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Nephropathy associated with sickle cell anemia: An autologous immune complex nephritis
Nephropathy Associated with Sickle Cell Anemia: An Autologous Immune Complex Nephritis II. Clinicopathologic
VICTORIAN0
Study of Seven Patients
PARDO, M.D.
JOSE STRAUSS,
M.D.
HELMUT KRAMER, M.D.
l
Miami, Florida TAKESHI OZAWA,
M.D.+
RAWLE M. MCINTOSH, M.D.z Denver, Colorado
From the Department of Pathology, the Veterans Administration Hospital and the University of Miami Medical Center; the Department of Pediatrics, the University of Miami School of Medicine, Miami, Florida; and the Departments of Pediatrics and Medicine, the University of Colorado Medical Center, Denver, Colorado. This study was supported by Veterans Administration Research: Colorado Sickle Cell Treatment and Research Center (House Bill No. 1578); U.S. Public Heafth Service Grant GR 5 501 RR05351 and NIH General CRC Grant RR.69. Requests for reprints should be addressed to Dr. Rawle M. McIntosh. University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220. Manuscript accepted May 1, 1975. Fellow in Nephrology, Veterans Administration Hospital, Miami, Florida. + Fellow in Nephrology, University of Cotorado Medical Center, Denver, Colorado. t Established Investigator of the American Heart Association. l
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A variety of renal structural and functional abnormalities have been associated with sickle cell disease. To define the relationship between the hemoglobinopathy and glomerular disease, clinicopathologic correlations, renal morphologic, ultrastructural immunohistologic and functional studies were performed on seven patients with clinical and laboratory evidence of glomerular disease. In addition, immunologic studies including isolation and characterization of cryoprecipitable immune complexes, and determination of immunoglobulin, total complement and complement component levels, and antibody titers to several antigens were performed in an attempt to define the etiologic and pathogenic mechanisms of the renal disease and its relationship to sickle cell anemia. Proteinuria was present in all patients. The nephrotic syndrome, hypertension, hematuria and renal insufficiency were found in more than one half the patients. All patients had membranoproliferative glomerulonephritis of varying degree; glomerular basement membrane splitting, electron dense deposits in the glomerulus; interstitial fibrosis, tubular atrophy and hemosiderin deposits were frequent. lmmunoglobulln complement components (classic complement pathway) and renal tubular epithelial antigen were distributed in a granular pattern along the glomerular basement membranes of all patients studied by these methods. Cryoprecipitable complexes of renal tubular epithelial antigen-antibody to renal tubular epithelial antigen as well as antibody to renal tubular epithelial antigen were detected in the circulation of some patients. There was no serologic evidence of activation of the alternate complement pathway. These studies demonstrated an immune deposit normocomplementemic nephritis associated with sickle cell anemia; they further support our hypothesis that the relationship is more than coincidental, and is mediated by glomerular deposition of immune complexes of renal tubular epithellal antigen-antibody to renal tubular epithelial antigen, the antigen possibly released after tubular damage secondary to oxygenation and hemodynamic alterations related to sickle cell disease.
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NEPHROLOGY
Renal manifestations hyposthenuria
and
of sickle unilateral
cell disease hematuria
such as
have
been
studied extensively [l-6]. Morphologic findings reported include congestion, hemorrhage, fibrosis and necrosis of the renal medulla [ 7,8]. On the other hand, with the exception of lesions of apparently minor clinical significance, such as glomerular enlargement and mild hypercellularity [9], information on the glomerular involvement in patients with overt renal disease such as the nephrotic syndrome is limited. Berman and Schreiner [lo] studied a series of patients in whom the nephrotic syndrome was associated with sickle cell disease and were the first to postulate a causal relationship. Subsequently, additional instances of this association have been reported [ 1 l-l 61. However, in these investigations the nature of the glomerular lesions has not been clearly defined, in some instances due to the difficulty in defining the structural changes using only light microscopy, and in other cases due to the difficulty in studying advanced stages at autopsy. In a recent report which included electron microscopic examination of three patients with sickle cell disease and the nephrotic syndrome, McCoy [ 171 demonstrated a mesangioproliferative glomerulonephritis with circumferential mesangial extension into the capillary loops (membranoproliferative glomerulonephritis). Similar findings were reported by Antonovych [ 181 and Elfenbein et al. [ 191 in ten additional subjects. The relationship between glomerulonephritis and sickle cell disease has been unclear. Recently, we described [ 201 a patient with sickle cell disease and immune-complex glomerulonephritis and presented evidence that the immune deposit nephropathy was secondary to circulating complexes of renal tubular epithelial antigen and specific antibody deposited in the renal glomerulus. We postulated that the release of antigen was due to tubular damage, secondary to hemodynamic or renal oxygenation abnormalities related to sickle cell disease. In the present study we describe renal morphologic, immunohistologic and ultrastructural investigations in seven patients with glomerular disease. In these studies we attempt to define the relationship between sickle cell disease and immune deposit nephropathy and include clinicopathologic correlations. MATERIALS
AND METHODS
Seven patients the study.
with sickle cell disease were included in
CASE REPORTS 1. Sickle cell anemia was diagnosed in this patient at two years of age by hemoglobin electrophoresis. After
Case
ASSOCIATED
WITH SICKLE CELL ANEMIA-PARDO
ET AL
repeated hemolytic crises, multiple blood transfusions and an episode of “hepatitis,” the patient was admitted to the hospital at age five with hematuria, edema and hypertension following beta hemolytic streptococcal impetigo. Renal function was normal and proteinuria was absent. A percutaneous renal biopsy was performed. At age 14 he was again hospitalized because of proteinuria, hematuria and cholelithiasis. Renal function was normal (creatinine clearance 150 ml/min/ 1.73 m*) and he was normotensive. Percutaneous renal biopsy was repeated. The patient is now 15 years old. Renal function is still normal, but proteinuria and hematuria persist. He is now receiving diuretics. 2. The nephrotic syndrome developed when the patient was nine years of age, and he was hospitalized. Renal function was normal (creatinine clearance 117 ml/ min/ 1.73 m*). He had pneumonia in the middle lobe of the right lung. He was normotensive, and hematuria was absent. An antistreptolysin 0 titer was 333 Todd U, and throat culture revealed Streptococcus viridans. A percutaneous renal biopsy was performed. At 11 years of age the patient was readmitted with persistent nephrotic syndrome and treated with cyclophosphamide and corticosteroids without any improvement. This patient had a large amount of fetal hemoglobin and he has been relatively free of of symptoms associated with sickle cell disease. The nephrotic syndrome has persisted, hematuria and hypertension are absent. There has been no deterioration in renal function. Case
Case 3. In this patient, the diagnosis of sickle cell disease was made when he was six months of age: it was confirmed by hemoglobin electrophoresis. He had multiple episodes of hemolytic crises, abdominal pain, upper respiratory tract infections and pneumonia. At age 12, the nephrotic syndrome developed and he was hospitalized. Renal function was markedly impaired (creatinine clearance 15 ml/min/ 1.73 m*). Hematuria was present, and the patient was normotensive. Intravenous pyelography revealed bilateral renal enlargement with pyelocalcyceal distortion; a venacavagram showed a patent left renal vein with a questionable decrease in renal blood flow on the right.
A percutaneous renal biopsy was performed, and therapy with cyclophosphamide and corticosteroids was instituted. Renal function deteriorated rapidly and the patient died two months later. An autopsy was performed. Case 4. In this patient, the diagnosis of sickle cell disease was made when he was two years of age. At age 25, after multiple hemolytic crises, cholelithiasis. respiratory tract infections, hepatitis and numerous blood transfusions, she presented to the hospital with the nephrotic syndrome, hematuria, hypertension and impaired renal function (creatinine clearance 40 ml/min/ 1.73 m*). A percutaneous renal biopsy was performed, and therapy with cyclophosphamide and corticosteroids was instituted. Renal function deteriorated progressively and 18 months later she was hospitalized with uremia. Hemodialy-
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NEPHROLOGY ASSOCIATED WITH SICKLE CELL ANEMIA-PAR00
TABLE I __-
Summary of Clinical Data -~------_-_-~ Data Case1
Age* Age at diagnosist Sex
Case2
ET AL
-___-__ Case3
14 2
10 9
13 0.5
M -
M +
M +
8-20 -
17 +
Nephrotic syndrome Proteinuriat Hematuria Renal insufficiency Inferior venacavagram Hypertension
ND -
Admissions
10
1.2 -t -
+§ -
+ 1
15
NOTE: + = renal vein thrombosis; ND = not done. * Age in years when biopsied. t Age at which diagnosis of sickle cell disease was made.
sis was instituted. An inferior venacavagram revealed no abnormalities. Three months later the patient died. An autopsy was performed. Case 5. The diagnosis of sickle cell disease was made in this patient at three years of age. After multiple admissions for sickle cell crises, pneumonia, meningitis and blood transfusions, hypertension was detected at age 14. She was hospitalized at this time and found to have the nephrotic syndrome, hematuria and azotemia. Percutaneous renal biopsy was performed and antihypertensive therapy instituted. Renal function deteriorated rapidly and the patient died three months later. An autopsy was performed. Immunologic and immunopathologic studies on this patient have been reported recently [ 201. Case 6. In this patient the diagnosis of sickle cell disease was made at nine years of age when she suffered a mild cerebral artery thrombosis. She experienced several painful crises and upper respiratory tract infections. At age 26, she was hospitalized with uremia, nephrotic syndrome and hematuria. Hemodialysis was instituted and she died during this period. An autopsy was performed. Case 7. This patient was found to have sickle cell disease at the age of 6 months. After numerous admissions for hemolytic crises, ulcers and transfusions, she was found to have microscopic hematuria and proteinuria at age 18 years. Two years later hypertension was detected. At this time the patient had hematuria and proteinuria. Creatinine clearance was 117 mVmirVl.73 m* body surface area. A percutaneous renal biopsy was performed. Clinical data on these seven patients are summarized in Table I. Renal function was assessed prospectively or retrospectively in all patients by urinalysis, creatinine clearance (Ccr), quantitative urine protein excretion, urine osmolality and determinations of blood urea nitrogen as well as serum creatinine, sodium, chloride, carbon dioxide, calcium, phosphorous, magnesium, cholesterol and osmolality. Roentgenologic studies included intravenous pyelography and in some cases inferior venacavagrams.
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Case4
Case5
Case6
Case7
28 2 F + 6.4 +
14 3 F + 4.2 + +$
26 9 M ? 10.2
20 0.5 F -
+a + 20
+ 10
+§ ND 2
1.0 + + 20
$ g in 24 hours. § Autopsy performed. In some patients quantitative immunoglobulin levels (IgM, IgG and IgA), total serum complement, complement components (Cl, C2, C3, C4, C5, C6 and C7) as well as properdin were determined. Serum cryoglobulin isolation and characterization as well as detection of circulating antibody to renal tubular epithelial antigen were studied in the serums obtained from some of the patients. Hepatitis B antigen and antibody titers as well as antistreptolysin 0 titers, rheumatoid factor, antinuclear antibody and antiDNA were studied in the serum and cryoprotein of several of these patients. These methods have been described in detail previously [20-241. Clinical observations included examination for the presence of hypertension, edema, flank tenderness and a history of flank pain or gross hematuria. Data on the number and nature of crises, hospital admissions and therapeutic measures including transfusions were carefully collected. Percutaneous renal biopsy was performed twice in one of the seven patients and once in five patients. Renal tissue was obtained from four patients at the time of autopsy. In three of these, a percutaneous renal biopsy had been performed previously. Renal tissue was studied by light microscopy, immunofluorescence and electron microscopy. Hlstology and Electron Microscopy. For light microscopy, tissue was fixed in 10 per cent buffered formaldehyde and embedded in paraffin or in 3 per cent glutaraldehyde and embedded in butoxy-ethanol-glycol methacrylate (Polysciences Inc., Warrington, Pa.) for thin sectioning. Sections were stained with hematoxylin and eosin, periodic acid-Shiff, Jones periodic acid silver methenamine and Perl’s method for the detection of iron was used. For electron microscopy, the tissue was fixed in 3.5 per cent glutaraldehyde in phosphate buffer, postfixed in 2 per cent osmium tetroxide in the same buffer, dehydrated and embedded in Epone 812 and cut in a Sorvall MT-2 ultramicrotome. Semithin (1 or 2) sections were stained with 1 per cent alkaline toluidine blue for the selection of glomeruli, and thin sections were stained with uranyl acetate and lead citrate. lmmunohlstologlc Studies. lmmunofluorescence studies were performed utilizing the antiserums to human IgM, IgA, IgG, Clq, C4, C2. C5, C3 proactivator (C3PA). fibrinogen,
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TABLE I I
WITH SICKLE CELL ANEMIA-PARDO
ET AL.
Light and Electron Microscopic Findings Glomeruli
Case Case 1 Case 1 Case 2 Case 3 Case 3 Case 4 Case 4 Case 5 Case 5 Case 6 Case 7
-__ NOTE:
(B’) (B*) (B) (6) (A) (B) (A) (B) (A) (A) (B)
~~__~_.
A = autopsy;
Glomerular Basement Membrane Splitting
Electron Dense Deposits
Glomerular Sclerosis -
3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+
ND + + ND -
3+ 3+ 3+ 3+ 3+ 2+ 2+
++
Tubules Atrophy
-
-
2+ 3+ 3+ 3+ 3+ 2+ 2+
2+ 3+ 3+ 3+ 3+ 2+ 2+
Hemosiderin 3+ 2+ l+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+
B = biopsy; ND = not done.
renal tubular antigen and rabbit antiserums to hepatitis B antigen, as well as to streptococcal protoplast membrane, streptococcal M protein and whole Streptococcus. Fluorescein-labelled aggregated human IgG was used for glomerular localization of rheumatoid factor. These procedures have been described in detail previously [ 201. Staining for renal tubular epithelial antigen as well as hepatitis B surface antigen and Streptococcal antigens was performed before and after frozen cryostat sections were treated with 3M potassium iodide, 3M potassium cyanate or O.OlM citrate buffer pli 3.2 and phosphate buffered saline solution pH 7.2 before and after absorption with the respective antigens as previously described [20]. RESULTS Light and electron rized in Table II.
Interstitial Fibrosis
microscopic
findings are summa-
Light Microscopy. Generalized glomerular lesions were observed early in the course of the disease. There was an increase in mesangial matrix and nuclei, the glomerular basement membranes appeared thickened and some nuclei were observed along the inner peripheral contour of the capillary loops (Figure 1). With the periodic acid-Schiff and Jones periodic acid silver methenamine stains a splitting or reduplication of the basement membrane was found in the areas of apparent basement membrane thickening (Figure 2). Hemosiderin granules were rare in the glomeruli. Tubules exhibited different degrees of hemosiderin deposition. Vascular intimal thickening and/or hyalinization were minimal in early cases. In advanced stages, usually associated with bio-
Figure 1. Case 1. Representative glomerulus from patient presents an increase in mesangkl nuclei and some nuclei may be seen also in the peripheral portions of the loops. Refringent granules corresponding to hemosiderin appear in the cytoplasm of proximal tubular cells. Hematoxylin and eosin stain; original magnification X 350, reduced by 33 per cent.
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Figure 2. Case 3. Glomerulus from patient exhibits an increase in mesangial matrix and a reduplicated basement membrane in many areas. There are sickled erythrocytes in the capillary lumen. Jone ‘s silver methenamine stain; original magnification X 350, reduced by 24 per cent.
chemical manifestations of renal failure, many glomeruli also exhibited changes ranging from sclerosis to complete obsolescence. These glomeruli appeared larger than those seen in most cases of sclerosing and proliferative glomerulonephritis due to other etiologic factors. An increase in interstitial fibrous tissue and tubular atrophy and dilatation was also observed (Figure 3). At autopsy in Case 3 an old thrombosis of the renal vein including intraparenchymatous branches and inferior vena cava was found. In Cases 4, 5 and 6 there was no evidence of venous thrombosis.
Electron Microscopy. On electron microscopy of the early lesions, rows of mesangial cytoplasmic processes were sandwiched between the lamina densa at the filtration surface of the capillary and the endothelium. A line of basement membrane-like material was present under the endothelium and ran a parallel course to the outer basal lamina in a circumferential fashion (Figure 4). This ultrastructural appearance of a double lamina densa resembles the apparent splitting of the basement membrane observed in the light microscopic preparations.
Figure 3. Case 3. Autopsy tissue from patient showing interstitial fibrosis, tubular dilatation and atrophy, hypercenubr glomeruli with perigomerular fibrosis and moderate arteriosclerosis. Uematoxyiin and eosin stain; original magnification X 350, reduced by 34 per cent. November 1975
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Figure 4. Case 1. Mesangial ceil.. (M) extend into the peripheral capMary loop and an additional lamina densa (arrows) is present between these ceils and the endothelium (EN). Small electron dense deposits (arrowheads) occupy the inner aspect of the original lamina densa (LO). L = capillary lumen. EP = epithekm. Uranyl acetate and lead citrate stain; original magnification X 6,125, reduced by 35 per cent. Inset: upper right portion of the figure presents in detail, at higher magnification, the capillary wall X 13,725, reduced by 35 per cent.
Electron dense deposits on the inner aspect of the lamina densa were seen in four patients (Figure 4, inset). Markedly electron dense (60 to 80 A) particles with an internal tetrad substructure were observed free in the cytoplasm of mesangial and epithelial cells. There were also larger membrane bound conglomerates of high electron density which represented siderosomes. These predominated in visceral epithelial and mesangial cells. Advanced glomerular lesions displayed a thickened and wrinkled lamina densa with marked increase in mesangial matrix and collapse or obliteration of the capillary lumen. In these instances the mesangioproliferative reactions were similar to the peripheral mesangial interposition seen in “chronic” sclerosing and proliferative glomerulonephritis in some glomeruli. Immunohistology. The results of immunohistologic studies are shown in Table Ill. IgG and C3 were detected in a granular pattern along the glomerular basement membrane and the
mesangium of all patients studied (Figure 5). IgM was also demonstrated in several of the cases. Clq, C4 and C2 were present but C3PA and properdin were not detected in patients investigated with these antiserums. Renal tubular antigen was localized in the same pattern as immunoglobulins and C3 in two of the subjects studied (Figure 6). Renal tubular antigen was also localized along the proximal tubules, and staining for renal tubular epithelial antigen was abolished by absorption with the antigen. Streptococcal antigens, albumin and hepatitis B antigen and transferin were absent in all the patients studied. lmmunopathologic studies including characterization of antibody eluted from diseased glomeruli have been reported in detail in one of these cases [ 201. Serologic Studies. Cryoproteins were isolated from the serum of three of the four patients studied. The cryoprecipitates contained IgG, IgM and immunoreactive renal tubular epithelial antigen. The IgG was shown to be antibody to renal tubular epithelial antigen and the IgM antibody to IgG and renal tubular epi-
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NEPHFWLOGYASSOCIATED WITH SICKLE CELL ANEMIA-PARDO
TABLE I I I
lmmunohistologic Studies
Case Case Case Case Case NOTE:
1 3 5 7
ET AL.
(B2) (6) (A) (B)
IgG
IgM
+ + + +
+ +
RTE = Renal tubular
epithelial
IgA
C/q
c3
-
ND ND + +
+ + + +
antigen;
c4 ND ND + +
ND = not done;
thelial antigen. Detailed studies on the cryoprotein characterization have been reported previously [21]. Antibody to renal tubular epithelial antigen was detected in the serum of two of the subjects investigated including the one in whom cryoproteins were not identified. Serum complement and complement components as well as properdin levels were normal in the four patients studied. Rheumatoid factor titers were elevated in two patients, and p hemolytic Streptococcus was isolated from one. ANA and anti-DNA antibodies were not present in any patient. Hepatitis B antigen was not detected in the serum of three of the three subjects tested. COMMENTS There has been an increasing number of reports of glomerular disease associated with sickle cell disease since the original observation of Berman and Schreiner [lo]. The similarity of the histologic and electron microscopic findings in recent reports suggests that this association is not fortuitous but that there may be a common pathogenic mechanism. Sickle cell disease may produce glomerular lesions through multiple pathogenic mechanisms.
B = biopsy
5. Case 1. Ghnnerular distribution of IgG in basement membrane demonstrated by dkect immunofluorescence using antihuman IgG fluorescein conjugated serum. Or@inal magnification X 350, reduced by 33 per cent.
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ND ND + +
C3PA ND ND -
tissue: A = autopsy
P
RTE -.
__-ND ND -
ND ND + +
tissue.
Sickling and sludging of erythrocytes within glomerular and postglomerular capillaries may result in stasis and anoxemia. The latter have been considered in the pathophysiology of the nephrotic syndrome associated with heart failure and renal vein thrombosis, but the glomerular alterations observed in these diseases [25,26] differ from those seen in sickle cell disease. Moreover, although instances of renal vein thrombosis related to sickle cell disease have been reported [27,28], in only one of our patients was bilateral renal vein thrombosis demonstrated and in this patient a negative venacavagram was obtained after the manifestations of renal involvement had been documented. A pathogenic mechanism by toxic effect of the extensive iron deposits observed in patients with this disease has been postulated in the renal disease [ 171. However, there is no correlation between the amount of these deposits, which is minimal in the glomeruli, and the presence of renal disease in patients with sickle cell disease. Our patient (Case l), who presented the most extensive hemosiderin deposition, has been followed for six years without any deterioration in his renal status. Furthermore, experimental glomerular lesions produced by iron overload [29-311 do not resemble those encountered in our
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NEPHROLOGY ASSOCIATED WITH SICKLE CELL ANEMIA-PARDO
study. In addition, the glomerular involvement in hemochromatosis is related to the development of diabetes and not to the degree of iron content. Phagocytosis of fragmented sickled erythrocytes by mesangial cells and subsequent stimulation of these cells has been considered an important factor by Antonovych [ 181 and Elfenbein et al. [ 191. But partial circumferential mesangial interposition may be encountered in patients with sickle cell disease in the absence of any clinical manifestation of glomerular disease 119,321. The finding of electron dense deposits in four of the seven patients and the localization of IgG and C3 in the glomeruli of four of our four patients investigated by immunofluorescence suggest the presence of immune-deposits. In the experimental animal as well as in man, immune-complex disease may be secondary to autologous, exogenous or endogenous antigens. In acute and chronic experimental serum sickness, Dixon et al. [33] have demonstrated that the development and progression of glomerulonephritis is related to the physiochemical properties of immune-complexes which are associated with the host’s immune response. The increased incidence of bacterial infections [34] and exposure to exogenous antigens from multiple blood transfusions suggest that these patients may have an immune-complex glomerulonephritis related to microbial or blood products antigens. The association between streptococcal [35] and other bacterial infections as well as hepatitis B antigenemia with immune-complex glomerulonephritis is well documented [ 36-391. The splenic dysfunction [40,41] and abnormalities
ET AL.
of the reticuloendothelial system as well as the impaired immunity observed in patients with sickle cell disease [41-461 may render them more susceptible to persistent progressive glomerulonephritis as encountered with low antibody producers in the persistent progressive experimental serum sickness model of Dixon et al. [33]. Furthermore, reduced clearance of immune-complexes by a defective reticuloendothelial system may facilitate localization of the complexes in the kidney. Local factors such as impacted red cell masses in the capillary loops or mesangial proliferation may also influence glomerular perfusion or filtration and facilitate deposition and localization of the immune-complexes. There was no evidence of hepatitis B antigenemia associated nephritis in any of our patients. Although there was evidence of streptococcal infection in two, the histologic and ultrastructural changes observed were not the characteristic lesions described in poststreptococcal glomerulonephritis. The membranoproliferative changes are similar to those described in hypocomplementemic membranoproliferative glomerulonephritis associated with the activation of the alternate pathway mechanism [47,48]. However, none of our patients showed serologic or immunohistologic evidence of an alternate pathway mechanism. It is of interest that in the large series of patients with hypocomplementemic nephritis reported by Mandalenakis et al. [48] none of the patients was black. Despite the lack of characteristic anatomic tubular lesions, functional tubular defects have been observed with frequency in sickle cell disease [4,6]. Edgington and co-workers [49] described gous immune-complex glomerulonephritis
an autoloin rats in-
Figure 6. Case 7. Representative glomerulus from patient stained fQr renal tubular antigen (RTE). Gram&r deposits are located along the glomerular capillary wall. Original magnification X 400. reduced by 24 per cent.
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duced by complexes of renal tubular antigen and specific antibody. Alterations of the tubules in these patients secondary to hypoxia, hemodynamic disturbances or thrombosis could possibly lead to release and subsequent sensitization to the antigen. Also, iron deposits in tubules may alter cellular constituents and render them antigenic in a manner similar to that postulated for the membranous glomerulopathy associated with the administration of gold salts in rheumatoid arthritis [50,5 11. The demonstration of renal tubular antigen and antibody in the serum of some of these patients, as well as the localization of this antigen with IgG and C3 on the glomerular basement membrane, further suggest an autologous immune-complex pathogenesis for the membranoproliferative glomerulonephritis associated with sickle cell anemia. In addition these investigations support our previous hypothesis in which the mechanism was defined by detailed rhvestigations in one case. Renal tubular epithelial antigenanti-renal tubular epithelial antigen immune deposit disease has not been previously observed in membranoproliferative glomerulonephritis either in patients with hemoglobin S-S or hemoglobin A-A. Furthermore most of our patients with membranoproliferative glomerulonephritis do not have S hemoglobin. The patients described here comprise less than 2 per cent of our patients with membranoproliferative glomerulonephritis. This autologous immune deposit nephritis has been described in a few patients with membranous nephropathy not reportedly associated with sickle cell disease [52,53]. We have found this antigen and antibody in the circulation and in the
glomeruli in association with IgG and C3 of three patients with renal cell carcinoma [54], one with membranous nephropathy associated with renal vein thrombosis, one with sickle cell trait and mild proliferative nephritis, and three patients with sickle cell disease with proliferative nephritis, as well as in preliminary studies in gold, cadmium and mercury nephropathy in man and experimental animals. In addition, in other preliminary studies on black patients, this antibody sometimes associated with antigen has only been detected in patients with hemoglobin S, with or without clinical or morphologic evidence of renal disease. In sickle cell disease, the morphologic finding associated with renal tubular epithelial antigen and antibody in the serum and glomerulus is not always membranoproliferative. As mentioned, four patients not included here had proliferative lesions. It is of interest that in our experience other autologous (renal tubular epithelial antigen-anti-renal tubular epithelial antigen) immune deposit nephritis has been observed in diseases in which there is possible renal tubular damage, such as by hemodynamic and/or oxygenation factors in renal vein thrombosis, toxic injury such as by heavy metals (gold, mercury or cadmium) or diseases of presumably renal tubular origin as in renal cell carcinoma. Further statistical analysis and retrospective and prospective studies are required to determine the incidence of renal tubular epithelial antigen related immune complex disease in patients with sickle cell hemoglobin. However, the present studies strongly suggest a more than coincidental relationship and support our hypothesis.
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34. 35. 36.
37.
of the glomerulus in sickle cell anemia with and without nephrotic syndrome. Am J Pathol 77: 357, 1974. Strauss J, Pardo V, Koss M, et al.: Nephropathy associated with sickle cell anemia: an autologous immune-complex nephritis. I. Studies on the nature of the glomerular bound antibody and antigen identification in a patient with sickle cell disease and immune deposit glomerulonephritis. Am J Med 58: 382, 1975. Strauss J. Koss M, Griswold W, McIntosh RM: Cryoprecipitable circulating serum autologous immune complexes in nephropathy associated with sickle cell disease. Ann Intern Med 81: 114, 1974. McIntosh RM, Kaufman DB, Kulvinskas C, Grossman BJ: Cryoglobulins. I. The nature, incidence and clinical significance of cryoglobulins in glomerulonephritis. J Lab Clin Med 75: 95. 1970. McIntosh RM, Kaufman DB, Kulvinskas C: Cryoglobulins. II. The biological and chemical properties of cryoglobulins in acute post-streptococcal glomerulonephritis. Int Arch Allerg Appl lmmunol 41: 700, 1971. McIntosh RM, Griswold WR, Chernack WB, Williams G. Strauss J, Kaufman DB, Koss MN, McIntosh JR, Cohen R, Weil R IV: Cryoglobulins. Ill. Further studies on the nature, incidence, clinical, diagnostic prognostic and immunopathogenic significance of cryoproteins in renal disease. Q J Med 64: 286, 1975. Squire JR, Blainey JD, Hardwicke J: The nephrotic syndrome. Br Med Bull 13: 43, 1951. Rosenman E, Pollack VE, Pirani C: Renal vein thrombosis in the adult. Clinical and pathologic studies based on renal biopsies. Medicine 47: 269, 1968. Strom T, Muehrcke RC, Smith RD: Sickle cell anemia with the nephrotic syndrome and renal vein obstruction. Arch intern Med 129: 104, 1972. Heptinstall RH: Pathology of the Kidney, 2nd ed.. Liile Brown & Co., Boston, 1974, p 1152. Ellis JT: Glomerular lesions and the nephrotic syndrome in rabbits given saccharated iron oxide intravenously. J Exp Med 103: 127. 1956. Ellis JT: Glomerular lesions in rabbits with experimentally induced proteinuria as disclosed by electron microscopy. Am J Pathol 34: 559, 1958. Dachs S, Churg J: Iron nephropathy. Fed Proc 24: 619, 1965. Pitcock JA. Muirhead EE, Hatch FE, et al.: Early renal changes in sickle cell anemia. Arch Pathol 90: 403, 1970. Dixon FJ, Feldman JD, Vazquez JJ: Experimental glomerulonephritis. The pathogenesis of a laboratory model resembling the spectrum of human glomerulonephritis. J Exp Med 122: 99, 1965. Barrett-Connor E: Bacterial infection and sickle cell anemia. Medicine (Baltimore) 50: 97, 1971. Rammelkamp CH: Microbiologic aspects of glomerulonephritis. J Chronic Dis 5: 28, 1957. Combes B, Statstny P, Shorey J, et al.: Glomerulonephritis with deposition of Australia antigen-antibody complexes in glomerular basement membrane. Lancet 2: 234, 1971 Myers BD. Griffel B, Naveh D. et al.: Membranoproliferative
38.
39.
40. 41.
42. 43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
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glomerulonephritis associated with persistent viral hepatitis Am J Clin Pathol 60: 222, 1973. Kohler PF, Cronin RE, Hammond W, et al.: Chronic membranous glomerulonephritis caused by Hepatitis B antigen-antibody complexes. Ann Intern Med 81: 448, 1974. Gyorkey F, Min KW. Gyorkey P: Renal changes in hepatitis associated antigenemia with hematuria. Immunofluorescence and electronmicroscopic study. Am J Pathol 66: 58a, 1972. Pearson HA, Spencer RP, Cornelius EA: Functional asplenia in sickle cell anemia. N Engl J Med 281: 923, 1969. Panos G, Rothenberg SP, Guy R: Correlation of low serum IgM levels with absence of functional splenic tissue in sickle cell disease syndromes. Am J Med 57: 542, 1974. Rosen FS: Sickle cell disease and the properdin system. N Engl J Mad 288: 845, 1973. Winkelstein JA. Drachman RH: Deficiency of pneumococcal opsonizing activity in sickle cell disease. N Engl J Med 279: 459, 1968. Shulman ST, Bartlett J, Clyde WA, et al.: The unusual severity of mycoplasmal pneumonia in children with sickle cell disease. N Engl J Med 287: 164, 1972. Schwarz AD, Pearson HA: Impaired antibody response to intravenous immunization in sickle cell anemia. Pediatr Res 6: 145, 1972. Johnston RB, Newman SL, Struth AG: Serum opsonins and the alternate pathway in sickle cell disease. N Engl J Med 288: 803. 1973. West CD, McAdams AJ, McConville JM. et al.: Hypocomplementemic and normocomplementemic persistent (chronic) glomerulonephritis. Clinical and pathologic characteristics. J Pediatr 67: 1089, 1968. Mandalenakis N, Mendoza N, Pirani CL, et al.: Lobular glomerulonephritis and membranoproliferative glomerulonephritis. Medicine (Baltimore) 50: 318, 1971. Edgington TS, Glassock RJ. Dixon FJ: Autologous immune complex nephritis induced with renal tubular antigen. I. Identification and isolation of the pathogenic antigen. J Exp Med 127: 55, 1968. Nagi AH, Alexander F, Barabas AZ: Gold nephropathy in rats. Light and electron microscopic studies. Exp Mol Pathol 16: 354, 1971. Tornroth T. Skrifvars B: Gold nephropathy prototype of membranous glomerulonephritis. Am J Pathol 75: 573, 1974. Naruse T, Kitamura K, Miyakawa Y, Shibata S: Deposition of renal tubular epithelial antigen along the glomerular capillary walls of patients with membranous glomerulonephritis. J lmmunol 110: 1163, 1973. Naruse T, Miyakawa Y, Kitamura K, Shibata S: Membranous glomerulonephritis mediated by renal tubular epithelial antigen-antibody complex. J Allerg Clin lmmunol 54: 311, 1974. Ozawa, T, Pluss R, Lather J, Boedecker E, Guggenheim S, Hammond W, McIntosh R: Endogenous immune complex nephropathy associated with malignancy, I. Studies on the nature and immunopathogenic significance of glomerular bound antigen and antibody, isolation and characterization of tumor specific antigen and antibody and circulating immune complexes. Cl J Med 64: 563, 1975.
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VICTORIAN0
Study of Seven Patients
PARDO, M.D.
JOSE STRAUSS,
M.D.
HELMUT KRAMER, M.D.
l
Miami, Florida TAKESHI OZAWA,
M.D.+
RAWLE M. MCINTOSH, M.D.z Denver, Colorado
From the Department of Pathology, the Veterans Administration Hospital and the University of Miami Medical Center; the Department of Pediatrics, the University of Miami School of Medicine, Miami, Florida; and the Departments of Pediatrics and Medicine, the University of Colorado Medical Center, Denver, Colorado. This study was supported by Veterans Administration Research: Colorado Sickle Cell Treatment and Research Center (House Bill No. 1578); U.S. Public Heafth Service Grant GR 5 501 RR05351 and NIH General CRC Grant RR.69. Requests for reprints should be addressed to Dr. Rawle M. McIntosh. University of Colorado Medical Center, 4200 East Ninth Avenue, Denver, Colorado 80220. Manuscript accepted May 1, 1975. Fellow in Nephrology, Veterans Administration Hospital, Miami, Florida. + Fellow in Nephrology, University of Cotorado Medical Center, Denver, Colorado. t Established Investigator of the American Heart Association. l
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A variety of renal structural and functional abnormalities have been associated with sickle cell disease. To define the relationship between the hemoglobinopathy and glomerular disease, clinicopathologic correlations, renal morphologic, ultrastructural immunohistologic and functional studies were performed on seven patients with clinical and laboratory evidence of glomerular disease. In addition, immunologic studies including isolation and characterization of cryoprecipitable immune complexes, and determination of immunoglobulin, total complement and complement component levels, and antibody titers to several antigens were performed in an attempt to define the etiologic and pathogenic mechanisms of the renal disease and its relationship to sickle cell anemia. Proteinuria was present in all patients. The nephrotic syndrome, hypertension, hematuria and renal insufficiency were found in more than one half the patients. All patients had membranoproliferative glomerulonephritis of varying degree; glomerular basement membrane splitting, electron dense deposits in the glomerulus; interstitial fibrosis, tubular atrophy and hemosiderin deposits were frequent. lmmunoglobulln complement components (classic complement pathway) and renal tubular epithelial antigen were distributed in a granular pattern along the glomerular basement membranes of all patients studied by these methods. Cryoprecipitable complexes of renal tubular epithelial antigen-antibody to renal tubular epithelial antigen as well as antibody to renal tubular epithelial antigen were detected in the circulation of some patients. There was no serologic evidence of activation of the alternate complement pathway. These studies demonstrated an immune deposit normocomplementemic nephritis associated with sickle cell anemia; they further support our hypothesis that the relationship is more than coincidental, and is mediated by glomerular deposition of immune complexes of renal tubular epithellal antigen-antibody to renal tubular epithelial antigen, the antigen possibly released after tubular damage secondary to oxygenation and hemodynamic alterations related to sickle cell disease.
The American Journal ol Medkine
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NEPHROLOGY
Renal manifestations hyposthenuria
and
of sickle unilateral
cell disease hematuria
such as
have
been
studied extensively [l-6]. Morphologic findings reported include congestion, hemorrhage, fibrosis and necrosis of the renal medulla [ 7,8]. On the other hand, with the exception of lesions of apparently minor clinical significance, such as glomerular enlargement and mild hypercellularity [9], information on the glomerular involvement in patients with overt renal disease such as the nephrotic syndrome is limited. Berman and Schreiner [lo] studied a series of patients in whom the nephrotic syndrome was associated with sickle cell disease and were the first to postulate a causal relationship. Subsequently, additional instances of this association have been reported [ 1 l-l 61. However, in these investigations the nature of the glomerular lesions has not been clearly defined, in some instances due to the difficulty in defining the structural changes using only light microscopy, and in other cases due to the difficulty in studying advanced stages at autopsy. In a recent report which included electron microscopic examination of three patients with sickle cell disease and the nephrotic syndrome, McCoy [ 171 demonstrated a mesangioproliferative glomerulonephritis with circumferential mesangial extension into the capillary loops (membranoproliferative glomerulonephritis). Similar findings were reported by Antonovych [ 181 and Elfenbein et al. [ 191 in ten additional subjects. The relationship between glomerulonephritis and sickle cell disease has been unclear. Recently, we described [ 201 a patient with sickle cell disease and immune-complex glomerulonephritis and presented evidence that the immune deposit nephropathy was secondary to circulating complexes of renal tubular epithelial antigen and specific antibody deposited in the renal glomerulus. We postulated that the release of antigen was due to tubular damage, secondary to hemodynamic or renal oxygenation abnormalities related to sickle cell disease. In the present study we describe renal morphologic, immunohistologic and ultrastructural investigations in seven patients with glomerular disease. In these studies we attempt to define the relationship between sickle cell disease and immune deposit nephropathy and include clinicopathologic correlations. MATERIALS
AND METHODS
Seven patients the study.
with sickle cell disease were included in
CASE REPORTS 1. Sickle cell anemia was diagnosed in this patient at two years of age by hemoglobin electrophoresis. After
Case
ASSOCIATED
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ET AL
repeated hemolytic crises, multiple blood transfusions and an episode of “hepatitis,” the patient was admitted to the hospital at age five with hematuria, edema and hypertension following beta hemolytic streptococcal impetigo. Renal function was normal and proteinuria was absent. A percutaneous renal biopsy was performed. At age 14 he was again hospitalized because of proteinuria, hematuria and cholelithiasis. Renal function was normal (creatinine clearance 150 ml/min/ 1.73 m*) and he was normotensive. Percutaneous renal biopsy was repeated. The patient is now 15 years old. Renal function is still normal, but proteinuria and hematuria persist. He is now receiving diuretics. 2. The nephrotic syndrome developed when the patient was nine years of age, and he was hospitalized. Renal function was normal (creatinine clearance 117 ml/ min/ 1.73 m*). He had pneumonia in the middle lobe of the right lung. He was normotensive, and hematuria was absent. An antistreptolysin 0 titer was 333 Todd U, and throat culture revealed Streptococcus viridans. A percutaneous renal biopsy was performed. At 11 years of age the patient was readmitted with persistent nephrotic syndrome and treated with cyclophosphamide and corticosteroids without any improvement. This patient had a large amount of fetal hemoglobin and he has been relatively free of of symptoms associated with sickle cell disease. The nephrotic syndrome has persisted, hematuria and hypertension are absent. There has been no deterioration in renal function. Case
Case 3. In this patient, the diagnosis of sickle cell disease was made when he was six months of age: it was confirmed by hemoglobin electrophoresis. He had multiple episodes of hemolytic crises, abdominal pain, upper respiratory tract infections and pneumonia. At age 12, the nephrotic syndrome developed and he was hospitalized. Renal function was markedly impaired (creatinine clearance 15 ml/min/ 1.73 m*). Hematuria was present, and the patient was normotensive. Intravenous pyelography revealed bilateral renal enlargement with pyelocalcyceal distortion; a venacavagram showed a patent left renal vein with a questionable decrease in renal blood flow on the right.
A percutaneous renal biopsy was performed, and therapy with cyclophosphamide and corticosteroids was instituted. Renal function deteriorated rapidly and the patient died two months later. An autopsy was performed. Case 4. In this patient, the diagnosis of sickle cell disease was made when he was two years of age. At age 25, after multiple hemolytic crises, cholelithiasis. respiratory tract infections, hepatitis and numerous blood transfusions, she presented to the hospital with the nephrotic syndrome, hematuria, hypertension and impaired renal function (creatinine clearance 40 ml/min/ 1.73 m*). A percutaneous renal biopsy was performed, and therapy with cyclophosphamide and corticosteroids was instituted. Renal function deteriorated progressively and 18 months later she was hospitalized with uremia. Hemodialy-
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TABLE I __-
Summary of Clinical Data -~------_-_-~ Data Case1
Age* Age at diagnosist Sex
Case2
ET AL
-___-__ Case3
14 2
10 9
13 0.5
M -
M +
M +
8-20 -
17 +
Nephrotic syndrome Proteinuriat Hematuria Renal insufficiency Inferior venacavagram Hypertension
ND -
Admissions
10
1.2 -t -
+§ -
+ 1
15
NOTE: + = renal vein thrombosis; ND = not done. * Age in years when biopsied. t Age at which diagnosis of sickle cell disease was made.
sis was instituted. An inferior venacavagram revealed no abnormalities. Three months later the patient died. An autopsy was performed. Case 5. The diagnosis of sickle cell disease was made in this patient at three years of age. After multiple admissions for sickle cell crises, pneumonia, meningitis and blood transfusions, hypertension was detected at age 14. She was hospitalized at this time and found to have the nephrotic syndrome, hematuria and azotemia. Percutaneous renal biopsy was performed and antihypertensive therapy instituted. Renal function deteriorated rapidly and the patient died three months later. An autopsy was performed. Immunologic and immunopathologic studies on this patient have been reported recently [ 201. Case 6. In this patient the diagnosis of sickle cell disease was made at nine years of age when she suffered a mild cerebral artery thrombosis. She experienced several painful crises and upper respiratory tract infections. At age 26, she was hospitalized with uremia, nephrotic syndrome and hematuria. Hemodialysis was instituted and she died during this period. An autopsy was performed. Case 7. This patient was found to have sickle cell disease at the age of 6 months. After numerous admissions for hemolytic crises, ulcers and transfusions, she was found to have microscopic hematuria and proteinuria at age 18 years. Two years later hypertension was detected. At this time the patient had hematuria and proteinuria. Creatinine clearance was 117 mVmirVl.73 m* body surface area. A percutaneous renal biopsy was performed. Clinical data on these seven patients are summarized in Table I. Renal function was assessed prospectively or retrospectively in all patients by urinalysis, creatinine clearance (Ccr), quantitative urine protein excretion, urine osmolality and determinations of blood urea nitrogen as well as serum creatinine, sodium, chloride, carbon dioxide, calcium, phosphorous, magnesium, cholesterol and osmolality. Roentgenologic studies included intravenous pyelography and in some cases inferior venacavagrams.
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Case4
Case5
Case6
Case7
28 2 F + 6.4 +
14 3 F + 4.2 + +$
26 9 M ? 10.2
20 0.5 F -
+a + 20
+ 10
+§ ND 2
1.0 + + 20
$ g in 24 hours. § Autopsy performed. In some patients quantitative immunoglobulin levels (IgM, IgG and IgA), total serum complement, complement components (Cl, C2, C3, C4, C5, C6 and C7) as well as properdin were determined. Serum cryoglobulin isolation and characterization as well as detection of circulating antibody to renal tubular epithelial antigen were studied in the serums obtained from some of the patients. Hepatitis B antigen and antibody titers as well as antistreptolysin 0 titers, rheumatoid factor, antinuclear antibody and antiDNA were studied in the serum and cryoprotein of several of these patients. These methods have been described in detail previously [20-241. Clinical observations included examination for the presence of hypertension, edema, flank tenderness and a history of flank pain or gross hematuria. Data on the number and nature of crises, hospital admissions and therapeutic measures including transfusions were carefully collected. Percutaneous renal biopsy was performed twice in one of the seven patients and once in five patients. Renal tissue was obtained from four patients at the time of autopsy. In three of these, a percutaneous renal biopsy had been performed previously. Renal tissue was studied by light microscopy, immunofluorescence and electron microscopy. Hlstology and Electron Microscopy. For light microscopy, tissue was fixed in 10 per cent buffered formaldehyde and embedded in paraffin or in 3 per cent glutaraldehyde and embedded in butoxy-ethanol-glycol methacrylate (Polysciences Inc., Warrington, Pa.) for thin sectioning. Sections were stained with hematoxylin and eosin, periodic acid-Shiff, Jones periodic acid silver methenamine and Perl’s method for the detection of iron was used. For electron microscopy, the tissue was fixed in 3.5 per cent glutaraldehyde in phosphate buffer, postfixed in 2 per cent osmium tetroxide in the same buffer, dehydrated and embedded in Epone 812 and cut in a Sorvall MT-2 ultramicrotome. Semithin (1 or 2) sections were stained with 1 per cent alkaline toluidine blue for the selection of glomeruli, and thin sections were stained with uranyl acetate and lead citrate. lmmunohlstologlc Studies. lmmunofluorescence studies were performed utilizing the antiserums to human IgM, IgA, IgG, Clq, C4, C2. C5, C3 proactivator (C3PA). fibrinogen,
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Light and Electron Microscopic Findings Glomeruli
Case Case 1 Case 1 Case 2 Case 3 Case 3 Case 4 Case 4 Case 5 Case 5 Case 6 Case 7
-__ NOTE:
(B’) (B*) (B) (6) (A) (B) (A) (B) (A) (A) (B)
~~__~_.
A = autopsy;
Glomerular Basement Membrane Splitting
Electron Dense Deposits
Glomerular Sclerosis -
3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+ 3+
ND + + ND -
3+ 3+ 3+ 3+ 3+ 2+ 2+
++
Tubules Atrophy
-
-
2+ 3+ 3+ 3+ 3+ 2+ 2+
2+ 3+ 3+ 3+ 3+ 2+ 2+
Hemosiderin 3+ 2+ l+ 2+ 3+ 2+ 2+ 2+ 2+ 2+ 2+
B = biopsy; ND = not done.
renal tubular antigen and rabbit antiserums to hepatitis B antigen, as well as to streptococcal protoplast membrane, streptococcal M protein and whole Streptococcus. Fluorescein-labelled aggregated human IgG was used for glomerular localization of rheumatoid factor. These procedures have been described in detail previously [ 201. Staining for renal tubular epithelial antigen as well as hepatitis B surface antigen and Streptococcal antigens was performed before and after frozen cryostat sections were treated with 3M potassium iodide, 3M potassium cyanate or O.OlM citrate buffer pli 3.2 and phosphate buffered saline solution pH 7.2 before and after absorption with the respective antigens as previously described [20]. RESULTS Light and electron rized in Table II.
Interstitial Fibrosis
microscopic
findings are summa-
Light Microscopy. Generalized glomerular lesions were observed early in the course of the disease. There was an increase in mesangial matrix and nuclei, the glomerular basement membranes appeared thickened and some nuclei were observed along the inner peripheral contour of the capillary loops (Figure 1). With the periodic acid-Schiff and Jones periodic acid silver methenamine stains a splitting or reduplication of the basement membrane was found in the areas of apparent basement membrane thickening (Figure 2). Hemosiderin granules were rare in the glomeruli. Tubules exhibited different degrees of hemosiderin deposition. Vascular intimal thickening and/or hyalinization were minimal in early cases. In advanced stages, usually associated with bio-
Figure 1. Case 1. Representative glomerulus from patient presents an increase in mesangkl nuclei and some nuclei may be seen also in the peripheral portions of the loops. Refringent granules corresponding to hemosiderin appear in the cytoplasm of proximal tubular cells. Hematoxylin and eosin stain; original magnification X 350, reduced by 33 per cent.
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Figure 2. Case 3. Glomerulus from patient exhibits an increase in mesangial matrix and a reduplicated basement membrane in many areas. There are sickled erythrocytes in the capillary lumen. Jone ‘s silver methenamine stain; original magnification X 350, reduced by 24 per cent.
chemical manifestations of renal failure, many glomeruli also exhibited changes ranging from sclerosis to complete obsolescence. These glomeruli appeared larger than those seen in most cases of sclerosing and proliferative glomerulonephritis due to other etiologic factors. An increase in interstitial fibrous tissue and tubular atrophy and dilatation was also observed (Figure 3). At autopsy in Case 3 an old thrombosis of the renal vein including intraparenchymatous branches and inferior vena cava was found. In Cases 4, 5 and 6 there was no evidence of venous thrombosis.
Electron Microscopy. On electron microscopy of the early lesions, rows of mesangial cytoplasmic processes were sandwiched between the lamina densa at the filtration surface of the capillary and the endothelium. A line of basement membrane-like material was present under the endothelium and ran a parallel course to the outer basal lamina in a circumferential fashion (Figure 4). This ultrastructural appearance of a double lamina densa resembles the apparent splitting of the basement membrane observed in the light microscopic preparations.
Figure 3. Case 3. Autopsy tissue from patient showing interstitial fibrosis, tubular dilatation and atrophy, hypercenubr glomeruli with perigomerular fibrosis and moderate arteriosclerosis. Uematoxyiin and eosin stain; original magnification X 350, reduced by 34 per cent. November 1975
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Figure 4. Case 1. Mesangial ceil.. (M) extend into the peripheral capMary loop and an additional lamina densa (arrows) is present between these ceils and the endothelium (EN). Small electron dense deposits (arrowheads) occupy the inner aspect of the original lamina densa (LO). L = capillary lumen. EP = epithekm. Uranyl acetate and lead citrate stain; original magnification X 6,125, reduced by 35 per cent. Inset: upper right portion of the figure presents in detail, at higher magnification, the capillary wall X 13,725, reduced by 35 per cent.
Electron dense deposits on the inner aspect of the lamina densa were seen in four patients (Figure 4, inset). Markedly electron dense (60 to 80 A) particles with an internal tetrad substructure were observed free in the cytoplasm of mesangial and epithelial cells. There were also larger membrane bound conglomerates of high electron density which represented siderosomes. These predominated in visceral epithelial and mesangial cells. Advanced glomerular lesions displayed a thickened and wrinkled lamina densa with marked increase in mesangial matrix and collapse or obliteration of the capillary lumen. In these instances the mesangioproliferative reactions were similar to the peripheral mesangial interposition seen in “chronic” sclerosing and proliferative glomerulonephritis in some glomeruli. Immunohistology. The results of immunohistologic studies are shown in Table Ill. IgG and C3 were detected in a granular pattern along the glomerular basement membrane and the
mesangium of all patients studied (Figure 5). IgM was also demonstrated in several of the cases. Clq, C4 and C2 were present but C3PA and properdin were not detected in patients investigated with these antiserums. Renal tubular antigen was localized in the same pattern as immunoglobulins and C3 in two of the subjects studied (Figure 6). Renal tubular antigen was also localized along the proximal tubules, and staining for renal tubular epithelial antigen was abolished by absorption with the antigen. Streptococcal antigens, albumin and hepatitis B antigen and transferin were absent in all the patients studied. lmmunopathologic studies including characterization of antibody eluted from diseased glomeruli have been reported in detail in one of these cases [ 201. Serologic Studies. Cryoproteins were isolated from the serum of three of the four patients studied. The cryoprecipitates contained IgG, IgM and immunoreactive renal tubular epithelial antigen. The IgG was shown to be antibody to renal tubular epithelial antigen and the IgM antibody to IgG and renal tubular epi-
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NEPHFWLOGYASSOCIATED WITH SICKLE CELL ANEMIA-PARDO
TABLE I I I
lmmunohistologic Studies
Case Case Case Case Case NOTE:
1 3 5 7
ET AL.
(B2) (6) (A) (B)
IgG
IgM
+ + + +
+ +
RTE = Renal tubular
epithelial
IgA
C/q
c3
-
ND ND + +
+ + + +
antigen;
c4 ND ND + +
ND = not done;
thelial antigen. Detailed studies on the cryoprotein characterization have been reported previously [21]. Antibody to renal tubular epithelial antigen was detected in the serum of two of the subjects investigated including the one in whom cryoproteins were not identified. Serum complement and complement components as well as properdin levels were normal in the four patients studied. Rheumatoid factor titers were elevated in two patients, and p hemolytic Streptococcus was isolated from one. ANA and anti-DNA antibodies were not present in any patient. Hepatitis B antigen was not detected in the serum of three of the three subjects tested. COMMENTS There has been an increasing number of reports of glomerular disease associated with sickle cell disease since the original observation of Berman and Schreiner [lo]. The similarity of the histologic and electron microscopic findings in recent reports suggests that this association is not fortuitous but that there may be a common pathogenic mechanism. Sickle cell disease may produce glomerular lesions through multiple pathogenic mechanisms.
B = biopsy
5. Case 1. Ghnnerular distribution of IgG in basement membrane demonstrated by dkect immunofluorescence using antihuman IgG fluorescein conjugated serum. Or@inal magnification X 350, reduced by 33 per cent.
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ND ND + +
C3PA ND ND -
tissue: A = autopsy
P
RTE -.
__-ND ND -
ND ND + +
tissue.
Sickling and sludging of erythrocytes within glomerular and postglomerular capillaries may result in stasis and anoxemia. The latter have been considered in the pathophysiology of the nephrotic syndrome associated with heart failure and renal vein thrombosis, but the glomerular alterations observed in these diseases [25,26] differ from those seen in sickle cell disease. Moreover, although instances of renal vein thrombosis related to sickle cell disease have been reported [27,28], in only one of our patients was bilateral renal vein thrombosis demonstrated and in this patient a negative venacavagram was obtained after the manifestations of renal involvement had been documented. A pathogenic mechanism by toxic effect of the extensive iron deposits observed in patients with this disease has been postulated in the renal disease [ 171. However, there is no correlation between the amount of these deposits, which is minimal in the glomeruli, and the presence of renal disease in patients with sickle cell disease. Our patient (Case l), who presented the most extensive hemosiderin deposition, has been followed for six years without any deterioration in his renal status. Furthermore, experimental glomerular lesions produced by iron overload [29-311 do not resemble those encountered in our
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study. In addition, the glomerular involvement in hemochromatosis is related to the development of diabetes and not to the degree of iron content. Phagocytosis of fragmented sickled erythrocytes by mesangial cells and subsequent stimulation of these cells has been considered an important factor by Antonovych [ 181 and Elfenbein et al. [ 191. But partial circumferential mesangial interposition may be encountered in patients with sickle cell disease in the absence of any clinical manifestation of glomerular disease 119,321. The finding of electron dense deposits in four of the seven patients and the localization of IgG and C3 in the glomeruli of four of our four patients investigated by immunofluorescence suggest the presence of immune-deposits. In the experimental animal as well as in man, immune-complex disease may be secondary to autologous, exogenous or endogenous antigens. In acute and chronic experimental serum sickness, Dixon et al. [33] have demonstrated that the development and progression of glomerulonephritis is related to the physiochemical properties of immune-complexes which are associated with the host’s immune response. The increased incidence of bacterial infections [34] and exposure to exogenous antigens from multiple blood transfusions suggest that these patients may have an immune-complex glomerulonephritis related to microbial or blood products antigens. The association between streptococcal [35] and other bacterial infections as well as hepatitis B antigenemia with immune-complex glomerulonephritis is well documented [ 36-391. The splenic dysfunction [40,41] and abnormalities
ET AL.
of the reticuloendothelial system as well as the impaired immunity observed in patients with sickle cell disease [41-461 may render them more susceptible to persistent progressive glomerulonephritis as encountered with low antibody producers in the persistent progressive experimental serum sickness model of Dixon et al. [33]. Furthermore, reduced clearance of immune-complexes by a defective reticuloendothelial system may facilitate localization of the complexes in the kidney. Local factors such as impacted red cell masses in the capillary loops or mesangial proliferation may also influence glomerular perfusion or filtration and facilitate deposition and localization of the immune-complexes. There was no evidence of hepatitis B antigenemia associated nephritis in any of our patients. Although there was evidence of streptococcal infection in two, the histologic and ultrastructural changes observed were not the characteristic lesions described in poststreptococcal glomerulonephritis. The membranoproliferative changes are similar to those described in hypocomplementemic membranoproliferative glomerulonephritis associated with the activation of the alternate pathway mechanism [47,48]. However, none of our patients showed serologic or immunohistologic evidence of an alternate pathway mechanism. It is of interest that in the large series of patients with hypocomplementemic nephritis reported by Mandalenakis et al. [48] none of the patients was black. Despite the lack of characteristic anatomic tubular lesions, functional tubular defects have been observed with frequency in sickle cell disease [4,6]. Edgington and co-workers [49] described gous immune-complex glomerulonephritis
an autoloin rats in-
Figure 6. Case 7. Representative glomerulus from patient stained fQr renal tubular antigen (RTE). Gram&r deposits are located along the glomerular capillary wall. Original magnification X 400. reduced by 24 per cent.
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duced by complexes of renal tubular antigen and specific antibody. Alterations of the tubules in these patients secondary to hypoxia, hemodynamic disturbances or thrombosis could possibly lead to release and subsequent sensitization to the antigen. Also, iron deposits in tubules may alter cellular constituents and render them antigenic in a manner similar to that postulated for the membranous glomerulopathy associated with the administration of gold salts in rheumatoid arthritis [50,5 11. The demonstration of renal tubular antigen and antibody in the serum of some of these patients, as well as the localization of this antigen with IgG and C3 on the glomerular basement membrane, further suggest an autologous immune-complex pathogenesis for the membranoproliferative glomerulonephritis associated with sickle cell anemia. In addition these investigations support our previous hypothesis in which the mechanism was defined by detailed rhvestigations in one case. Renal tubular epithelial antigenanti-renal tubular epithelial antigen immune deposit disease has not been previously observed in membranoproliferative glomerulonephritis either in patients with hemoglobin S-S or hemoglobin A-A. Furthermore most of our patients with membranoproliferative glomerulonephritis do not have S hemoglobin. The patients described here comprise less than 2 per cent of our patients with membranoproliferative glomerulonephritis. This autologous immune deposit nephritis has been described in a few patients with membranous nephropathy not reportedly associated with sickle cell disease [52,53]. We have found this antigen and antibody in the circulation and in the
glomeruli in association with IgG and C3 of three patients with renal cell carcinoma [54], one with membranous nephropathy associated with renal vein thrombosis, one with sickle cell trait and mild proliferative nephritis, and three patients with sickle cell disease with proliferative nephritis, as well as in preliminary studies in gold, cadmium and mercury nephropathy in man and experimental animals. In addition, in other preliminary studies on black patients, this antibody sometimes associated with antigen has only been detected in patients with hemoglobin S, with or without clinical or morphologic evidence of renal disease. In sickle cell disease, the morphologic finding associated with renal tubular epithelial antigen and antibody in the serum and glomerulus is not always membranoproliferative. As mentioned, four patients not included here had proliferative lesions. It is of interest that in our experience other autologous (renal tubular epithelial antigen-anti-renal tubular epithelial antigen) immune deposit nephritis has been observed in diseases in which there is possible renal tubular damage, such as by hemodynamic and/or oxygenation factors in renal vein thrombosis, toxic injury such as by heavy metals (gold, mercury or cadmium) or diseases of presumably renal tubular origin as in renal cell carcinoma. Further statistical analysis and retrospective and prospective studies are required to determine the incidence of renal tubular epithelial antigen related immune complex disease in patients with sickle cell hemoglobin. However, the present studies strongly suggest a more than coincidental relationship and support our hypothesis.
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