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6 renal ablation in rats
Kidney International, Vol. 55 (1999), pp. 945–955
Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats FREDDY ROMERO, BERNARDO RODRI´GUEZ-ITURBE, GUSTAVO PARRA, LUISANDRA GONZA´LEZ, JAIME HERRERA-ACOSTA, and EDILIA TAPIA Renal Service and Laboratory, Hospital Universitario and Instituto de Investigaciones Biome´dicas, Maracaibo, Venezuela, and the Department of Nephrology and Renal Laboratory, Instituto Nacional de Cardiologı´a Ignacio Chavez, Me´xico City, Me´xico
tration with CD43- and ED1-positive cells in glomeruli and interstitium was two to five times lower in MMF-treated rats (P , 0.01). Expression of adhesion molecules CD18 and CD11b was similarly reduced. Conclusion. MMF ameliorates the progressive renal damage in the remnant kidney after 5/6Nx. This effect is associated with a reduction in the infiltration of lymphocytes and monocytes, whereas glomerular hypertrophy and systemic hypertension are unchanged.
Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats. Background. Extensive renal ablation is associated with progressive sclerosis of the remnant kidney. Because lymphocytes and monocytes accumulate in the remnant kidney, it is likely that they play a role in the renal scarring. Therefore, we treated rats with 5/6 nephrectomy (5/6Nx) with mycophenolate mofetil (MMF), a drug that has an antiproliferative effect and that suppresses the expression of intercellular adhesion molecules. Methods. Sprague-Dawley rats with 5/6Nx received MMF (30 mg · kg21 · day21 by daily gastric gavage, N 5 15) or vehicle (N 5 16). Ten additional rats were sham operated. All rats were fed a 30% protein diet. Body weight, serum creatinine, and urinary protein excretion were determined weekly. Lipid peroxidation, as a measure of oxidative stress observed by urinary malondialdehyde determinations, was performed every two weeks. Histologic studies were done in the remnant kidney four weeks (9 rats from the vehicle-treated group, 7 rats from the MMF group, and 5 sham-operated rats) and eight weeks after surgery (the remaining rats). Glomerular volume, sclerosis in glomeruli (segmental and global) and interstitium (semiquantitative scale), infiltrating lymphocytes and macrophages (CD43- and ED1-positive cells), and expression of adhesion molecules (CD54, CD18, and CD11b) were analyzed. Results. MMF treatment prevented the progressive increment in serum creatinine and the proteinuria observed in the 5/6 nephrectomized rats during the eight weeks of observation (P , 0.01). Weight gain was comparable in the MMF-treated and sham-operated rats, whereas weight gain was decreased in untreated 5/6 nephrectomized rats. Excretion of malondialdehyde increased after surgery but returned sooner to control levels in the MMF-treated rats. Increments in glomerular size and mean arterial blood pressure induced by renal ablation were not modified by MMF treatment. Eight weeks after surgery, segmental sclerosis was present in 48.4 6 8.35% (6 sd) glomeruli in the vehicle-treated group versus 25 6 10.5% in the MMF-treated group (P , 0.001). Interstitial fibrosis was reduced significantly with MMF treatment (P , 0.001). Infil-
The progression of renal damage resulting from reduced nephron mass has been extensively studied in the 5/6Nx model in the rat, as described originally by Shimamura and Morrison [1]. Classic studies by Hostetter et al demonstrated that this experimental model was characterized by progressive glomerulosclerosis and chronic renal failure [2]. The remnant nephron units developed glomerular hypertension, increased single nephron glomerular filtration rate, and glomerular hypertrophy, and over the previous 15 years, all of these factors have been postulated to play a pathogenetic role in the progressive scarring of the kidney [3]. Surviving nephrons have increased oxygen consumption and are subjected to oxidant stress [4]. Because the initial event in the subtotal renal ablation model is an increase in glomerular pressure, treatment strategies have usually been directed to correct this hemodynamic disturbance, specifically, to reduce intraglomerular pressure. Correction of this pathophysiologic event and normalization of permselectivity properties of the glomerular capillary barrier [5] are major mechanisms underlying the protective effect on renal function observed with angiotensin-converting enzyme inhibition and low-protein diet in several models of renal disease in the experimental animal and in humans [6]. Less commonly, treatments have been directed to modify late events in the development of glomerulosclerosis, that is, events deriving from the hemodynamic and hypertrophic glomerular changes. For example, heparin
Key words: sclerosis, remnant kidney, scarring, adhesion molecules, proteinuria, interstitial fibrosis. Received for publication July 20, 1998 and in revised form October 19, 1998 Accepted for publication October 20, 1998
1999 by the International Society of Nephrology
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Romero et al: Mycophenolate mofetil therapy in renal ablation
treatment, which prevents microthrombosis [7], as well as Ticlopidine, a drug with a wide spectrum of inhibitory actions on platelets and platelet–cell interactions, retards the progression of renal failure in the remnant kidney model [8]. More recently, Shimizu et al have reported that administration of Pirfenidone, a compound that prevents and reverses extracellular matrix accumulation, ameliorates the histologic damage in the remnant kidney of the rat with 5/6Nx [9]. Largely undefined is the role played by infiltrating lymphocytes and macrophages in the development of glomerulosclerosis triggered by a reduction of nephron mass. There is limited information on lymphocyte accumulation in this model. However, monocyte/macrophage infiltration occurs early [10, 11], and their numbers increase fourfold to fivefold in the glomeruli and tubulointerstitial areas of the remnant kidney [12, 13]. Therefore, it is likely that these cells play a significant role in the subsequent development of fibrosis. In fact, cytokines produced by infiltrating cells appear to mediate renal scarring in this experimental model [10]. Mycophenolate mofetil (MMF) is a new immunosuppressant agent in which the active component, mycophenolic acid, is an inhibitor of inosine 59-monophosphate dehydrogenase, which controls the synthesis of guanosine triphosphate [14, 15]. MMF depletes guanosine triphosphate pools in lymphocytes and monocytes and suppresses the de novo synthesis of purines, thereby exerting a selective and reversible antiproliferative activity on these cells. In addition, the expression of intercellular adhesion molecules, which is critical for the migration of leukocytes from circulation and their subsequent accumulation in the tissues, is suppressed by this drug [16]. Therefore, we decided to investigate whether treatment with MMF would reduce the renal cellular infiltration and the expression of adhesion molecules, as well as the oxidant-induced lipid peroxidation observed after 5/6Nx, and whether these modifications would be associated with improvement in the progression of renal disease. We found that deterioration of renal function and proteinuria, which is characteristic of this model, was prevented and that the hallmark histologic changes were markedly improved. Because the hematologic, liver, and renal toxicity of MMF are relatively minor [17, 18] and this drug is used with relative safety in the long-term treatment of renal transplantation, MMF could be potentially useful to retard the progressive damage associated with reduced nephron mass in humans. METHODS Design of the study and experimental groups Studies were done in 50 male Sprague-Dawley rats weighing 200 to 280 g at the start of the experiments. Throughout the study, all rats were fed ad libitum with
rat chow containing 30% protein (Purina, St. Louis, MO, USA) and tap water. After obtaining blood samples for serum creatinine and 24-hour urine for determination of proteinuria and urinary malondialdehyde excretion, 40 rats had 5/6Nx (mentioned later here), and 10 rats had a sham operation. Nine rats in the group that had 5/6Nx died during surgery or in the following 48 hours. Five days after surgery, the surviving rats in the 5/6Nx group were randomly subdivided in two groups, one of which received MMF (5/6NxMMF group, N 5 15) the other one received vehicle (5/6Nx, N 5 16). Four of these rats died in the subsequent four weeks (two from the 5/6NxMMF group and two from the 5/6Nx group). Histologic studies were done four and eight weeks after surgery. At the four-week endpoint, nine rats from the 5/6Nx group, seven rats from the 5/6NxMMF group, and five sham-operated rats were sacrificed. The remaining rats (5 rats from the shamoperated group, 5 rats from the 5/6Nx group, and 6 rats from the 5/6NxMMF group) were sacrificed eight weeks after surgery. Body weight, serum creatinine, and urinary protein excretion were determined weekly. Urinary malondialdehyde (MDA) determinations were done every two weeks. Histologic and immunohistologic studies were done in remnant kidneys and in sham-operated animals at the time of sacrifice. Dose of mycophenolate mofetil Preliminary studies were done to determine the maximal tolerated dose of MMF. Doses higher than 30 mg · kg21 · day21 were usually associated with diarrhea; consequently, this dose was chosen. Daily gastric gavage was used to administer 30 mg · kg21 · day21 of MMF diluted in 200 ml of water to the rats of the 5/6NxMMF group. Because mycophenolate (CellCept; Roche Pharmaceutical Co., Caracas, Venezuela) is insoluble in water, we prepared each dose individually and agitated the mixture vigorously to obtain a suspension, and then the suspension was immediately given to the rat before the MMF precipitated. Rats in the 5/6Nx group and sham groups received the same volume of water by the same route of administration daily. Surgical procedures Subtotal renal ablation was done in a single surgical procedure by subcapsular removal of the right kidney and selective infarction of two thirds of the left kidney by ligation of the posterior and one or two of the anterior extrarenal branches of the renal artery, to leave approximately one sixth of the total kidney tissue mass. Sham operations consisted in laparotomy and manipulation of the kidneys and renal pedicle without destruction of renal tissue.
Romero et al: Mycophenolate mofetil therapy in renal ablation
Determination of lipid peroxidation We used the method of Ohkawa et al to determine thiobarbituric acid reactive substances, an index of lipid peroxidation [19]. MDA was measured in 24-hour urine centrifuged at 2000 r.p.m. and processed the same day. Four hundred microliters of the sample were added to a reaction mixture consisting of 200 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.5% thiobarbituric acid, and 400 ml of bidistilled water. This mixture was heated at 958C in water bath for 60 minutes. After removal from the water bath, 1 ml of water and 5 ml N-butanol-pyridine were added, and the mixture was agitated and centrifuged at 2000 g for 15 minutes. The upper organic layer was pipetted, and the absorbance of this fraction was read at 532 nm in a Shimatzu model UV21100 S spectrophotometer (Kyoto, Japan). Malondialdehyde bis-dimethyl acetal was used as external standard. Results were expressed as nmol of MDA excreted in 24 hours. Laboratory determinations Proteinuria was determined in 24-hour urine collections by the sulfosalicylic acid method and the serum creatinine by modification of the Jaffe´ reaction in autoanalyzer methodology. Tissue preparation From every animal, coronal sections of remnant renal tissue were obtained at the time of sacrifice. One fragment was fixed in 15% formalin, and 10 were embedded in Paraplast Plus (Monoject, Sherwood Medical Scientific Division, St. Louis, MO, USA); the rest were included in Tris buffered saline (TBS) freezing medium (Triangle Biomedical Sciences, Durham, NC, USA) and were immediately frozen in dry ice and acetone and were stored at 2708C. Light microscopy studies Paraffin-embedded sections were stained with hematoxylin eosin, Masson’s Gomori’s stain, and periodic acid-Schiff reagent (PAS). Coronal sections cut at 4 mm thickness, including superficial and juxtamedullary glomeruli, were evaluated. Evaluation of histologic damage. Sections were examined by two investigators without previous knowledge of the experimental group of the animal from which the tissue was taken. At least 10 sections were analyzed for each kidney, and the evaluation that was assigned represented the mean values of these sections. Agreement between the two observers was within 10%. Evaluation was done as follows. Glomerular sclerosis. Glomerular lesions were evaluated by determining the number of normal glomeruli and the number with either segmental or total glomerular
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sclerosis in superficial and juxtamedullary sections. Glomerulosclerosis was evaluated in PAS-stained tissue and was defined as the increment of PAS-positive material, with loss of cellular elements, collapse of capillary lumina, and entrapment of amorphous hyaline material associated or not with adhesions to Bowman’s capsule. Sclerosis involving more than 80% of the glomerular tuft was considered global, and sclerosis involving less than 80% was considered segmental. No attempt was made to subclassify the segmental lesions in accordance with the percentage involvement of the glomerulus. At least 100 glomeruli were evaluated per biopsy. Results are expressed as the percentage glomeruli examined (more than 100 per biopsy) showing segmental or global sclerosis. Tubulointerstitial lesions. Severity of tubular dilation and atrophy, as well as tubulointerstitial infiltration (hematoxylin and eosin staining) and fibrosis (trichrome staining), were evaluated individually, determining the percentage in the biopsy section affected by the corresponding finding with the help of a grid-fitted eyepiece. Gradation of damage (0 to 51) was done by modifying the scale used by Zoja et al as follows: 0 5 changes in less than 10% of the histologic section; 11 5 changes in up 20% of the section; 21 5 changes in up to 40% of the section; 31 5 changes in up to 60% of the section; 41 5 changes in up to 80% of the section; 51 5 changes in more than 80% of the section [8]. The average score obtained in the sections of each biopsy (10 to 15 sections per biopsy) was used as the score of the biopsy. Immunofluorescence studies Identification of intercellular adhesion molecules was done in frozen 3 mm sections by indirect immunofluorescence technique, as described in previous publications [20, 21] using the monoclonal antibodies listed later. As secondary antibody we used fluorescein-conjugated affinity-pure F(ab9)2 rat antimouse IgG with minimal cross reactivity with rat proteins (Accurate Chemical & Scientific Corp., Westbury, CT, USA) at a concentration of 60 mg/ml (1:50). Ethidium bromide was used for nuclear staining. The following monoclonal antibodies were used: antiCD43 (Accurate Chemical & Scientific Corp., Westbury, NY, USA; mouse IgG1 clone W3/13, distribution in T lymphocytes, concentration 5 5 mg/ml); anti-ED1 (Accurate Chemical & Scientific; mouse IgG1, distribution in monocytes, concentration 5 30 mg/ml); anti-ICAM1 (CD54; Seikegaku Corp. Tokyo, Japan; IgG1, distribution in endothelial cells, monocytes, T and B lymphocytes, epithelial cells, dendritic cells, mesangial cells; concentration 5 10 mg/ml); anti-CD11b (Biosource International, Camarillo, CA, USA; mouse IgG2 A, distribution in monocytes/macrophages, neutrophils; concentration 5 5 mg/ml) and anti-LFA1 (CD18; Seikegaku Corp.; IgG1,
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Romero et al: Mycophenolate mofetil therapy in renal ablation
distribution in lymphocytes, monocytes, neutrophils; concentration 5 10 mg/ml). Double-staining procedures were done as follows: The monoclonal antibody (mAb) used to identify the integrins (mAb anti-CD18 or anti-CD11b) was applied to the tissue (concentrations listed earlier here) and was incubated in a humid chamber at room temperature for 45 minutes. Afterward, tetramethylrhodamine isothiocyanate (TRIC)-conjugated, affinity-pure F(ab9)2 rat antimouse IgG, with minimal cross-reaction with rat proteins (Accurate Chemical & Scientific) was added. After washing for 10 minutes with PBS 0.01 m, 0.15 m NaCl (one change of PBS) and 100 ml of blocking solution (PBS 0.01 m, 0.15 m NaCl containing 10% fetal calf serum and 10% goat serum) were added, incubated for 10 minutes, and washed as described earlier here. Afterward, the tissues were incubated with the mAb used to identify the leukocyte populations (mAb anti-CD43 or mAb antiED1 at the dilutions described earlier) and were then reacted with 50 ml of fluorescein-conjugated, affinitypure F(ab9)2 rat antimouse IgG (20 mg/ml) with minimal cross-reaction with rat proteins (Accurate Chemical & Scientific). As a control for the double-staining procedure, five tissues were double stained with CD18 and ED1, as described earlier here. Tissues were examined with a Zeiss microscope with epifluorescence system, phase contrast, and appropriate filters. Cells with red nuclei surrounded by a green cytoplasmatic membrane were counted as positive. Counting was done with the help of a grid-fitted eyepiece. Within the glomerular tuft, staining results were expressed as positive cells per glomerular cross-section, with the exception of CD54 staining that was expressed as a 11 to 41 score, described previously [17], which takes into account the number of positive cells and the intensity of the fluorescent staining. In the interstitial areas, results were expressed as positive cells per mm2. Glomerular volume At least 100 glomeruli in each kidney tissue were examined, and the glomerular diameter was measured using a microscopic scale attached to the ocular lens. Then, the mean random cross-sectional glomerular area (AG) was calculated, and the glomerular volume (VG) was determined using the following formula: VG 5 (b/k 3 AG)3/2 where b 5 1.38 is the shape coefficient for spheres, the idealized shape of the glomerular tuft, and k 5 1.1 is the size distribution coefficient [22]. Statistical analysis Results are expressed as mean 6 sd. Comparisons between groups were done by one-way analysis of variance followed by Tukey-Kramer post-tests. When differences in the standard deviation of the groups under analysis were significant, nonparametric Mann–Whitney rank
Fig. 1. Serum creatinine concentration in sham-operated animals (h) and rats with reduction in renal mass, untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). Data from five to eight weeks correspond to five animals in each group; otherwise, the values represent 10 to 16 animals in each group (Methods section, experimental design). Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.05, **P , 0.01, ***P , 0.001 vs. sham. Bars are standard deviation. The differences between the 5/6Nx group and the MMF treated group are also significant (P , 0.001) after the fourth week.
test or Kruskal Wallis tests with Dunn’s multiple comparison post-test were used. Differences were considered significant when P was less than 0.05. RESULTS Figure 1 shows the serial changes in serum creatinine concentration observed in the sham-operated and in the rats with 5/6 reduction in renal mass with (5/6NxMMF) and without (5/6Nx) treatment with MMF. The untreated 5/6Nx group of rats experienced a progressive rise in serum creatinine (P , 0.01 vs. baseline) from the second week up to the seventh to eighth week after nephrectomy when it appeared to reach a plateau, at levels of 156 6 6.61 mmol/liter, approximately 2.4 times above control levels. The levels of serum creatinine the 5/6NxMMF group and in the sham-operated group were lower than in the untreated 5/6Nx rats (P , 0.001 in Fig. 1). In contrast, the 5/6NxMMF group had essentially similar serum creatinine levels than the sham-operated group the first four weeks after surgery. Thereafter, the level of the creatinine in 5/6NxMMF group was slightly above the levels in the sham-operated group but was steady at each time interval (Fig. 1). Urinary protein excretion is shown in Figure 2. The animals receiving MMF (5/6NxMMF) and the shamoperated group showed essentially similar levels of proteinuria throughout the eight-week observation period. Urinary protein excretion in these groups (5/6NxMMF group and sham-operated group) is unchanged from the baseline levels. In contrast, the untreated animals with reduced renal mass (5/6Nx group) showed a progressive increment in proteinuria.
Romero et al: Mycophenolate mofetil therapy in renal ablation
Fig. 2. Urinary protein excretion in sham-operated animals (h) and rats with reduction of renal mass untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). Data from five to eight weeks correspond to five animals in each group; otherwise, the values represent 10 to 16 animals in each group (Methods section, experimental design). Bars are standard deviation. Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.01 and **P , 0.001 vs. sham and MMF group.
All animal groups had similar weights (g) at the beginning of the experiment (5/6Nx group 5 255 6 11.42, 5/6NxMMF group 5 236.9 6 10.09, sham-operated group 5 255 6 5.00) and gained weight during the period of observation. However, the 5/6NxMMF group and the sham-operated groups gained weight similarly at four weeks (5/6NxMMF group 5 320 6 6.70, sham-operated group 5 315 6 4.30) and at eight weeks (5/6Nx,MMF group 5 409 6 2.45, sham-operated group 5 411.3 6 2.40); in contrast, the 5/6Nx group gained less weight at four weeks and eight weeks (286.1 6 9.44 and 338 6 8.00, respectively, P , 0.01). Mean arterial blood pressure at the time of sacrifice (4 weeks after surgery) was 121.1 6 10.33 in the shamoperated rats, 165.5 6 16.5 in the 5/6Nx group (P , 0.001 vs. sham), and 158.3 6 15.4 in the 5/6NxMMF group (P , 0.01 vs. sham). Figure 3 shows the urinary excretion of MDA. Both groups of nephrectomized rats (5/6Nx group and 5/6NxMMF group) had their urinary excretion of MDA doubled after surgery; however, the group treated with MMF showed a progressive decrease in urinary MDA starting three to four weeks after surgery, reaching control levels during the fifth to sixth week. In contrast, the untreated animals (5/6Nx group) increased the MDA excretion nine times, peaking at the fourth week and decreasing thereafter (Fig. 3). The histologic observations are shown in Table 1. The glomerular volume is higher in the two groups of rats with reduced renal mass. There were no significant differences in the glomerular hypertrophy in the 5/6Nx group and the 5/6NxMMF group. Glomerular sclerosis, both segmental and global, was present in a small percentage
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Fig. 3. Urinary malondialdehyde (MDA) excretion in sham-operated animals (h) and rats with reduction of renal mass untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). From five to eight weeks, all groups consisted of five animals each; otherwise, all values represent at least 10 animals in each group (Methods section, experimental design). Bars are standard deviation. Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.001 vs. sham; **P , 0.01 vs. sham. The difference between the 5/6Nx group and the 5/6NxMMF group at three to four weeks and at five to six weeks is also significant (P , 0.001).
of the glomeruli in sham-operated animals and increased 13 times at four weeks and 17 times at eight weeks after 5/6Nx in the untreated group (5/6Nx group). Eight weeks after renal ablation, segmental glomerulosclerosis in rats treated with MMF (5/6NxMMF group) was 25 6 10.5%, half of the value in untreated rats at the same period of time (P , 0.001). Tubular dilation and atrophy, as well as tubulointerstitial infiltration and fibrosis, were substantially reduced in the 5/6NxMMF group. In contrast, the untreated nephrectomized animals (5/6Nx group) had increasing severity of tubular and interstitial damage, involving 40% to 80% of the examined remnant renal tissue after eight weeks (Table 1). Figure 4A shows typical sclerotic changes in glomeruli and interstitium in untreated animals, in contrast with animals treated with MMF (Fig. 4B). Cells expressing adhesion molecules (CD54, CD18, and CD11b) and CD43- and ED1-positive cells in the glomeruli and in tubulointerstitial areas are shown in Tables 2 and 3, respectively, as well as in Figure 4 C–F, and Figure 5. In biopsies examined four and eight weeks after surgery, the number of lymphocytes (CD43-positive cells) and macrophages (ED1-positive cells) in the glomeruli (Table 2) and interstitium (Table 3) was not statistically different in sham-operated rats and in rats treated with MMF (5/6NxMMF group). In contrast, the untreated 5/6Nx group showed values three to four times higher (Table 2). Four weeks after surgery, the untreated 5/6Nx group
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Romero et al: Mycophenolate mofetil therapy in renal ablation Table 1. Histological observations 4 weeks after surgery
VG lm3 3 10 6 Glomerular sclerosis Segmental % Global % Tubular dilation & atrophy (0–5) Interstitial infiltration (0–5) Interstitial fibrosis (0–5)
8 weeks after surgery
Sham (N 5 5)
5/6 Nx (N 5 9)
5/6 Nx 1 MMF (N 5 7)
Sham (N 5 5)
5/6 Nx (N 5 5)
5/6 Nx 1 MFF (N 5 5)
1.61 6 0.10
2.23 6 0.43
2.27 6 0.34
1.81 6 0.42
2.61 6 0.21
2.58 6 0.90
3.0 6 0.71 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00
38.7 6 4.33 7.57 6 3.65 2.89 6 0.78 1.56 6 0.73 1.67 6 0.50
21.7 6 9.41a 4.29 6 6.45 0.71 6 0.76a 1.0 6 0.89 0.42 6 0.54a
2.8 6 0.83 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00
48.4 6 8.35 20.6 6 15.27 4.2 6 0.45 3.8 6 1.48 2.0 6 0.71
25.0 6 10.51a 6.20 6 7.95b 0.8 6 0.84a 1.2 6 2.16 0.4 6 0.89b
Segmental and global glomerular sclerosis expressed as the percent of the glomeruli examined presenting the corresponding lesion. Tubulointerstitial damage was graded according to the percent of the biopsy affected by the corresponding finding (see Methods section). N corresponds to the number of renal biopsies in each group; the value in each biopsy is the mean of 10-15 sections. Statistical significances between 5/6NxMMF vs. 5/6Nx are: a P , 0.001, b P , 0.05. The percentage glomeruli showing global sclerosis in the 5/6Nx group is higher at 8 weeks than at 4 weeks (P , 0.001). Data in untreated 5/6Nx are different (P ranging from ,0.05 to ,0.001) from data in sham operated rats.
had increased expression of adhesion molecules CD45, CD18, and CD11b (Tables 2 and 3). Eight weeks after surgery, the expression of CD54 in the untreated 5/6Nx group had decreased, whereas in contrast, CD11b had more than doubled in tubulointerstitial areas (Table 3). Sham-operated animals and 5/6Nx rats treated with MMF had essentially similar expression of CD54, CD18, and CD11b at four weeks (Table 2). At eight weeks, the only significant difference between the sham-operated and 5/6Nx group was a reduction in the interstitial infiltration of cells expressing CD54 in the MMF-treated group (Table 3). DISCUSSION The 5/6Nx model in the rat has been widely used to evaluate the effect of treatment strategies on the progressive scarring of the kidney that is associated with extensive renal ablation. Because the initial effects of the reduction of nephron mass involves hypertension and hyperfiltration of the remnant nephron units, a vast majority of research efforts have been directed to determine if the correction of the altered hemodynamics improves the renal damage. Understandably, drugs that lower systemic and glomerular pressure are among the most prominently studied [23–26]. Other treatment strategies have been investigated less frequently. Pukerson et al showed that thrombotic mechanisms are important in the progression of the glomerular lesion and that heparin had a beneficial effect [7]. Suppression of platelet adhesion and platelet-derived factors was a treatment modality chosen by Zoja et al, who found that Ticlopidine, a drug with a wide spectrum of antiplatelet effects, improved the progressive renal damage of the remnant kidney [8]. Interestingly, the beneficial effects of this treatment occur without modification of the systemic blood pressure [8], which also appears to be the case in the improvement induced with MMF reported here, as the blood pressure levels in the
5/6Nx and the 5/6NxMMF groups were similarly elevated with respect to the sham-operated group. The doses of MMF used in this work are not expected to have a significant antiplatelet effect or to decrease the numbers of circulating leukocytes, but this possibility was not ruled out in this study. Other recent studies addressing the final common pathway by which hemodynamic alterations induce renal scarring are those of Shimizu et al, who used Pirfenidone, a drug that prevents and reverses collagen accumulation [9]. They found attenuation of the chronic renal failure in the rats with 5/6Nx [9]. Suppression of lymphocyte activity and monocyte/ macrophage infiltration is yet another strategy of potential use for amelioration of the progressive renal damage resulting from renal ablation. Macrophages are known to participate in the inflammatory reaction, producing a number of soluble mediators, including cytokines, proteases, and free oxygen radicals [27], and several studies have shown that macrophage infiltration and proliferation are prominent findings in the remnant kidney [11– 13, 28]. Furthermore, Schiller et al have recently demonstrated that mechanical injury resulting from glomerular capillary hypertension induces up-regulation of monocyte chemoattractant protein-1 (MCP-1) [10]. Interestingly, a low-protein diet modulates the expression of MCP-1 [10], which suggests that protein restriction, in addition to improving glomerular hemodynamics in the remnant nephrons, could have beneficial effects resulting from inhibition of macrophage infiltration. Mycophenolate mofetil is the morpholinoethytl ester of mycophenolic acid, and it inhibits inosine 59-monophosphate dehydrogenase, which controls the de novo synthesis of guanosine triphosphate [29]. This drug has a relatively specific effect on lymphocytes because other cells have alternative purine synthesis pathways not blocked by MMF. In the experimental setting of experimental renal ablation, MMF is a drug well suited for the
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Fig. 4. Changes observed by light microscopy and lymphocyte and macrophage infiltration of remnant kidneys four weeks after 5/6Nx. Severe segmental sclerosis (approximately 60% of the glomerular tuft) observed in untreated rat (receiving vehicle; A) is contrasted with essentially normal glomerular appearance in rat treated with MMF (B). In addition, interstitial fibrosis is evident in untreated rat and absent in the biopsy from the rat treated with MMF (Mason’s Trichromic stain, original magnification &time;400). Immonuhistology showing remnant kidney tissue stained with mAb anti-CD43 (C, untreated rat; D, rat treated with MMF) and with mAb anti-ED1 (E, untreated rat; F, rat treated with MMF). Original magnification was 3630.
purpose of testing the effects of suppressing macrophage infiltration and proliferation for several reasons. First, MMF depletes the pools of guanosine triphosphate not only in lymphocytes but also in monocytes and thereby decreases the synthesis of fucose and mannose of mem-
brane glycoproteins, which are ligands of the selectin adhesion molecules in the vascular endothelium [29]; in addition, MMF inhibits up-regulation of CD18 in lymphocytes [30]. Consequently, the infiltration of monocytes, as well as lymphocytes, is inhibited by this drug
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Romero et al: Mycophenolate mofetil therapy in renal ablation Table 2. Cellular infiltration in the glomeruli 4 weeks
Positive cells per gcs CD43 ED1 CD54 CD18 CD11b
8 weeks
Sham (N 5 5)
5/6Nx (N 5 9)
5/6NxMFF (N 5 7)
Sham (N 5 5)
5/6Nx (N 5 5)
5/6NxMFF (N 5 5)
1.14 6 0.27 0.74 6 0.11 1.27 6 0.13 0.98 6 0.27 1.04 6 0.60
3.72 6 1.30 3.14 6 0.48 2.21 6 0.79 4.44 6 1.45 4.75 6 2.18
1.46 6 0.94b 0.97 6 0.27b 0.86 6 0.18a 1.94 6 1.02a 1.30 6 0.22a
0.46 6 0.31 0.86 6 0.27 0.85 6 0.12 1.21 6 0.14 1.07 6 0.70
3.47 6 0.54 2.28 6 0.37 0.54 6 0.11 1.30 6 0.25 3.50 6 0.94
1.15 6 0.56a 1.26 6 0.44b 1.08 6 0.08a 0.80 6 0.24b 0.83 6 0.16a
N corresponds to the number of biopsies studied; the value in each biopsy represents the mean of 10-15 sections. Statistical significances between 5/6Nx MMF and 5/6 Nx are: a P , 0.001, b P , 0.01. In the 5/6Nx group, the differences between the 4th and 8th week in CD54 and CD18 are significant (P , 0.01). The data of untreated 5/6Nx are significantly different (P ranging from ,0.05 to ,0.001) from the data in sham operated rats.
Table 3. Cellular infiltration in the interstitium 4 weeks Positive cells per mm2 CD43 ED1 CD54 CD18 CD11b
Sham (N 5 5) 68.4 6 56.5 35.2 6 7.6 252.0 6 27.5 25.4 6 10.8 32.0 6 28.4
8 weeks
5/6Nx (N 5 9)
5/6NxMFF (N 5 7)
Sham (N 5 5)
5/6Nx (N 5 5)
5/6NxMFF (N 5 5)
135.6 6 64.2 194.4 6 61.2 531.6 6 216 229.6 6 132 121.2 6 43.1
59.2 6 39.3c 34.0 6 9.6a 314.4 6 124c 72.6 6 31.2b 25.2 6 11.9
36.8 6 17.3 56.0 6 11.6 261.6 6 44.5 41.6 6 12.5 21.6 6 17.8
431.2 6 59.2 140.0 6 22.8 128.8 6 15.3 74.4 6 19.9 352.8 6 105
91.7 6 59.9a 75.6 6 4.91a 73.6 6 42.6c 75.0 6 27.9 36.1 6 21.8a
N corresponds to the number of biopsies studied; the value in each biopsy represents the mean of 10-15 sections. Statistical differences between 5/6Nx MMF and 5/6Nx are: a P , 0.001, b P , 0.01, c P , 0.05. In the 5/6Nx group, the differences between the 4th and 8th week in CD54 and CD18 are significant (P , 0.01). The data in the untreated 5/6Nx group are significantly different (p ranging from ,0.05 to ,0.001) from the data in the sham operating group, except for CD18 positive cells at 8 weeks.
[30–32]. Second, MMF exerts a long-term inhibition of the production of lymphocyte and macrophage-derived cytokines [33], a valuable characteristic in the prevention of chronic scarring in the remnant kidney [34]. Third, MMF, in contrast to other antirejection drugs, is not associated with renal toxicity [15] and is reasonably well tolerated as a long-term treatment [17, 18]. Treatment with MMF completely prevented the proteinuria induced by renal ablation. This protective effect is maintained for at least eight weeks (Fig. 2). To our knowledge, there is no direct effect of MMF on glomerular permselectivity. Reduction of proteinuria, noted in anecdotal reports of several kidney diseases treated with this drug [35], is presumed to be a consequence of its immunosuppressant properties, reduction of cellular infiltration, and decrease in cytokine production. Renal function is also markedly improved by MMF treatment. As reported by others, the 5/6Nx group presents a progressive rise in serum creatinine, whereas in contrast, the rats with 5/6 reduction in renal mass treated with MMF showed only a modest increment in serum creatinine that remained at steady levels from the fourth to the eighth week (Fig. 1). The histologic findings are in accordance with the renal function data. The number of glomeruli with segmental and global sclerosis is substantially reduced with MMF treatment. Moreover, the percentage of glomeruli with these changes (Fig. 5 A, B) remains essentially unchanged
after four weeks in the 5/6NxMMF group, whereas in contrast, it increases in the untreated 5/6Nx group (Table 1). Tubulointerstitial changes are ameliorated importantly in the MMF-treated group (P . 0.05 vs. sham; Table 1), in contrast with the changes seen in the 5/6Nx group (Fig. 5 A, B). The observed beneficial effect on renal scarring is likely the result of direct and indirect actions of MMF because cytokine “cross-talk” between macrophages and intrinsic renal cells is central to the cell proliferation and matrix expansion that leads to glomerulosclerosis and interstitial fibrosis [36]. Careful studies by Floege et al and Kliem et al have shown that in the remnant kidney model, proliferation of intrinsic glomerular and tubulointerstitial cells is an early event in the development of renal scarring; macrophage infiltration appears later and contributes to amplify the inflammatory response [12, 13]. To our knowledge, there is no evidence of a direct suppressive effect of MMF on cytokine production by intrinsic renal cells. However, this drug reduces cell adhesion molecules in endothelial cells [16], and there is a well-defined inhibition of the production of all lymphocyte and macrophage-derived cytokines and growth factors. Specifically, platelet-derived growth factor, interleukin 2, tumor necrosis factor a, and transforming growth factor-b are profoundly reduced [33]. Suppression of macrophage-derived platelet-derived growth factor should prevent further activation of the mesangial
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Fig. 5. Intercellular adhesion molecules in remnant kidneys four weeks after 5/6Nx. Intense interstitial infiltration of cells expressing CD18 in kidney from untreated rat (A) is contrasted with the negative appearance of the kidney from a rat treated with MMF (B). Intense glomerular expression of CD54 in untreated 5/6Nx rat (C) contrasts with moderate expression of CD54 in 5/6Nx rats treated with MMF (D).
cells, in addition to limiting amplification of the proliferative reaction of infiltrating cells. Suppression of macrophage-derived transforming growth factor-b should have a beneficial effect on glomerulosclerosis by limiting the synthesis and favoring the degradation of extracellular matrix by renal metalloproteinases [36, 37]. An additional influence that may induce synthesis of chemokynes is the generation of reactive oxygen species. Nath, Croatt and Hostetter have shown that oxidant stress is increased in the 5/6Nx model [4], and our studies, in addition to confirming their findings, have shown that urinary MDA is significantly reduced by MMF treatment from the first to the sixth week after nephrectomy (Fig. 3). This effect of MMF is likely the result of suppression of the inflammatory infiltrate, but by itself, a reduction in reactive oxygen species would reduce the expression of MCP-1, RANTES, and ICAM-1 in the mesangial cells [reviewed in 34]. In addition, a decrease in oxidative stress would eliminate a stimulus for apoptosis, which is an early event in the remnant kidney model [38].
It is interesting that a similar increment in glomerular volume was found in the 5/6Nx and the 5/6NxMMF groups. This finding firstly suggests that the hemodynamic disturbances that trigger glomerular hypertrophy are similar in both groups of rats with renal ablation, and secondly suggests that renal hypertrophy is unaffected by MMF treatment. This is in contrast with the reduction in the renal hypertrophy and hyperplasia associated with other immunosuppressive drugs such as cyclosporine A [39]. Lymphocyte (CD43-positive cells) and macrophage (ED1-positive cells) infiltration is comparable in the MMF-treated group and the sham-operated group at four weeks after surgery. In contrast, the 5/6Nx group shows the cellular infiltration of the remnant kidney reported previously by others [10–13, 28]. These findings indicate that MMF administration was capable of effectively counteracting the infiltration and proliferative stimuli triggered by the glomerular hypertension. Expression of adhesion molecules has not been stud-
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Romero et al: Mycophenolate mofetil therapy in renal ablation
ied in the renal ablation model. All adhesion molecules tested increased several times in the remnant kidney (glomeruli and interstitium) in the 5/6Nx rats examined four weeks after nephrectomy. In the following month, the expression of CD54 and CD18 in glomeruli and interstitium fell to levels comparable to those present in shamoperated rats (Tables 2 and 3). The reasons for the reduction in expression of CD54 and CD18 are not clear, but it is likely that progressive scarring of the kidney is associated with a decrease in the number of CD54-positive cells, which includes endothelial cells, lymphocytes, and neutrophils. As shown in Table 3, the rats from the 5/6Nx group showed a minor reduction (P . 0.05) in tubulointerstitial ED1-positive cells at four and eight weeks after nephrectomy (194 6 61.2 and 140 6 22.8 cells per mm2, respectively), whereas the CD11b-positive cells almost triple in number (Table 3). Because CD11b stains granulocytes in addition to monocytes and macrophages, we interpret these findings as indicative of an increment in nonmacrophage leukocyte infiltration after eight weeks. Interestingly, the numbers of CD11b-positive cells in rats treated with MMF are comparable to the sham-operated animals (Tables 2 and 3). The relatively stable number of macrophages from the fourth to the eight weeks in the remnant kidney observed by us is in contrast with the findings in a recent report by Yang et al [28], who found that ED1-positive cells increased progressively up to 16 weeks after nephrectomy. Their study did not use a high-protein diet [28], and consequently, the tubulointerstitial damage and the increment in serum creatinine progressed at a lower rate than in this study. It is possible that this explains, at least in part, the discrepancy. These studies confirm recent preliminary reports from our group [40] and Fujihara et al [41], and suggest that MMF may be useful in the treatment of progressive renal failure associated with reduced renal mass. Yet to be determined is whether MMF treatment would give comparable or additional beneficial effects to those obtained by other treatment modalites in this experimental model. The route of administration, as well as the dose of MMF in this study, is comparable to those used routinely in humans with relatively minor side-effects. Therefore, consideration may be given to trials with this drug in the clinical setting of progressive renal failure. ACKNOWLEDGMENTS The authors are grateful for financial support from Asociacio´n de Amigos del Rin˜o´n, Maracaibo, Venezuela. Parts of this article were presented in the 30th Annual Meeting of the American Society of Nephrology (San Antonio, Texas, November 2–5, 1997) and were published in abstract form (Romero et al, J Am Soc Nephrol 8, 628A, 1997). Reprint requests to Bernardo Rodrı´guez-Iturbe, Apartado Postal 1430, Maracaibo 4001-A, Venezuela. E-mail: [email protected]
REFERENCES 1. Shimamura T, Morrison AB: A progressive glomerulosclerosis occurring in partial five-sixths nephrectomy. Am J Pathol 79:95– 101, 1975 2. Hostetter TH, Olson JL, Venkatachalam MA, Brenner BM: Hyperfiltration of remnant nephrons: A potentially adverse response to renal ablation. Am J Physiol 241:F85–F93, 1981 3. Zatz R: Haemodynamically mediated glomerular injury: The end of a 15-year-old controversy? Curr Opin Nephrol Hypertens 5:468– 475, 1996 4. Nath K, Croatt AJ, Hostetter TH: Oxygen consumption and oxidant stress in surviving nephrons. Am J Physiol 258(Renal Fluid Electrolyte Physiol 27):F1354–F1362, 1990 5. Olson JL, Hostetter TH, Rennke HG, Brenner BM, Venkatachalam MA: Altered glomerular permselectivity and progressive sclerosis following extreme ablation of renal mass. Kidney Int 22:112–126, 1982 6. Brenner BM, Lawler EV, Mackenzie HS: The hyperfiltration theory: A paradigm shift in nephrology. Kidney Int 49:1774–1777, 1996 7. Pukerson M, Hoffsten PE, Klahr S: Pathogenesis of the glomerulopathy associated with renal infarction in rats. Kidney Int 9:407– 417, 1976 8. Zoja C, Perico N, Bergamelli A, Pasini M, Morigi M, Dadan J, Belloni A, Bertani T, Remuzzi G: Ticlopidine prevents renal disease progression in rats with reduced renal mass. Kidney Int 37:934–942, 1990 9. Shimizu T, Fukagawa M, Kuroda T, Hata S, Iwasaki Y, Nemoto M, Shirai K, Yamauchi S, Margolin S, Shmizu F, Kurokawa K: Pirfenidone prevents collagen accumulation in the remnant kidney in rats with partial nephrectomy. Kidney Int 52:S239–S243, 1997 10. Schiller B, Moran J: Focal glomerulosclerosis in the remnant kidney model: An inflammatory disease mediated by cytokines. Nephrol Dial Transplant 12:430–437, 1997 11. Wu LL, Yang N, Roe CJ, Cooper ME, Gilbert CR, Atkins RC, Lan HY: Macrophage and myofibroblast proliferation in remnant kidney: Role angiotensin II. Kidney Int 52:S221–S225, 1997 12. Floege J, Burns MW, Alpers CE, Yoshimura A, Pritzl P, Gordon K, Seifert RA, Bowen-Pope DF, Couser WG, Johnson RJ: Glomerular cell proliferation and PDGF expression precede glomerulosclerosis in the remnant kidney model. Kidney Int 41:297– 309, 1992 13. Kliem V, Johnson RJ, Alpers CE, Yoshimura A, Couser WG, Koch KM, Floege J: Mechanisms involved in the pathogenesis of tubulointerstitial fibrosis in 5/6-nephrectomized rats. Kidney Int 49:666–678, 1996 14. Allison AC, Eugui EM: Mycophenolate mofetil a rationally designed immunosuppressive drug. Clin Transplant 7:96–112, 1993 15. Sollinger HW: Mycophenolate mofetil. Kidney Int 48(Suppl 52):S14–S17, 1995 16. Hauser IA, Johnson DR, Thevenod F, Goppelt-Strube M: Effect of mycophenolic acid on TNF alpha-induced expression of cell adhesion molecules in human venous endothelial cells in vitro. Br J Pharmacol 122:1315–1322, 1997 17. European Mycophenolate Mofetil Cooperative Study Group: Placebo-controlled study of mycophenolate mofetil combined with cyclosporin and corticosteroids for prevention of acute rejection. Lancet 345:1321–1325, 1995 18. Schiff MH, Gloldblum R, Rees MMC: 2-Morpholinoethyl mycophenolic acid (ME-MPA) in the treatment of refractory rheumatoid arthritis (RA). (abstract) Arthritis Rheum 33:s155, 1990 19. Ohkawa H, Ohishi N, Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 95:351–358, 1979 20. Parra G, Romero M, Henri´quez-La Roche C, Pineda R, Rodri´guez-Iturbe B: Expression of adhesion molecules in poststreptococcal glomerulonephritis. Nephrol Dial Transplant 9:1412–1417, 1994 21. Parra G, Moreno P, Rodri´guez-Iturbe B: Glomerular proliferative activity and T lymphocyte infiltration in acute serum sickness. Clin Immunol Immunopathol 82:299–302, 1997 22. Weibel ER: Stereological Methods: Practical Methods for Biologi-
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31. Heeman U, Azuma H, Schmid C, Philipp T, Tilney N: Effect of mycophenolic acid mofetil on acute rejection of kidney allograft in rats. Clin Nephrol 5:355–359, 1996 32. Laurent AF, Dumont S, Poindron P, Muller CD: Mycophenolic acid suppresses protein N-linked glycosilation in human monocytes and their adhesion to endothelial cells and to some substrates. (abstract) Exp Hematol 24:59–67, 1996 33. Nadeau KC, Azuma H, Tilney NL: Sequential cytokine expression in renal allografts in rats immunosuppressed with maintenance cyclosporine or mycophenolate mofetil. Transplantation 62:1362– 1366, 1966 34. Schena FP, Gesualdo L, Grandaliano G, Montinaro V: Progression of renal damage in human glomerulonephritides: Is there sleight of hand in winning the game? Kidney Int 52:1439–1457, 1997 35. Briggs WA, Choi MJ, Scheel PJ Jr: Successful mycophenolate mofetil treatment of glomerular disease. Am J Kidney Dis 31:213– 217, 1998 36. Schlo¨ndorff D: The role of chemokines in the initiation and progression of renal disease. Kidney Int 47(Suppl):S44–S47, 1995 37. Roberts WA, Noble NA: Transforming growth factor-b in tissue fibrosis. N Engl J Med 331:1286–1292, 1994 38. Sugiyama H, Kashihara N, Makino H, Yamasaki I, Ota Z: Apoptosis in glomerular sclerosis. Kidney Int 49:103–111, 1996 39. Batlle DC, Gutterman C, Keilani T, Peces R, Lapointe M: Effect of cyclosporine A on renal function and kidney growth in the uninephrectomized rat. Kidney Int 37:21–28, 1990 40. Romero F, Parra G, Rodri´guez-Iturbe B, Gonza´lez L: Mycophenolate mofetil (MMF) prevents the renal damage induced by 5/6 nephrectomy in the rat. (abstract) J Am Soc Nephrol 8:628A, 1997 41. Fujihara CK, Malheiros DMAC, Oliveira SG, Antunes GR, Zatz R, Noronha I: Mycophenolate mofetil attenuates renal injury in the remnant kidney. (abstract) J Am Soc Nephrol 8:615A, 1997
Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats FREDDY ROMERO, BERNARDO RODRI´GUEZ-ITURBE, GUSTAVO PARRA, LUISANDRA GONZA´LEZ, JAIME HERRERA-ACOSTA, and EDILIA TAPIA Renal Service and Laboratory, Hospital Universitario and Instituto de Investigaciones Biome´dicas, Maracaibo, Venezuela, and the Department of Nephrology and Renal Laboratory, Instituto Nacional de Cardiologı´a Ignacio Chavez, Me´xico City, Me´xico
tration with CD43- and ED1-positive cells in glomeruli and interstitium was two to five times lower in MMF-treated rats (P , 0.01). Expression of adhesion molecules CD18 and CD11b was similarly reduced. Conclusion. MMF ameliorates the progressive renal damage in the remnant kidney after 5/6Nx. This effect is associated with a reduction in the infiltration of lymphocytes and monocytes, whereas glomerular hypertrophy and systemic hypertension are unchanged.
Mycophenolate mofetil prevents the progressive renal failure induced by 5/6 renal ablation in rats. Background. Extensive renal ablation is associated with progressive sclerosis of the remnant kidney. Because lymphocytes and monocytes accumulate in the remnant kidney, it is likely that they play a role in the renal scarring. Therefore, we treated rats with 5/6 nephrectomy (5/6Nx) with mycophenolate mofetil (MMF), a drug that has an antiproliferative effect and that suppresses the expression of intercellular adhesion molecules. Methods. Sprague-Dawley rats with 5/6Nx received MMF (30 mg · kg21 · day21 by daily gastric gavage, N 5 15) or vehicle (N 5 16). Ten additional rats were sham operated. All rats were fed a 30% protein diet. Body weight, serum creatinine, and urinary protein excretion were determined weekly. Lipid peroxidation, as a measure of oxidative stress observed by urinary malondialdehyde determinations, was performed every two weeks. Histologic studies were done in the remnant kidney four weeks (9 rats from the vehicle-treated group, 7 rats from the MMF group, and 5 sham-operated rats) and eight weeks after surgery (the remaining rats). Glomerular volume, sclerosis in glomeruli (segmental and global) and interstitium (semiquantitative scale), infiltrating lymphocytes and macrophages (CD43- and ED1-positive cells), and expression of adhesion molecules (CD54, CD18, and CD11b) were analyzed. Results. MMF treatment prevented the progressive increment in serum creatinine and the proteinuria observed in the 5/6 nephrectomized rats during the eight weeks of observation (P , 0.01). Weight gain was comparable in the MMF-treated and sham-operated rats, whereas weight gain was decreased in untreated 5/6 nephrectomized rats. Excretion of malondialdehyde increased after surgery but returned sooner to control levels in the MMF-treated rats. Increments in glomerular size and mean arterial blood pressure induced by renal ablation were not modified by MMF treatment. Eight weeks after surgery, segmental sclerosis was present in 48.4 6 8.35% (6 sd) glomeruli in the vehicle-treated group versus 25 6 10.5% in the MMF-treated group (P , 0.001). Interstitial fibrosis was reduced significantly with MMF treatment (P , 0.001). Infil-
The progression of renal damage resulting from reduced nephron mass has been extensively studied in the 5/6Nx model in the rat, as described originally by Shimamura and Morrison [1]. Classic studies by Hostetter et al demonstrated that this experimental model was characterized by progressive glomerulosclerosis and chronic renal failure [2]. The remnant nephron units developed glomerular hypertension, increased single nephron glomerular filtration rate, and glomerular hypertrophy, and over the previous 15 years, all of these factors have been postulated to play a pathogenetic role in the progressive scarring of the kidney [3]. Surviving nephrons have increased oxygen consumption and are subjected to oxidant stress [4]. Because the initial event in the subtotal renal ablation model is an increase in glomerular pressure, treatment strategies have usually been directed to correct this hemodynamic disturbance, specifically, to reduce intraglomerular pressure. Correction of this pathophysiologic event and normalization of permselectivity properties of the glomerular capillary barrier [5] are major mechanisms underlying the protective effect on renal function observed with angiotensin-converting enzyme inhibition and low-protein diet in several models of renal disease in the experimental animal and in humans [6]. Less commonly, treatments have been directed to modify late events in the development of glomerulosclerosis, that is, events deriving from the hemodynamic and hypertrophic glomerular changes. For example, heparin
Key words: sclerosis, remnant kidney, scarring, adhesion molecules, proteinuria, interstitial fibrosis. Received for publication July 20, 1998 and in revised form October 19, 1998 Accepted for publication October 20, 1998
1999 by the International Society of Nephrology
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treatment, which prevents microthrombosis [7], as well as Ticlopidine, a drug with a wide spectrum of inhibitory actions on platelets and platelet–cell interactions, retards the progression of renal failure in the remnant kidney model [8]. More recently, Shimizu et al have reported that administration of Pirfenidone, a compound that prevents and reverses extracellular matrix accumulation, ameliorates the histologic damage in the remnant kidney of the rat with 5/6Nx [9]. Largely undefined is the role played by infiltrating lymphocytes and macrophages in the development of glomerulosclerosis triggered by a reduction of nephron mass. There is limited information on lymphocyte accumulation in this model. However, monocyte/macrophage infiltration occurs early [10, 11], and their numbers increase fourfold to fivefold in the glomeruli and tubulointerstitial areas of the remnant kidney [12, 13]. Therefore, it is likely that these cells play a significant role in the subsequent development of fibrosis. In fact, cytokines produced by infiltrating cells appear to mediate renal scarring in this experimental model [10]. Mycophenolate mofetil (MMF) is a new immunosuppressant agent in which the active component, mycophenolic acid, is an inhibitor of inosine 59-monophosphate dehydrogenase, which controls the synthesis of guanosine triphosphate [14, 15]. MMF depletes guanosine triphosphate pools in lymphocytes and monocytes and suppresses the de novo synthesis of purines, thereby exerting a selective and reversible antiproliferative activity on these cells. In addition, the expression of intercellular adhesion molecules, which is critical for the migration of leukocytes from circulation and their subsequent accumulation in the tissues, is suppressed by this drug [16]. Therefore, we decided to investigate whether treatment with MMF would reduce the renal cellular infiltration and the expression of adhesion molecules, as well as the oxidant-induced lipid peroxidation observed after 5/6Nx, and whether these modifications would be associated with improvement in the progression of renal disease. We found that deterioration of renal function and proteinuria, which is characteristic of this model, was prevented and that the hallmark histologic changes were markedly improved. Because the hematologic, liver, and renal toxicity of MMF are relatively minor [17, 18] and this drug is used with relative safety in the long-term treatment of renal transplantation, MMF could be potentially useful to retard the progressive damage associated with reduced nephron mass in humans. METHODS Design of the study and experimental groups Studies were done in 50 male Sprague-Dawley rats weighing 200 to 280 g at the start of the experiments. Throughout the study, all rats were fed ad libitum with
rat chow containing 30% protein (Purina, St. Louis, MO, USA) and tap water. After obtaining blood samples for serum creatinine and 24-hour urine for determination of proteinuria and urinary malondialdehyde excretion, 40 rats had 5/6Nx (mentioned later here), and 10 rats had a sham operation. Nine rats in the group that had 5/6Nx died during surgery or in the following 48 hours. Five days after surgery, the surviving rats in the 5/6Nx group were randomly subdivided in two groups, one of which received MMF (5/6NxMMF group, N 5 15) the other one received vehicle (5/6Nx, N 5 16). Four of these rats died in the subsequent four weeks (two from the 5/6NxMMF group and two from the 5/6Nx group). Histologic studies were done four and eight weeks after surgery. At the four-week endpoint, nine rats from the 5/6Nx group, seven rats from the 5/6NxMMF group, and five sham-operated rats were sacrificed. The remaining rats (5 rats from the shamoperated group, 5 rats from the 5/6Nx group, and 6 rats from the 5/6NxMMF group) were sacrificed eight weeks after surgery. Body weight, serum creatinine, and urinary protein excretion were determined weekly. Urinary malondialdehyde (MDA) determinations were done every two weeks. Histologic and immunohistologic studies were done in remnant kidneys and in sham-operated animals at the time of sacrifice. Dose of mycophenolate mofetil Preliminary studies were done to determine the maximal tolerated dose of MMF. Doses higher than 30 mg · kg21 · day21 were usually associated with diarrhea; consequently, this dose was chosen. Daily gastric gavage was used to administer 30 mg · kg21 · day21 of MMF diluted in 200 ml of water to the rats of the 5/6NxMMF group. Because mycophenolate (CellCept; Roche Pharmaceutical Co., Caracas, Venezuela) is insoluble in water, we prepared each dose individually and agitated the mixture vigorously to obtain a suspension, and then the suspension was immediately given to the rat before the MMF precipitated. Rats in the 5/6Nx group and sham groups received the same volume of water by the same route of administration daily. Surgical procedures Subtotal renal ablation was done in a single surgical procedure by subcapsular removal of the right kidney and selective infarction of two thirds of the left kidney by ligation of the posterior and one or two of the anterior extrarenal branches of the renal artery, to leave approximately one sixth of the total kidney tissue mass. Sham operations consisted in laparotomy and manipulation of the kidneys and renal pedicle without destruction of renal tissue.
Romero et al: Mycophenolate mofetil therapy in renal ablation
Determination of lipid peroxidation We used the method of Ohkawa et al to determine thiobarbituric acid reactive substances, an index of lipid peroxidation [19]. MDA was measured in 24-hour urine centrifuged at 2000 r.p.m. and processed the same day. Four hundred microliters of the sample were added to a reaction mixture consisting of 200 ml of 8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid (pH 3.5), 1.5 ml of 0.5% thiobarbituric acid, and 400 ml of bidistilled water. This mixture was heated at 958C in water bath for 60 minutes. After removal from the water bath, 1 ml of water and 5 ml N-butanol-pyridine were added, and the mixture was agitated and centrifuged at 2000 g for 15 minutes. The upper organic layer was pipetted, and the absorbance of this fraction was read at 532 nm in a Shimatzu model UV21100 S spectrophotometer (Kyoto, Japan). Malondialdehyde bis-dimethyl acetal was used as external standard. Results were expressed as nmol of MDA excreted in 24 hours. Laboratory determinations Proteinuria was determined in 24-hour urine collections by the sulfosalicylic acid method and the serum creatinine by modification of the Jaffe´ reaction in autoanalyzer methodology. Tissue preparation From every animal, coronal sections of remnant renal tissue were obtained at the time of sacrifice. One fragment was fixed in 15% formalin, and 10 were embedded in Paraplast Plus (Monoject, Sherwood Medical Scientific Division, St. Louis, MO, USA); the rest were included in Tris buffered saline (TBS) freezing medium (Triangle Biomedical Sciences, Durham, NC, USA) and were immediately frozen in dry ice and acetone and were stored at 2708C. Light microscopy studies Paraffin-embedded sections were stained with hematoxylin eosin, Masson’s Gomori’s stain, and periodic acid-Schiff reagent (PAS). Coronal sections cut at 4 mm thickness, including superficial and juxtamedullary glomeruli, were evaluated. Evaluation of histologic damage. Sections were examined by two investigators without previous knowledge of the experimental group of the animal from which the tissue was taken. At least 10 sections were analyzed for each kidney, and the evaluation that was assigned represented the mean values of these sections. Agreement between the two observers was within 10%. Evaluation was done as follows. Glomerular sclerosis. Glomerular lesions were evaluated by determining the number of normal glomeruli and the number with either segmental or total glomerular
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sclerosis in superficial and juxtamedullary sections. Glomerulosclerosis was evaluated in PAS-stained tissue and was defined as the increment of PAS-positive material, with loss of cellular elements, collapse of capillary lumina, and entrapment of amorphous hyaline material associated or not with adhesions to Bowman’s capsule. Sclerosis involving more than 80% of the glomerular tuft was considered global, and sclerosis involving less than 80% was considered segmental. No attempt was made to subclassify the segmental lesions in accordance with the percentage involvement of the glomerulus. At least 100 glomeruli were evaluated per biopsy. Results are expressed as the percentage glomeruli examined (more than 100 per biopsy) showing segmental or global sclerosis. Tubulointerstitial lesions. Severity of tubular dilation and atrophy, as well as tubulointerstitial infiltration (hematoxylin and eosin staining) and fibrosis (trichrome staining), were evaluated individually, determining the percentage in the biopsy section affected by the corresponding finding with the help of a grid-fitted eyepiece. Gradation of damage (0 to 51) was done by modifying the scale used by Zoja et al as follows: 0 5 changes in less than 10% of the histologic section; 11 5 changes in up 20% of the section; 21 5 changes in up to 40% of the section; 31 5 changes in up to 60% of the section; 41 5 changes in up to 80% of the section; 51 5 changes in more than 80% of the section [8]. The average score obtained in the sections of each biopsy (10 to 15 sections per biopsy) was used as the score of the biopsy. Immunofluorescence studies Identification of intercellular adhesion molecules was done in frozen 3 mm sections by indirect immunofluorescence technique, as described in previous publications [20, 21] using the monoclonal antibodies listed later. As secondary antibody we used fluorescein-conjugated affinity-pure F(ab9)2 rat antimouse IgG with minimal cross reactivity with rat proteins (Accurate Chemical & Scientific Corp., Westbury, CT, USA) at a concentration of 60 mg/ml (1:50). Ethidium bromide was used for nuclear staining. The following monoclonal antibodies were used: antiCD43 (Accurate Chemical & Scientific Corp., Westbury, NY, USA; mouse IgG1 clone W3/13, distribution in T lymphocytes, concentration 5 5 mg/ml); anti-ED1 (Accurate Chemical & Scientific; mouse IgG1, distribution in monocytes, concentration 5 30 mg/ml); anti-ICAM1 (CD54; Seikegaku Corp. Tokyo, Japan; IgG1, distribution in endothelial cells, monocytes, T and B lymphocytes, epithelial cells, dendritic cells, mesangial cells; concentration 5 10 mg/ml); anti-CD11b (Biosource International, Camarillo, CA, USA; mouse IgG2 A, distribution in monocytes/macrophages, neutrophils; concentration 5 5 mg/ml) and anti-LFA1 (CD18; Seikegaku Corp.; IgG1,
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distribution in lymphocytes, monocytes, neutrophils; concentration 5 10 mg/ml). Double-staining procedures were done as follows: The monoclonal antibody (mAb) used to identify the integrins (mAb anti-CD18 or anti-CD11b) was applied to the tissue (concentrations listed earlier here) and was incubated in a humid chamber at room temperature for 45 minutes. Afterward, tetramethylrhodamine isothiocyanate (TRIC)-conjugated, affinity-pure F(ab9)2 rat antimouse IgG, with minimal cross-reaction with rat proteins (Accurate Chemical & Scientific) was added. After washing for 10 minutes with PBS 0.01 m, 0.15 m NaCl (one change of PBS) and 100 ml of blocking solution (PBS 0.01 m, 0.15 m NaCl containing 10% fetal calf serum and 10% goat serum) were added, incubated for 10 minutes, and washed as described earlier here. Afterward, the tissues were incubated with the mAb used to identify the leukocyte populations (mAb anti-CD43 or mAb antiED1 at the dilutions described earlier) and were then reacted with 50 ml of fluorescein-conjugated, affinitypure F(ab9)2 rat antimouse IgG (20 mg/ml) with minimal cross-reaction with rat proteins (Accurate Chemical & Scientific). As a control for the double-staining procedure, five tissues were double stained with CD18 and ED1, as described earlier here. Tissues were examined with a Zeiss microscope with epifluorescence system, phase contrast, and appropriate filters. Cells with red nuclei surrounded by a green cytoplasmatic membrane were counted as positive. Counting was done with the help of a grid-fitted eyepiece. Within the glomerular tuft, staining results were expressed as positive cells per glomerular cross-section, with the exception of CD54 staining that was expressed as a 11 to 41 score, described previously [17], which takes into account the number of positive cells and the intensity of the fluorescent staining. In the interstitial areas, results were expressed as positive cells per mm2. Glomerular volume At least 100 glomeruli in each kidney tissue were examined, and the glomerular diameter was measured using a microscopic scale attached to the ocular lens. Then, the mean random cross-sectional glomerular area (AG) was calculated, and the glomerular volume (VG) was determined using the following formula: VG 5 (b/k 3 AG)3/2 where b 5 1.38 is the shape coefficient for spheres, the idealized shape of the glomerular tuft, and k 5 1.1 is the size distribution coefficient [22]. Statistical analysis Results are expressed as mean 6 sd. Comparisons between groups were done by one-way analysis of variance followed by Tukey-Kramer post-tests. When differences in the standard deviation of the groups under analysis were significant, nonparametric Mann–Whitney rank
Fig. 1. Serum creatinine concentration in sham-operated animals (h) and rats with reduction in renal mass, untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). Data from five to eight weeks correspond to five animals in each group; otherwise, the values represent 10 to 16 animals in each group (Methods section, experimental design). Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.05, **P , 0.01, ***P , 0.001 vs. sham. Bars are standard deviation. The differences between the 5/6Nx group and the MMF treated group are also significant (P , 0.001) after the fourth week.
test or Kruskal Wallis tests with Dunn’s multiple comparison post-test were used. Differences were considered significant when P was less than 0.05. RESULTS Figure 1 shows the serial changes in serum creatinine concentration observed in the sham-operated and in the rats with 5/6 reduction in renal mass with (5/6NxMMF) and without (5/6Nx) treatment with MMF. The untreated 5/6Nx group of rats experienced a progressive rise in serum creatinine (P , 0.01 vs. baseline) from the second week up to the seventh to eighth week after nephrectomy when it appeared to reach a plateau, at levels of 156 6 6.61 mmol/liter, approximately 2.4 times above control levels. The levels of serum creatinine the 5/6NxMMF group and in the sham-operated group were lower than in the untreated 5/6Nx rats (P , 0.001 in Fig. 1). In contrast, the 5/6NxMMF group had essentially similar serum creatinine levels than the sham-operated group the first four weeks after surgery. Thereafter, the level of the creatinine in 5/6NxMMF group was slightly above the levels in the sham-operated group but was steady at each time interval (Fig. 1). Urinary protein excretion is shown in Figure 2. The animals receiving MMF (5/6NxMMF) and the shamoperated group showed essentially similar levels of proteinuria throughout the eight-week observation period. Urinary protein excretion in these groups (5/6NxMMF group and sham-operated group) is unchanged from the baseline levels. In contrast, the untreated animals with reduced renal mass (5/6Nx group) showed a progressive increment in proteinuria.
Romero et al: Mycophenolate mofetil therapy in renal ablation
Fig. 2. Urinary protein excretion in sham-operated animals (h) and rats with reduction of renal mass untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). Data from five to eight weeks correspond to five animals in each group; otherwise, the values represent 10 to 16 animals in each group (Methods section, experimental design). Bars are standard deviation. Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.01 and **P , 0.001 vs. sham and MMF group.
All animal groups had similar weights (g) at the beginning of the experiment (5/6Nx group 5 255 6 11.42, 5/6NxMMF group 5 236.9 6 10.09, sham-operated group 5 255 6 5.00) and gained weight during the period of observation. However, the 5/6NxMMF group and the sham-operated groups gained weight similarly at four weeks (5/6NxMMF group 5 320 6 6.70, sham-operated group 5 315 6 4.30) and at eight weeks (5/6Nx,MMF group 5 409 6 2.45, sham-operated group 5 411.3 6 2.40); in contrast, the 5/6Nx group gained less weight at four weeks and eight weeks (286.1 6 9.44 and 338 6 8.00, respectively, P , 0.01). Mean arterial blood pressure at the time of sacrifice (4 weeks after surgery) was 121.1 6 10.33 in the shamoperated rats, 165.5 6 16.5 in the 5/6Nx group (P , 0.001 vs. sham), and 158.3 6 15.4 in the 5/6NxMMF group (P , 0.01 vs. sham). Figure 3 shows the urinary excretion of MDA. Both groups of nephrectomized rats (5/6Nx group and 5/6NxMMF group) had their urinary excretion of MDA doubled after surgery; however, the group treated with MMF showed a progressive decrease in urinary MDA starting three to four weeks after surgery, reaching control levels during the fifth to sixth week. In contrast, the untreated animals (5/6Nx group) increased the MDA excretion nine times, peaking at the fourth week and decreasing thereafter (Fig. 3). The histologic observations are shown in Table 1. The glomerular volume is higher in the two groups of rats with reduced renal mass. There were no significant differences in the glomerular hypertrophy in the 5/6Nx group and the 5/6NxMMF group. Glomerular sclerosis, both segmental and global, was present in a small percentage
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Fig. 3. Urinary malondialdehyde (MDA) excretion in sham-operated animals (h) and rats with reduction of renal mass untreated (5/6Nx, j) and treated with mycophenolate mofetil (5/6NxMMF, d). From five to eight weeks, all groups consisted of five animals each; otherwise, all values represent at least 10 animals in each group (Methods section, experimental design). Bars are standard deviation. Abbreviations are: B, baseline; weeks, weeks after surgery. *P , 0.001 vs. sham; **P , 0.01 vs. sham. The difference between the 5/6Nx group and the 5/6NxMMF group at three to four weeks and at five to six weeks is also significant (P , 0.001).
of the glomeruli in sham-operated animals and increased 13 times at four weeks and 17 times at eight weeks after 5/6Nx in the untreated group (5/6Nx group). Eight weeks after renal ablation, segmental glomerulosclerosis in rats treated with MMF (5/6NxMMF group) was 25 6 10.5%, half of the value in untreated rats at the same period of time (P , 0.001). Tubular dilation and atrophy, as well as tubulointerstitial infiltration and fibrosis, were substantially reduced in the 5/6NxMMF group. In contrast, the untreated nephrectomized animals (5/6Nx group) had increasing severity of tubular and interstitial damage, involving 40% to 80% of the examined remnant renal tissue after eight weeks (Table 1). Figure 4A shows typical sclerotic changes in glomeruli and interstitium in untreated animals, in contrast with animals treated with MMF (Fig. 4B). Cells expressing adhesion molecules (CD54, CD18, and CD11b) and CD43- and ED1-positive cells in the glomeruli and in tubulointerstitial areas are shown in Tables 2 and 3, respectively, as well as in Figure 4 C–F, and Figure 5. In biopsies examined four and eight weeks after surgery, the number of lymphocytes (CD43-positive cells) and macrophages (ED1-positive cells) in the glomeruli (Table 2) and interstitium (Table 3) was not statistically different in sham-operated rats and in rats treated with MMF (5/6NxMMF group). In contrast, the untreated 5/6Nx group showed values three to four times higher (Table 2). Four weeks after surgery, the untreated 5/6Nx group
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Romero et al: Mycophenolate mofetil therapy in renal ablation Table 1. Histological observations 4 weeks after surgery
VG lm3 3 10 6 Glomerular sclerosis Segmental % Global % Tubular dilation & atrophy (0–5) Interstitial infiltration (0–5) Interstitial fibrosis (0–5)
8 weeks after surgery
Sham (N 5 5)
5/6 Nx (N 5 9)
5/6 Nx 1 MMF (N 5 7)
Sham (N 5 5)
5/6 Nx (N 5 5)
5/6 Nx 1 MFF (N 5 5)
1.61 6 0.10
2.23 6 0.43
2.27 6 0.34
1.81 6 0.42
2.61 6 0.21
2.58 6 0.90
3.0 6 0.71 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00
38.7 6 4.33 7.57 6 3.65 2.89 6 0.78 1.56 6 0.73 1.67 6 0.50
21.7 6 9.41a 4.29 6 6.45 0.71 6 0.76a 1.0 6 0.89 0.42 6 0.54a
2.8 6 0.83 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00 0.0 6 0.00
48.4 6 8.35 20.6 6 15.27 4.2 6 0.45 3.8 6 1.48 2.0 6 0.71
25.0 6 10.51a 6.20 6 7.95b 0.8 6 0.84a 1.2 6 2.16 0.4 6 0.89b
Segmental and global glomerular sclerosis expressed as the percent of the glomeruli examined presenting the corresponding lesion. Tubulointerstitial damage was graded according to the percent of the biopsy affected by the corresponding finding (see Methods section). N corresponds to the number of renal biopsies in each group; the value in each biopsy is the mean of 10-15 sections. Statistical significances between 5/6NxMMF vs. 5/6Nx are: a P , 0.001, b P , 0.05. The percentage glomeruli showing global sclerosis in the 5/6Nx group is higher at 8 weeks than at 4 weeks (P , 0.001). Data in untreated 5/6Nx are different (P ranging from ,0.05 to ,0.001) from data in sham operated rats.
had increased expression of adhesion molecules CD45, CD18, and CD11b (Tables 2 and 3). Eight weeks after surgery, the expression of CD54 in the untreated 5/6Nx group had decreased, whereas in contrast, CD11b had more than doubled in tubulointerstitial areas (Table 3). Sham-operated animals and 5/6Nx rats treated with MMF had essentially similar expression of CD54, CD18, and CD11b at four weeks (Table 2). At eight weeks, the only significant difference between the sham-operated and 5/6Nx group was a reduction in the interstitial infiltration of cells expressing CD54 in the MMF-treated group (Table 3). DISCUSSION The 5/6Nx model in the rat has been widely used to evaluate the effect of treatment strategies on the progressive scarring of the kidney that is associated with extensive renal ablation. Because the initial effects of the reduction of nephron mass involves hypertension and hyperfiltration of the remnant nephron units, a vast majority of research efforts have been directed to determine if the correction of the altered hemodynamics improves the renal damage. Understandably, drugs that lower systemic and glomerular pressure are among the most prominently studied [23–26]. Other treatment strategies have been investigated less frequently. Pukerson et al showed that thrombotic mechanisms are important in the progression of the glomerular lesion and that heparin had a beneficial effect [7]. Suppression of platelet adhesion and platelet-derived factors was a treatment modality chosen by Zoja et al, who found that Ticlopidine, a drug with a wide spectrum of antiplatelet effects, improved the progressive renal damage of the remnant kidney [8]. Interestingly, the beneficial effects of this treatment occur without modification of the systemic blood pressure [8], which also appears to be the case in the improvement induced with MMF reported here, as the blood pressure levels in the
5/6Nx and the 5/6NxMMF groups were similarly elevated with respect to the sham-operated group. The doses of MMF used in this work are not expected to have a significant antiplatelet effect or to decrease the numbers of circulating leukocytes, but this possibility was not ruled out in this study. Other recent studies addressing the final common pathway by which hemodynamic alterations induce renal scarring are those of Shimizu et al, who used Pirfenidone, a drug that prevents and reverses collagen accumulation [9]. They found attenuation of the chronic renal failure in the rats with 5/6Nx [9]. Suppression of lymphocyte activity and monocyte/ macrophage infiltration is yet another strategy of potential use for amelioration of the progressive renal damage resulting from renal ablation. Macrophages are known to participate in the inflammatory reaction, producing a number of soluble mediators, including cytokines, proteases, and free oxygen radicals [27], and several studies have shown that macrophage infiltration and proliferation are prominent findings in the remnant kidney [11– 13, 28]. Furthermore, Schiller et al have recently demonstrated that mechanical injury resulting from glomerular capillary hypertension induces up-regulation of monocyte chemoattractant protein-1 (MCP-1) [10]. Interestingly, a low-protein diet modulates the expression of MCP-1 [10], which suggests that protein restriction, in addition to improving glomerular hemodynamics in the remnant nephrons, could have beneficial effects resulting from inhibition of macrophage infiltration. Mycophenolate mofetil is the morpholinoethytl ester of mycophenolic acid, and it inhibits inosine 59-monophosphate dehydrogenase, which controls the de novo synthesis of guanosine triphosphate [29]. This drug has a relatively specific effect on lymphocytes because other cells have alternative purine synthesis pathways not blocked by MMF. In the experimental setting of experimental renal ablation, MMF is a drug well suited for the
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Fig. 4. Changes observed by light microscopy and lymphocyte and macrophage infiltration of remnant kidneys four weeks after 5/6Nx. Severe segmental sclerosis (approximately 60% of the glomerular tuft) observed in untreated rat (receiving vehicle; A) is contrasted with essentially normal glomerular appearance in rat treated with MMF (B). In addition, interstitial fibrosis is evident in untreated rat and absent in the biopsy from the rat treated with MMF (Mason’s Trichromic stain, original magnification &time;400). Immonuhistology showing remnant kidney tissue stained with mAb anti-CD43 (C, untreated rat; D, rat treated with MMF) and with mAb anti-ED1 (E, untreated rat; F, rat treated with MMF). Original magnification was 3630.
purpose of testing the effects of suppressing macrophage infiltration and proliferation for several reasons. First, MMF depletes the pools of guanosine triphosphate not only in lymphocytes but also in monocytes and thereby decreases the synthesis of fucose and mannose of mem-
brane glycoproteins, which are ligands of the selectin adhesion molecules in the vascular endothelium [29]; in addition, MMF inhibits up-regulation of CD18 in lymphocytes [30]. Consequently, the infiltration of monocytes, as well as lymphocytes, is inhibited by this drug
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Romero et al: Mycophenolate mofetil therapy in renal ablation Table 2. Cellular infiltration in the glomeruli 4 weeks
Positive cells per gcs CD43 ED1 CD54 CD18 CD11b
8 weeks
Sham (N 5 5)
5/6Nx (N 5 9)
5/6NxMFF (N 5 7)
Sham (N 5 5)
5/6Nx (N 5 5)
5/6NxMFF (N 5 5)
1.14 6 0.27 0.74 6 0.11 1.27 6 0.13 0.98 6 0.27 1.04 6 0.60
3.72 6 1.30 3.14 6 0.48 2.21 6 0.79 4.44 6 1.45 4.75 6 2.18
1.46 6 0.94b 0.97 6 0.27b 0.86 6 0.18a 1.94 6 1.02a 1.30 6 0.22a
0.46 6 0.31 0.86 6 0.27 0.85 6 0.12 1.21 6 0.14 1.07 6 0.70
3.47 6 0.54 2.28 6 0.37 0.54 6 0.11 1.30 6 0.25 3.50 6 0.94
1.15 6 0.56a 1.26 6 0.44b 1.08 6 0.08a 0.80 6 0.24b 0.83 6 0.16a
N corresponds to the number of biopsies studied; the value in each biopsy represents the mean of 10-15 sections. Statistical significances between 5/6Nx MMF and 5/6 Nx are: a P , 0.001, b P , 0.01. In the 5/6Nx group, the differences between the 4th and 8th week in CD54 and CD18 are significant (P , 0.01). The data of untreated 5/6Nx are significantly different (P ranging from ,0.05 to ,0.001) from the data in sham operated rats.
Table 3. Cellular infiltration in the interstitium 4 weeks Positive cells per mm2 CD43 ED1 CD54 CD18 CD11b
Sham (N 5 5) 68.4 6 56.5 35.2 6 7.6 252.0 6 27.5 25.4 6 10.8 32.0 6 28.4
8 weeks
5/6Nx (N 5 9)
5/6NxMFF (N 5 7)
Sham (N 5 5)
5/6Nx (N 5 5)
5/6NxMFF (N 5 5)
135.6 6 64.2 194.4 6 61.2 531.6 6 216 229.6 6 132 121.2 6 43.1
59.2 6 39.3c 34.0 6 9.6a 314.4 6 124c 72.6 6 31.2b 25.2 6 11.9
36.8 6 17.3 56.0 6 11.6 261.6 6 44.5 41.6 6 12.5 21.6 6 17.8
431.2 6 59.2 140.0 6 22.8 128.8 6 15.3 74.4 6 19.9 352.8 6 105
91.7 6 59.9a 75.6 6 4.91a 73.6 6 42.6c 75.0 6 27.9 36.1 6 21.8a
N corresponds to the number of biopsies studied; the value in each biopsy represents the mean of 10-15 sections. Statistical differences between 5/6Nx MMF and 5/6Nx are: a P , 0.001, b P , 0.01, c P , 0.05. In the 5/6Nx group, the differences between the 4th and 8th week in CD54 and CD18 are significant (P , 0.01). The data in the untreated 5/6Nx group are significantly different (p ranging from ,0.05 to ,0.001) from the data in the sham operating group, except for CD18 positive cells at 8 weeks.
[30–32]. Second, MMF exerts a long-term inhibition of the production of lymphocyte and macrophage-derived cytokines [33], a valuable characteristic in the prevention of chronic scarring in the remnant kidney [34]. Third, MMF, in contrast to other antirejection drugs, is not associated with renal toxicity [15] and is reasonably well tolerated as a long-term treatment [17, 18]. Treatment with MMF completely prevented the proteinuria induced by renal ablation. This protective effect is maintained for at least eight weeks (Fig. 2). To our knowledge, there is no direct effect of MMF on glomerular permselectivity. Reduction of proteinuria, noted in anecdotal reports of several kidney diseases treated with this drug [35], is presumed to be a consequence of its immunosuppressant properties, reduction of cellular infiltration, and decrease in cytokine production. Renal function is also markedly improved by MMF treatment. As reported by others, the 5/6Nx group presents a progressive rise in serum creatinine, whereas in contrast, the rats with 5/6 reduction in renal mass treated with MMF showed only a modest increment in serum creatinine that remained at steady levels from the fourth to the eighth week (Fig. 1). The histologic findings are in accordance with the renal function data. The number of glomeruli with segmental and global sclerosis is substantially reduced with MMF treatment. Moreover, the percentage of glomeruli with these changes (Fig. 5 A, B) remains essentially unchanged
after four weeks in the 5/6NxMMF group, whereas in contrast, it increases in the untreated 5/6Nx group (Table 1). Tubulointerstitial changes are ameliorated importantly in the MMF-treated group (P . 0.05 vs. sham; Table 1), in contrast with the changes seen in the 5/6Nx group (Fig. 5 A, B). The observed beneficial effect on renal scarring is likely the result of direct and indirect actions of MMF because cytokine “cross-talk” between macrophages and intrinsic renal cells is central to the cell proliferation and matrix expansion that leads to glomerulosclerosis and interstitial fibrosis [36]. Careful studies by Floege et al and Kliem et al have shown that in the remnant kidney model, proliferation of intrinsic glomerular and tubulointerstitial cells is an early event in the development of renal scarring; macrophage infiltration appears later and contributes to amplify the inflammatory response [12, 13]. To our knowledge, there is no evidence of a direct suppressive effect of MMF on cytokine production by intrinsic renal cells. However, this drug reduces cell adhesion molecules in endothelial cells [16], and there is a well-defined inhibition of the production of all lymphocyte and macrophage-derived cytokines and growth factors. Specifically, platelet-derived growth factor, interleukin 2, tumor necrosis factor a, and transforming growth factor-b are profoundly reduced [33]. Suppression of macrophage-derived platelet-derived growth factor should prevent further activation of the mesangial
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Fig. 5. Intercellular adhesion molecules in remnant kidneys four weeks after 5/6Nx. Intense interstitial infiltration of cells expressing CD18 in kidney from untreated rat (A) is contrasted with the negative appearance of the kidney from a rat treated with MMF (B). Intense glomerular expression of CD54 in untreated 5/6Nx rat (C) contrasts with moderate expression of CD54 in 5/6Nx rats treated with MMF (D).
cells, in addition to limiting amplification of the proliferative reaction of infiltrating cells. Suppression of macrophage-derived transforming growth factor-b should have a beneficial effect on glomerulosclerosis by limiting the synthesis and favoring the degradation of extracellular matrix by renal metalloproteinases [36, 37]. An additional influence that may induce synthesis of chemokynes is the generation of reactive oxygen species. Nath, Croatt and Hostetter have shown that oxidant stress is increased in the 5/6Nx model [4], and our studies, in addition to confirming their findings, have shown that urinary MDA is significantly reduced by MMF treatment from the first to the sixth week after nephrectomy (Fig. 3). This effect of MMF is likely the result of suppression of the inflammatory infiltrate, but by itself, a reduction in reactive oxygen species would reduce the expression of MCP-1, RANTES, and ICAM-1 in the mesangial cells [reviewed in 34]. In addition, a decrease in oxidative stress would eliminate a stimulus for apoptosis, which is an early event in the remnant kidney model [38].
It is interesting that a similar increment in glomerular volume was found in the 5/6Nx and the 5/6NxMMF groups. This finding firstly suggests that the hemodynamic disturbances that trigger glomerular hypertrophy are similar in both groups of rats with renal ablation, and secondly suggests that renal hypertrophy is unaffected by MMF treatment. This is in contrast with the reduction in the renal hypertrophy and hyperplasia associated with other immunosuppressive drugs such as cyclosporine A [39]. Lymphocyte (CD43-positive cells) and macrophage (ED1-positive cells) infiltration is comparable in the MMF-treated group and the sham-operated group at four weeks after surgery. In contrast, the 5/6Nx group shows the cellular infiltration of the remnant kidney reported previously by others [10–13, 28]. These findings indicate that MMF administration was capable of effectively counteracting the infiltration and proliferative stimuli triggered by the glomerular hypertension. Expression of adhesion molecules has not been stud-
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Romero et al: Mycophenolate mofetil therapy in renal ablation
ied in the renal ablation model. All adhesion molecules tested increased several times in the remnant kidney (glomeruli and interstitium) in the 5/6Nx rats examined four weeks after nephrectomy. In the following month, the expression of CD54 and CD18 in glomeruli and interstitium fell to levels comparable to those present in shamoperated rats (Tables 2 and 3). The reasons for the reduction in expression of CD54 and CD18 are not clear, but it is likely that progressive scarring of the kidney is associated with a decrease in the number of CD54-positive cells, which includes endothelial cells, lymphocytes, and neutrophils. As shown in Table 3, the rats from the 5/6Nx group showed a minor reduction (P . 0.05) in tubulointerstitial ED1-positive cells at four and eight weeks after nephrectomy (194 6 61.2 and 140 6 22.8 cells per mm2, respectively), whereas the CD11b-positive cells almost triple in number (Table 3). Because CD11b stains granulocytes in addition to monocytes and macrophages, we interpret these findings as indicative of an increment in nonmacrophage leukocyte infiltration after eight weeks. Interestingly, the numbers of CD11b-positive cells in rats treated with MMF are comparable to the sham-operated animals (Tables 2 and 3). The relatively stable number of macrophages from the fourth to the eight weeks in the remnant kidney observed by us is in contrast with the findings in a recent report by Yang et al [28], who found that ED1-positive cells increased progressively up to 16 weeks after nephrectomy. Their study did not use a high-protein diet [28], and consequently, the tubulointerstitial damage and the increment in serum creatinine progressed at a lower rate than in this study. It is possible that this explains, at least in part, the discrepancy. These studies confirm recent preliminary reports from our group [40] and Fujihara et al [41], and suggest that MMF may be useful in the treatment of progressive renal failure associated with reduced renal mass. Yet to be determined is whether MMF treatment would give comparable or additional beneficial effects to those obtained by other treatment modalites in this experimental model. The route of administration, as well as the dose of MMF in this study, is comparable to those used routinely in humans with relatively minor side-effects. Therefore, consideration may be given to trials with this drug in the clinical setting of progressive renal failure. ACKNOWLEDGMENTS The authors are grateful for financial support from Asociacio´n de Amigos del Rin˜o´n, Maracaibo, Venezuela. Parts of this article were presented in the 30th Annual Meeting of the American Society of Nephrology (San Antonio, Texas, November 2–5, 1997) and were published in abstract form (Romero et al, J Am Soc Nephrol 8, 628A, 1997). Reprint requests to Bernardo Rodrı´guez-Iturbe, Apartado Postal 1430, Maracaibo 4001-A, Venezuela. E-mail: [email protected]
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