EFFECT OF MYCOPHENOLATE MOFETIL ON THE PROGRESSION OF ADRIAMYCIN NEPHROPATHY

Abstract

In order to assess the effects of mycophenolate mofetil (MMF) on the development of adriamycin-induced nephropathy, the development of this nephropathy in rats treated with MMF was compared to that in non-treated animals (group ADR + V) over 28 weeks. At weeks 8, 16 and 20, 24-h proteinuria of the treated group statistically differed from that of the non-treated group. However, no significant difference in proteinuria was observed thereafter between the groups. At the end of the experiment, there was no significant difference between both groups regarding the frequency of glomerular lesion (Group ADR + V: Md = 35%, P25 = 20%, P75 = 68%; Group ADR + MMF: Md = 27%, P25 = 9.5%, P75 = 54%); tubulointerstitial lesion index (Group ADR + V: Md = 7, P25 = 1.5, P75 = 9; Group ADR + MMF: Md = 8, P25 = 2, P75 = 9); glomerulosclerosis area (group ADR + V = 2779 μm², P25 = 751.8 μm², P75 = 3115 μm²; Group ADR + MMF = 1147 μm², P25 = 3969.7 μm², P75 = 1560 μm²); and, interstitial fibrosis area (Group ADR + V: Md = 218200 μm², P25 = 78670 μm², P75 = 282700 μm² group ADR + MMF: Md = 136000, P25 = 25010, P75 = 255800 μm²). In conclusion, MMF caused a temporary reduction in proteinuria but did not change the severity of the renal lesion observed after 28 weeks.

• Adriamycin nephropathy
• Mycofenolate mofetil
• MMF
• Proteinuria
• Tubulointerstitial fibrosis
• Glomerulosclerosis

INTRODUCTION

Over the last decades, it has become more and more evident that tubulointerstitial lesion is one of the principal causes of renal lesion. Rats with adriamycin-induced nephropathy, besides glomerulosclerosis, display severe tubulointerstitial lesions, which are characterized by tubular atrophy and dilatation and cellular infiltrate. Initially, this infiltrate is formed mainly by macrophages. However, as the disease evolves, its composition changes in such a way that, after a few weeks, lymphocytes T are found to predominate [1-3].

The use of immunosuppressive drugs such as cyclosphosphamide [[4]] or cyclosporine [[5]], has been associated with reduction in the severity of the nephropathy. Moreover, in nude mice, the inoculation of adriamycin provokes mild glomerular lesions but gives no rise to proteinuria [[6]]. These observations suggest the immune system plays an important role in the development of adriamycin-induced nephropathy.

Among the several immunosuppressive drugs available, mycophenolate mofetil (MMF) is an agent in which the active component, mycophenolic acid, is an inhibitor of inosine 5′-monophosphate dehydrogenase, which controls the synthesis of guanosine triphosphate [7-9]. MMF depletes guanosine triphosphate pools in lymphocytes and suppresses the de novo synthesis of purines thereby exerting a selective and reversible antiproliferative activity on these cells [10-11].

The purpose of the present work was to investigate the role of mycophenolate mofetil in the progression of adriamycin-induced nephropathy.

MATERIALS AND METHODS

Experimental Design

Forty-eight Wistar male rats, initially weighing 160–200 g, were divided into 4 groups (Group ADR + V; Group ADR + MMF; Group S; and, Group S + MMF) with 12 animals each. Throughout the study, the animals were kept in boxes, which housed 4 to 5 rats each, and were fed “ad libitum” on a normal-protein diet and water.

Following a 28-week observation period, all animals were sacrificed. Blood samples were collected for the determination of serum albumin. The kidneys were removed and weighed, and renal tissue samples were processed for light microscopy, morphometry and determination of cortical hydroxyproline content.

Nephritis Induction

On day zero of the experiment, the animals of groups ADR +& V and ADR + MMF were injected iv 3 mg/kg/body weight of adriamycin (Adriblastina, Farmitalia – Carlo Erba, Brazil) while the animals of Group S and Group S + MMF received saline (3mL/kg/body weight) by the same route.

Treatment

Starting at week 5, Group ADR + MMF and Group S + MMF daily received, by gavage, 40 mg/kg of mycophenolate mofetil (SINTEX, Palo Alto, California, USA). From week 8 up to the end of the experiment, this dose was reduced to 20 mg/kg. The medication was dispersed in vehicle containing distilled water, 0.5% carboxymethylcellulose (Duas Rodas Industrial LTDA, Jaraguá do Sul, Santa Catarina, Brazil), 0.4% polysorbate 80 (Tween 80, Merck, Rio de Janeiro, Brazil), 0.9% benzyl alcohol (Merck, Rio de Janeiro, Brazil) and 0.9% sodium chloride (Merck, Rio de Janeiro, Brazil). The dose used was chosen based on reports, which demonstrated an immunosuppressive action in the proliferation of lymphocytes T [10-11].

For 24-h urine collection, the animals were housed in metabolic cages and allowed free access to water but not to food. Total urinary protein level was determined by the sulfosalicylic method and serum creatinine by Jaffe reaction.

At the end of the experiment, the content of hydroxyproline in the renal cortex was determined by the method of Switzer [[12]] and serum albumin by the bromocresol green method.

Renal Histology

For light microscopy, kidney fragments were fixed in Dubosq-Brazil solution, embedded in paraffin and cut into 4-μm sections. The fragments were stained with hematoxylin-eosin and trichome Masson's stain.

The renal fragments obtained at sacrifice were examined by two investigators without prior knowledge of the source of the samples studied. The frequency of glomerular sclerosis (GSF) was assessed according to the percent of affected glomeruli found in 100 glomeruli sequentially observed. For the determination of the tubulointerstitial lesion index (TILI), tubular atrophy and dilatation, inflammatory infiltrate and interstitial fibrosis were scored individually from 0 to 3+ according to intensity. TILI is the sum of + attributed to each lesion.

For the measurement of glomerular sclerosis area (GSA), 100 consecutive glomeruli were examined and the area of each glomerulus with sclerosing lesions was determined. Interstitial fibrosis (IF) was measured in the whole slide. The software KS 300 (Kontron Elektronik, Germany) was used in this analysis.

Statistical Analysis

Parametric data are expressed as mean ± mean standard error and non-parametric data as medians (Md), percentile 25 (P25) and percentile 75 (P75).

Comparison of sequential data (weight and 24-h proteinuria) was done by profile analysis. The means of the data obtained only once (serum albumin, hydroxyproline in the renal cortex) were compared by analysis of variance for totally randomized experiments. Contrasts between pairs of means were analyzed by Turkey's test.

Renal histology data (TILI, percentage of glomeruli with lesions, glomerular area, glomerulosclerosis and interstitial fibrosis) were compared by the Kruskall-Wallis test for independent samples.

Statistical significance was set at p < 0.05.

RESULTS

At the beginning of the experiment, the body weight of the nephrotic animals was lower than that of the controls (Group S = 187,33 ± 1.39 g and Group S + MMF = 186.10 ± 3.01 versus Group ADR + V = 180.18 ± 2.25 g and Group ADR + MMF = 178.64 ±1.48 g, p < 0.05). During the experiment, all animals gained weight. However, at the end of the experiment, the animals of Group ADR + V weighed less than those in the remaining groups (ADR + V = 509.64 ± 10.72 versus ADR + MMF =542.27 ± 7.02g, S = 572.08 ± 16.53 and S + MMF = 554.10 ± 10.93g, p < 0.05).

The 24-h proteinuria in all the experimental groups is showed in Figure 1 Throughout the experiment proteinuria was higher in the nephrotic groups than in the healthy control groups. Mean proteinuria in Group S (range = 7.19 ± 1.03g to 12.67 ± 1.59g) and group S + MMF (range = 6.69 ± 0.69g to 12.70 ± 1.84g) did not differ.

Figure 1. Twenty-four-hour urinary protein of the four groups. Data expressed in means ± SEM.

In the treated nephrotic animals (Group ADR + MMF), proteinuria was significantly different from that in the non-treated nephrotic rats (Group ADR + V) at weeks 8; 16 and 20. After that there was no significant difference between both groups.

At the end of the experiment (28 weeks), serum albumin in Group ADR + V (3.65 ± 0.18g/dL) significantly differed from that in Group S (4.56 ± 0.18 g/dL) whereas no significant difference was observed among Group ADR + MMF (4.02 ± 0.19g/dL); Group S + MMF (4.43 ± 0.18g/dL); and Group S (Table 1). The median of the renal weight/body weight relationship of Group ADR + V (0.87, P25 = 0.67, P75 = 0.98) was significantly different from that observed in Group S (0.52, P25 = 0.49, P75 = 0.54). There was no significant difference among groups ADR + MMF, S + MMF and S (Table 1). The concentration of hydroxyproline in the cortex of the animals of Group ADR + V (4.56 ± 0.53 μg/mg renal cortex) significantly differed from that observed in Group S (2.90 μg/mg renal cortex ±0.10 μg/mg renal cortex) which did not statistically differ from the remaining groups (S + MMF = 3.48 ± 0.32 μg/mg renal cortex; ADR + MMF = 3.32 ± 0.33 μg/mg renal cortex) (Table 1).

Table 1. Serum Albumin, Kidney Weight (KW) Corrected by Body Weight (BW), Hidroxiproline Concentration in the Kidney Cortex

Group	Serum Albumin (g/dL)	(KW/BW) × 100 Md (P25–P75)	Hidroxiproline (μg/mg Renal Cortex)
S	4.56 ± 0.18	0.52 (0.49–0.54)	2.90 ± 0.10
S + MMF	4.43 ± 0.18	0.55 (0.53–0.56)	3.48 ± 0.32
ADR + V	3.65 ± 0.18^*	0.87 (0.67–0.98)^*	4.56 ± 0.53^*
ADR + MMF	4.02 ± 0.19	0.75 (0.58–0.89)	33.24 ± 0.33

^*p < 0.05 vs S.

Semiquantitative histological analysis of the kidneys revealed that in the groups, which received no adriamycin, glomerular lesion hardly occurred whereas among the nephrotic animals, the frequency of affected glomeruli was 30% (Group ADR + V: Md = 35%, P25 = 20%, P75 = 68%; Group ADR + MMF: Md = 27%, P25 = 9.5%, P75 = 54%; p > 0.05, Figure 2A). TILI of all groups is shown in Figure 2B where it may be observed that in the groups receiving no adriamycin TILI ranged from 0 to 4+ (Group S: Md = 0, P25 = 0, P75 = 1; Group S + MMF: Md = 0, P25 = 0, P75 = 3; p > 0.05) whereas in the nephrotic groups it varied from 1 to 12+ (Group ADR + V: Md = 7, P25 = 1.5, P75 = 9; Group ADR + MMF: Md = 8, P25 = 2, P75 = 9; p > 0.05).

Figure 2. A) Frequency of glomerular lesions B) tubulointerstitial lesion index on the four groups (TILI).

GSA (μm²) in Group S (Md = 69.4, P25 = 0, P75 = 207) and Group S + MMF (Md = 0, P25 = 0, P75 = 55) did not differ. However, the values observed in these 2 groups were statistically different from those found in the nephrotic groups. GSA was 2779 μm²in group ADR + V, (P25 = 751.8 μm², P75 = 3115 μm²) and 1147 μm² in Group ADR + MMF (P25 = 3969.7 μm², P75 = 1560 μm²) but this difference was not statistically significant (table 2).

Table 2. Glomerular Sclerosis Area (GSA) and Tubulointerstitial Fibrosis Area (TIFA) in the Four Studied Groups. Data Expressed as Median, Percentil 25 and Percentil 75

Group	GSA (μm^2) Md (P25–P75)	TIFA (μm^2) Md (P25–P75
S	69.4 (0–207)	0 (0–145)
S + MMF	0 (0–55)	70 (0–478)
ADR + V	2779 (751–3115)^*	218200 (78670–282700)^*
ADR + MMF	1147 (396–1560)^*	136000 (25010–255800)^*

^*p < 0.05 vs group S.

TIFA was 0 in Group S (P25 = 0, P75 =145 μm²) and Group S + MMF (P25 =, P75 = 478). The differences between these groups were not statistically different.

Group ADR + V displayed a large area of interstitial fibrosis (Md = 218200 μm², P25 = 78670 μm², P75 = 282700 μm²) and the same occurred with group ADR + MMF(Md = 136000 μm², P25 = 25010 μm², P75 = 255800 μm²). However, no statistically significant difference was observed between both groups. (table 2).

DISCUSSION

The data show that rats with adriamycin-induced nephropathy treated with mycophenolate mofetil presented temporary proteinuria reduction but no changes in the severity of the histologic lesion. It has been reported to reduce the severity of the nephropathy in experimental glomerulonephritis mediated by the immune system [13-16] and in the nephropathy induced by the ablation of the renal mass [17-18]. However, the reason why adriamycin-induced nephropathy has not responded to mycophenolate remains unclear. It does not seem to be dose-related because the doses used in studies of the renal ablation model were similar to those utilized with the adriamycin-induced nephropathy.

In present work, the treatment was started five weeks after the nephropathy induction whereas in the investigations that demonstrated mycophenolate mofetil was efficient, the treatment was introduced immediately after nephropathy was induced [17-18]. In the renal ablation model, it was also observed that whenever treatment was introduced late, the natural development of the disease remained unchanged [[19]]. Thus, it is probable that the protective effect of mycophenolate was not observed due to its late introduction.

The possibility that the action of MMF in the renal mass ablation model is as well transient cannot be disregarded because in the investigations in which this model was used the observation period lasted up to 8 weeks [17-18], which is much less than the 28 weeks of the present work.

In this study, the rats with adriamycin-induced nephropathy, when treated with MMF, showed transitory proteinuria reduction. A similar response has been observed when other drugs such as allopurinol [[20]] and ticlopidine [[21]] were used. Cyclosporine, introduced 15 days after nephritis induction, reduced proteinuria. However, when it was introduced late, it did not change the natural development of the disease [[5]]. These data suggest that in this model the renal lesion mechanisms differ according to the development stage of the disease.

ACKNOWLEDGMENT

This study was supported by FAPESP and CNPq.