Gabl, F.

Kopsa, H.

Lapin, A.

Zazgornik, J.

Zyman, H.

Clinical Chemistry and Laboratory Medicine (CCLM)

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La p in et al.: Monitoring of urinary proteins 221J. Clin. Chem. Cün. Biochem.Vol. 24, 1986, pp. 221-225© 1986 Walter de Gruyter & Co.Berlin · New YorkMonitoring of Urinary Proteins by SDS Electrophoresis in KidneyTransplant PatientsBy A. Lapin, H. Zyman, F. GabiInstitut für Klinische Chemie und Laboratoriumsdiagnostik\ j H. Kopsa and J. ZazgornikL Medizinische UniversitätsklinikMedizinische Fakultät, Universität Wien, Vienna, Austria\l (Received September 28/November 29, 1985)Summary: Sodium dodecyl sulphate (SDS) electrophoresis of urinary proteins was used routinely for monitor-ing more than 80 kidney transplant recipients äs out-patients during one year. Special attention was paid tothe question of whether this method can help the clinieian to differentiate between a graft rejection reactionand Cyclosporin A-induced nephrotoxic damage. Two cases are presented showing the time course ofproteimiric patterns together with serum creatinine, total urinary protein and, in one case, the blood levelof Cyclosporin A. Changes of proteinuric patterns signalled the start of rejection and/or nephrotoxicity. Ahomogeneous collective of 33 kidney recipients (first transplantation) with chronic glomerulonephritis äs abasic disease was specially selected. In this collective, the distribution of proteinuric patterns among patientsand its dependence on immunosuppresive therapy was investigated. During the observation period of 7months, two proteinuric patterns, "tubular" and "mixed weak", were found exclusively in Cyclosporin A-treated patients. We ascribe this finding to nephrotoxic effect of Cyclosporin A. We found the SDSelectrophoresis of urinäry proteins to be a useful, non-invasive method for monitoring Cyclosporin A-treatedkidney transplant patients.Überwachung von Harnproteinen mit SDS-Elektrophorese bei nierentransplantierten PatientenZusammenfassung: Die Natriumdodecylsulfat-(SDS-) Elektrophorese von Harnproteinen wird seit einemJahr zur routinemäßigen Überwachung von mehr als 80 ambulanten Nierentransplantierten angewendet.Insbesondere sollte geklärt werden, ob diese Methode für den Kliniker eine Hilfe beim Erkennen und derDifferentiaidiagnose zwischen Cyclosporin -induzierter nephrotoxischer Einwirkung und Abstoßungsreak-tion bietet. Es wurden zwei Fälle präsentiert, bei denen neben zeitlichen Änderungen des Proteinuriemustersauch Verläufe von Kreatinin im Serum, Protein im Harn und, in einem Fall, Konzentration von CyclosporinA im Blut dargestellt sind. Änderungen im Verlauf des Proteinuriemusters signalisierten jeweils beginnendeAbstoßungsreaktionen bzw. nephrotoxische Einwirkungen von Cyclosporin A. Ein homogenes Kollektiv von33 Nierentransplantierten (erste Transplantation) mit chronischer Glomerulonephritis als Basiserkrankungwurde speziell ausgesucht. Es wurde in Abhängigkeit von immunsuppresiver Therapie die Verteilung desjeweiligen Proteinuriemusters auf die einzelnen Patienten untersucht. In 7 Monaten Beobachtungszeit wurdenbei den Cyclosporin -behandelten Patienten exklusiv zwei Proteinuriemuster gefunden: „tubular" und„gemischt—schwach". Wir schreiben diesen Befund der Cyclosporin -induzierten nephrotoxischen Einwir-kung zu. Wir fanden die SDS-Elektrophorese von Harnproteinen als eine wertvolle Methode, die hilfreichbei nicht-invasiver Überwachung von nierentransplantierten Patienten eingesetzt werden kann.J. Clin. Chem. Clin. BipcheraL / Vol. 24,1986 / No. 4222 Lapin et al.: Monitoring of urinary proteinsIntroductionThe immunosupressant "Cyclosporin A" (cyclospo-rin) is widely used to prevent allograft rejection oforgan transplants (l, 2). However, in view of thedanger of serious side effects, it is necessary to mea-sure the circulating levels of cyclosporin, in order toadjust the dosage to the optimal level (3, 4). Thenephrotoxic side effect of cyclosporin is a mostdangerous complication in kidney transplantationand therefore requires special attention (4—8). Thedecision äs to whether graft rejection and/or nephro-toxicity are responsible for the restriction of the func-tionning of the transplanted kidney is a current clini-cal problem (8). Sodium dodecyl sulphate (SDS) elec-trophoresis of urinary proteins can be useful for thedetection of cyclosporin-induced nephrotoxicity. Thismethod provides Information about the distributionof molecular weights of proteins in pathological ur-ines. The molecular weight spectrum of urinary pro-teins is, to a, large extent, indicative of the affectedpart of the glomerulo-tubular apparatus of the kidney(9, 10). High molecular weight proteins are assignedto glomerular damage, low molecular weight proteinsto tubular damage. SDS electrophoresis permits con-tinuous and non invasive monitoring, and is es-pecially suitable for detecting and distinguishing be-tween changes in proteinuric patterns, which indicatethe onset of damage due either to cyclosporin-in-duced nephrotoxic damage, or to a rejection.Materials and MethodsPatientsWe have used SDS electrophoresis of urinary proteins routinelyover a period of one year for the weekly check up of kidneygraft recipients äs out patients of our nephrological unit. Morethan 80 renal transplant patients havefbeen examined in thisway. From these, a homogeneous group was selected, who hadreceived only one transplant and whose original complaint hadbeen chronic glomerulonephritis (tab. l, top). Their urines wereanalysed by SDS electrophoresis at l to 4 week intervals düring7 months. Seventeen of these patients were treated with lowdoses of steroids and cyclosporin and another 16 patientsrecieved conventional immunosupressive therapy consisting ofazathiaprine and prednisolone.Cyclosporin dosageWe started with a daily intravenous cyclosporin dose of 5 mg/kg body weight, given continuously düring the first 3^-5 daysafter surgery. This regimen was changed to an oral dose of 15mg/kg body weight per day divided in 2 or 3 equal portioiis.Depending on the cyclosporin concentrations in the blood thedaily cyclosporin dose was then reduced to a maintenancedose of 5—8 mg/kg. The conventional therapy consisted ofazathioprine (2 mg/kg · day) and prednisolone (10 ml per day),or an alternative schedule of 10 or 25 mg prednisolone everysecond day. For controls, the findings of SDS-electrophoresiswere compared with the same kind of analysis of urines from17 -transplanted patients, suffering from chronic glomeru-lonephritits.Samples and SDS electrophoresisFour hour urine samples were collected under sterile conditionsand submitted on the same day to SDS electrophoresis orTab. 1. Patients with the basic renal disease, chronic glomerulonephritis.Immunosuppressive therapyNumber of patientsMean age (years)Mean time aftertransplantation (years)Observation timeTransplant patientscorticosteroid + cyclosporin1744.9 ± 6.41.6 ±0.5Transplant patients · ····azathioprine -h prednisolone1646.9 ± 12.68.4 + 3.7Non-transplant patientsnone1744.1 ± 12.9—7 months in 1 to 4 weeks intervalsDistr ibution (n) of proteinuric patterns among patientsProteinuric patteraPhysiologicalGlomerularselectivemedium selectivenon-selectiveTubularMixedweakmediumintensive1215724671757500.354541001i'7J. Clin. Chem. Clin. Bic-chem. / VoL 24» 1986,/ No. 4Lapin et al.: Monitoring of urinary proteins 223stored deep frozen (—20 °C) until analysed. Deep freezing didnot affect the result of the SDS electrophoresis. No preser-vatives were added to samples and no concentration procedureswere necessary.An adapted horizontal SDS electrophoresis was used permittingsimultaneous analysis of 22 samples s described elsewhere (11).Results and DiscussionUsing the SDS electrophoresis we were able to moni-tor acute s well s chronic changes of kidney graftfunction.Evaluation of proteinuric pattern in the SDS electro-phoresisTable 2 shows a list of proteins found usually in pathologicalurine samples with their molecular weights. These data, re-ported in the literature (10, 12) were verified by our ownimmunoblotting experiments. According to their pathophysiol-ogical origin two groups of urinary proteins can be distinguish-ed:ι — glomerular proteins cornprising albumin and proteins withl Mr > 67000— tubular proteins with MT < 67000Tab. 2 Urinary proteinsGlomerular proteins1800001500001300007700067000apolipoprotein Aimmunoglobulinpolymeric albumintransferrinalbuminMr Tubular proteins540004400040000210001500011800ott-antitrypsincc2-acid glycoprotein2-glycoproteinretinol binding proteinlysozyme2-microglobulinWe differentiated four main proteinuric patterns with somesubdivisions:1. physiologlcal: total protein below 0.15 g/l2. glomerular: containing only glomerular proteins subdividedinto— selective (containing only albumin)— medium selective (containing only albumin and transferrin)— non selective (containing albumin, transferrin and immuno-globulin)3. tubular: containing albumin and proteins with MT < 67000,with total protein > 0.15 g/l4. mixed: proteinuric pattern, consisting of glomerular andtubular; according total protein subdivided into:— weak (0.15 to 0.3 g/l total protein)— medium (0.3 to 1.0 g/l total protein)— intensive (more than 1.0 g/l total protein)J. Clin. Chem. Clinu Biochem. / Vol. 24,1986 / No. 4Case lFigure l illustrates the time course of the pattern ofurinary proteins, serum creatinine and total urinaryprotein of a 17 year old girl with diabetes type I. Shereceived a kidney graft and developed acute rejectionafter 5 weeks (35 days). The proteinuric patternchanged within this time from "glomerular unselec-tive" to "mixed intensive". The change of proteinuricpattern was accompanied by elevation of serum crea-tinine (28th day), both observed 4 weeks before thedefinitive increases of total urinary protein (39th day).On the 43rd day the kidney graft was removed.Case 2In figure 2, the time course of the proteinuric pattern,serum creatinine and cyclosporin blood levels can beseen. A 35 year old man with chronic glomeru-lonephritis received a kidney graft under treatmentwith low doses of corticosteroids and cyclosporin.In the 10th and 12Λ week after transplantation tworejection episodes were observed, accompanied by achange of proteinuric pattern towards "mixed inten-sive". Later, after recovery after the 21st week, a"tubular" proteinuric pattern was observed togetherwith a continuous elevation of serum creatinine (upto 372 μπιοΐ/ΐ in the 27Λ week). Since the cyclosporinblood level was near the suggested upper therapeuticlimit (3, 4) between the 24th and 27th week, we canInterpret the occurrence of "tubular" proteinuria sa cumulative transient toxic effect of cyclosporin ontubular cells, s recently reported in patho-morpho-logic studies (4, 5, 7).Patients with chronic glomerulonephri t is sa basic diseaseTable l (bottom) indicates the results of SDS electro-phoresis of urine samples during 7 months monitor-ing. Comparing the number of patients, in whichdifferent proteinuric patterns were found, we can seethat two types were found exclusively in patientstreated by low dose corticosteroids and cyclosporin:- "tubular" (in 3 of 16 patients)- "mixed weak" (in 8 of 16 patients)224 Lapin et al.: Monitoring of urinary proteinsA/r C=> O CD C3 C=> CD C2> iS. «94000e> c—| 67000°" 4300030000l 20000,2 144001000E±500o+1007 8 9 10 II 12 13 28 36 37 38 39 41 42 43Oays öfter transplantationFig. 1. All year old kidney transplant girl witb diabetes type I.Time course of proteinuric pattern (top) together withserum creatinine and total urinary protein (bottom).w 9400— f 67000°" 4300030000ö 20000•2' 144003 400« 300*?200!E os l100o "6 7 8 JO 11 12 J8 19 2\ 2 3 2 4 2 5 2 6 2 7 29Weeks öfter transplöntation l\^ ~^.—*·*"""* \ !\+—+.«../ V V + V^+^^.|^ >.|Xl"yAf'SIc_- >«-·2 »?1000-^ -—500 c -^f!Fig. 2. A 35 year old man, with chronic glomerular nephritisäs basic disease, receiviiig bis first kidney graft. Timecourse of proteinuric pattern together with serum crea-tinine total urinary protein and blood level of cyclospo-rin.The distribution of proteinuric types in patientstreated by conventional therapy (azathioprine andprednisolone) is comparable to that of the controlgroup of non transplant patients with chronic glo-merulonephritis; moreover, tubular and mixed weakproteinuric types do not occur in these two groups.The results agree with recently postulated nephro-toxic effects of cyclosporine (7), where the tubule isbelieved to be the primary site of an injury that isinitially reversible. Multiple injury of tubules thenleads to degeneration of entire nephrons.This pathogenetic mechanism seems to correlate withthe SDS electrophoretic findings: tubular injury leadsto low molecular weight proteins in the urine. De-generation of a few entire nephrons can be seen äs acause of "mixed weak" proteinuria. In conclusion wecan say that SDS electrophoresis of urinary proteinsprovides useful, plausible and illustrative Informationfor the clinician, especially with regard the changingStatus of patient. In addition, the method is non-invasive.Moreover, the diagnosis of tubular or glomerularproteinuria by SDS electrophoresis can be based en-tirely on the occurrence of a characteristic pattern ofurinary proteins. This can be seen äs a main advan-tage in comparison with other methods, which mea-sure a single parameter such äs urinary ß-2-microglo-bulin or certain enzymes. The widely used measüre-ment of ß-2-microglobulin äs a marker for tubulardamage is limited, because bf its instability in acidurine samples (9). The measurement of enzymes inurine is üseful for the diagnosis of tubular damage,but this measurement is of low significance in thedifferentiation between a rejection episode and cyclo-sporin töxicity in renal transplant patients.According to our studies, SDS electrophoresis is aüseful accessory diagnostic method for differentiatingbetween kidney graft rejection and the nephrotoxicside effect of cyclosporin A.AcknowledgementWe tbank to Dr. Ch. Mannhalter for immunqblotting assaysand Dr. J. Schindler for providing measurements of CyclosporinA in blood.We are greatful to Mrs. B. Neidhard for preparing thescript.J. Clin. Chem. Clin. Biochem. / Vol. 24,1986 / No. 4Lapin et ai.: Monitoring of urinary proteins 225References1. Canadian multicentre transplant study group (1983) N.Engl. J. Med. 309, 809-815.2. Beveridge, T. (1983) Transplant. Proc. 15, 433-437.3. Bowers, L. D. & Canafox, D. M. (1984) Ther. Drug Monit.6, 142-147.4. Thiel, G., Harder, F., Lörtscher, R., Brünisholz, M., Land-mann, J., Brunner, F., Follath, F., Wenke, M. & Mihatsch,M. (1983) Klin. Wochenschr. 61, 991-1000.5. Clane, R. Y., Thirn, S., McMaster, R, Craddoch, G. N.,White, D. J. G., Evans, D. B., Dünn, D. C., Pentlow,B. D. & Rolles, R. (1978) Lancet //, 1323-1327.6. Klintmalm, G. B. G., Iwatsuki, S. & Starzl, T. E. (1981)Lancet /, 470-471.7. Myers, D. H., ROSS, J., Newton, L., Luetscher, J. &Perlroth, M. (1984) N. Engl. J. Med. 311, 699-705.8. Taube, D. H., Williams, D. G., Hartley, B., Rudge, G. J.,Neild, G. H., Cameron, J. S., Ogg, G. S. & Welch, K. I.(1985) Lancet //, 171-174.9. Hardwicke, J. (1984) Clin. Nephrol. 21, 50-53.10. Boesken, W. H. (1981) In: Electrophoresis' 81 (Allen,R. C. & Arnaud, R, eds.) pp. 259-270, Walter de Gru-yter & Co, Berlin, New York.11. Lapin, A., Ogunyemi, E. O., Zyman, H., Gabi, F. & Zaz-gornik, J. (1985) J. Clin. Chem. Clin. Biochem. 23,777-780.12. Lubega, J. (1983) Clin. Chim. Acta 128, 151-168.DDr. Alexander LapinInstitut für Klinische Chemieund Laboratoriumsdiagnostikder Universität WienLazarettgasse 14A-1090 Wien;; J. Clin. Chem. Clin. Biochem. / Vol. 24,1986 / No. 4

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Monitoring of Urinary Proteins by SDS Electrophoresis in Kidney Transplant Patients

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Monitoring of Urinary Proteins by SDS Electrophoresis in Kidney Transplant Patients

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