The Fas - Apoptosis Stimulating Fragment - ligand (Fas-L) is responsible for initiating cell apoptosis, which may be modulated by cross-linking with the Fas receptor. Soluble Fas (sFas) blocks Fas-L and subsequently inhibits apoptosis, causing damage to the glomeruli and usually accelerating the progression of glomerulonephritis (1, 2). The cytotoxic actions mediated via Fas-L may contribute to inflammatory immune responses (3) and Fas-L expression positively correlates with the stage of kidney injury (4). The mRNAs of Fas and Fas-L were also increased in the glomerulus after anti-glomerular basement membrane antibody administration in rats (3).

Primary glomerulonephritis (GN) is an immune-mediated disease affecting both the glomeruli and interstitium with or without an infiltration of inflammatory cells associated with protein overabsorption by renal tubules (5). Proliferative (PGN) and non-proliferative (NPGN) types of GN were historically differentiated by the presence of leukocyte infiltration, cell proliferation and an imbalance of glomerular extracellular matrix turnover (the predominance of its production or degradation), as well as their involvement in the injury of kidney structures (6). Proliferative primary glomerulonephritis comprises mesangial proliferative GN (MesGN), membrano-proliferative GN (MPGN) and immunoglobulin A nephropathy (IgAN), while non-proliferative GN consists of minimal change disease (MCD), focal glomerulosclerosis (FSGS), and membranous nephropathy (MN) (6). In general, proliferative GN may be characterized by glomerular fibrosis - a complicated process initiating cell cytotoxicity and involving many factors, including Fas (1-3, 7, 8). Once induced by Fas an apoptosis helps to prevent local foci of atrophy caused by progressive fibrosis affecting mainly renal tubules (1-3, 7, 8). This process if inhibited by over-synthesized sFas may accelerate kidney injury. End-stage renal disease is an unfavorable outcome of fibrosis enhancement in primary glomerulonephritis. The introduction of a course of immunosuppressive therapy (IS) composed of corticosteroids and cyclophosphamide significantly improved GN outcome but did not exclude resistance to such routine treatment (9-13).

Due to the unpredictable course of GN and response to therapy, treatment individualization and optimization is often impossible. Hence, the search for more reliable predictors which might allow an adjusted and adequate immunosuppressive treatment scheme to be identified for individuals continues. This study was conducted to determine whether in severe courses of GN, sFas may be regarded as a complementing factor when deciding to introduce aggressive treatment in sensitive cases, or disqualify it to prevent IS complications. The aims of the study were:

The study was conducted from autumn 2002 to spring 2010 year, in 84 patients (45 males and 39 female) mean age 41.44 ± 13.25 yr with primary glomerulonephritis. The control group consisted of 18 age-matched healthy subjects. The structure of the study group (including glomerulonephritis type divisions) and the control group is shown in Table 1. Only patients with a severe course of GN i.e. nephrotic range proteinuria or deterioration of kidney function, reflected in an increase of serum creatinine concentration by more than 50% of baseline values, despite steroid treatment were included in this study. The diagnosis was established based on clinical symptoms, laboratory tests, accessory investigations and kidney biopsy.

All of the biopsy specimens were evaluated by light and immunofluorescence microscopy in Department of Kidney Pathology Medical University of Lodz. In addition, the percentage of interstitial volume was calculated using a computerized morphometric analysis of interstitium quantity (14, 15). The degree of glomerulosclerosis (GSC), based on number of involved glomeruli, and the intraglomerular extent of sclerosis were semiquantitatively estimated according to a scoring system proposed by Fuiano et al. (16). The GSC was graded on a four-point scale: normal (0), mild (1), moderate (2), and severe (3). The type of primary glomerulonephritis was identified by microscopic evaluation, and mesangio-proliferative GN was diagnosed after the exclusion of other proliferative glomerulonephritis (14-17).

Also, the urinary excretion of proteins, expressed in milligrams per milligram of creatinine in urine (mg/mg Cr) and total serum proteins, were measured.

All participants were treated with antihypertensive drugs (angiotensin converting enzyme inhibitors or angiotensin receptor blocker and calcium channel blockers) to establish and maintain blood pressure values according to recommendations (18, 19). In addition, statins were introduced to stabilize the lipid profile (atorvastatin at a mean dose of 20 mg/day). The primary aim was to reduce low density lipoprotein (LDL) serum concentration to below 100 mg/dL (20). Those variables were assessed at baseline and serially throughout the treatment to monitor the status of the patients. Finally all tests were repeated after 1 yr (± 3 months) of immunosuppressive treatment.

All subjects received an identical immunosuppressive protocol which consisted of initial pulse therapy with methylprednisolone (a calculated aggregate dose 1,000 mg per 20 kg body weight, administered 1,000 mg in every other day) followed by oral prednisone (25-30 mg/day) and cyclophosphamide in 6 monthly given pulses 0.6 g/b.m². The cumulative dose of cyclophosphamide did not exceed 6 grams. Before the initiation of treatment, the potential foci of infection was diagnosed and eliminated as a routine in all participants. A treatment scheme including pulses of steroids and cyclophosphamide (CPH) was restricted to cases of progressive GN with severe disease course (decreasing of eGFR) and was chosen as a rescue protocol. The introduction of cyclophosphamide as primary treatment is controversial in different types of GN although many authors describe it offers greater benefits than other immunosuppressive agents (21-32). CPH pulse treatment combined with steroids is regarded as well tolerated and effective, especially in steroid-dependent or corticosteroid resistant, severe nephrotic syndrome or in GN with progressive worsening of eGFR and diffuse microscopic lesions, irrespective of primary glomerulonephritis type (21-32).

To assess the influence of immunosuppressive therapy on cytokine activity, the blood and urine of healthy participants, as well as pre- and post-treatment blood and urine samples from patients, were prospectively collected to EDTA (ethylenediaminetetraacetic acid) tubes, centrifuged and stored at -70℃ until analysis. When completed, the serum concentrations and urinary excretion of soluble Fas were measured by enzyme-linked immunosorbent assay (ELISA) using commercial immunoassays according to manufacturer's instructions (Biosource® Europe S.A., Nivelles, Belgium). All tests were performed in a certified local laboratory.

To evaluate the potential of sFas in predicting GN outcomes after one year of treatment, patients were divided retrospectively into two subgroups according to their response to the therapy: R (Responders; proteinuria < 0.5 g/day, e.g. < 6 mg/mg urine Cr and improved or stable kidney function - serum creatinine change within a range of 15%), NR (Non-Responders; proteinuria > 0.5 g/day, e.g. > 6 mg/mg urine Cr and/or deterioration of kidney function - over 15% increase of serum creatinine concentration). When completed, the division allowed sFas to be evaluated retrospectively at baseline in R and NR. The structure of subgroups, divided according to type of primary glomerulonephritis, is presented in Table 1. No differences in distribution between subgroups R and NR were noticed (Table 1). No comorbidities, including diabetes, obesity or severe infections, which may affect the final evaluation in both subgroups were noted. The gender structure in the subgroups and the control group was homogenous. The study group and the control group were sex and age matched.

To verify the potency of sFas measurements to predict GN favorable outcomes and to indicate sFas abnormal urine excretion pointing the worse pre-treatment prognosis, the cut off value was calculated using ROC curve.

In the present study, only participants with severe proteinuria and/or an increase of serum creatinine were selected. The unsuccessful use of initial isolated steroid therapy (i.v. pulses) necessitated the need for combined treatment with cyclophosphamide in the study cohort. Although this immunosuppressive protocol exceeds the scope of standard therapy for some types of primary glomerulonephritis, many authors suggest its efficacy (21-32). However, the patients to be treated according this therapeutic strategy must be carefully selected, and only subjects with a severe course of GN should be chosen (21-32). In this study, one third of non-proliferative glomerulonephritis and nearly 40% of the patients with proliferative GN, despite the intensive treatment, had no improvement in proteinuria and/or kidney function after therapy prolonged to 1 yr. This appears to confirm the observation that the clinical course of GN still remains unpredictable in a significant number of the patients and therefore no uniform therapeutic strategy based on routine clinical and biochemical assessments can be proposed (32-34). The initial introduction of not radical immunosuppressive schemes may be not sufficient, hence more aggressive therapeutic strategies in those particularly complicated cases merit further investigation (21-32).

However serum sFas in the study group was significantly higher than in the healthy participants and elevated in comparison to other studies (35-37). Pre and post-treatment sFas serum concentrations showed no differences, irrespective of the type of glomerulonephritis. In the present study, highly elevated serum sFas values seem to be associated with a significant deterioration of kidney function in majority of participants. In contrast, in other studies on primary glomerulonephritis, sFas results were obtained mainly in patients with normal or only slightly impaired kidney function (35-37). Identical sFas values obtained in healthy participants in other studies, implying that the chosen methodology is correct (35-37).

Urinary sFas excretion has been occasionally evaluated (37). In the present study, urinary sFas was found to be lower after treatment than before in both the proliferative and non-proliferative GN groups. The decrease in sFas urinary excretion after the introduction of immunosuppressive therapy seems to be a good prognosis of favorable treatment outcome however the pooled result was still higher than the mean urinary sFas in Responders with PGN.

To confirm the potential of baseline sFas measurements in predicting the prognosis of GN, the subdivision into Responders and Non-Responders subgroups was needed. The retrospective analysis of gathered material showed that in proliferative and non-proliferative primary glomerulonephritis, no differences between R and NR subgroups were found with respect to pre-treatment biopsy findings and biochemical data.

The only parameter that varies between the Responders and Non-Responders subgroups is the urinary excretion of sFas in the proliferative GN group. In the Responders subgroup, the action of urinary sFas was comparable with that of urinary sFas described by Kacprzyk (37). In Non-Responders, sFas urinary excretion values were even lower than in patients with IgA nephropathy (37). Although, in R and NR subgroups all post-treatment urinary sFas were lower than pre-treatment, still the lowest was noted in responders with proliferative GN. The presented study results suggested that lower sFas urinary excretion may be linked with a good prognosis, and increased excretion with the risk of glomerulonephritis progression. This is in accord with other studies (37). Also the correlations between urinary sFas and post-treatment creatinine and proteinuria shown here in the proliferative GN group, which are positive in Non-Responders as well as in Responders, highlight the potential value of sFas measurements.

To verify the potency of sFas measurements in predicting GN the cut off value of sFas urinary excretion was calculated using ROC curve. It indicated that sFas urinary excretion was lower only in Responders (proliferative type of primary glomerulonephritis).

sFas inhibits apoptosis and creates the conditions conducive to uncontrolled glomerular and mesangial cell proliferation. It decreases the number of TUNNEL-positive cells in the glomeruli, promoting its progressive injury in the mechanism of apoptosis-proliferation deregulation (36, 37). The significantly increased pre-treatment values for urinary excretion of sFas in Non-Responders with proliferative GN seem to support this.

However, in some types of primary glomerulonephritis, proteinuria is regarded as an independent risk factor and a predictor of renal function deterioration; in some cases, it is not in fact accurate at baseline (38). In others, creatinine clearance, hypertension and severity of biopsy pathological lesions play the key role (39). Surprisingly, in this study, risk factors traditionally regarded as contributory for treatment outcome were found to be unreliable.

The decision to introduce immunosuppressive therapy is strictly based on patient general status, clinical and biochemical parameters. The clinician, basing his decision on own experience and guidelines, is obliged to individualize the treatment to achieve high effectiveness and avoid therapy disadvantages. It appears that the pre-treatment examinations of urinary sFas may become an additional argument in the optimization of treatment. Nevertheless, further studies evaluating sFas in glomerulonephritis, especially the proliferative type, are needed.

In this study, only a selected group of patients with a severe course of GN were evaluated and due to this limitation the number of participants is relatively small. Additionally, all participants were selected to undergo an immunosuppressive treatment protocol selected by the center and not widely recruited, but this was undoubtedly efficient in severe cases of glomerulonephritis (17-28). Although these disadvantages may detract from the results of post-treatment sFas evaluation, and a retrospective analysis (R and NR subdivisions) may be presumed to be less significant than a prospective trial, multivariate logistic evaluation using sFas as an exclusive contributory predictor of therapy outcomes should not be underestimated.

In conclusion, both sFas serum concentration and urinary excretion were highly elevated in patients. The immunosuppressive treatment reduced sFas urinary excretion in proliferative and non-proliferative primary glomerulonephritis. The study results suggest that the lower urinary sFas may be linked with favorable therapy outcomes in patients with proliferative primary glomerulonephritis, however further evaluations are needed.