Histomorphological and clinical study of primary and secondary glomerulopathies in Southeast Serbia ( 20-year period of analysis )

Background/Aim. Epidemiological studies of renal biopsies have been performed to follow up the incidence of glomerular diseases on a specified territory and to compare the obtained results with results from other regions. The aim of this study was to analyze the frequency of certain histopathophysiological types of glomerular diseases on the territory of Southeast Serbia. Methods. In a 20-year period (1986–2006), 316 kidney biopsies were performed in patients with clinicall signs of impaired renal function, in Southeast Serbia. On average 1.6 biopsies were made per year per 100 000 inhabitants. Results. Biopsies of adult patients represented 88% of all biopsies, biopsies in children (aged under 18 years) represented 8%, while biopsies of elderly patients (more than 60 years) represented 4% of all biopsies. The predominance of male patients was described with male/female ratio of 1.4. The most frequent clinical manifestation in patients at the time of biopsy were nephrotic syndrome (42.5%), and asymptomatic proteinuria and/or hematuria (31.3%) and nephritic syndrome (14.9%). The most common glomerular disease was IgA nephropathy with an incidence of 21.5% of total biopsy diagnosed glomerulopathies, followed by: membranous glomerulonephritis (12.6%), focal segmental proliferative and sclerosing glomerulonephritis (10.7%), lupus nephritis (8.4%), nephroangiosclerosis (7.0%), mesangio-proliferative glomerulonephritis (6.1%), minimal change disease (2.8%), mesangiocapillary glomerulonephritis (2.3%). Conclusion. The frequency of certain histopathologic findings significantly correlated with data from studies that we used for comparison, with the exception of minimal change disease whose incidence in our study was smaller.


Introduction
Glomerulonephritis (GN) is a bilateral, non-bacterial, non-suppurative inflammation of kidney which primarily affects glomerulus and then the other kidney structures.
Histological features are the most important criterion for the nomenclature and classification of glomerular disease.In addition to localization, the histological characteristics define the nature of glomerular lesion as well as cell proliferation, deposit of immune complexes or extracellular components lesions.
Immune processes underlie most cases of primary GN, but they are also present in many secondary events in the glomerulus.Therefore, the division of etiopathologic mechanisms of glomerulopathy is reduced to immunological and non-immunological 1 .
Glomerular lesion is clinically expressed with a relatively small number of signs and symptoms.There are five main clinical syndromes which may be manifested in glomerular disease: acute GN syndrome (acute nephritic syndrome), syndrome of rapidly progressive GN, nephrotic syndrome, asymptomatic urinary abnormalities (asymptomatic proteinuria and hematuria) and the syndrome of chronic GN.
The diagnosis of glomerular disease involves a complete understanding of the clinical condition of the patient, laboratory diagnostics, immunological and morphological studies.Diagnosis based only on the clinical manifestations may be unsuccessful because different glomerular diseases are characterized by similar or the same clinical presentation.On the other hand, the same glomerular disease may be presented differently in different patients 2 .
Therapeutic approach, response to therapy and prognosis, depend on the type of GN.The frequency of certain forms of GN is different in different geographical areas and associated with genetic and environmental factors.
This retrospective study was conducted with the aim to determine the frequency of certain types of glomerular disease in Southeast Serbia on the basis of which may be possible to predict the response to therapy and prognosis.

Methods
During a 20-year period (1986-2006), 316 kidney biopsies were performed in patients with clinically and laboratory shown signs of some primary or secondary glomerulopathy (nephritic or nephrotic syndrome, syndrome of rapide progressive GN and asymptomatic hematuria or proteinuria).All biopsies were done at the Institute of Nephrology and Hemodialysis in Clinical Center Niš and the whole tissue biopsy material was processed in the histopathological laboratory of this institute.The histopathologist has always taken biopsy material from a patient and prepared it for light microscopic analysis, immunofluorescence and if necessary for electron microscopy.
The same method for taking biopsy material was always used -blind percutaneous renal biopsy with previous intravenous pyelography or ultrasound examination.In parallel to light microscopy, immunofluorescence with IgG, IgA, IgM, C1q, C3, fibrinogen, albumin, kappa and lambda chains was always done.Electron microscopy was performed in 3-4% of cases when it was possible (during the difficult economic crisis) and when indications were present (other morphological examination aroused suspicion of a change in the GBM structure; existence of deposit which could not been precisely localize in immunofluorescence findings).
Of the total number of samples, 214 biopsies had positive histopathological findings in the glomeruli, while 102 findings were not included in this study.Only those cases who clearly indicated the existence of glomerulopathy were included.The patients whose clinical presentation and laboratory findings did not fit into diagnosis of glomerulopathy or tubulointerstitial changes were clinically and histopathologicaly confirmed, were not included in this study.Among samples which were not included in the study, 26 had negative glomerular findings, 50 of them did not have enough material for analysis, and in 26 of them findings in tubulointerstitial system were dominant (chronic pyelonephritis, acute tubular necrosis, balkan endemic nephropathy, etc).
Immunological tests included measurements of serum immunoglobulins concentration, complement fractions in serum, immune complexes while the presence of certain autoantibodies were detected by direct immunofluorescence.Immunoglobulins and complement fractions were measured by the nephelometric method (II BEN nefelometar -Dade-Behring, Magdeburg, Germany).Immune complexes were measured by the deposition of immunoglobulins with polyethylene glycol (PEG-6000).The presence of autoantibodies in biopsy specimens was performed by direct immunofluorescence with autoantibodies labeled with fluorescein (FITC).
Other analysis have included detailed medical history, routine biochemical tests as well as other relevant testing.The following findings have been specifically analyzed: duration of clinical manifestations of impaired kidney function, previous diseases, edema, the appearance of micro-or macrohematuria, blood pressure, finding of proteinuria, hypoproteinemia and hipoalbuminemia findings, and elevated serum cholesterol and triglycerides.
The data were analyzed by descriptive statistics using the program Sigma Stat.
Age and gender distributions are shown in Table 1.Age data were available for 204 patients.Most of the patients were aged 31-50 years, a total of 98 patients (61 males and 37 females), that was 45.8% of the total number of biopsies with positive findings.Within this age group, most were male patients with primary proliferative GN (33% or 33.7%).
Clinical presentation of the patients with GN is shown in Table 2. Of the 7 described syndromes, the most common was nephrotic syndrome in 91 (42.5%) patients.Of a total number, 140 biopsied patients had pathological findings of the primary forms of GN, which was 65.4%, of which the proliferative forms were much more frequent (Table 2).Secondary forms of GN had 22 (10.3%)patients, non-immunological glomerular lesions (diabetes, amyloidosis, and hypertensive changes) were recorded at 22 (10.3%)patients, while in the other group, terminally damaged renal parenchyma (end stage renal diseases-ESRD) occurred in 13 (6.1%) of the cases.
Primary nonproliferative forms of GN were found in 41 of 214 patients (29.3% of all the patients with primary GN).The most frequent nonproliferative GN was membranous GN, followed by focal segmental sclerosis and minimal change disease (Table 3).
Membranous GN was present in 27 patients (7 females and 20 males) of which hypertension was registered in 13 (severe stages in 4), edema in 23, hematuria in 22, while nephrotic proteinuria was present in 15 of the patients.The  findings of immunofluorescence of IgG were positive in 25 of the patients.Detection of primary proliferative GN was present in 99 patients of 214 which is 70.7% of the biopsied patients with findings of primary glomerulonephritis.The most common form of primary proliferative GN was IgA nephropathy, then focal segmantal praliferative and sclerosing GN, mesangioproliferative GN, endoteliomezangial GN, mesangiocapillary GN and rapidly progressive GN (Table 3).
IgA nephropathy was diagnosed in 46 patients (14 females and 32 males).The youngest patients belonged to the age group of 11-20 years while the oldest were between 51 and 60 years.Hypertension was present in 21, edema in 10, the finding of hematuria was present in all the patients, while proteinuria greater than 3.5 g/L was present in 8 patients with this finding.The largest number of light microscopy findings belonged to the group with focal segmentalproliferative and sclerosing GN.Second most frequent findings matched to focal segmental proliferative GN with mesangial area lesions.Diffuse changes in the glomeruli, such as diffuse proliferative and diffuse proliferative and sclerosing GN were registrated in a much lower number of cases.Extracapillary proliferation with less than 50% of affected glomeruli was observed in only one biopsy.Considering immunofluorescence findings all the patients of this group were positive for IgA.
Secondary forms of GN were registered in 22 out of 214 biopsied patients, which is 10.3%.The definitive diagnosis in these cases was confirmed by other immunological tests (primarily by determining the presence of serum autoantibodies).Out of 22 patients 18 had systemic lupus erythematosus, and 4 showed light microscopic signs of focal proliferative and sclerosing GN with changes in the skin and positive immunofluorescence finding, which is definitely pointing on Henoch-Schonlein purpura (Table 3).
Lupus nephritis was present in 18 cases.All the patients were females, aged 17 to 48 years.Hypertension was registrated in 7 of the patients, edema in 13, proteinuria and hematuria in all the patients.Serum levels of C3 and C4 were reduced in most patients, while immune complexes and antinuclear antibodies were present in 9 of the patients to whom these measurements were made.Non-immunological and other glomerulopathies were diagnosed in 52 patients, which is 24.3%.Nephroangiosclerosis was diagnosed in 15 of the patients, glomerular lesion in diabetic nephropathy in 4 patients and glomerular lesions in amyloidosis in 3 patients (Table 3).Other biopsy material presented 14% of the biopsied patients with positive findings and spoke in support of end-stage GN (13 patients), Alport syndrome (2 patients), postpartal thrombotic microangiopathy (1 patient), status after kidney transplantation (1 patient) or it was not possible to come out of any known entity.

Discussion
Epidemiology of glomerular disease often shows some peculiarities related to the geographic area, requiring a regional monitoring and mutual comparison of morbidity in different territories.This study presents the frequency of some glomerular diseases diagnosed by biopsy of renal tissue in the period of 20 years in Southeast Serbia (population over 1 million).
A large number of biopsy samples with insufficient material is probably due to technical reasons of biopsy procedure (blind percutaneous renal biopsy) and in some degree disparage the preciseness of results.According to our results, 1.6 biopsies were performed per 100 000 inhabitants per year.This number is several times lower than the rates present in Western European countries like Spain 3 , Italy 4 , France 5 , and the Czech Republic 6 , or Australia 7 , but is similar to the rate of biopsies in Romania 8 , whose registry is one of the few renal biopsy registers of Eastern Europe, and to the rate of biopsies obtained in another single centre study conducted in Serbia, for the same period in a much larger sample 9 .
Biopsies in adult patients represented 88% of all biopsies, biopsies in children (aged under 18 years) represented 8%, while the biopsies in elderly patients (more than 60 years) represented 4% of all biopsies (Table 1).This relationship between the number of biopsies of children and the elderly is quite different from the relationship presented in the Spanish registry of renal biopsies, where renal biopsy in children represented 7% and 22% in the elderly 3 .These results show predominance of adults in the biopsied patients and that a large number of elderly patients and children were treated on the basis of clinical manifestations.
The predominance of male patients is reported with male/female ratio of 1.4, which is consistent with data from other international studies 3,7,10 .From this, we can exclude secondary forms of glomerulonephritis, in which the predominance of females was present, what can be explained by a much higher incidence of systemic lupus in women (Table 1).However, male/female ratio is significantly different from the results obtained in another single centre study conducted in Serbia, for the same period in a much larger sample 9 .
The most frequent clinical presentations in patients at the time of biopsy were nephrotic syndrome (42.5%), asymptomatic proteinuria and/or hematuria (31.3%) and nephritic syndrome (14.9%).Other syndromes were significantly less frequent (Table 2).These data are consistent with the results from the Spanish 3 and Czech register 6 , as well as with data from a Japanese study with 1850 biopsied patients.Also, data from another single centre study conducted in Serbia were similar, except that the incidence of nephrotic syndrome showed more pronounced dominance 9 .However, the obtained data differ the results from the Italian registry where asymptomatic urinary abnormalities were more frequent than nephrotic syndrome 4 and the results from the Romanian registry where asymptomatic urinary abnormalities were less frequent than all other syndromes 8 (Table 4).
According to the literature and larger registers 4,[11][12][13] , a difference between glomerular pathology in children, adult and elderly population is clearly visible.With the increase of the average number of years of biopsied patients reduces the incidence of primary GN and increases the incidence of secondary forms of glomerular disease 3 .The results of this study confirm the distribution of certain glomerular patologic entities by the age of the group, although with less certainty, possibly due to the relatively small number of biopsied patients (Table 2).
Similar to other registers, primary GN was proven in about two thirds of biopsied patients 5,6 , while the secondary form and non-immunological GN were equally presented with one tenth of biopsied patients.
The most common was IgA nephropathy with an incidence of 21.5% of total diagnosed glomerulopathies.This frequency is most similar to the frequency of this entity in the Netherlands 14 and Spain 3 , and significantly lower than reported in other European studies 4,6,8,12 .The most frequent clinical presentation among this patients was asymptomatic proteinuria and/or hematuria.All the patients with this histopathological diagnosis in our study were younger than 50 years and only 4 of them were younger than 19 years.
Second most frequent glomerular disease, with an incidence of 12.6% was membranous glomerulopathy.The patients in this group had a higher incidence of developed nephrotic syndrome than all other histopathological groups.In this group, histopathological finding of focal segmental proliferative and sclerosing GN with an incidence of 10.7%, was three times as frequent as isolated sclerosing form of this finding.
Despite the fact that a small number of biopsies was performed in children, the incidence of minimal change disease was significantly lower (2.8%) than in other European and world studies 3,4,7,15 .Conservative treatment until symptoms withdrawal and lack of technical conditions for electron microscopy during the bombing of Serbia could be just some of the reasons for these results.
In the group of secondary GN, lupus nephritis as a dominant finding with the frequency of 8.4% is consistent with all known registries.At the time of biopsy these patients presented predominantlly nephrotic syndrome and asympto-matic proteinuria and/or hematuria.The most frequent histological form of lupus nephritis was diffuse proliferative lupus nephritis (class IV) registered in 12 out of 18 patients, followed by focal segmental lupus nephritis (class III) in 4 patients, mesangial lupus nephritis (Class II) in 1 patient, lupus nephritis without histological changes (Class I) in 1 patient, while patients with membranousand (Class V) and endstage lupus nephritis (Class VI) was not registrated.Although in a small sample and with lower frequency of mesangial lupus nephritis, these those are similar to results obtained by Dimitrijevic et al. 16 on a sample of 311 patients with lupus nephritis.In all the patients immunofluorescence findings were positive primarily for IgG and C3 and in the lower percentage on IgA, IgM and C4.All of 18 biopsy specimens with this findings belong to women aged between 19 and 50 years, except in two cases of 17 and 14 years of age (Table 3).

Conclusion
The obtained results lead to a conclusion that in the region of Southeast Serbia the rate of biopsied patients in order to obtain accurate histological diagnosis of clinically manifested glomerular disorders is significantly lower than in European countries.Also, the age distribution of the biopsied patients is significantly different compared to western European countries due to a small number of renal biopsies in children and in elderly.Primary proliferative forms of glomerular disease is a dominant finding in the study group.The frequency of respective histopathologic findings significantly correlated with data from studies that we used for comparison, with the exception of minimal change disease whose incidence in our study is lower.These data are a significant contribution to the epidemiological analisys of glomerular disease in Serbia and represent a database for comparison and improvement of renal diseases treatment.