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2016Vol. 2 No. 2: 15

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Journal of Hepatitis ISSN: 2471-9706

DOI: 10.21767/2471-9706.100015

Case Report

Golubovska O1,Herasun B2,Kondratiuk L1, Nepomnyashchiy V3, Bezrodna O1,Sukach M1 and Hrytsko R2

1 BogomoletsNationalMedicalUniversity,Ukraine

2 DanyloHalytskyLvivNationalMedicalUniversity,Lviv,Ukraine

3 StateInstitution"InstituteofNephrologyNAMSofUkraine",Ukraine

Corresponding author: HerasunB

gerab@3g.ua

DepartmentofInfectiousDiseases,DanyloHalytskyLvivNationalMedicalUniversity,Lviv,Ukraine.

Tel: +38 096 8487785

Citation: GolubovskaO,HerasunB,KondratiukL,etal.ClinicalandMorphologicalFeaturesofKidneys’DamageinPatientswithChronicHepatitisC.JHep.2016,2:2.

The Aim of our Study To evaluate frequency, clinical and laboratory symptoms ofkidneys’damageinpatientswithCHC.

Introduction Chronic hepatitis C (CHC) is considered as a systemic disease,with damage of not only the liver, but also with a variety of

extrahepatic manifestations. The leading place among thembelongs tomixed cryoglobulinemia. Since the discovery of thehepatitisCvirus(HCV)in1988ithasbeenidentifiedasthemaincause of mixed cryoglobulinemia, as it was found in 90% ofpatientswithcryoglobulinemia[1,2].Theclassicmanifestationsof Cryoglobulinemic Syndrome (CGS) is weakness, arthralgiaand purpura (Meltzer triad). Other symptoms include damageof other systems such as kidneys, peripheral (less frequently

AbstractTheaimofthestudywastoevaluatefrequency,typesandrenalmanifestationsin patients with chronic hepatitis C. Our research included 109 patients withchronic hepatitis C. They were examined and treated at the Department ofInfectiousDiseasesofBogomoletsNationalMedicalUniversityandSI"InstituteofNephrology,NAMSofUkraine."RenalmanifestationsofchronichepatitisCwerediagnosedin12.8%ofcases.

Main clinical and laboratory manifestations included general weakness (wasdetectedin14(100%)patients),arterialhypertension(wasobservedin11(78.6%)patients),edema(wasobservedin9(64.3%)patients),proteinuria(wasobservedin14(100%)patients)andmicrohematuria(wasobservedin11(78.6%)patients),nephroticsyndromewasobservedin9(64.3%)patients.Arthralgiawasobservedin3 (21.4%)patients,andpurpura in1 (7.1%)patient,eGFR reducingwas in9(64.3%) patients. In 8 (66.7%) patients of our study elevated levels of serumcryoglobulins(frommildtohighlevels)weredetected.

Accordingtoourdatalong-termHCV-infectionandcirrhosiswereconsideredriskfactors for the development of extrahepatic immunological diseases, includingrenaldisease.

AnimportantroleofrenalbiopsyinthedifferentialdiagnosisanddeterminationoffurthertreatmentplanofpatientswithrenalimpairmentinchronichepatitisCwaspointedout.WenotedthatkidneydamageinpatientswithchronichepatitisCinmostofthecaseswereassociatedwiththecryoglobulinemicsyndrome.Weperformedrenalbiopsiesin12patientswithsymptomsofchronickidneydiseaseandcryoglobulinemicglomerulonephritiswasdetectedin7(58.3%)patients.Othertypesofkidneydamagedetected inour study includedmembranoproliferativeglomerulonephritiswithout cryoglobulinemia,membranous nephropathy, focalsegmentalglomerulosclerosis,mesangioproliferativeglomerulonephritis.

Keywords: ChronichepatitisC;Cryoglobulinemicsyndrome;Membranoproliferativeglomerulonephritis

Clinical and Morphological Features of Kidneys’ Damage in Patients with Chronic

Hepatitis C

Received: February23,2016; Accepted: September15,2016; Published: September22,2016

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Journal of Hepatitis ISSN: 2471-9706

central)nervoussystem,gastrointestinaltract, lungs,etc.[3,4].RenaldiseaseinpatientswithHCV–infectionisconsideredtobeaprovenfact[5,6].ThemostcommonisMembranoproliferativeGlomerulonephritis(MbPGN),whichisassociatedwiththeIItypeofcryoglobulinemia(cryoglobulinemicGlomerulonephritis(GN).Less commonMbPGNwithout cryoglobulinemia,MembranousGlomerulonephritis(MGN),FocalSegmentalGlomerulosclerosis(FSGS), mesangioproliferative glomerulonephritis, fibrillaryglomerulonephritis and imunotaktoid glomerulopathy [7-17]. There are also other atypical cases of kidneys’ damagein HCV–infection–IgA nephropathy, rapidly progressiveglomerulonephritis, thrombotic microangiopathy [18-20].Isolated cases of albuminuria without kidney damage in HCVinfection were also described [16]. In general, the prognosisfor HCV–associated glomerulonephritis remains poor, not onlybecauseoftheprogressionofkidneys’diseaseanddevelopmentoflivercirrhosis,butalsothroughthehighriskofcardiovascularcomplications.

MethodsThe study was conducted at the Department of InfectiousDiseases of Bogomolets National Medical University and atthe Department of Nephrology and Dialysis of SI "Institute ofNephrologyofNAMSofUkraine."Weobserved109patientswithCHC. Patient selection criteriawas diagnosed chronic hepatitisC,accordingtotheclassificationofchronichepatitis,proposedattheInternationalCongressofGastroenterology(LosAngeles,1994) [21] which was verified by the identification of specificserological and molecular genetic markers of HCV. CGS wasdiagnosedaccording to the recommendationsof theEuropeanAssociationfortheStudyofLiver(EASL,2012)basedonpresencein serum of mixed cryoglobulins (CGs), antibodies to HCV,positive rheumatoid factor (RF), reduction of complement C4,morphological (leukocytoclastic vasculitis, infiltration of tissuesbymonoclonalB-cells)andclinical(purpura,fatigue,arthralgia,MbPGN,peripheralneuropathy)criteria[22].

Clinicalexamination,generallaboratorytests,biochemicaltests,serological and molecular genetic studies in dynamics wereperformedinaccreditedcommerciallaboratories.

Thecryoglobulinslevelsweredetectedusingspectrophotometrymethod by calculating the difference of the optical density ofthesolutionof serum inabuffer (pH=8.6)at4°Cand37°C [4].Glomerular filtration rate (GFR) was estimated according toMDRD[23].

Morphological study of renal samples were performed in 12patients. Presence of clinical and/or laboratorymanifestationsof chronic kidney disease in patients with chronic hepatitis Cfortheperiodof3monthsorlongerwerethemainindicationsforrenalbiopsy.Renalsampleswereobtainedbypercutaneousneedlebiopsyofthekidneybyastandardtechniqueunderthesupervisionoftheultrasoundscanner.Weusedthemethodsoflight,immunofluorescentandtransmissionelectronmicroscopy.For light microscopy paraffin sections were stained withhematoxylin and eosin,Masson's trichrome, Congo red, Schiffreagent. Jones silver stainingwas also used. Directmethod ofCoonswithFITC labeledpolyclonalantibodies to IgA, IgG, IgM,

and light chains κ and λ, fibrinogen and complement fractionsC1qandC3 indilution1:10 - 1:60 (DAKO,Denmark)wasusedonfrozensectionsofkidneysfortheimmunofluorescencestudy.FluorescencemicroscopeOlympus BX-51 (Japan)was used forthe light and immunofluorescence studies. The material forthe electron-microscopic studywas embedded in EPON-aralditmixtureusinganultramicrotomeLKB-III(Sweden)forproductionofsemithinandultrathinsections.Semithinsectionswerestainedwithtoluidineblue.Ultrathinsectionswerecontrastedwith2%solution of uranil acetate and plumbum citrate and examinedwith an electronmicroscope TEM-125K (SELMI,Ukraine) at anacceleratingvoltageof60kV.Morphologicaldiagnosiswasbasedonmorphologicalclassificationofrenaldiseasesfornephrologypractice,2010[23].

Statistical analysis of the results was performed by usingdescriptivestatistics.Calculationsoftheresultswereperformedusingthesoftwarepackage"Statistica6.0".

ResultsWe observed 109 patients with CHC. Renal lesions werediagnosedin14(12.8%)patients.BaselinecharacteristicsofthepatientspresentedinTable 1.

Laboratory tests showed next results: 9 (64.3%) patientswere anemic, 7 (50.0%) patients had elevated ESR level, 5(35.7%) patients had increased creatinine level, 6 (42.9%)patients had increased urea level, 10 (71.4%) patients hadhypoproteinemiaandhypoalbuminemia,9(64.3%)patientshadhypercholesterolemia, 9 (64.3%)patientshaddecreasedeGFR.ItwasnotedthathyperbilirubinemiaandincreasedALTandASTlevelthatindicatetheactivityofhepatitisweredetectedonlyin3(21.4%)and1(7.1%)patientsrespectively.

Resultsoftheurinalysesshowedthatallpatientshadproteinuriawhich ranged from 0.24 g/l to 9.5 g/l. Erythrocyturia (from10 erythrocytes to solid in sight) was observed in 11 (78.6%)patients.Leykocyturiawasobservedin9(64.3%),cylindruria–in12(85.7%)patients.Nephroticsyndromeoccurredin9(64.3%)patients.

Thus, according to the results of clinical and laboratoryexamination the symptoms of kidney damage in patientswithCHCweredominantandtheactivityofhepatitiswasminimalinthemajorityofthesepatients.However,itshouldbeemphasizedthathepaticfibrosisF3-F4wasdetectedin5(35.7%)patients.

Age,years 40,3±12,6Sexratio(M/F) 8/6

EstimateddurationofHCV-infection,years 10,2±3,7Generalweakness 14Hypertension 11

Lowerextremitiesandfaceswelling(edema) 9Drymucousmembranes 2

Arthralgia 3Purpura 1

Dyspepticsyndrome 2

Table 1.BaselinecharacteristicsofpatientswithCHCandrenallesions,n=14.

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Renalneedlebiopsywithmorphologicalstudywasperformedin12 patientswith chronic kidney disease. In other twopatientswith transient proteinuria were no indications for biopsy.CryoglobulinemicGNwasdiagnosedin7of12patients(58.3%)according to the results of morphological studies of renalspecimens.TwopatientshadMbPGNwithoutcryoglobulinemia.Membranousnephropathy,FSGSandmesangioproliferativeGNwasdiagnosedin1patientrespectively.

Increased levels of serumCGs (frommild to high levels)werediagnosed in 8 (66.7%) patients. Seven of these patients hadcryoglobulinemicGNandonepatienthadFSGS.

An increased size of the glomeruli with global endocapillaryhypercellularity and excess of mesangial matrix was observedin all cases of HCV-associated MbPGN with and withoutcryoglobulinemia using light and electron microscopic study.Diffusethickeningoftheglomerularbasementmembrane(GBM)wasdifferentlyexpressedindifferentcapillaryloops.ThickeningpartsofGBMatthetestsamplesstainedwithSchiffreagentandJones silver impregnation looked like double-contoure (Figure 1a). Infiltration by mononuclear cells of varying degrees ofseveritywereobservedin4(33.3%)cases.Cellularandfibrous-cellular crescentsweredetected in2 (16.7%)cases.Numeroussubendothelial and mesangial electron-dense deposits wereidentified using electron-microscopic study. They were smallanddiscreteandalsolargeandelongated.Additionalmembraneatthepericapillarypartwasoftenseenaswellas interpositionof mesangial cells. Subepithelial electron-dense deposits wereobserved in 2 (16.7%) cases. The fusion of small processes ofpodocyteswasnotedasacharacteristicfeature.

The focal tubular atrophy was detected in 6 (50.0%) patientsand accumulation of foamy macrophages was found in theinterstices of 3 (25.0%) patients. The areas of fibrosis weredetectedin5(41.6%)patients,focalinflammatoryinfiltration-in7(58.3%)patients.2(16.7%)patientshadfeaturesofmyointimalhyperplasiaofsmallarteries.

AcharacteristicfeatureofcryoglobulinemicGNwasthepresenceofeosinophilicandPAS-positivehyalinethrombiinthelumenofglomerularcapillaries.Theywereeithersingle(insomecapillaryloopsoftheglomeruli)ornumerous(inmostoftheglomerularloops) (Figure 1b). The expressive glomerular mononuclearcells infiltrationwasalsofound.Dataobtainedduringelectron-microscopicexaminationshowedthepresenceofsubendothelial,mesangial and intraluminal electron-dense deposits. In somecases itwaspossible todetect theirmicrotubuleor crystalloidstructurebyhighmagnification.

ImmunofluorescenceinvestigationinallcasesofHCV-associatedMbPGNshowedsmallandlargegranullardepositsofC3,IgMandIgGalongthecapillarywallsandatthemesangium.ThehighestintensityofluminescencewastypicalforC3andIgM(Figures 1c-1e).IntraluminaldepositsincasesofcryoglobulinemicGNwerepositiveforIgMandIgG,aswellasforC3.Immunofluorescenceinvestigation data of patients with HCV-associated MbPGN isshowninFigure 2.

DiscussionRenaldiseasecandevelopwithina fewyearsorevendecadesafterinfectionwithHCV.Accordingtotheliteraturedata,themostcommon form of kidneys’ damage in CHC is cryoglobulinemicMbPGN (PbPGN type I), which is usually associated with typeII of cryoglobulinemia [5-10]. Usually MbPGN is diagnosed inpatientswithlong-termHCV-infection.InourstudythedurationofHCV-infectionwas10.2±3.7yearsandtheaverageageofthepatientswas40.3±12.6years.AccordingtotheliteraturedataMbPGNtypeIisoftendiagnosedinpatientswithHCV-associatedlivercirrhosis[24,25].HepaticfibrosisF3-F4wasdiagnosedin5(35.7%)patientsinofourresearch.Thus,long-termHCV-infectionandhepaticfibrosisF3-F4areriskfactorsforthedevelopmentofkidneydamageinpatientswithCHC.

Clinical course of the disease in patients with impaired renalfunction usually include other symptoms of cryoglobulinemia:purpura, arthralgia, fatigue, which were also seen in patientsof this study (general weakness – in 14 (100%) patients,arthralgia- in3(21.4%)patients,purpura- in1(7.1%)patient.According to the literature data, symptoms of kidney damageapproximately occur in one third of patients with CGS. Renalsymptoms of cryoglobulinemia include nephrotic syndrome,isolated proteinuria (that can be the only symptom of kidneydamageinCHCforalongtime),microscopichematuria,80%ofpatientsdevelophypertension [26]. Inour study,hypertensionwas diagnosed in 11 (78.6%) patients, swelling of the lowerextremities and face - in 9 (64.3%) patients, proteinuria wasfound in all patients, microhematuria–in 11 (78.6%) patients.Nephroticsyndromeoccurredin9(64.3%)patients.

Theresultsofthisstudyalsoconfirmstudiesofforeignauthors:the majority of patients–7 (58.3%)–with chronic hepatitisC and kidney lesions were diagnosed morphologically withcryoglobulinemicGN,themajorpathogeneticlinkofwhichisthedepositionoftheCGinthecapillariesandglomerularmesangial.However, therewereother typesof renal injury in this study–MbPGN without cryoglobulinemia (in case of which thedepositionofimmunecomplexes(HCVantigens,IG,complementfragments)occurredinthemesangium);MGN(incaseofwhichthesubepithelialdepositionof immunecomplexes(antigensofHCV, IG, complement fragments) develops); FSGS (associatedwith direct damage of podocytes caused by hepatitis C virus)andmesangioproliferativeGN(causedbythedirectactionofthehepatitis C virus on themesangium through TLR-3 orMMP-2)[27,28].

ConclusionsThus, our study showed that the renal manifestations of HCVinfection appear in 12.8% of cases. The main clinical andlaboratorymanifestationsinpatientsofourstudyweregeneralweakness, hypertension, lower extremities and face swelling,proteinuriaandmicrohematuria,nephroticsyndromeandeGFRreduction.Wealso observed such symptoms as arthralgia andpurpura. In our study levels of serum CGswere elevated in 8(66.7%)patients(frommildtohighlevels).

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HCV– associated MbPGN. A. MbPGN without cryoglobulinemia. Double–contoure of GBM. Jones silver impregnation. 400Xmagnification. B. cryoglobulinemic MbPGN. Double–contoure of GBM and intraluminal deposits. Jones silver impregnation.400Xmagnification.C.MbPGNwithoutcryoglobulinemia.Subendothelialandmesangialgranulardeposits.Immunofluorescenceinvestigation.IgM.400Xmagnification.D.CryoglobulinemicMpGN.Intraluminaldeposits.Immunofluorescenceinvestigation.IgM.400Xmagnification.E.CryoglobulinemicMpGN.Microtubuledeposits.Theelectron–microscopicexamination.2400Xmagnification.

Figure 1

4

3

2

1

0IgA IgG IgM kLC 1LC C1q C3

Cryoglobulinemic GN MbPGN without cryoglobulinemiaAverage luminescence intensity of immunoglobulins and complement in patientswithHCV– associatedMbPGN, the results ofimmunofluorescencestudies,n=9.

Figure 2

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Journal of Hepatitis ISSN: 2471-9706

Theresultsofourstudyshowedthatthelong-termHCV-infectionand cirrhosis were risk factors for the development of renallesions.

Needlekidneybiopsy is important in thedifferentialdiagnosis,since clinical manifestations of kidney damage are usually

nonspecific.Accordingtotheresultsofourstudy, themajorityof renal lesions in chronic hepatitis C was linked to the CGS,ascryoglobulinemicGNwas found in7 (58.3%)of12patients.Therewerealsoothertypesofkidneydamage–amembranousnephropathy,MbPGNwithoutcryoglobulinemia,focalsegmentalglomerulosclerosisandmesangioproliferativeGN.

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