For clarification of the pathogenetic role of viral infectionin chronic glomerulonephritis, the renal effects of CoxsackieB4 virus (CB4) were examined in hyper-IgA (HIGA) mice. In experiment1, HIGA mice (n = 75) were inoculated intravenously with liveCB4 and inactivated CB4 once a month from 1 to 12 mo of age.In experiment 2, HIGA mice (n = 45) were inoculated intravenouslywith live CB4 and inactivated CB4 once at 6 wk of age. In experiment3, 60 mice were inoculated intravenously with carbon and liveor inactivated CB4 once at 6 wk of age. Mice in the controlgroup were inoculated with vehicle. The kidneys were extirpatedfrom five mice of each group killed with time after inoculationfor histologic evaluation. The scores for mesangial IgA deposition,PCNA-positive cells, and matrix at 20 wk were higher in micewith live CB4 than in mice with inactivated CB4 or without CB4.On electron microscopic examination, swelling and detachmentof endothelial cells from 24 h after inoculation and increaseof serum IFN- concentration were found in mice with live CB4.Many carbon particles were present in peripheral and centralzones of the mesangium from 5 to 10 d in mice with carbon andlive CB4. These results suggest that CB4 provokes exacerbationof renal pathologic findings in HIGA mice via endothelial injury,IFN- production, and dysfunction of the mesangial pathway.
Primary IgA nephropathy (IgAN) first was reported in 1968 byBerger and Hinglais and is characterized clinically by microhematuriaand proteinuria and histologically by the deposition of IgA(1,2). There have been many reports on the relationship betweenviral infection and renal injury (3–10). Clinically, weoften have noted that viral infection provoked transient proteinuria,hematuria, and renal dysfunction. The role of viruses in thepathogenesis and exacerbation of human renal diseases is notwell understood, although it is likely that in acute infections,viruses have direct cytopathic effects on the renal tissue,whereas chronic long-standing infections may exert cytopathiceffects via immune complexes (11).
Recently, viral antigens were detected in renal tissues frompatients with IgAN and viral DNA by PCR or in situ hybridization(12–16). Enteroviruses are established or suspected causativeagents in numerous diseases. In particular, group B coxsackieviruses (CB), which are enteroviruses, have been implicatedin several diseases, including pancreatitis, insulin-dependentdiabetes, myocarditis, and myositis (3–6). They also havebeen reported to be causative agents in some renal diseases.For example, induction of experimental nephritis by coxsackieB4 virus (CB4) first was described by Sun et al. (17), and subsequentreports indicated relationships of CB4 with renal diseases inhumans and experimental animals. However, there have been noreports on exacerbation of nephritis by viral infections orthe mechanism of such exacerbation. Conversely, there have beenreports on a model mouse of IgAN. Muso et al. (18) establishedan inbred murine model of IgAN, a high IgA strain (HIGA) ofddY mice, by selective mating of pooled crude ddY mice thathave been reported to develop spontaneously mesangioproliferativeglomerulonephritis with glomerular IgA deposition. HIGA miceexhibited constantly high serum levels of IgA from 10 to 60wk of age with polymeric IgA dominant mesangial deposition andenhanced extracellular matrix accumulation. In this investigation,to examine the renal effects of CB4 in IgAN, we evaluated renalinjury after viral inoculation in HIGA mice.
Disease Model
Animal experiments were performed using female inbred HIGA mice(Japan SLC, Inc., Shizuoka, Japan) that were 5 wk of age. Micewere allowed free access to normal mouse chow and tap water.All animal experiments were performed according to the InstitutionalAnimal Care and Use Committee guidelines of Fukushima MedicalUniversity School of Medicine. Virus-infected mice were maintainedin separate rooms from control mice.
Virus
CB4 was prepared by methods previously described by Yoshidaet al. (19).
Experimental Protocol Experiment 1: Administration of Live and Inactivated CB4 Once a Month from 2 to 12 Mo of Age.
Seventy-five mice were inoculated intravenously with 0.3 mlof 107 TCD50 per 0.1 ml of live CB4 (group A) and inactivated(group B) CB4 once a month from 1 to 12 mo of age. Mice in thecontrol group (group C) were inoculated with the supernatantof Vero cells that were not infected with CB4 in a similar manner.
These mice were killed once every 10 wk from 10 to 50 wk ofage under chloroform anesthesia. Twenty-four-hour urine sampleswere collected at 10, 20, 30, 40, and 50 wk of age. Five micein each group were killed at 10, 20, 30, 40, and 50 wk of age.
Experiment 2: Intravenous Administration of Live and Inactivated CB4 Once to Mice at 6 Wk of Age.
Forty-five mice were inoculated intravenously with 0.3 ml of107 TCD50 per 0.1 ml of live (group 1) or inactivated (group2) CB4 once at 6 wk of age. Mice in the control group (group3) were inoculated with the supernatant of Vero cells that werenot infected with CB4 in a similar manner. These mice were killedat 6 h, 24 h, or 5 d after administration under chloroform anesthesia.
Experiment 3: Intravenous Administration of Colloidal Carbon and CB4 Once to Mice at 12 Wk of Age.
Sixty mice were inoculated intravenously with 6 mg/0.1 ml carbon(Pilot) and 0.3 ml of 107 TCD50 per 0.1 ml of live (group 1)or inactivated (group 2) CB4 once at 6 wk of age. Mice in thecontrol group (group 3) were inoculated with the supernatantof Vero cells that were not infected with CB4 in a similar manner.Five mice in each group were killed at 12 h, 36 h, 5 d, and10 d after administration. In all experiments, after cardiacpuncture for blood sampling, the kidneys were removed, weighed,cut into portions, and used for assessment by light microscopy(LM), immunofluorescence (IF), immunohistochemical microscopy(IHM), and electron microscopy (EM).
Laboratory Investigation
Serum creatinine (Scr), serum blood urea nitrogen, and urinarycreatinine (Ucr) levels were measured. From the Scr valves,Ucr, 24-h urine volume, and body weight at sacrifice, the 24-hendogenous creatinine clearance (Ccr) was calculated using thefollowing formula:
Histologic Examination
When the rats were killed, one kidney was excised from eachrat and divided into three parts for examination by LM, IF,IHM, and EM.
LM
The renal tissue was fixed in buffered formalin and embeddedin paraffin for LM examination. Sections 2 to 3 µm thickthen were stained individually with hematoxylin-eosin, periodicSchiff, and periodic acid–silver methenamine and observedunder a light microscope. Three observers, blind to treatments,semiquantitatively graded extracellular matrix accumulationin each quadrant in 50 glomeruli per kidney on a scale from0 to 3 using the following scales: 0, no increase in mesangialmatrix; 1, slight increase in mesangial matrix; 2, moderateincrease in mesangial matrix; and 3, nearly confluent appearanceof mesangial matrix. Each score reflects changes in the extentrather than in the intensity of mesangial matrix staining. Cellularcrescent formation score was calculated as the percentage ofglomeruli with cellular crescent formation among all glomeruli.
IHM
The paraffin-embedded sections were dewaxed and incubated sequentiallywith normal goat serum (1:20 dilution) for 20 min, mAb to mouse-smooth muscle actin (-SMA; 1A4; Dako, Glostrup, Denmark; 1:50dilution), mouse PCNA (19A2; Coulter, Hialeah, FL; 1:50 dilution),and mouse macrophage (MCAP497; UK-Serotec Ltd., Tokyo, Japan;1:100 dilution) for 1 h, and finally with horseradish peroxidase–conjugatedgoat anti-mouse Ig (EnVision; Dako Japan Co., Kyoto, Japan)for 1 h. The peroxidase reaction product was visualized with0.5 mg/ml 3'-diaminobenzidine tetrahydrochloride/0.01% hydrogenperoxide as a substrate. For all biopsies, negative controlsinvolved substitution of the primary antibody by equivalentconcentrations of an irrelevant murine mAb or normal rabbitIgG. For each biopsy, >50 cross-sections of consecutive corticalglomeruli with diameters of at least 100 µm were evaluated.Mean numbers of MCAP497-positive cells of glomeruli per biopsywere calculated. For evaluation of immunoperoxidase stainingfor PCNA and -SMA, each glomerulus was grade semiquantitatively,as described previously (20).
Semiquantitative Analysis of the Distribution of Carbon Particles within Glomeruli
The degree of the distribution of carbon particles with in glomeruliis shown in Figure 1. Glomerular tuft was divided into threearbitrary zones, from surrounding capillaries (grade S) throughmesangial areas (grade M) to lacis area (grade L). The proportionof these grade S, M, and L from 12 h to 10 d of age was calculatedin groups 1, 2, and 3.
Figure 1. Schematic representation of glomerular zones.
IF, EM, and In Situ Hybridization
The renal tissue specimens were prepared by methods that weredescribed previously by Yoshida et al. (19).
Measurement of Each Cytokine, Serum IgA, and Neutralizing Antibody
We measured IL-4 and IFN- concentrations in serum samples withcommercially available ELISA kits (Endogen, Inc., Woburn, MA)and IgA concentrations in serum samples with a radial immunofusionplate (Serotec Ltd., Oxford, UK). Anti-CB4 neutralizing antibodywas measured by the microtitration technique after incubationat 56°C for 30 min.
Urinalysis
Urine protein concentrations were determined by colorimetricassay (BioRad, Oakland, CA) using BSA as a standard. Occultblood was measured using N-Multistix SG-L (Bayer-Sankyo Co.,Tokyo, Japan). Presence of occult blood was graded negativeor positive.
Statistical Analyses
Values are the means ± SD. Statistical analysis was performedon a Macintosh computer with a software package for statisticalanalysis (Stat View; Abacus Concepts, Berkeley, CA). Differencesamong groups in laboratory data were assessed by the Mann-Whitneyrank sum test or Wilcoxon signed rank test or contingency tables(2). Correlations were evaluated using Fisher r test. Findingsof P < 0.05 were considered significant.
Results of Experiment 1 Comparison of Pathologic Findings among Groups. Comparison of IF Findings among Groups.
The degree of deposition of Ig (e.g., IgG, IgM, IgA) and C3are shown in Figure 2. The degrees of deposition of IgA at 20wk were higher in group A than in groups B and C (2.1 ±0.5 versus 0.8 ± 0.4 [P < 0.01] and 2.1 ± 0.3versus 0.6 ± 0.5 [P < 0.01], respectively). The degreesof deposition of IgA from 10 to 30 wk of age were higher ingroup A than in groups B and C. The depositions of IgA are shownin Figure 3. Glomerular IgA deposition was significantly shownat 20 wk of age in group A (Figure 3A), and glomerular IgA depositionwas almost never shown at 20 wk of age in group C (Figure 3B).The degree of deposition of IgA did not differ between groupsB and C. After 40 wk of age, the degree of deposition of IgAdid not differ among the three groups.
Figure 3. (A) Glomerular IgA deposition was significantly shown at 20 wk of age in group A. (B) Glomerular IgA deposition almost never was shown at 20 wk of age in group C. (C) At 20 wk, strong positive signals were seen in mesangial cells of group A. Magnification, x400.
LM Findings.
The proportions of crescent formation from 10 to 50 wk of agewere higher in group A than in groups B and C (Figure 4, A andD). The scores for PCNA-positive cells at 20 wk were higherin group A than in groups B and C (1.6 ± 0.3 versus 0.8± 0.3 [P < 0.01] and 1.6 ± 0.3 versus 0.9 ±0.3 [P < 0.01], respectively). The scores for PCNA-positivecells from 10 to 20 wk were higher in group A than in groupsB and C. The scores for -SMA–positive cells at 20 wk werehigher in group A than in groups B and C (1.7 ± 0.4 versus0.8 ± 0.3 [P < 0.05] and 1.6 ± 0.3 versus 0.9± 0.4 [P < 0.05], respectively). The scores for -SMA–positivecells from 10 to 20 wk were higher in group A than in groupsB and C. Proportions of crescent formation, scores for PCNA-positivecells, and scores for -SMA–positive cells did not differbetween groups B and C (Figures 4 through 6). Mesangial proliferationand PAS-positive depositions were found more frequently in groupA than in groups B and C (Figure 4, D and E).
Figure 4. Light microscopy (LM) findings (periodic acid-Schiff [PAS] stain). (A) There was a slight increase in mesangial cell and crescent formation in a glomerulus at 10 wk of age in group A. (B) There was a marked increase in mesangial cell and some PAS-positive depositions in a glomerulus at 30 wk of age in group A. (C) There was a moderate increase in mesangial cell and extracellular matrix in a glomerulus at 50 wk of age in group A. (D) There is a slight increase in mesangial cell in a glomerulus in group C. (E) There was a moderate increase in mesangial cell and a few PAS-positive depositions in a glomerulus at 30 wk of age in group C. (F) There was a slight increase in mesangial cell and a moderate increase in extracellular matrix in a glomerulus at 50 wk of age in group C. Magnification, x400.
Figure 6. (A) PCNA -positive cells were found frequently in the glomeruli at 20 wk in group A. (B) PCNA-positive cells were not seen in the glomeruli at 20 wk in group C. (C) -SMA–positive cells were expressed frequently in the glomeruli at 20 wk in group A. (D) -SMA–positive cells were not expressed in the glomeruli at 20 wk in group C.
Matrix scores increased from 40 to 50 wk in each group. Matrixscores at 30 wk were higher in group A than in groups B andC (1.6 ± 0.4 versus 1.0 ± 0.3 [P < 0.05] and1.6 ± 0.4 versus 1.1 ± 0.3 [P < 0.05], respectively).Matrix scores from 20 to 40 wk of age were higher in group Athan in groups B and C (Figure 4, C and F). Matrix scores didnot differ between groups B and C. The mean number of MCAP497-positivecells at 20 wk was higher in group A than in groups B and C(4.2 ± 1.6 versus 0.6 ± 0.4 [P < 0.01] and4.2 ± 1.6 versus 0.4 ± 0.4 P < 0.01, respectively).The mean number of MCAP497-positive cells from 10 to 20 wk washigher in group A than in groups B and C.
Comparison of EM Findings among the Three Groups.
The degree of electron-dense mesangial deposition from 10 wkto 30 wk was higher in group A than in groups B and C. The degreeof electron-dense mesangial deposition did not differ betweengroups B and C.
In Situ Hybridization.
At 20 wk, strong positive signals were seen in mesangial cellsof group A, and positive signals were not seen in mesangialcells of groups B and C (Figure 3C).
Comparison of Laboratory Findings and Serum Cytokine Levels among Groups.
Urinary protein from 10 and 50 wk of age was detected in groupA and was not detected in groups B and C. At 30 wk, urinaryprotein (mg/d) increased mildly in group A compared with groupsB and C (1.9 ± 0.8 versus 0.1 ± 0.0 [P < 0.05]and 1.9 ± 0.8 versus 0.1 ± 0.0 [P < 0.05],respectively) (Table 1). From 10 and 50 wk of age, hematuriawas not detected in all groups. There were no significant differencesin body weight and Ccr among the three groups (Table 1, Figure 7).
Figure 7. Comparison of serum IFN-, IL-4 concentrations, and neutralization antibody titer of sera among the three groups.
Serum IgA concentrations (mg/dl) increased markedly after 30wk in all three groups and did not differ among them (147 ±19, 156 ± 24, and 151 ± 22 in groups A, B, andC, respectively). Serum IFN- concentration increased from 10to 40 wk in group A, and was hardly detected in groups B andC (Figure 7). Serum IL-4 concentration increased from 10 to50 wk in groups A, B, and C (Figure 7), with no differencesin concentration among the three groups. Serum anti-CB4 virusneutralizing antibody titers in groups A and B were elevatedgradually, and no elevation in group C was noted (Figure 7).
Results of Experiment 2 Comparison of Pathologic Findings among Groups.
On LM examination, proliferation score and matrix score didnot differ among the three groups. On EM examination, swellingand detachment of endothelial cells from 3 h to 5 d after inoculationwere found in all mice of group 1, and none of them from 3 hto 5 d were found in all mice of groups 2 and 3 (Figure 8).Swelling and detachment of endothelial cells was more frequentlyfound in group 1 than in groups 2 and 3 (Figure 8, A and B).
Figure 8. Pathologic findings in experiment 2. There were swelling and detachment of endothelial cells at 24 h of age after inoculation in Group 1. Magnifications: x800 in A; x2500 in B.
Results of Experiment 3 Comparison of the Proportion of Grades S, M, and L from 12 H to 10 D of Age among Three Groups.
The proportions of grade S from 12 h to 10 d and those of gradeM from 5 to 10 d were higher in group 1 than in groups 2 and3. The proportions of grade M from 12 h and those of grade Lfrom 36 h to 10 d were higher in group 1 than in groups 2 and3 (Figure 9).
In group 1, large carbon particles were present in surroundingcapillary lumina from 12 (Figure 10A) to 36 h (Figure 10B) afteradministration. Many carbon particles were present in the peripheraland central zones of the mesangium from 5 (Figure 10C) to 10d (Figure 10D) after administration.
Figure 10. Comparison of the proportion of grades S, M, and L between groups 1 and 3. (A) Large carbon particles were present in surrounding capillary lumina from 12 h after administration in group 1. (B) Large carbon particles were still present in capillary lumen, and mesangium contained small amounts of carbon particles at 36 h after administration in group 1. (C) Many carbon particles were present in the peripheral and central zones of the mesangium from 5 d after administration in group 1. (D) Many carbon particles were still present in the peripheral and central zones of the mesangium from 10 d after administration in group 1. (E) Large carbon particles were present in surrounding capillary lumina at 12 h after administration in group 3. (F) Moderate numbers of carbon particles were present in the peripheral zone of the mesangium at 36 h after administration in group 3. (G) Many carbon particles were present in the peripheral and central zones of the mesangium and lacis area at 5 d after administration in group 3. (H) Most carbon particles had disappeared from the mesangium and lacis area at 10 d after administration in group 3.
In groups 2 and 3, large carbon particles were present in surroundingcapillary lumina at 12 h after administration (Figure 10E).Moderate numbers of carbon particles were present in the peripheralzone of the mesangium at 36 h after administration (Figure 10F).Many carbon particles were present in the peripheral and centralzones of the mesangium and lacis area at 5 d after administration(Figure 10G). Almost all carbon particles had disappeared fromthe mesangium and lacis area at 10 d after administration (Figure 10H).
Our findings showed that CB4 promoted IgA deposition in mesangiallesions and increased mesangial cell and matrix proliferationin glomeruli of HIGA mice. Endothelial cell injury and crescentformation were found more frequently in HIGA mice with liveCB4 than in HIGA mice without CB4 and HIGA mice with inactivatedCB4.
There have been many reports on the relationship between viralinfection and renal injury. Clinically, renal signs and symptomsfrequently are preceded by episodes of upper respiratory tractinfection and/or gastrointestinal infection, suggesting thatviral infection as the cause of IgAN.
Gregory et al. (10) revealed that human cytomegalovirus participatedin the pathogenesis of IgAN. Iwama et al. (16) reported thatEpstein-Barr virus (EBV)-specific DNA in renal biopsies wasdetected by PCR in seven (58%) of 12 patients with IgAN, three(50%) of six patients with membranous nephropathy, none (0%)of 10 patients with minor glomerular abnormalities, and two(100%) of two patients with focal segmental lesions. We reportedpositive PCR for the presence of enteroviral RNA for three of10 patients with IgAN and that enteroviral infection may playa role in the mechanism of onset or evolution of IgAN (12).
CB4, which is in the group of enteroviruses and minimum RNAviruses without lipid, is a common agent of gastrointestinalinfection and also is associated with more serious diseasessuch as myocarditis or myositis. CB4 has been reported as thecausative agent in some renal diseases (3,4,17). Yoshida etal. (19) reported that CB4, when inoculated repeatedly intomice, induces lesions that are similar to IgAN. However, HIGAmice are reported as a model mouse of IgAN. Muso et al. (18)developed HIGA mice by selective mating of ddY mice that exhibitedhigh serum levels of IgA. At 25 wk of age, HIGA mice spontaneouslydevelop high levels of serum IgA along with glomerular IgA deposition.In addition, the expression of TGF- in the kidney was shownto be increased in these mice (21). Moreover, HIGA mice showa remarkable glomerular deposition of matrix components suchas fibronectin and collagen IV (22). These findings suggestthat HIGA mice may provide a valuable model for studying themechanisms of chronic sclerosis developing types of IgAN. However,glomerular crescent formation, which sometimes is observed inddY mice, was decreased in HIGA animals (21). In addition, HIGAmice had no hematuria and no change of GFR and the renal immunohistologydid not show increased C3 deposition in contrast to IgAN. BecauseHIGA mice are an animal model, they may not be the same as typicalhuman IgAN at all.
In this study, we found that CB4 promoted IgA deposition inmesangial lesions and increased mesangial cell and matrix proliferationin glomeruli of HIGA mice. Endothelial cell injury and crescentformation were found more frequently in HIGA mice with liveCB4 than in HIGA mice without CB4 and HIGA mice with inactivatedCB4. These findings suggested that CB4 infection provoked pathologicchanges in HIGA mice.
The mechanism of exacerbation of renal injury by viral infectionstill is unclear. However, there has been some speculation regardingthe mechanism of exacerbation of renal injury by CB4 infection.With recent advances in immunopathologic and molecular biologictechnique, it has become possible to clarify the mechanismsof induction of glomerulonephritis by viruses. There are twoexplanations for the renal injury. First, direct injury by virusescan cause renal injury. In our study, the detachment and swellingof endothelial cells were found more frequently in HIGA micewith CB4 than in HIGA mice without CB4 and HIGA mice with inactivatedCB4. These findings showed that CB4 directly injured glomerularendothelial cells. In addition, serum IFN- concentration inHIGA mice with live CB4 was higher than those in HIGA mice withoutCB4 and HIGA mice with inactivated CB4. IFN- is the major cytokinesecreted from T-helper 1 cells and promotes cellular immunityincluding macrophage and natural killer cell (23). CB4 promotedIgA deposition in mesangial lesions and mesangial cell and matrixproliferation in glomeruli of HIGA mice, and crescent formationand infiltration by macrophages were observed more frequentlyin HIGA mice with live CB4. IFN- might have caused these pathologicchanges. Therefore, direct injury and mediators such as IFN-that are produced by viral infection in the glomerulus may playimportant roles in the cause of these pathologic changes.
Second, dysfunction of the mesangial pathway that is inducedby viruses can cause renal injury (24,25). As to a functionalpathway between the mesangium and juxtaglomerular apparatus,Leiper et al. (24) reported that iron-dextran complex as tracerparticles initially was taken into the matrix channels of themesangium from which it progressed over the course of 8 h tothe matrix of the juxtaglomerular apparatus and intercellularspaces of the macula densa and mentioned mesangial pathway,which is a continuous functional pathway from the glomerularcapillary lumen to the macula densa cells of the distal tubulefor material that is taken up by the mesangium.
In our study, many carbon particles were present in peripheraland central zones of the mesangium from 5 to 10 d in HIGA micewith live CB4 after administration. These findings suggest thatmesangial pathway function was decreased in HIGA with CB4. Inaddition, IgA concentrations did not differ among the groups.These findings suggest that low clearance of IgA by CB4 infectionmight cause an increase in deposition of mesangial IgA.
There has been some speculation concerning renal injury by immunecomplexes (17,19). Immune complexes consist of antigen and antibody,and the antigen may be virus or renal tissue that is injuredby viral infection. Deposition of these immune complexes inthe mesangium and basement membrane can cause renal injury.However, in our study, results of pathologic examination didnot differ between HIGA without CB4 and HIGA with inactive CB4.Therefore, immune complexes that are induced by inactive CB4may not cause renal disease in our model.
Figure 5. Comparison of the proportions of crescent formation, scores for PCNA-positive cells, scores for -smooth muscle actin (-SMA)–positive cells, matrix scores, and mean number of MCAP497-positive cells among the three groups. aP < 0.05; bP < 0.01.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
concentration were found in mice with live CB4. Many carbon particles were present in peripheral and central zones of the mesangium from 5 to 10 d in mice with carbon and live CB4. These results suggest that CB4 provokes exacerbation of renal pathologic findings in HIGA mice via endothelial injury, IFN-
-smooth muscle actin (
2). Correlations were evaluated using Fisher r test. Findings of P < 0.05 were considered significant. 
in the kidney was shown to be increased in these mice (21). Moreover, HIGA mice show a remarkable glomerular deposition of matrix components such as fibronectin and collagen IV (22). These findings suggest that HIGA mice may provide a valuable model for studying the mechanisms of chronic sclerosis developing types of IgAN. However, glomerular crescent formation, which sometimes is observed in ddY mice, was decreased in HIGA animals (21). In addition, HIGA mice had no hematuria and no change of GFR and the renal immunohistology did not show increased C3 deposition in contrast to IgAN. Because HIGA mice are an animal model, they may not be the same as typical human IgAN at all. 
