MATERIAL AND METHODS
The study was conducted on 102 consecutive Fatients in whom suitable renal biopsy tissues were obtained by the Department of Nephrology, Jinnah Postgraduate Medical Centre, from January 1988 to August 1988. There were 73 patients with primary and 29 with secondary glomerulonephritis. All the patients were evaluated clinically and hospitalized for percutaneous needle biopsies. The biopsy tissues were sliced into three fragments; for light, immunofluorescence and electron microscopy.
The renal biopsy specimens were fixed in 10% neutral buffered formalin, routinely processed, embedded in paraffin and sectioned at 2 U thickness. The sections were stained with haematoxylin and eosin (H&E), periodic acidschiff (PAS), periodic acid silver- methenamine (PASM) and Masson’s trichrome. Biopsies containing at least 5 glomeruli were considered adequate for evaluation.
The biopsy specimens were snap frozen in liquid nitrogen after covering with OCT cryoprotectant and stored at -25°C until they were transported in dry ice to the Department of Pathology, National University of Singapore where they were stored at -70°C. Cryostat sections were cut at a 4u and prepared for direct immunofluorescence microscopy according to the procedure described by Sinniah et al11 . FITC antibody preparations used for the study included rabbit antisera to human IgG, IgA, IgD, IgE, IgM, IgA secretory piece, C3, C1q, C4, fibrinogen, HBs antigen, HBc antigen, Kappa and Lambda light chains and albumin (Hoechst-Behring Laboratories). Sections were examined using the Zeiss (universal) large fluorescence microscope with 200 W/4 Li super pressure mercury lamp and a III RS epi-fluorescence condenser. The primary (exciter) filter was blue-violet Zeiss 487707 with an excitation range of 450-490nm, the secondary (barrier) filter was orange filter LP520 Zeiss 467873 with a cut off at 520nm. For black and white photography, Kodak plus-x, 125 ASA, 35mm film was used. The intensity was graded subjectively from 0 to + 3; 0 being negative and + 3 maximum intensity (mild 1+; moderate 2+, and marked 3+) fluorescence.
Specimens were fixed in 4% glutaraldehyde at 4°C, postfixed in 1% Dalton’s chrome-osmium fixative, processed and embedded in Araldite. Thin sections cut at 60-90 mu were stained with uranyl acetate and lead citrate. The sections were examined and photographed in a Philips 400T electron microscope.
Criteria for histological diagnosis
The definitions and classification of the glomerular lesions described in the present study were based on the uniform system of classification for glomerular diseases, proposed by the World Health Organization Committee12.
Of the 102 cases of primary glomerulonephritis, eight (7.8%) were shown to have significant amounts of IgA deposits in the glomeruli, but 2 of them were excluded from the study as one had associated diabetes mellitus and the other eclampsia associated nephropathy. The remaining 6 (5.9%) cases classified as idiopathic or primary IgA nephropathy were analysed. The immunofluorescence findings summarizing the presence of immunoproteins in these 6 cases are shown in Table 1.
IgA mesangial deposits as the predominant or sole immunoglobulin were found in all the cases. In addition IgM was present in 5 (83.3%), IgG in 2 (33.3%), C1q in 4 (66.6%), C3 & C4 in 3 cases (50%) each, and fibrin in 2 (33.3%) of the cases. IgA of grade 1+ was found in 1(16.1%) patient as the sole immunoglobulin. Five (83.3%) patients showed grades 2 + to 3+ IgA, whereas the intensity of fluorescence was not as marked with the other immunoglobulins, complements and fibrin. In 4 of 5 cases with IgM of grade 2+ to 3+ intensity and in the 2 cases with IgG of grade 2 + intensity, the fluorescence of the immunoglobulins did not exceed that of IgA. The constant site of IgA deposition was in the mesangium (Figure 1).
In 3 (50%) cases, IgA and other proteins were also found in the paramesangium and in some of the peripheral capillary loops. IgA was found in extra-glomerular vessels in 3 (50%) cases, while IgM was seen in 2(33.3%) and IgG in 1(16.6%) of the cases. IgA tubular casts were seen in 1 case. The complement components, C3, C1q & C4, showed a distribution similar to that of IgA in the glomerulus. C3 was found in extraglomerular vessels in 2 (33.3%) cases, usually in the juxtaglomerular arterioles. C1q was present in the blood vessels in one case while C4was not found in any patient. All the 6 cases were negative for IgA secretory piece, HBsAg and HBcAg.
The histological changes involving the glomeruli, tubules, interstitium and blood vessels are summarised in Table II.
All the cases showed mesangial widening, best seen with PAS, Masson’s trichrome and silver stains. There was a wide range of glomerular lesions, comprising of minor change, focal and/or segmental lesions, diffuse, mesangial cell proliferff tion and diffuse sclerosing glomerulonephritis. Minor change showed widening of mesangium and a maximum of upto 3 nuclei per mesangial area in the peripheral capillary loops. One (16.6%) case belonged to this category (Figure 2),
and one (16.6%) kase had focal glomerulonephritis with segmentaLsclerosis (Figure 3),
focal being defined as a lesion involving some but not all glomeruli (<80%). It also showed inflammatory cells comprising macrophages and polvmorphs in some glomeruli. Diffuse proliferative glomerulonephritis involving all or nearly all (>80%) glomeruli was seen in 2 (333%) cases, with superimposed lesions of segmental sclerosis in both and adhesions to Bowman’s capsule in one (Figure 4).
Mesangial deposits were detected in one (16.6%) patient as discrete droplets (Figure 4) identified with H & B, PAS, PASM and Masson’s trichrome stains. Global sclerosis was frequently observed, and in 2 (33.3%) cases was extensive (>80%), and was classified as diffuse sclerosing glomerulonephritis Superimposed lesion of segmental cellular crescent was seen in one case. Thbulointerstitial lesions of tubular necro¬sis, atrophy, interstitial fibrosis and inflammation were subjectively graded as mild, moderate and severe lesions. Four cases (66.6%) showed mild to moderate tubulointerstitial damage (TID), one (16.6%) mild, and 3 (50%) cases had moderate tubulointerstitial damage (Figure 5).
An attempt was made to correlate the glomerular pathology to the tubulointerstitial damage (TID). We observed that all cases with a moderate degree of TID had focal or diffuse sclerosing glomerulonephritis. One patient with mild TID belonged to the category of moderate diffuse mesangial proliferative glomerulone-phritis with focal and segmental sclerosis as super imposed lesions. The two patients with minor change IgA nephropathy and diffuse mild njesangial proliferation with superimposed focal and segmental sclerosis did not show any TID. Vascular narrowing due to arteriolosclerosis was seen in 5 (83.4%) cases ranging from mild in one (16.6%), moderate in one (16.6%) to severe in 3(50%) cases (Figure 5). The patient with minor change IgA nephropathy showed no vasculopathy.
Electron microscopy was performed in the six cases of idiopathic mesangial IgA nephritis. Electron dense deposits (EDD) were found in the glomerular mesangium in all cases (Figure 6).
Paramesangial extensions and deposits in the basement membranes of some peripheral capillary loops was seen in 2(33.3%) cases (Figure 7).
Another constant feature was the increase in mesangial matrix with basement membrane like material. Mesangial cell changes, mainly in thç form of increased rough endoplasmic reticulum and mitochondria was seen in cases with diffuse mesangial cell proliferation and focal or diffuse sclerosing glomerulonephritis. In cases with diffuse sclerosing glomerulonephritis additional changes of mesangial cell atrophy was seen. Collagen fibrils were seen in the mesangium in the cases of diffuse mesangial proliferation, and in sclerosing glomerulonephritis (Figure 7). All the cases showed areas of obliteration and broadening of foot processes with formation of microvilli. No inflammatory cell or platelet was seen in the capillary lumen. No double contour of capillary loops or tubuloreticular particles were seen.
Clinical features (Table III)
The male to female ratio was 2:1, and the patients’ ages ranged from 10 to 50 years. Five patients presented with nephrotic syndrome with accompanying microhaematuria in 4; and one presented with proteinuria/microhaematuria with 11-20 rbc per HPF. Creatinine clearance of <30ml/min was seen in two cases of diffuse sclerosing glomerulonephritis. Four cases also had hypertension at the time of presentation. An attempt was made to correlate glomerular lesions with clinical presentations. All the patients who presented with nephrotic syndrome bad severe glomerular pathology, with diffuse mesangial proliferation, with superimposed segmental sclerosis, focal or diffuse sclerosing glomerulonephritis.
IgA nephropathy has been recognised as a distinct form of primary glomerular disease. The diagnosis is determined solely by immunofluorescence microscopy. The presence of IgA as the predominant immunoglobulin in the mesangium in patient with no systemic disease differentiates in patients with no systemic disease differentiates it from other diseases in which glomerular glomerular deposition of IgA occurs, as in systemic lupus erythematosus13, Henoch-Schonlein Purpura14 and alcoholic liver cirrhosis15-16. In France and Italy IgA nephropathy is found in 20-25% of the adult patients with idiopathic glomerulonephritis4,17. A higher incidence of 33.7% was reported from Singapore18, and between 35-40% from Japan19-20. However, lower incidence ranging from 1.5 to 10% have been published from Britain, the United States and Canada6,8,21-22. In our series the primary IgA nephropathy incidence of 5.9% is quite low compared to other Asian Pacific countries. A part from geographical variation, it may be related in part to the criteria of patient inclusion or selection for renal biopsy. However, larger number of cases have to be analysed to determine the true prevalence of this disease in Pakistan. The majority of our patients were young, with a predominance of males as reported by others4-8. We found a higher incidence of nephrotic syndrome than other workers18,21, which could partly be due to the selection of patients for biopsy, and also patients coming late for treatmen23. The initial reports termed the lesion IgA-IgG mesangial nephropathy2,4-5,7,9,17,19. An IgA-IgG combination was seen in approximately 50% of the cases18,22. We observed an IgA-IgM combination was seen in 83.3% of the cases studied. The frequency of associated IgM is the highest report in the world literature. IgM was found in 60-70% of cases by many workers from North America (USA) 8,24 and from Northern Europe (Holland) 25,26, whereas in all reports from Southern Europe (France, Italy and Spain), from Asian (Japan and Singapore), and from Australia the prevalence is lower27. This discrepancy probably reflects a difference in the immunological response27, possibly due to geographical differences related to environmental and/or genetic susceptibility factors18,28. Another distinctive feature is the high frequency of early complement components of the classical pathway of C3 activation. The high frequency of C1q (83.3%) and C4 (50%), with grade 3+ staining in one case each is also the highest frequency reported so far29. This suggests that along with the activation of complement C3 through the alternate pathway by aggregated IgA, the classical pathway is activated through IgG and IgM, in IgA nephropathy as shown by other workers30,31. Electron microscopic changes of mesangial deposits with peripheral extensions, increased mesangial matrix and mesangial cell organelles, and formation of collagen fibrils with cellular atrophy have already been described in greater detail32,33. The tubulointerstitial damage and arteriolosclerosis with glomerular damage represent greater degree of damage with poor prognosis. This paper shows that IgA nephropathy exists in Pakistan, and more work needs to be done to determine the extent of the disease, the clinical presentations, and the possible causes of the disease(s).
1. Berger, J. and Hinglais, N. Les depots intercapillaires d’ IgA- IgG. J. Urol. Nephrol., 1968; 74 : 694.
2. Berger, J. IgA glomerular deposits in renal disease. Transplant Proc., 1969; 1 : 939.
3. Maintz, J., Elema, J.D., Hennington, B., Blaker, F. and Bunger, P. A special form of chronic glomerulonephritis IgA-IgG nephropathy. Dtuch Med. Wschensch., 1972; 97 1527.
4. Morel-Maroger, L., Leathem, A. and Richet, G. Glomerular abnormalities in nonsystemic diseases. Relationship between findings by light microscopy and immunofluorescence in 433 renal biopsyspecimens. Am. J. Med., 1972; 53: 170.
5. Levy, M., Beaufils, H., Gubler, M.C. and Habib, R. Idiopathic recurrent macroscopic hematuria and mesangial lgA-IgG deposits in children (Berger’s disease). Clin. Nephrol., 1973; 1 : 63.
6. Lowance, D.C., Mullins, J.D. and McPhaul, U. Immunoglobulin-A- associated glomerulonephritis. Kidney Int., 1973; 3: 167.
7. McEnery, P.T., McAdams, A.J. and West, C.D. Glomerular morphology, natural history and treatment of children with IgA- IgG mesangial nephropathy, in glomerulonephritis. Kincaid-Smith, Mathew and Becker (eds), New York, Wiley, 1973.
8. McCoy, RC., Abramowsky, C.R. and Tisher, C.C. IgA nephropathy. Am. J. Pathol., 1974; 76: 123.
9. Ueda, Y., Sakai, 0., Yamagata, M., Saito, T., Kitajima, T., Suzuki, T., Kusuma, Y., Kawamura, K., and Mitome, T. Mesangial IgA-lgG deposit nephritis (in Japanese). J. Clin. Sci., 1974; 10: 1421.
10. Sinniah, R., Pwee, U.S., and Lim, C.H. Renal glomerular lesions in patients with asymptomatic microscopic haematuria - proteinuria discovered on a routine medical examination, in Proceedings of the Colloquium in Nephrology, Supplement. Ann. Acad. Med., 1975; 4: 11-16.
11. Sinniah, R., Pwee, H.S., and Lint, C.H. Glomerular lesions in asymptomatic microscopic hematuria discovered on routine medical examination. Clin. Nephrol., 1976; 5 : 216.
12. Churg, J., in Collaboration with L.H. Sobin and pathologists and nephrologists in 14 countries: Renal disease: classification and atlas of glomerular diseases. Tokyo. Igakushoin, 1982.
13. Sinniah, R. and Feng, P.H. Lupus nephritis; correlation between light, electron microscopic and immunofluores cent findings and renal function. Clin. Nephrol., 1976; 6-340.
14. Sinniah, R., Feng, P.H.. and Chen, B.T.M. Henoch Schölein syndrome; a clinical and morphological study of renal biopsies. Clin. Nephrol., 1978; 9: 219.
15. Callard, P., Feldmann, G., Prandi, D., Belair, M.F., Maundet, C., Weiss, Y., Druet, P., Benhamou, J.P. and Bariety, J. Immune complex type glomerulonephritis in cirrhosis of liver. Am. J. Pathol., 1975; 80: 329.
16. Sinniah, R. Heterogeneous IgA glomerulonephropathy in liver cirrhosis. Flistopathology, 1984; 8: 947.
17. Di Belgiojoso, G.B., Tarantino, A., Civati, 0., Limido, D. and Minetti, L. Glomerulonefrite A depositi inter capillari di IgA- IgG: Studio clinico e morfologico di 63 casi. Rincera Clin. Lab., 1973; 3: 30.
18. Sinniah, R., Javier, A. R. and Ku, G. The pathology of mesangial IgA nephritis with clinical correlation. Histopathology, 1981; 5 :469.
19. Sakai, 0., Yamagata, M., Kitajima, T., Suzuki, T. and Ueda,Y. Mesangial IgA-IgG deposits nephritis (abstract No. 352). VIth Int. Congr. Nephrol., 1975, florence.
20. Shirai, T., Tomino, Y., Sato, M., Yoshiki, T. and Itoh, T. IgA nephropathy: clinicopathology and immunopathology. Contr. Nephrol., Karger, Basel, 1978; 9 : 88.
21. Sissons, J.G., Woodrow, D.F., Curtis, J.R., Evans, D.J., Gower, P.E., Sloper, J.C., and Peters, D.K. Isolated glomerulonephritis with mesangial IgA deposits. Br. Med. J., 1975; 3: 611.
22. Alexander, F., Barabas, A.Z. and Jack, R.G. IgA nephropathy. Hum. Pathol., 1977; 8: 173.
23. Jamal, Q., Jafarey, N.A. and Naqvi, A.J. A review of 1508 percutaneous renal biopsies. JPMA., 1988; 38: 272.
24. Zimmerman, SW. and Burkholder, P.M. Immunoglobulin A nephropathy. Arch. Intern. Med., 1975;135: 1217.
25. Van der Feet, J., Arisz, L., Brentjens, J.P.H., Marrink, J. and Hoedemacker, Pi. The clinical course of IgA nephropathy in adults. Clin. Nephrol., 1977; 8 : 335.
26. Van de Putte, L.B.A., de Ia Riviere, G.B. and Van Breda Vriesman, P.J.C. Recurrent or persistent haematuria. Sign of mesangial immune-complex deposition. N. EngI. J. Med., 1974; 290:1165.
27. D’Amico, 0. Idiopathic mesangial IgA nephropathy. In: Bertani, T., Remuzzi, G. (eds). Glomerular injury 300 years after Morgagni Milan: Wichtig Editore, 1983;p205-228.
28. Sinniah, R. IgA mesangial nephropathy: Berger’s disease. Am. J. Nephrol., 1985; 5 : 73.
29. Sinniah, R. The pathology of IgA nephropathy, in IgA nephropathy, Edited by Clarkson, A.R. Boston. Mar tinus Nighoff Publishing 1987, p.66.
30. Tomino, Y., Endoh, M., Nomoto, Y. and Sakai, H. Double immunofluorescence studies of immunoglobulins, complement components and their control proteins in patients with IgA nephropathy. Acta Pathol. Jpn., 1982; 32: 251.
31. TorAino, Y. Complement system in IgA nephropathy. Tokai J. Exp. Clin. Med., 1980; 5: 15.
32. Sinniah, R. and Churg, J. Effect oPIgA deposits on the glomerular mesangium in Berger’s disease. Ultrastruct. Pathol., 1983; 4: 9.
33. Sinniah, R. Idiopathic IgA mesangial nephropathy; histological features. Contrib. Nephrol., 1984; 40: 156.