4) 62 (8 8) 63 (9-2)

220 Patients were examined out of 497 screened.*Crohn's disease activity index > 150; colitis activity index >4. These were obtained from diariescompleted by the patients one week before and the week after urinary analysis. As expected,patients with active disease received larger daily doses of 5-ASA than patients with inactiveinflammatory bowel disease (p<0-0001). UC=Ulcerative colitis; CD=Crohn's disease.

40

30-I:R

1010

None s1.5 >1.5-3.0 >3.0

5-ASA (g/day)Figure 1: Point prevalence of tubular proteinuria andincreased urine alkaline phosphatase (AP) concentrationsunder 5-ASA treatment. The prevalence of tubularproteinuria (open bars, p=0-0086) as well as of increasedurinary AP activity (>0 9 U/mmol creatinine, grey bars,p=0 001) increases with daily dosages of5-ASA.Interestingly, 11% ofpatients with inflammatory boweldisease who did not receive any 5-ASA in the two monthperiod preceeding the study showed tubular proteinuria and5% had an increasedAP activity in urine. Allp valueswere calculatedfor comparison between the >3 0 gldaygroup and the group receiving no 5-ASA.

tubular proteinuria detected by SDS-PAGE incomparison with 9-1% in inactive patients(12/132). The height of clinical disease activityscore was not found to correlate with theoccurrence of either a pathological SDS-PAGEresult or raised urinary AP concentrations.Serum soluble interleukin-2 receptor concen-trations correlated with disease activity, but nostatistical relation with the occurrence oftubular proteinuria or raised urinary AP con-centrations was seen (data not shown). Un-fortunately, the influence of disease activity(active disease v remission) could not bestatistically separated from 5-ASA dosagesbecause of a highly significant relation betweenactivity variables and 5-ASA doses (p<0 0001)both for patients with ulcerative colitis orCrohn's disease. Differences between ulcer-ative colitis and Crohn's disease or betweendifferent groups (disease location and extent,previous resections) did not reach statisticalsignificance.

URINARY ALKALINE PHOSPHATASE AND

y-GLUTAMYLTRANSFERASE ACTITIESBoth AP and GGT are sensitive markers ofdamage to the proximal tubular epithelium ofthe kidney. The results are expressed asmarker/creatinine concentrations to standard-ise for differences in timing and amount of theurine collection. We found a dose dependentincrease in urinary AP and GGT activities withincreasing 5-ASA load with differences fromnormal controls reaching significance in thegroup of patients receiving 23-0 g 5-ASA/day(Fig 2). The data suggest that AP values areincreased much further above normal thanGGT values.

ROUTINE KIDNEY FUNCTION INDICES

Excretion rates of total protein, albumin,NAG, and ax-MG were investigated. No statis-tical differences were seen between different

763

Schreiber, Hamling, Zehnter, Howaldt, Daerr, Raedler, Kruis

3-:0)

None <1.5 >1.5-3.0 u3.0

5-ASA (g/day)

Figure 2: Urinary alkaline phosphatase (AP) andy-glutamyltransferase (GGT) activity in patients withinflammatory bowel disease with 5-ASA treatment.Urinary activities ofGGT (open bars) andAP (grey bars)are shown. Values have been related to urinary creatinineexcretion to compensate for sampling errors. High dailydosages of5-ASA are related to increased activities ofGGT(*p<005) andAP (tp<002). Dotted lines show uppernormal ranges for GGT andAP urinary activities.

treatment groups or normal controls andpatients with inflammatory bowel diseasetreated with 5-ASA. Creatinine clearance andpH values were very variable, and again no

statistical differences were seen betweennormal controls and different 5-ASA treatmentgroups. No correlation was seen betweencreatinine clearance and either cumulativelifetime dose or daily dose of 5-ASA (data notshown).

CUMULATIVE 5-ASA EXPOSURE AND TUBULAR

PROTEINURIA

Cumulative 5-ASA exposure was assessed by a

questionnaire. Lifetime exposure rates to5-ASA seemed to be related (p<O0O1) to an

increased prevalence of tubular membraneprotein excretion (Fig 3). No significantcorrelation could be established between AP or

GGT values and lifetime 5-ASA dosage.Moreover, no correlation was seen betweencumulative dose and creatinine clearance,NAG values, total protein excretion, albuminexcretion, and cd-MG values.

40

35

30

S 25

C 20

15

10

0) 0co *~ 0 0

-DII- IA

5-ASA Lifetime exposure (g)Figure 3: Point prevalence of tubular proteinuria in relationto lifetime 5-ASA intake. Point prevalence oftubularproteinuria is related to the lifetime intake of5-ASA(p<001). Interestingly a tubularproteinuria is also seen in9% ofpatients with inflammatory bowel disease who hadnever received 5-ASA in their lives. The prevalence ofproteinuria with a tubular pattern in the SDS-PAGE is lessthan 1% in the control population (data not shown).

DiscussionAcute salicylate induced tubular nephro-toxicity has been seen in patients taking aspirinoverdoses or large doses of sodium salicylate.4It is debated whether chronic analgesic nephro-pathy, which is associated with consumptionof large amounts of phenacetin, is associatedwith salicylate intake as well.27 Moreover, 4-aminosalicylic acid (4-ASA), a structurallyrelated molecule which has been used for thetreatment of tuberculosis over many yearsin high systemic doses, did not generate achronic clinical problem with drug inducednephrotoxicity.28 In high doses, however, casereports indicate acute nephrotoxicity, inparticular in patients with pre-existing renaldamage.28 29 The evidence for 5-ASA orsulphasalazine (SASP) induced clinically rel-evant renal toxicity is rare. In rats and dogs,high doses of 5-ASA (far beyond those used inhuman treatment) were capable of inducingacute tubular and papillary necrosis whengiven over six to 12 months,7 30 whereas a fourweek treatment did not induce any changes.3'

In this study we have assessed sensitiveindices of renal function such as classificationof microproteinuria by SDS-PAGE to detectearly signs of tubular dysfunction. In a pointprevalence study in 223 patients with inflam-matory bowel disease we found an increasedprevalence of tubular membrane protein ex-cretion, which was related to both 5-ASA doseand disease activity. The detectable excretion oftubular membrane proteins - confirmed byincreased AP activity to originate most likelyfrom proximal tubular epithelial cells - ispathological. It often precedes the clinicalmanifestation of chronic drug induced damageto the kidney.38 39 This study confirms theearlier work by Zehnter et al, which was con-ducted in fewer patients. Zehnter et al reportedthat a pathological SDS-PAGE showingtubular proteinuria was found only in patientstreated with 5-ASA, but not in patients withinflammatory bowel disease receiving noaminosalicylates. Moreover, the authors alsofound raised urinary AP and GGT concen-trations indicating the proximal tubular epi-thelium as the source of urinary protein ex-cretion.40 Riley et al described a group ofpatients with quiescent ulcerative colitis, inwhom renal dysfunction marker excretionvalues were above the 95th percentile forhealthy volunteers, thereby raising the possi-bility of mild glomerular and tubular dys-function in these patients.'0 Recent studies haveconfirmed the presence of alterations of sensi-tive markers of renal dysfunction in patientswith inflammatory bowel disease, although norelation between aminosalicylate dose, orextent or duration of disease was seen.8 9 4 42

Moreover, abnormalities in renal functiontests were seen also in non-aminosalicylatetreated patients4' and an influence of immuno-logical disease activity on sensitive indices ofrenal function was suggested.9 Recently, renallesions resulting in signs of tubular dysfunctionwere proposed to be an extraintestinalmanifestation of inflammatory bowel disease.43In a point prevalence study like the one

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Tubular proteinuria in inflammatory bowel disease

presented here no clear distinction between theinfluence of disease activity and drug treatmentcan be made, if increased dosage of the anti-inflammatory drug used correlates highly withdisease activity. The data presented by us maytherefore be indicative of either a druginduced, dose dependent or an inflammationinduced, time dependent process capable ofimpacting on kidney function in the long term.High doses of 5-ASA may contribute to thesigns of kidney tubule dysfunction seen in thestudy. However, 11% of non-5-ASA treatedpatients with inflammatory bowel disease werealso excreting tubular proteins. Inflammatorymediators, including TNF-ot, which areincreased in inflammatory bowel disease,4-50may play an important part in inducing tubulardysfunction.9 Our study confirms the data pre-sented by Riley et allo as well as others thatNAG, x 1-MG, creatinine clearance, albumin,and total protein excretion as well as routinekidney function indices are not raised inaminosalicylate treated quiescent patients withinflammatory bowel disease. These markers, aswell as routine kidney function indices, whichhave been obtained as safety indices inremission maintenance studies using 5-ASAcontaining drugs,'1-5 may be too insensitive todetect early signs of tubular damage.52 53Moreover, the negative study by Riley et all'investigated patients with quiescent ulcerativecolitis. Tubular dysfunction, however, may berelated to both disease activity and drug.A recent series of case reports indicate that

acute renal toxicity may occur in humans withboth SASP and 5-ASA treatment, in particularwith delayed release mesalazine formulations.Clinical presentations have been typically withacute renal failure, acute nephritis, ornephrotic syndrome.32 Renal biopsies usuallyshow signs of tubulointerstitial nephritis.33 34 54Although most case reports indicate re-versibility after cessation of the drug, in somecases permanent clinical kidney dysfunctionhas been reported.33 3 54 In all cases submittedto the pharmaceutical manufacturers36 acuterenal damage was reported independent ofdosage and treatment duration. Therefore,recent case reports may reflect acute allergicevents rather than chronic toxicity. Thishypothesis is supported by a case report inwhich rechallenge with both sulphasalazineand mesalazine drugs has been undertaken andin which a dose independent relapse of haema-turia was found.37 The recent case reports ofacute tubulointerstitial damage caused byaminosalicylates therefore most likely reflect adifferent pathophysiology than the findingsreported in this study. The cases of acute renalfailure associated with aminosalicylate therapymay describe a dose independent allergiccondition, which may represent a clinical entitydifferent from the findings reported in thisstudy. Further prospective studies arewarranted to dissect the potential influence ofdisease activity from 5-ASA treatment on renaltubular dysfunction. In particular the prospec-tive evaluation of long term use of amino-salicylate drugs with different absorptionkinetics (slow release mesalazine formulations

(Pentasa), double molecules (olsalazine-dipentum, SASP, balsalazide), and delayedrelease mesalazines (Salofalk, Claversal, Asa-col)) may be of importance in dissecting theinfluence of a systemic load with 5-ASA fromthat of intestinal inflammation. Particular careshould be taken in monitoring renal functionindices (creatinine) in patients when furtherincreasing long term 5-ASA dosages for treat-ment. However, at present no clinical data havebeen presented suggesting that chronic treat-ment with aminosalicylates is unsafe unless ahypersensitivity reaction occurs.

Parts of this work have been presented at the 95th annualmeeting of the American Gastroenterological Association, 1994,New Orleans, LA, USA.We thank Dr J Armbrecht from SmithKline Beecham GmbH

(Germany), C Oerns from Pharmacia A/S (Sweden), DrTauschel from Dr Falk Pharma GmbH (Germany), and Dr MLarak from Ferring GmbH (Germany) for the helpfuldiscussion of our findings. The contribution of detailed humantoxicology and drug toxicity/side effect reports by SmithKlineBeecham (Germany/United Kingdom) and Pharmacia(Sweden) is gratefully acknowledged. Parts of this work havebeen supported by a grant of the Deutsche Forschungs-gemeinschaft (DFG Schr 512/1-2).

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