Thrombotic Microangiopathy in the HIV-2-Infected Macaque
1999; Elsevier BV; Volume: 155; Issue: 2 Linguagem: Inglês
10.1016/s0002-9440(10)65161-9
ISSN1525-2191
AutoresFrank Eitner, Yan Cui, Kelly L. Hudkins, Ann Marie Schmidt, Ted Birkebak, Michael B. Agy, Shiu-Lok Hu, William R. Morton, David Anderson, Charles E. Alpers,
Tópico(s)HIV-related health complications and treatments
ResumoThrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2287, in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4+ PBMC (<350/μl) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2287, seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2287, was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism. Thrombotic microangiopathy (TMA) has been increasingly reported in human immunodeficiency virus (HIV)-infected humans over the past decade. The pathogenesis is unknown. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques (Macaca nemestrina), infected intravenously with a virulent HIV-2 strain, HIV-2287, in addition to that of four uninfected control macaques. Necropsies were performed between 12 hours and 28 days after infection. HIV-2 antigen was detectable in peripheral blood mononuclear cell (PBMC) cocultures in all animals after 10 days of HIV-2 infection; a rapid decline in CD4+ PBMC (<350/μl) was seen in five of six animals 21 days and 28 days after infection. No macaque developed features of clinical AIDS. Typical lesions of human HIV-associated nephropathy were undetectable. Six of the 27 HIV-2-infected macaques demonstrated both histological TMA lesions (thrombi in glomerular capillary loops and small arteries, mesangiolysis) and ultrastructural lesions (mesangiolysis, subendothelial lucency, platelet thrombi in glomerular capillary lumina). Extrarenal thrombi were detected in the gastrointestinal and adrenal microvasculature of macaques that had developed renal TMA. None of the control animals demonstrated features of renal TMA at necropsy. In a retrospective analysis of kidneys obtained from 39 additional macaques infected with HIV-2287, seven cases demonstrated TMA. In situ hybridization showed no detectable HIV-2 RNA in kidney sections of 65/66 HIV-2-infected macaques, including all 13 TMA cases. Expression of the chemokine receptor CXCR4, the putative coreceptor for HIV-2287, was absent in intrinsic renal cells in all HIV-2-infected macaques. The HIV-2-infected macaque may be a useful model of human HIV-associated TMA. Our data do not support a role of direct HIV-2 infection of intrinsic renal cells as an underlying mechanism. Renal thrombotic microangiopathy (TMA) was first described in an AIDS patient by Boccia et al in 1984.1Boccia RV Gelmann EP Baker CC Marti G Longo DL A hemolytic-uremic syndrome with the acquired immunodeficiency syndrome.Ann Intern Med. 1984; 101: 716-717Crossref PubMed Scopus (69) Google Scholar Subsequently TMA has been reported in several hundred HIV-infected humans worldwide and may be the second most common renal injury associated with HIV infection.2Hymes KB Karpatkin S Human immunodeficiency virus infection and thrombotic microangiopathy.Semin Hematol. 1997; 34: 117-125PubMed Google Scholar, 3Thompson CE Damon LE Ries CA Linker CA Thrombotic microangiopathies in the 1980s, clinical features, response to treatment, and the impact of the human immunodeficiency virus epidemic.Blood. 1992; 80: 1890-1895PubMed Google Scholar, 4Ucar A Fernandez HF Byrnes JJ Lian EC Harrington Jr, WJ Thrombotic microangiopathy and retroviral infections: a 13-year experience.Am J Hematol. 1994; 45: 304-309Crossref PubMed Scopus (74) Google Scholar, 5Bell WR Chulay JD Feinberg JE Manifestations resembling thrombotic microangiopathy in patients with advanced human immunodeficiency virus (HIV) disease in a cytomegalovirus prophylaxis trial (ACTG 204).Medicine (Baltimore). 1997; 76: 369-380Crossref PubMed Scopus (108) Google Scholar, 6Maslo C Peraldi MN Desenclos JC Mougenot B Cywiner-Golenzer C Chatelet FP Jacomet C Rondeau E Rozenbaum W Sraer JD Thrombotic microangiopathy and cytomegalovirus disease in patients infected with human immunodeficiency virus.Clin Infect Dis. 1997; 24: 350-355Crossref PubMed Scopus (65) Google Scholar, 7Ruggenenti P Lutz J Remuzzi G Pathogenesis and treatment of thrombotic microangiopathy.Kidney Int Suppl. 1997; 58: S97-S101PubMed Google Scholar HIV-associated nephropathy (HIVAN), characterized by focal and segmental glomerulosclerosis (FSGS) and a collapsing glomerulopathy, has been identified as the single most common cause of chronic renal insufficiency in HIV-infected humans.8Bourgoignie JJ Pardo V The nephropathology in human immunodeficiency virus (HIV-1) infection.Kidney Int Suppl. 1991; 35: S19-S23PubMed Google Scholar, 9Humphreys MH Human immunodeficiency virus-associated glomerulosclerosis.Kidney Int. 1995; 48: 311-320Crossref PubMed Scopus (95) Google Scholar, 10Winston JA Klotman PE Are we missing an epidemic of HIV-associated nephropathy?.J Am Soc Nephrol. 1996; 7: 1-7PubMed Google Scholar, 11D'Agati V Appel GB HIV infection and the kidney.J Am Soc Nephrol. 1997; 8: 138-152PubMed Google Scholar The TMA injury involves damage to the endothelial lining of glomerular capillaries and arterial microvessels, with activation of the plasma coagulation cascade, causing thromboses in these vessels.12Remuzzi G Ruggenenti P The hemolytic uremic syndrome.Kidney Int. 1995; 48: 2-19Crossref PubMed Scopus (230) Google Scholar Occlusive thrombi in the renal microvasculature, detachment of endothelial cells from the basement membrane, and mesangiolysis are typical findings in HIV-associated TMA. TMA presents clinically as hemolytic uremic syndrome (HUS) or thrombotic thrombocytopenic purpura (TTP), depending on the presence of extrarenal manifestations, and patients may have features of thrombocytopenia, hemolytic anemia, and renal failure.2Hymes KB Karpatkin S Human immunodeficiency virus infection and thrombotic microangiopathy.Semin Hematol. 1997; 34: 117-125PubMed Google Scholar, 13Rao TK Renal complications in HIV disease.Med Clin North Am. 1996; 80: 1437-1451Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar The pathogenesis of HIV-associated TMA is poorly understood. 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In this study, we report on a nonhuman primate model infected by a well-characterized strain of HIV-2, HIV-2287.31Watson A McClure J Ranchalis J Scheibel M Schmidt A Kennedy B Morton WR Haigwood NL Hu SL Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.AIDS Res Hum Retroviruses. 1997; 13: 1375-1381Crossref PubMed Scopus (57) Google Scholar, 32Looney DJ McClure J Kent SJ Radaelli A Kraus G Schmidt A Steffy K Greenberg P Hu SL Morton WR Wong-Staal F A minimally replicative HIV-2 live-virus vaccine protects M. nemestrina from disease after HIV-2(287) challenge.Virology. 1998; 242: 150-160Crossref PubMed Scopus (35) Google Scholar Pigtailed macaques (Macaca nemestrina) infected by HIV-2287 predictably and rapidly develop clinical features characteristic of human HIV-1 infections. A significant proportion of these animals develop renal and systemic TMA that is morphologically similar to TMA as it occurs in humans. These lesions develop despite an apparent lack of HIV infection of renal parenchymal cells, although systemic infection of hematopoietic cells can be readily demonstrated. Additional studies to identify the presence of chemokine coreceptors needed to support cellular infection by HIV-1 in humans suggest the absence of these receptors on renal cells,33Eitner F Cui Y Hudkins KL Anderson DM Schmidt A Morton WR Alpers CE Chemokine receptor (CCR5) expression in human kidneys and in the HIV infected macaque.Kidney Int. 1998; 54: 1945-1954Crossref PubMed Scopus (58) Google Scholar, 34Eitner F Cui Y Hudkins KL Alpers CE Chemokine receptor (CXCR4) mRNA expressing leukocytes are increased in human renal allograft rejection.Transplantation. 1998; 66: 1551-1557Crossref PubMed Scopus (34) Google Scholar which may account for the difficulty in demonstrating the direct infection of such cells by strains of HIV-1. We believe the characterization of a model of HIV-associated TMA offers a major opportunity to understand this important disease process. Pigtailed macaque monkeys infected by HIV-2287, recovered from macaque F89287 after a second passage of a human isolate HIV-2EHO, develop a reliable and very reproducible immunodeficiency syndrome with many features of human HIV disease. We prospectively analyzed the renal pathology and function of 27 pigtailed macaques, experimentally infected intravenously with a virulent HIV-2 strain, HIV-2287. Necropsies were performed at 12 hours and at days 1, 2, 4, 6, 10, 14, 21, and 28 (n = 3, each time point) after infection. Renal pathology was evaluated at necropsy by light and electron microscopy. Renal function was assessed at necropsy by measurement of serum-creatinine, urea, and urinary protein excretion. Renal pathology and function were examined prospectively in four uninfected control macaques matched for age and sex. Furthermore, kidney tissues obtained from 39 pigtailed macaques experimentally infected with HIV-2287 that were involved in viral dissemination studies were analyzed retrospectively. HIV-2 RNA was detected in kidney tissues by an in situ hybridization technique. The expression of the HIV infection coreceptor CXCR4 in kidney tissues was analyzed by in situ hybridization. Twenty-seven pigtailed macaques, Macaca nemestrina (ages 1.2–3.1 years, median 2.1 years; 11 male, 16 female) were inoculated intravenously with 50 TCID50 (50% tissue culture infectious doses) HIV-2287. All macaques involved in this study were colony-born and weighed between 2.1 and 4.5 kg (median 3.0 kg) at necropsy. Before inoculation, each animal was determined to be clinically healthy by physical examination and complete blood cell count. All animals in this study were screened and determined to be negative for simian immunodeficiency virus (SIV) and simian retrovirus (SRV) coinfection before HIV-2 inoculation. This was determined by antibody, cell culture, and polymerase chain reaction (PCR) assays. The macaques were euthanized at 12 hours and on day 1, 2, 4, 6, 10, 14, 21, 28 (n = 3, each time point. after inoculation. The animals were sedated with an intramuscular injection of ketamine-HCl (10 mg/kg body weight) followed by an intravenous overdose of pentabarbital. Complete necropsy examinations were performed on all macaques. All study protocols and procedures were reviewed and approved by the Washington Regional Primate Research Center and the University of Washington Animal Care and Use Committee. Four uninfected, untreated, clinically healthy pigtailed macaques, M. nemestrina (ages 1.4–2.6 years; three male, one female. were euthanized and served as control animals. Kidney tissue specimens were obtained from 39 additional macaques experimentally infected by HIV-2287. These animals were involved in previous studies investigating HIV-2 dissemination kinetics and pathogenesis. Protocols of these studies have been published previously.31Watson A McClure J Ranchalis J Scheibel M Schmidt A Kennedy B Morton WR Haigwood NL Hu SL Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.AIDS Res Hum Retroviruses. 1997; 13: 1375-1381Crossref PubMed Scopus (57) Google Scholar, 32Looney DJ McClure J Kent SJ Radaelli A Kraus G Schmidt A Steffy K Greenberg P Hu SL Morton WR Wong-Staal F A minimally replicative HIV-2 live-virus vaccine protects M. nemestrina from disease after HIV-2(287) challenge.Virology. 1998; 242: 150-160Crossref PubMed Scopus (35) Google Scholar All 39 macaques (ages 0.3–6.4 years, median 1.9 years; 16 male, 23 female) were colony-born and weighed between 0.4 and 9.3 kg (median 2.9 kg) at necropsy. The macaques were euthanized between 63 and 556 days (median 165 days) after HIV-2 infection. HIV-2287 was derived by serial passage of HIV-2EHO (originally obtained from Dr. Luc Montagnier, Pasteur Institute) in M. nemestrina. Animal F87265 was inoculated intravenously with 106 TCID (tissue culture infective dose) cell-free HIV-2EHO as well as 107 autologous peripheral blood mononuclear cells (PBMCs. infected in vitro. Ten milliliters of whole blood obtained from macaque F87265 44 weeks after infection was transfused into animal F89071, and then 10 ml of blood from animal F89071 at 20 weeks after infection was inoculated into F89287. The HIV-2287 challenge stock was derived from coculture of lymph node mononuclear cells of animal F89287 with fresh stimulated allogenic macaque PBMCs. Virus stocks were prepared as clarified supernatants (3000 × g for 20 minutes at 4°C) and aliquoted and stored at −80°C until use. In the prospective part of this study, all 27 macaques were inoculated intravenously with 50 TCID50 of the virus stock solution. HIV-2 infected PBMC were detected by a quantitative coculture assay as described.31Watson A McClure J Ranchalis J Scheibel M Schmidt A Kennedy B Morton WR Haigwood NL Hu SL Early postinfection antiviral treatment reduces viral load and prevents CD4+ cell decline in HIV type 2-infected macaques.AIDS Res Hum Retroviruses. 1997; 13: 1375-1381Crossref PubMed Scopus (57) Google Scholar, 35Hollinger FB Bremer JW Myers LE Gold JW McQuay L Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group.J Clin Microbiol. 1992; 30: 1787-1794PubMed Google Scholar Briefly, freshly isolated macaque PBMCs or lymphocytes were serially diluted in triplets, starting with 106 cells, and cocultivated with fresh human CD8-positive T-cell-depleted phytohemagglutinin-activated PBMCs. Cultures were incubated for 14 days, and the presence of virus was detected using an HIV-2 p27 antigen capture assay. Titers were calculated as the maximum dilution of cells that gave positive cultures and reported as numbers of positive cells/106Maslo C Peraldi MN Desenclos JC Mougenot B Cywiner-Golenzer C Chatelet FP Jacomet C Rondeau E Rozenbaum W Sraer JD Thrombotic microangiopathy and cytomegalovirus disease in patients infected with human immunodeficiency virus.Clin Infect Dis. 1997; 24: 350-355Crossref PubMed Scopus (65) Google Scholar PBMCs. Samples of serum, EDTA-plasma, and urine were obtained from each macaque before experimental euthanasia. Complete blood count, serum-creatinine, urea, and urinary protein excretion were measured using standard procedures. The CD4-positive T-lymphocyte subset was measured as described previously.36Frumkin LR Agy MB Coombs RW Panther L Morton WR Koehler J Florey MJ Dragavon J Schmidt A Katze MG Corey L Acute infection of Macaca nemestrina by human immunodeficiency virus type 1.Virology. 1993; 195: 422-431Crossref PubMed Scopus (43) Google Scholar Briefly, lymphocytes were stained with PE-conjugated anti-CD4 antibody (LEU 3a. Becton-Dickinson, San Jose, CA) and analyzed with a flow cytometer (FACScan/FACSort; Becton-Dickinson). Tissue samples of kidney, liver, lung, brain, adrenal gland, skin, gut, and lymph nodes were fixed in 10% phosphate-buffered formalin, embedded in paraffin, and sectioned. Sections of kidney tissue were routinely stained with hematoxylin and eosin, periodic acid-Schiff (PAS), and Jones's silver methenamine reagents, respectively. Tissue sections of all other organs were routinely stained for hematoxylin and eosin, PAS, and elastic van Gieson, respectively. Tissue for electron microscopy was fixed in half-strength Karnovsky's solution (1% paraformaldehyde and 1.25% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, pH 7.0). After fixation, tissue was postfixed in 1% osmium tetroxide for 2 hours, dehydrated in graded ethanols, and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate and examined with a Phillips 410 electron microscope (Phillips Export BV, Eindhoven, the Netherlands). Kidney tissue examination included at least two and usually three or more glomeruli per animal, as well as a survey of cortical and medullary interstitium, tubules, and blood vessels. A 1.7-kb sequence of DNA coding for HIV-2 gp120 (env) (nucleotides 6480–8335 of the HIV-2287 sequence) was subcloned into pCR II (Invitrogen) (kindly provided by Bristol-Myers Squibb, Seattle, WA), linearized with XhoI, and transcribed with Sp6 RNA polymerase for the antisense probe, or linearized with BamHI and transcribed with T7 RNA polymerase for the sense probe. Detailed protocols for the transcription reaction and the characterization of the specificity of this riboprobe have been described previously.33Eitner F Cui Y Hudkins KL Anderson DM Schmidt A Morton WR Alpers CE Chemokine receptor (CCR5) expression in human kidneys and in the HIV infected macaque.Kidney Int. 1998; 54: 1945-1954Crossref PubMed Scopus (58) Google Scholar A 1.1-kb sequence of DNA coding for human CXCR4 was subcloned into pcDNAI/amp (Invitrogen) (obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, originally provided by Dr. Nathaniel Landau),24Deng H Liu R Ellmeier W Choe S Unutmaz D Burkhart M Di Marzio P Marmon S Sutton RE Hill CM Davis CB Peiper SC Schall TJ Littman DR Landau NR Identification of a major co-receptor for primary isolates of HIV-1.Nature. 1996; 381: 661-666Crossref PubMed Scopus (3222) Google Scholar, 37Morgenstern JP Land H Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.Nucleic Acids Res. 1990; 18: 3587-3596Crossref PubMed Scopus (1910) Google Scholar linearized with HindIII, transcribed with Sp6 for the antisense probe or linearized with XbaI, and transcribed with T7 for the sense probe. Specificity of the CXCR4 antisense riboprobe has been demonstrated previously by Northern analysis and by in situ hybridization using a series of cell lines transfected with different members of the family of chemokine receptors.34Eitner F Cui Y Hudkins KL Alpers CE Chemokine receptor (CXCR4) mRNA expressing leukocytes are increased in human renal allograft rejection.Transplantation. 1998; 66: 1551-1557Crossref PubMed Scopus (34) Google Scholar HIV-2 RNA and CXCR4 mRNA were detected in tissue sections within situ hybridization techniques following protocols that we have previously used.33Eitner F Cui Y Hudkins KL Anderson DM Schmidt A Morton WR Alpers CE Chemokine receptor (CCR5) expression in human kidneys and in the HIV infected macaque.Kidney Int. 1998; 54: 1945-1954Crossref PubMed Scopus (58) Google Scholar, 38Alpers CE Hudkins KL Davis CL Marsh CL Riches W McCarty JM Benjamin CD Carlos TM Harlan JM Lobb R Expression of vascular cell adhesion molecule-1 in kidney allograft rejection.Kidney Int. 1993; 44: 805-816Crossref PubMed Scopus (67) Google Scholar, 39Alpers CE Davis CL Barr D Marsh CL Hudkins KL Identification of platelet-derived growth factor A and B chains in human renal vascular rejection.Am J Pathol. 1996; 148: 439-451PubMed Google Scholar Four-micron sections of formalin-fixed, paraffin-embedded tissue samples were rehydrated through xylene and graded ethanols, washed with 0.5× standard saline citrate (SSC) (1× SSC = 150 mM NaCl, 15 mM Na citrate, pH 7.0), and digested with proteinase K (5 μg/ml; Sigma) in Tris buffer for 30 minutes at 37°C. Several 0.5× SSC washes were followed by prehybridization for 2 hours in 100 μl of prehybridization buffer (0.3 mol/L NaCl, 20 mmol/L Tris (pH 8.0), 5 mmol/L EDTA, 1× Denhardt‘s solution, 10% dextran sulfate, 10 mmol/L dithiothreitol). The hybridizations were started by adding 500,000 cpm of35Hollinger FB Bremer JW Myers LE Gold JW McQuay L Standardization of sensitive human immunodeficiency virus coculture procedures and establishment of a multicenter quality assurance program for the AIDS Clinical Trials Group.J Clin Microbiol. 1992; 30: 1787-1794PubMed Google Scholar S-labeled riboprobe in 50 μl of prehybridization buffer and allowed to proceed overnight at 50°C. After hybridization, sections were washed with 0.5× SSC, and treated with RNase A (20 μg/ml, 30 minutes at 37°C), washed in 2× SSC (2 × 2 minutes), followed by three high-stringency washes in 0.1× SSC/0.5% Tween 20 (Sigma) for 40 minutes each at 50°C and several 2× SSC washes. After the tissue was dehydrated and air-dried, it was dipped in NTB2 nuclear emulsion (Kodak, Rochester, NY) and exposed in the dark at 4°C for 2 weeks (HIV-2) or 4 weeks (CXCR4). After developing, the sections were counterstained with hematoxylin and eosin, dehydrated, and coverslipped. Infection with HIV-2287 in this animal model results in a rapid and predictable decline in CD4-positive cells in the peripheral blood lymphocyte population. Blood samples obtained at necropsy demonstrated a decrease in CD4-positive cells below 350 cells/μl in five of six animals 21 days and 28 days after infection (Figure 1A). HIV-2-infected PBMCs were detectable in PBMC cocultures in 12/12 animals after 10 days of infection (Figure 1B). None of the 27 macaques involved in this study developed other features of clinical AIDS. Pathological examinations of renal tissue from the 27 macaques acutely infected with HIV-2 revealed TMA lesions in six animals. Renal TMA was detectable in two out of three animals as early as 12 hours after HIV-2 infection, and four out of nine macaques had developed TMA after 14 days of infection. None of the macaques euthanized between 1 and 10 days after HIV-2 infection demonstrated features of TMA at necropsy. Table 1 summarizes the histological and ultrastructural features of all 27 macaques.Table 1Pathologic Features in Macaques Acutely Infected with HIV-2 and in Uninfected Control MacaquesTime p.i.AnimalGlomerular and vascular histologyRenal interstitial inflammationElectron microscopyExtrarenal TMAHIV-2 infected 12 hoursK95267nspaVery focalTMANoJ95248nspaVery focaln.d.NoK96161TMA (focal)Mild, focalTMAGut, periadrenal 1 dayJ96226nspaNon.d.NoK94386nspaMild, focaln.d.NoJ96064nspaNon.d.No 2 daysJ96279nspaNon.d.NoJ95271nspaVery focaln.d.NoJ94456nspaNon.d.No 4 daysJ95314nspaNon.d.NoJ95227nspaFocaln.d.NoJ95263nspaNon.d.No 6 daysF95276nspaNon.d.NoK95280nspaNon.d.NoF95265nspaNon.d.No 10 daysJ96140nspaMild, focaln.d.NoJ96173nspaNon.d.NoM96111nspaNon.d.No 14 daysF94434Focal mesangiolysisMild, focalTMAPeriadrenalT96156nspaVery focaln.d.NoK96183nspaFocaln.d.No 21 daysF95269TMA (global)NoTMAGut, adrenalJ96174nspaNonspaNoM96162nspa*See text.Nonspa*See text.No 28 daysK95061TMA (focal)NoTMAGut, periadrenalJ95284TMA (focal)NoTMAGutT96194nspaNon.d.NoUninfectedJ96083nspaVery focalnspan.d.M95311nspaNonspan.d.J96086nspaMild, focalnspa*See text.n.d.F96092nspaVery focalnspa*See text.n.d.nspa, No specific pathologic abnormalities; n.d., not defined; p.i., postinfection.* See text. Open table in a new tab nspa, No specific
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