Multicenter Study on Hepatitis C Virus–Related Cryoglobulinemic Glomerulonephritis

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Multicenter Study on Hepatitis C Virus–RelatedCryoglobulinemic Glomerulonephritis

Dario Roccatello, MD,* Alessandro Fornasieri, MD,* Osvaldo Giachino, MD, Daniela Rossi, MD,Alessandra Beltrame, MD, Giovanni Banfi, MD, Roberto Confalonieri, MD, Antonio Tarantino, MD,

Sonia Pasquali, MD, Antonio Amoroso, MD, Silvana Savoldi, MD, Valeriana Colombo, MD,Carlo Manno, MD, Antonio Ponzetto, MD, Luigi Moriconi, MD, Antonello Pani, MD,

Roberto Rustichelli, MD, Giovanni Barbiano Di Belgiojoso, MD, Chiara Comotti, MD,and Maria Ida Quarenghi, MD

Background: Mixed cryoglobulinemia is a multisystem disorder associated strongly with hepatitis Cvirus (HCV) infection. The kidney frequently is involved, and glomerulonephritis represents the keyfactor affecting prognosis.

Methods: Clinical, serological, immunogenetic, and morphological data were collected retrospec-tively from medical records of 146 patients with cryoglobulinemic glomerulonephritis who underwentbiopsies in 25 Italian centers and 34 cryoglobulinemic controls without renal involvement.

Results: Eighty-seven percent of patients were infected with HCV; genotype 1b was more frequentthan genotype 2 (55% versus 43%). Diffuse membranoproliferative glomerulonephritis was the mostprevalent histological pattern (83%). Type II cryoglobulin (immunoglobulin M� [IgM�]/IgG) was detectedin 74.4% of cases. The remainder had type III (polyclonal IgM/IgG) cryoglobulins. A multivariate Coxproportional hazard model showed that age, serum creatinine level, and proteinuria at the onset of renaldisease were associated independently with risk for developing severe renal failure at follow-up. Overallsurvival at 10 years was about 80%. Kaplan-Meier survival curves were worsened by a basal creatininevalue greater than 1.5 mg/dL (�133 �mol/L), but were unaffected by sex and HCV infection. Cardiovas-cular disease was the cause of death in more than 60% of patients.

Conclusion: Data confirm the close association between mixed cryoglobulinemia and HCV infectionand between glomerulonephritis and type II cryoglobulin. Survival profiles are better than previouslyreported in the literature, probably because of improvement in therapeutic regimens. Causes of deathreflect this improvement in survival, with an increased prevalence of cardiovascular events comparedwith infectious complications and hepatic failure, which were predominant in the past.Am J Kidney Dis 49:69-82. © 2006 by the National Kidney Foundation, Inc.

INDEX WORDS: Mixed cryoglobulinemia; cryoglobulinemic glomerulonephritis; hepatitis C virus (HCV)–associated glomerulonephritis; membranoproliferative glomerulonephritis; cryoglobulins; HCV syndrome.

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ixed cryoglobulins are plasma proteinsthat precipitate reversibly at low tempera-

ures and are composed of immunoglobulins,

From the Centro Universitario di Ricerche di Immu-opatologia e Documentazione su Malattie Rare, Osped-le S.G. Bosco; SC Immunologia dei Trapianti, Aziendaspedaliera S.G. Battista; Dipartimento di Gastroentero-

ogia, Azienda Ospedaliera S.G. Battista, Molinette,orino; Divisione di Nefrologia e Dialisi, Ospedale S.arlo Borromeo; Divisione di Nefrologia e Dialisi, Osped-le Maggiore IRCCS; Unità Operativa di Nefrologia,ialisi e Terapia Medica del Trapianto Renale, Ospedaleiguarda Cà Granda; Unità Operativa di Nefrologia eialisi, Ospedale Sacco, Milano; Divisione di NefrologiaDialisi, Policlinico S. Orsola Malpighi, Bologna; UOAefrologia e Dialisi, Ospedale Civile Ciriè; Divisione diefrologia, Dialisi e Trapianti, Policlinico, Bari; Nefrolo-ia e Dialisi, Ospedale degl’Infermi, S. Miniato; Divi-ione di Nefrologia e Dialisi, Ospedale Regionale S.ichele, Cagliari; Divisione di Nefrologia, Arcispedale

. Maria Nuova, Reggio Emilia; UO Nefrologia e Dialisi,
spedale S. Chiara, Trento; and Divisione di Nefrologia,
merican Journal of Kidney Diseases, Vol 49, No 1 (January), 200

ncluding 1 with rheumatoid activity. Two typesf mixed cryoglobulins can be defined on theasis of the classification of Brouet et al.1 Type II

ialisi e Terapia del Trapianto Renale, Azienda Osped-liera S. Anna, Como, Italy.Received April 24, 2006; accepted in revised form Septem-

er 27, 2006.Originally published online as doi:10.1053/j.ajkd.2006.09.015

n November 28, 2006.*D.R. and A.F. were the principal investigators and writ-

rs; they equally contributed to this work.Support: None. Potential conflicts of interest: None.Address reprint requests to Dario Roccatello, MD, Centroultidisciplinare di Ricerche di Immunopatologia, Ospedale

.G. Bosco, L. go Donatore del Sangue 3 10154 Torino, Italy.-mail: [email protected] or to Alessandro Fornasieri,D, Divisione di Nefrologia e Dialisi, Ospedale S. Carlo Bor-

omeo, via Pio II 3, 20153 Milano, Italy. E-mail: [email protected]

© 2006 by the National Kidney Foundation, Inc.0272-6386/06/4901-0009$32.00/0

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ryoglobulins consist of 1 monoclonal immuno-lobulin (usually immunoglobulin M� [IgM�])ith rheumatoid activity against polyclonal IgGs,hereas type III cryoglobulins are characterizedy a combination of polyclonal immunoglobu-ins. Several mixed cryoglobulins, defined assecondary mixed cryoglobulins,” were detectedn patients with connective tissue disorders, lym-hoproliferative disorders, chronic infections,oninfectious hepatobiliary diseases, or immuno-ogically mediated glomerular diseases. Wheno underlying disease is present, cryoglobuline-ia is defined as “essential.” The clinical syn-

rome of mixed cryoglobulinemia was describedrst by Meltzer et al2 in 1966. It was character-

zed by purpura, weakness, and arthralgias, asell as glomerular lesions in some patients.everal subsequent reports further defined thisyndrome, indicating that its incidence varied inifferent geographic areas, with the majority ofases reported in Mediterranean countries:amely, Italy, France, Spain, and Israel.3,4

An important step toward understanding thisisease was achieved when hepatitis C virusHCV) infection was found in the majority ofatients with essential mixed cryoglobulinemiaf either type,5-11 suggesting that these caseshould no longer be considered essential.

Clinical symptoms range from mild palpableurpura, arthralgias, and fatigue to severe vascu-itis with skin necrosis and glomerulonephritisGN), as well as involvement of peripheral nerves,entral nervous system, gastrointestinal tract,ungs, and myocardium.12-16

In rheumatological surveys, patients with typeII mixed cryoglobulins outnumbered those withype II mixed cryoglobulins.2,12 Conversely, sur-eys based on renal involvement showed a greaterrevalence of type II mixed cryoglobulins, withgM� usually the monoclonal immunoglobu-in.17,18

In this multicenter retrospective study, the Italianroup of Renal Immunopathology collected clini-

al and serological data from 146 patients withiopsy-proven cryoglobulinemic GN recorded inhe Italian Registry of Renal Biopsies in 1995.he main criterion for enrollment into the studyas willingness to provide blood samples foriral genotyping studies and characterization ofryoglobulins. The aim of this study is to de-
cribe clinical and biological manifestations ob- s
erved in patients with GN secondary to mixedryoglobulinemia and investigate clinical, histo-ogical, and biological data that may influencerognosis.

METHODS

atients

Clinical and biological data from 146 patients with cryo-lobulinemic GN from 25 hospitals in 11 Italian regionsere collected retrospectively. Inclusion criteria were: (1)resence of purpura and/or arthralgias, (2) biopsy-provenN secondary to mixed cryoglobulinemia, and (3) liveatient at recruitment. With regard to serological and immu-ogenetic studies, 34 other patients with normal renal func-ion and absence of urinary abnormalities were used asontrols. Controls included 18 patients (7 men, 11 women)ith cryoglobulinemic syndrome and HCV-dependent liverisease (control group A) and 16 patients (7 men, 9 women)ith HCV-dependent liver disease without cryoglobuline-ic syndrome (control group B). Thorough clinical and

aboratory data from 1,249 visits (mean, 8.5 visits/patient;ange, 3 to 15 visits) were available for analysis.

iochemical Evaluation

Laboratory evaluation included a complete hemogram,erum chemistry profile (serum alanine aminotransferaseALT], aspartate aminotransferase, alkaline phosphatase,�-lutamyl transferase, cholinesterase, and serum electrophore-is) and determination of complement factors C3 and C4,heumatoid factor assay, and urine light chain determination.rine sediment and 24-hour proteinuria also were assessed

or each patient.

ryoglobulins and HCV

Cryoglobulins were measured by using different methodst the various centers. The most commonly used method wass follows: venous blood was collected in a warm syringe,ransferred into warmed tubes, and stored at 37°C until itlotted. After centrifugation at 37°C, serum was collectednd stored at 4°C for 1 week. The main differences withegard to sample processing among centers included system-tic use of warm syringes and volume of processed serumnd its storage time (up to 10 days), both of which cannfluence the extent of cryoglobulin precipitation.

After isolation and washing, the cryoprecipitate was quan-ified and expressed as a percentage of precipitate/serumolume (ie, cryocrit). Components of the cryoprecipitateere characterized by using immunofixation electrophore-

is. When immunofixation electrophoresis could not beerformed or results were unclear and biological materialas still available (20 cases), characterization of cryoglobu-

ins was performed at CMID Laboratories (Centro di Ricerchei Immunopatologia e Documentazione su malattie rare,niversity of Turin, Italy) using a commercially availableit that was sensitive enough to detect the presence ofligoclones (Criokit; Helena Labs, Milano, Italy). Serumas not available for 9 cases.HCV RNA, HCV genotypes, and viral charge were as-

essed for all patients at the Virology Institute of the Univer-

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HCV-Related Cryoglobulinemic Glomerulonephritis 71

ity of Milan, Italy. HCV RNA sequence was tested byeans of polymerase chain reaction (PCR) using nested

rimers of 5= untranslated region.19,20 HCV genotypes weredentified by using nested PCR amplification with type-pecific primers of the NS5 region and core, as describedlsewhere.21 Viral load was determined by using a signalmplified through branched DNA (Quantiplex, HCV RNA.0 Assay; Chiron Co, Emmerville, CA).22

istological Analysis

Histological specimens were available for all 146 patientsnd were representative enough to characterize patterns oflomerular involvement. Histological material was exam-ned independently by 2 investigators. Immunofluorescenceesults were available for all patients. A mean of 17 � 11.2lomeruli (average, 5 to 70 glomeruli) were observed bysing light microscopy. Patients with advanced sclerosisere excluded from the study (90% showed �20% glomer-lar hyalinosis). Enough histological material was availableor quantitative and semiquantitative evaluation of 130 of46 cases. This included the following features: (1) percent-ge of glomeruli with global or segmental sclerosis andxtracapillary proliferation; (2) amount of mesangial sclero-is, endocapillary proliferation and/or exudation, mesangialroliferation, diffuse thickening of the capillary wall, doubleontours, and endoluminal hyaline thrombi; (3) percentagef cortex with tubular atrophy, interstitial infiltrates, ornterstitial fibrosis; and (4) presence or absence of arteriolaryalinosis, intimal fibrosis, neointimal hyperplasia, arteritis,nd thrombotic microangiopathy.

Three main patterns were recognized: (1) mesangial pro-iferative GN characterized by mesangial expansion androliferation without exudation or endocapillary prolifera-

Table 1. Scoring System for Calc

Activity Index (maximum, 22)

Lesion Grading

ndocapillary proliferation 0-3

esangial proliferation 0-2

xtracapillary proliferation(% glomeruli)

�0 to �10 � 1�10 to �20 � 2

�20 � 3ouble contours/basementmembrane thickening

0-3

nterstitial infiltrate (% cortex) �0 to �30% � 1�30 to �60% � 2

�60% � 3ndoluminal thrombi (% glomeruli) �0 to �30% � 1

�30 to �60% � 2�60% � 3

rteritis 2 (if present)hrombi/microangiopathy 2 (if present)

ion, with segmental and irregular deposits of IgM, IgG, and s

3 in mesangial and paramesangial localizations upon immu-ofluorescence examination; (2) focal membranoprolifera-ive GN that had a typical immunohistologic pattern ofryoglobulinemic GN, but involved less than 50% of glo-eruli; and (3) diffuse membranoproliferative GN that had

he same lesions as the focal form, but involved more than0% of glomeruli.On the basis of these described features, arbitrary chronic

nd activity histological indices were calculated (Table 1).

linical Features

Clinical parameters were recorded at disease onset, bi-psy, and follow-up visits. They included extrarenal symp-oms (purpura, fever, arthralgia, myalgia, abdominal pain,utaneous ulcers, hepatosplenomegaly, pericarditis, neurop-thy, malaise, weight loss, fatigue, dyspnea, sicca syndrome,eizures, and Raynaud phenomenon), renal involvementefined according to the 5 stages of chronic kidney diseaseCKD) indicated by the National Kidney Foundation (NKF),23

roteinuria (protein in grams per 24 hours), hematuria (�3ed blood cells/high-power microscopic field), and hyperten-ion (blood pressure �140/90 mm Hg).

Renal syndromes were defined as follows. Urinary abnor-alities are persistent microscopic hematuria and/or protein-

ria with protein less than 3 g/24 h with normal (�90L/min [�1.50 mL/s]) glomerular filtration rate (GFR;KF CKD stage 1) or mild (60 to 90 mL/min [1.00 to 1.50L/s]; stage 2) or moderate (30 to 59 mL/min [0.50 to 0.98L/s]; stage 3) decrease in GFR, estimated by using theockcroft-Gault equation.24 Nephrotic syndrome is protein-ria with protein greater than 3.0 g/24 h and serum albuminevel of 3 g/dL or greater (�30 g/L).25 Acute nephriticyndrome is the presence of macroscopic hematuria or

Activity and Chronicity Indices

Sclerosis Index (maximum, 16)

Lesion Grading

bal sclerosis (% glomeruli) �0 to �10 � 1�10 to �20 � 2

�20 � 3mental sclerosis (% glomeruli) �0 to �10 � 1

�10 to �20 � 2�20 � 3

rotic crescents (% glomeruli) �0 to �10 � 1�10 to �20 � 2

�20 � 3sangial sclerosis 0-3

ular atrophy and interstitial fibrosiscortex)

�0 to �30% � 1�30 to �60% � 2

�60% � 3erosclerosis 1 (if present)

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ence of mild to severe GFR decrease. Chronic renal insuffi-iency is NKF CKD stages 3 and 4 (GFR, 15 to 29 mL/min0.25 to 0.48 mL/s]) with minimal urinary abnormalitiesproteinuria with protein � 0.3 g/24 h and � 3 red bloodells/high-power microscopic field). Acute renal failure isapid onset of severe decrease in GFR with or withoutliguria (�400 mL/d diuresis), albeit partially or completelyeversible. Flare-up is defined as the clinical conditioneading to an increase in maintenance immunosuppressiveherapy.

mmunogenetic Studies

These studies were performed on all cryoglobulinemicatients (both the nephropathic group of 146 patients andontrol groups A and B) and 109 healthy controls.

The restriction fragment length polymorphism (RFLP)ethod, chosen for low-resolution HLA-DR and HLA-DQ

yping, allowed for unequivocal determination of heterozy-osity for RFLP patterns that was not impaired by the lack ofetection of rare novel alleles associated with most PCR-ased methods. Polymerase chain reaction sequence specificligonucleotide typing also was used for high-resolutionyping when an RFLP allele deviated in frequency.

RFLP analysis for the identification of HLA-DRB, HLA-QA, and HLA-DQB allotypes was performed according torocedures suggested by the 10th International Workshopouthern blotting protocol.26 To simplify the nomenclaturef RFLP polymorphisms, each allotype was given the namef the current allele nomenclature.27

Oligonucleotide typing was performed for DRB1*11 sub-yping. The second exon encoding for the first polymorphicomain of the HLA-DRB1 gene was amplified by means ofCR from 0.5 �g of DNA samples using specific primersnd Taq polymerase according to the manufacturer’s recom-endations (Perkin Elmer Roche, Branchburg, NJ).Primer sequences and amplification protocol proposed by

he 11th International Histocompatibility Workshop weresed. Labeling of oligonucleotide probes with digoxigenin-i-deoxy-uridine diphosphate (Boehringer Mannheim, Mann-eim, Germany) was carried out as described elsewhere.28

ilters were hybridized at 54°C for 2 hours in a water bath.etection of digoxigenin-labeled nucleic acids was per-

ormed by using enzyme immunoassay with luminescencen a nylon membrane29 under conditions recommended byhe manufacturer (Boehringer Mannheim).

A pSSG4 probe subcloned in pVC19 from the recombi-ant phage Ch4A-H-IgG4-2 was used for switch regionFLP analysis. This probe, in conjunction with the HindIIInzyme, shows RFLPs in the switch regions flanking theGHC � 1 (SG1) and � 4 (SG4) genes. Three allelic fragments5.5, 5.4, and 5.0 kb) can be detected at the SG1 locus, and 5lleles (3.7, 3.8, 3.9, 4.0, and 4.1 kb) can be detected at theG4 locus.30

tatistical Analyses

Statistical analyses were performed using chi-square test.uantitative values, expressed as mean � SD, were com-ared by using Student t-test and Wilcoxon test.To assess the effect of biological markers on renal failure

azard, we used a proportional hazard Cox regression model t

ith multiple failure data, considering the event as a creati-ine level greater than 2.5 mg/dL (�221 �mol/L). Anyensored subject could reenter as an “at-risk” subject if aepeated measurement showed creatinine levels less than theelected cutoff value. After censoring for subjects withultiple events, time was calculated as years (with frac-

ions) between the first visit and censoring date.The following variables were included as predictors: age;

ex; basal creatinine level and proteinuria; cryocrit; C4 level;teroid, interferon, immunosuppressant, and plasmapheresisherapy; macroscopic hematuria; oliguria; HCV genotype;nd ALT values. All models were tested by means of thetepwise method using the STATA 7 Intercooler statisticalackage (Stata Corp, College Station, TX), with exclusionriteria of P of 0.10 or greater. Hazard ratios (HRs) with5% confidence intervals were reported for variables re-ained in the final model. Annual incidence rates of renalailure also were calculated by means of the person-yearsethod.Actuarial survival curves were established by using the

aplan-Meier method (Epi Info 3.3.2; Centers for Diseaseontrol and Prevention, Atlanta, GA). End points taken intoonsideration for statistical analyses were death and replace-ent of renal function by dialysis.

RESULTS

emographic Data

Mean age of the 146 patients (63 men, 83omen) at onset of cryoglobulinemia was 52.2 �3 years. Mean age at onset of renal disease was4.3 � 11.9 years and followed the onset of theisease by 2.6 � 4.4 years (range, 0 to 28 years).he interval between onset of renal symptomsnd renal biopsy was 19 months (range, 2 to 28.2onths). Mean follow-up from renal disease

nset was 8.1 years. Main features of the studyroup are listed in Table 2.

enal Symptoms at Presentation andeterioration of Renal Function

The most frequent presenting renal syndromeas subnephrotic proteinuria with microscopicematuria, with or without chronic renal insuffi-iency (41% of cases), followed by nephroticyndrome with or without chronic renal insuffi-iency (21%), acute nephritic syndrome (14%),hronic renal insufficiency without significantrinary abnormalities (12%), and acute renalailure (9%). Urinary abnormalities at presenta-ion were undefined in 2 patients (1 patient, CKDtage 2; 1 patient, CKD stage 3). Moreover, 8%f patients had macroscopic hematuria, and 6%resented with oliguria. As listed in Table 2, a
otal of 74.4% of nephritic patients who could be

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nalyzed for cryoglobulin typing had type IIryoglobulins (almost invariably formed by IgGnd IgM�) compared with 39% (7 of 18 patients)f control group A and 12.5% (2 of 16 patients)f control group B (P � 0.001). Among nephriticatients, 1 patient had polyclonal IgG/IgA (typeII) cryoglobulins and 3 patients had type II

Table 2. Main Features of the Study Group: SubjectsWith Cryoglobulinemic Nephritis

Parameter Frequency Percentage

exMen 63 43.2Women 83 56.8

ge at onset of nephropathy (y)�40 6 4.1140-49 12 8.2250-59 37 25.3460-69 59 40.41�70 32 21.92

ollow-up from onset ofnephropathy (y)

�1 13 8.901-3 17 11.643-5 23 15.755-10 50 34.2410-15 23 15.7515-20 17 11.64�20 3 2.05ryoglobulin typingType III 35 23.97Type II 102 69.86Not typed 9 6.16CV genotype1b 62 54.392a 49 42.98Others 3 2.63CV RNANegative 24 16.44Positive 114 78.08Unknown 8 5.48ephropathy onsetIsolated UA 42 29.37UA with CRI 17 11.89Pure NS 13 9.09NS with CRI 18 12.59ANS 20 13.99CRI 18 12.59ARF 13 9.09ND 2 1.40

Abbreviations: ANS, acute nephritic syndrome; ARF,cute renal failure; CRI, chronic renal insufficiency; ND,ndetermined urinary abnormalities at first detection ofFR reduction; NS, nephrotic syndrome; UA, urinary abnor-alities.

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According to the NKF CKD staging system, atnset of renal involvement, 14 patients (9.6%)ere in stage 1; 47 patients (32.2%), stage 2; 68atients (46.6%), stage 3; and 14 patients (9.6%),tage 4, whereas 3 patients (2.1%) presentingith acute renal failure had a GFR less than 15L/min (�0.25 mL/s; stage 5) and 1 patient was

n dialysis treatment.After 2 and 5 years of follow-up, values were

s follows: stage 1, 7.6% and 2.5%; stage 2,3.1% and 33.3%; stage 3, 46.6% and 44.4%;nd stage 4, 8.5% and 8.6%, respectively. Stagewas observed in 4.2% (including 2 patients on

ialysis therapy) after 2 years and 11.1% (includ-ng 5 patients on dialysis therapy) after 5 years.

Seventy-eight patients (53.4%) experiencedare-ups, which often were multiple (median, 1are-up; range, 1 to 4 flare-ups).

orphological Analysis of Kidneyiopsy Specimen andlinicopathologic Correlations

Ten patients had mesangial proliferative GN.he main lesions in this GN included global andiffuse slight mesangial matrix expansion andesangial cell proliferation. Focal endoluminal

hrombi of immunoglobulin material were foundn 3 cases.

Focal membranoproliferative GN was de-ected in 10 patients. Among them, proliferation,xudation, and thickening of the capillary wallere mild and irregularly distributed within the

ame glomerulus and among glomeruli. Endolu-inal thrombi were found in only 1 of these 10

atients.Diffuse membranoproliferative GN was de-

ected in 108 patients. Proliferative and exudativebnormalities usually were severe, with segmentalistribution in 15% of patients. Centrolobularclerosis was present in 50% of patients. Capil-ary wall abnormalities did not correlate withroliferative lesions. Double contour appearancef the capillary wall was present in 70% ofatients, and endoluminal thrombi were found inore than 50% of patients. Extracapillary prolif-

ration was present in 16% of patients. It isoteworthy that in addition to the common immu-ofluorescence pattern, the presence of IgA de-osits with prevalent mesangial and parietal local-
zation was observed in 4 patients.

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Interstitial leukocyte infiltration, usually focal,as present more often (60%) in the membrano-roliferative forms and correlated with intensityf proliferative glomerular lesions. Interstitialbrosis, usually focal, also was present in theembranoproliferative forms: 90% in focal and

0% in diffuse patterns compared with 50% inesangial proliferative GN. Arteriosclerotic le-

ions were present in 30% of patients, and noifferences were observed among various pat-erns. Arteritis was rare (5.5%).

No differences were found with regard to thehronicity index among the 3 histological forms.he index was mesangial proliferative GN, 4.3 �.8; focal membranoproliferative GN, 4.1 � 1.9;nd diffuse membranoproliferative GN, 4.0 �.3. Conversely, the activity index increased ashe number of proliferative and inflammatoryesions grew and was mesangial proliferativeN, 3.6 � 2.3; focal membranoproliferative GN,.8 � 2.5; and diffuse membranoproliferativeN, 8.4 � 9.0.Percentages of types II and III cryoglobulins

n the 3 histological groups did not differ. Ofote, 2 patients had membranous nephropathyharacterized by the same morphological charac-eristics as the primary form, with prevalentubepithelial deposits of IgM, IgG, and C3, buto proliferative changes.Main clinical features of the 3 main histologi-

al groups at the time of biopsy are listed inable 3. Serum creatinine levels were lower inatients with mesangial proliferative GN thanhe other groups, although the percentage of

Table 3. Clinica

Histological GroupsMesangial Prolifera

(n � 10)

ge (y) 51.5 � 13.3en/women 60/40ime from onset of nephropathy (mo) 13 � 21.2erum creatinine (mg/dL) 1.3 � 0.7erum creatinine �1.5 mg (%) 40roteinuria (g/24 h) 2.5 � 3.0ematuria (%) 100ypertension (%) 60C3 (�80 mg/dL) (%) 20C4 (�15 mg/dL) (%) 60ALT (�35 U/L) (%) 30

Note: Results expressed as mean � SD or percentages,o �mol/L, multiply by 88.4; C3 and C4 in mg/dL to g/L, mul

atients with renal failure was substantially the R

ame. Patients with diffuse membranoprolifera-ive GN showed higher levels of proteinuria andower C4 levels. Extrarenal symptoms and signsere observed more frequently in patients with aembranoproliferative pattern, whereas only

0% of patients with mesangioproliferative GNad extrarenal symptoms and signs.

xtrarenal Clinical Manifestations

Extrarenal clinical manifestations are listed inable 4. Among other symptoms, it is notewor-

hy that 4 patients with abdominal pain under-ent laparotomy that confirmed the presence ofesenteric vasculitis. Seventy-nine patients

ures at Biopsy

Focal Membranoproliferative(n � 10)

Diffuse Membranoproliferative(n � 108)

55.4 � 9.7 57.9 � 10.5340/60 43/57

28.4 � 37.1 18 � 24.62.5 � 2.2 2.6 � 1.8

30 332.9 � 2.9 4.0 � 9.7

100 10040 66.730 5260 9330 22

indicated otherwise. To convert serum creatinine in mg/dL0.01.

Table 4. Extrarenal Clinical Manifestations FromOnset to End of Follow-Up

DiseaseOnset

RenalBiopsy

LastFollow-Up

o. of patients 144 143 115urpura (%) 65.8 48.6 48.6ever (%) 16.4 17.1 15.8rthralgias (%) 39.0 30.1 34.9yalgias (%) 2.7 3.4 9.6bdominal pain (%) 8.2 7.5 11.0utaneous ulcers (%) 0.7 2.7 8.2epatosplenomegaly (%) 2.1 6.2 8.2ericarditis (%) 2.1 4.1 7.5europathy (%) 9.7 15.1 28.7alaise (%) 2.1 2.1 3.4eight loss (%) 2.7 1.4 3.4

atigue (%) 6.4 17.0 31.5yspnea (%) 2.1 2.1 2.1icca syndrome (%) 2.1 1.4 0eizures (%) 1.4 0 0

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54%) had definite signs of liver involvement.ifty-one patients underwent liver biopsy, whichhowed chronic active hepatitis in 85% of cases.igns of cirrhotic evolution were observed in1% of biopsy specimens. Regarding uncommoneurological disorders, hemorrhage, as an expres-ion of brain vasculitis, was documented in 1atient. Myocardial infarction, angina, and car-iac failure were absent at the onset of disease,ut became progressively more frequent untileaching 6.8% by the end of follow-up. Therevalence of hypertension (blood pressure140/95 mm Hg) at disease onset was 48%,hereas at the time of renal biopsy, it increased

o 74%. Keeping arterial pressure under controluring follow-up proved to be problematic de-pite adding 2 or 3 antihypertensive drugs (patho-ogical values were observed in 54% of theatients during �50% of follow-up visits).

erological Data

Data are listed in Table 5. Among study groupatients, 114 of 146 patients (78%) were HCVNA positive (HCV RNA�), 24 patients wereCV RNA negative (HCV RNA�), and 8 pa-

ients were HCV RNA not determined (ND).ight of 24 HCV RNA� patients had HCVntibodies (anti-HCV�), whereas 7 of 8 HCVNA ND patients were anti-HCV� and 1 patientas anti-HCV ND. Overall, 129 of 146 patients

88%) had at least 1 laboratory marker of HCVnfection, whereas 16 patients were completelyegative both for virus RNA and antibodies.epatitis B surface antigen test results were

vailable for 147 of 180 patients who made uphe 3 groups, and only 5 patients were hepatitis Burface antigen� (4 patients also were HCV

Table 5. Results

Study Group

Anti-HCV� Anti-HCV

CV RNA� 114CV RNA� 8 16CV RNA ND 7 1BsAg� 2 1enotype 1a 2enotype 1b 62enotype 2 49enotype 3a 1

Abbreviation: HBsAg, hepatitis B surface antigen.

NA�). u

HCV RNA genotype was assessed in 145atients. The most common genotypes were 1b56%) and 2 (42%). However, on the basis ofenotype, no statistically significant differencesere found among groups. With regard to clini-

al presentation, ALT level abnormalities wereess frequent and persistent in patients with geno-ype 2 (average ALT level, 30.9 � 21.3 U/L)han in patients with genotype 1b (58.7 � 39.8/L; P � 0.001).

mmunogenetics

Nephritic patients with cryoglobulinemic syn-rome had an increased frequency of DRB1*11ompared with 109 healthy controls (0.579 and.367; corrected p value [pc] � 0.0078; relativeisk, 2.4; confidence limits [CL], 1.4 to 3.9). Anpposite profile was shown for DRB1*7 (0.104ersus 0.256, respectively; pc � 0.0104; relativeisk, 0.33; CL, 0.17 to 0.65). The increasedrequency of DRB1*11 in nephritic patients withryoglobulinemic syndrome mainly was causedy HCV� individuals, for whom DRB1*11 wasignificantly more frequent than in HCV� sub-ects (0.614 versus 0.478; pc � 0.0026; relativeisk, 2.7; CL, 1.6 to 4.7). DRB1*14 was morerequent in those with 1b genotype comparedith genotype 2 HCV� patients (0.173 versus.016; pc � 0.0351). Nephritic patients withryoglobulinemia had a lower frequency ofRB1*15 than non-nephritic patients (0.068 ver-

us 0.39; pc � 0.000; relative risk, 0.11; CL, 0.04o 0.36).

herapy

Based on available data, we were able todentify some of the main therapeutic strategies

rological Study

Control Group

TotalAnti-HCV� Anti-HCV�

31 1452 261 92 5

219 8112 61

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sed for 124 of 146 nephritic patients (85%).

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reatment included corticosteroids (110 patients;ntravenous pulses in 33 patients), immunosup-ressive drugs (56 patients; cyclophosphamide,5 patients; azathioprine, 8 patients; chloram-uci, 2 patients; and cyclosporine, 1 patient), andnterferon (55 patients treated for at least 3onths; median, 12 months). Plasma exchange

5 to 14 procedures of exchange of 1 plasmaolume) was added to pharmacological treat-ent in 33 patients. Most patients (34 of 46

atients) with renal insufficiency (NKF CKDtages 3 and 4 and acute nephritic syndrome)nderwent aggressive treatment with corticoste-oids and immunosuppressive drugs that wasssociated with plasma exchange in the mostevere cases (stages 4 and 5 with acute nephriticyndrome or acute renal failure). Corticosteroidslone were used almost exclusively (38 of 40atients) in patients with isolated urinary abnor-alities or urinary abnormalities combined with

hronic renal insufficiency. Patients with ne-hrotic syndrome were treated with either corti-osteroids alone (10 patients) or corticosteroidslus immunosuppressive drugs (14 patients). In-erferon was used alone (10 patients) or in asso-iation (45 patients), mainly in patients withersistently high ALT levels or in the presence ofiver insufficiency. Six patients (9%) experi-nced a sustained viral response.

rognostic Factors

Clinical manifestations at disease onset, timef renal biopsy, and end of follow-up were con-idered possible prognostic factors either indi-idually or in association. Some correlated sig-ificantly, particularly arthralgias and fever andpleen and liver enlargement. No correlation wasound between single clinical manifestations and1) the presence or worsening of renal insufficiencydefined as a stable 0.5-mg/dL [44-�mol/L] in-

Table 6. Estimated Person-Years, Number of Events

Variable Person-Years

roteinuria �3 g/24 h 423.0roteinuria �3 g/24 h 100.3asal creatinine �2.5 mg/dL 504.0asal creatinine �2.5 mg/dL 19.2otal 523.3

Note: To convert serum creatinine in mg/dL to �mol/L, multiply b

rease in serum creatinine level) at the end ofollow-up, (2) HCV viremia levels, or (3) HCVenotype. Conversely, significant correlation wasound between number of flare-ups and renalnsufficiency at the end of follow-up (P � 0.004).

Single-Variable AnalysesCorrelations were searched for between renal

yndrome at presentation and serum creatinineevel at biopsy. Serum creatinine levels greaterhan 3.3 mg/dL (�292 �mol/L), together withhe presence of acute nephritic syndrome (P �.0001) and age older than 50 years (P � 0.01),ere associated significantly with developing

hronic renal insufficiency, dialysis therapy, oreath. The presence of hypertension at the timef renal biopsy significantly correlated with theresence of renal failure (P � 0.002) and worsen-ng of renal function (defined as stable increasen serum creatinine level of 0.5 mg/dL [44 �mol/]) at the end of follow-up (P � 0.016). The

ollowing variables did not show a predictiveong-term value: sex, HCV genotype, and treat-ent with steroids or interferon.

Multivariable AnalysesVariables that correlated independently with

isk for end-stage renal failure included age,erum creatinine level, and proteinuria at theime of renal biopsy.

Risk for renal insufficiency, calculated usinghe stepwise method, increased as patient ageHR, 1.05; 95% CL, 1.00 to 1.10), serum creati-ine level (HR, 1.43; CL, 1.23 to 1.68), androteinuria (HR, 1.15; CL, 1.02 to 1.30) at theime of renal biopsy increased. Person-years atisk, number of events, and incidence rates areisted in Table 6.

Kaplan-Meier survival analyses are shown inigs 1-3. Thirty-one deaths were recorded. The

nnual Incidence Rate Listed by Relevant Variables

ailures Rate (%)95% Confidence

Interval

18 4.3 2.7 6.89 9.0 4.7 17.2

20 4.0 2.6 6.27 36.4 17.4 76.4

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ain cause of death (19 patients) was cardiovas-ular (Table 7). Mean follow-up was 6.7 � 3.6ears (range, 1 to 16 years) for nonsurvivors,hereas it was 8.4 � 5.7 years (range, 1 to 26ears) for survivors. Mean age at death was 66 �2 years (range, 29 to 82 years), whereas meange of surviving patients at last follow-up was 63

10 years (range, 32 to 92 years). Overallurvival rates were about 90% at 5 years andbout 80% at 10 years. Survival was not affectedy sex or HCV status (ie, presence or absence ofCV infection). Basal creatinine level greater

han 1.5 mg/dL (�133 �mol/L) significantlyffected the survival probability. Kidney survivalrobability is shown in Fig 4 and was affected byex. Men had a significantly greater risk foreeding dialysis.

Figure 1. Kaplan-Meier sur-ival analyses according toasal creatinine values: 1.5g/dL or less (101 subjects)nd greater than 1.5 mg/dL (45ubjects). To convert serumreatinine in mg/dL to �mol/L,ultiply by 88.4.

DISCUSSION

It was estimated that about 170 million peopleorldwide are infected with HCV, with a sero-revalence rate of 1% in Western Europe, but 3%n some areas of the Mediterranean. Ten percento 40% of chronically infected patients haveirculating cryoglobulins, although clinical fea-ures of mixed cryoglobulinemia only develop inminority (�5%) of HCV� patients.31 Genetic

ackground may be an important factor in deter-ining who will eventually develop the disease.It presently is believed that HCV infects B

ymphocytes while infecting hepatocytes be-ause of the common expression of CD81 recep-ors.32 An HCV-dependent gene translocation33

hat protects lymphocytes from apoptosis sus-

Figure 2. Kaplan-Meier sur-
vival according to sex (83women, 63 men).

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ains the oligoclonal monotyping lymphoprolif-ration that occurs in patients with mixed cryo-lobulinemia.32 IgM� that share rheumatoidctivity toward anti-HCV polyclonal IgG formegacomplexes that are removed slowly by the

loodstream34,35 and deposited in glomeruli, alsoecause of the affinity of monoclonal IgM to theesangial matrix.36 Infected B lymphocytes also

re characterized by a decreased cell activationhreshold and widespread autoantibody produc-ion. This favors the development of a number ofmmune manifestations associated with HCVnfection that are assembled variably in clinicalictures collectively called “HCV syndrome.”37

herefore, patients with mixed cryoglobuline-ia also share a variety of associated immune

isorders, including thyroiditis, thrombocytope-ia, pulmonary fibrosis, and sicca syndrome.With specific regard to nephrological manifes-

ations, cryoglobulinemic nephritis is a unique ne-hropathy undoubtedly pathogenetically relatedo HCV infection.38 The relationship between

Table 7. Causes of Death in This Study and aPrevious Series

PresentStudy

Tarantinoet al17 1995

ardiovascular disease (%) 62 29epsis (%) 10 21epatic failure (%) 19 19eoplasia (%) 3 10dult respiratory distresssyndrome (%) 3 2

cnknown (%) 3 19

CV infection and cryoglobulinemic-associatedephritis is addressed specifically in the presenteport, which describes clinical characteristics,ause as related to viral specificities, and out-ome of a large cohort of patients with cryoglobu-inemic GN.

The incidence of cutaneous manifestations inhese patients did not differ from data reportedy other studies of similar sample sizes.1,2,13,16,39

urpura was the most common external sign andhowed an increase in incidence during follow-p. However, it often proved transient and spon-aneously regressed in one half of our patients.

Peripheral neuropathy certainly was underesti-ated. When it was systematically searched for

n a single reference center, it was found in all 19atients who underwent electrophysiological ex-mination. Peripheral neuropathy is thought toe the result of axonal ischemic damage causedy deposits of cryoprecipitable immunocom-lexes in the vasa nervorum. However, highevels of antineuronal IgG (anti-ganglioside GM1nd antisulfatide) recently were detected in pa-ients with mixed cryoglobulinemia and GN,hus suggesting that direct autoantibody reac-ions may have a role in affecting neuronaltructures.40

Central nervous system manifestations werebserved rarely (2% of cases). Hypertension wasignificantly more frequent in our cohort (�50%f patients) than in other reports.1,13,39 Hyperten-ion was a major risk factor in our patients.ardiovascular events were the most frequent

Figure 3. Kaplan-Meier sur-vival according to HCV status:HCV� (17 subjects) and HCV�

(129 subjects).

auses of death (61%) in our cohort of patients.

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Raynaud phenomenon certainly was underesti-ated in this study. Videocapillaroscopy was

sed to assess microcirculation in patients withryoglobulinemic GN, and almost invariablyhowed significant abnormalities, even in thebsence of Raynaud phenomenon.41

With regard to cause, about 90% of our pa-ients had a history of HCV infection, whereasnly 3% had been exposed to hepatitis B virusgenerally occurring as a coinfection). In addi-ion to hepatic involvement, no clinical or labora-ory differences could be detected between HCV�

129 cases) and HCV� (17 cases) cryoglobuline-ic patients with nephritis. This differs fromhat was observed in rheumatological and der-atological patients.16,42 During the last few

ears, several attempts were made to identify antiologic agent in the small sample of patientsot infected with HCV. At first, GB virus C,nother member of the Flaviviridae family, wasdentified as the possible agent in these pa-ients,43 but initial observations were not con-rmed.44 Some evidence for a role of TT viruses,DNA hepatotropic virus, are limited to coinfec-

ious conditions.45 Genotype 1 has a 70% preva-ence in patients with chronic hepatitis in most

estern countries. In Italy, the prevalence ofenotype 2 is close to that of genotype 1. Geno-ype distribution in nephritic patients and controlroups was similar. A prevalence of genotype 1bas especially evident in southern Italy and theorthwest, possibly because of postwar immigra-

Figure 4. Kaplan-Meier kid-ey survival according to sex83 women, 63 men).

ion from southern Italy to industrial areas. l

As expected by seroprevalence data, someenetic markers were identified in this study.RB1*11 was associated with nephritic involve-ent, whereas DRB1*15 was found to be protec-

ive against developing nephritis.In this study, as in others, the prevalence of

ype II cryoglobulins was significantly greater inephritic patients than non-nephritic controls74% versus 27%), showing that clonal restric-ion probably is important in renal involvement.

Despite the variety of clinical presentations,bout 70% of patients presented with diffuseembranoproliferative GN. Although both hema-

uria and hypertension were distributed equallymong various histological groups, serositis,epatosplenomegaly, leukopenia, peripheral neu-opathy, and cardiac involvement were observedn only the membranoproliferative pattern. More-ver, patients with diffuse membranoprolifera-ive GN showed stronger C4 hypocomple-entemia and greater levels of proteinuria.As shown in Fig 1, 10-year life expectancy

fter the onset of nephropathy was close to 80%,uch greater than in the 1995 study of Tarantino

t al.17 In our study, causes of death includedardiovascular disease, infections, hepatic fail-re, and neoplasia. Cardiovascular death wasredominant compared with results of the studyf Tarantino et al,17 in which the number ofeaths caused by infections and hepatic failurepproached the number of deaths caused byardiovascular disease (Table 7). This difference
ikely is the result of: (1) greater caution in the

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Roccatello et al80

se of immunosuppressive drugs, which weredministered for limited courses and most com-only for nephritic onset and flare-ups; (2) exten-

ive administration of antiviral agents, especiallyn patients with milder forms of GN; (3) availabil-ty of more potent antibiotics; and (4) improve-ent in survival, which led to the late occurrence

f coronary artery disease.The optimal therapeutic strategy for patients

ith cryoglobulinemic GN currently is unde-ned. The discovery of an association betweenryoglobulinemia and HCV infection promptedlinicians to try to control the disease by eradicat-ng the triggering infection. The antiviral treat-ent administered to the patients under studyas less potent (including only nonpegylated

nterferon) and administered at lower doses thanecommended by current protocols. This possi-ly is why only a minority of patients, ie, lesshan 10%, reached a sustained viral response.revalence of genotype 1b could be anothereason for ineffective virus eradication. How-ver, the condition of cryoglobulinemia per selso may be implied. Cryoglobulinemia favorsCV entrapment through non-neutralizing anti-odies inside megacomplexes that are phagocy-osed, but not adequately digested, by circulatingnd tissue phagocytes,35 thus ultimately becom-ng a virus reservoir.

During acute immunologic flares, antiviralreatment was not used alone. Steroids and immu-osuppressive drugs, mainly cyclophosphamide,nd plasma exchange usually were added.

Although our data are not sufficient to statisti-ally support this statement, we suspect that theetter survival profiles obtained during the lastecade (compared with profiles in previous re-orts) are the consequence of the aggressiveime-limited intervention on acute renal involve-ent, together with the long-term antiviral

herapy used to control moderate manifestationsf renal and extrarenal involvement.In conclusion, data collected in this multi-

enter study, which includes the biggest cohortf patients with cryoglobulinemic GN ever exam-ned worldwide, confirm the strict associationith HCV infection. Genotype 1b was morerevalent than genotype 2, although not signifi-antly. Significant prognostic variables includege, male sex, creatinine level and proteinuria at
he time of renal biopsy, number of syndromic r
elapses, and poor blood pressure control. HLA-RB1 polymorphism might be implicated in theatural history of mixed cryoglobulinemia. Riskor developing the disease after HCV infection isreater in patients with the DRB1*11, whereasRB1*15 protects cryoglobulinemic patients

rom renal involvement.

ACKNOWLEDGMENTItalian Study Group of Renal Immunopathology: the fol-

owing centers participated in patient recruitment (Referral/irector): Dipartimento di Gastroenterologia, Azienda Os-edaliera S.G.Battista, Molinette Torino (A. Ponzetto/M.izzetto); Divisione di Nefrologia e Dialisi, Ospedale S.arlo Borromeo, Milano (A. Fornasieri/G. D’Amico); UOefrologia, Azienda Ospedaliera Spedali Civili di Brescia

S. Savoldi/R. Maiorca); UO di Nefrologia, Dialisi e Terapiaedica del Trapianto Renale, Ospedale Niguarda Cà Grandailano (V. Colombo/G. Civati); CMID Ospedale G. Bosco

orino (D. Rossi/D. Roccatello); Divisione di Nefrologia,ialisi e Trapianti Policlinico, Bari (G. Giannico/F.P. Schena);O Nefrologia e Dialisi Universitaria Ospedale S. Chiaraisa (L. Moriconi/G. Barsotti); Divisione di Nefrologia eialisi, Ospedale Regionale S. Michele Cagliari (A. Pani/P.ltieri); Divisione di Nefrologia e Dialisi Ospedale Mag-iore IRCCS Milano (G. Banfi/C. Ponticelli); Divisione diefrologia Arcispedale S.Maria Nuova Reggio Emilia (R.ustichelli); UO di Nefrologia e Dialisi Ospedale Saccoilano (G. Barbiano Di Belgiojoso); Divisione di Nefrolo-

ia, Dialisi e Terapia del Trapianto Renale, Azienda Osped-liera S. Anna Como (M. Quarenghi/C. Grillo); UO Nefrolo-ia e Dialisi Ospedale S. Chiara Trento (C. Comotti/C.R.ovati); S.C. di Nefrologia e Dialisi, Azienda Ospedaliera dierugia (R. Brugnano/U. Buoncristiani); S.C.U. Nefrologia,ialisi e Trapianto Azienda Ospedaliera S.G. Battista, Moli-ette Torino (P. Stratta/G. Piccoli); S.C. Nefrologia e DialisiCentro Calcolosi Renale, Osp Mauriziano Umberto I,

orino (M. Bruno/A. Ramello); Divisione di Nefrologia emodialisi Fondazione Salvatore Maugeri Pavia (V. Pi-zza/S. Segagni); U.O. Nefrologia, Dialisi e Trapiantozienda Ospedaliera Bianchi Melacrino Morelli, Reggioalabria (C. Martorano/C. Zoccali); S.C. di Nefrologia eialisi, Azienda Ospedaliera di Lecco (F. Tentori/F. Lo-

atelli); S.C. di Nefrologia e Dialisi, Azienda Ospedaliera diuneo (G. Canepari/PM. Ghezzi); S.C. di Nefrologia eialisi, Ospedale degli Infermi Biella (P. Bajardi/P. Bajardi);.O. Nefrologia e Dialisi Azienda Ospedaliera di Meleg-ano (MI) (C. Grassi); U.O. Nefrologia e Dialisi Ospedaleatebenefratelli Roma (Selvaggi/ M.G. Chiappini); Clinicaefrologica, Dialisi e Trapianto Azienda Ospedaliera Univer-

itaria Genova (G. Garibotto/G. Deferrari); Language edit-ng: F. Perricone.

The authors thank Giuseppe D’Amico for careful readingf the manuscript and his precious suggestions.

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Multicenter Study on Hepatitis C Virus–Related Cryoglobulinemic Glomerulonephritis

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