Mycophenolate mofetil slows progression in anti-thy1-induced chronic renal fibrosis but is not additive to a high dose of enalapril
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《美国生理学杂志》
Department of Nephrology and Center of Cardiovascular Research, Charité University Medicine Berlin, Charité Campus Mitte, Humboldt University, Berlin, Germany
Department of Cell Biology and Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
ABSTRACT
Tubulointerstitial inflammation and fibrosis are hallmarks of chronic progressive renal diseases. To characterize the functional interaction between cell infiltration and matrix expansion, this study compared the immunosuppressant mycophenolate mofetil (MMF), intended as primarily anti-inflammatory intervention, the angiotensin-converting enzyme inhibitor enalapril, intended as primarily an anti-fibrotic drug, and a combination of both as anticipated anti-inflammatory/anti-fibrotic intervention. The model used was anti-thy1-induced chronic-progressive glomerulosclerosis (cGS) in the rat, where a brief anti-thy1-induced glomerular injury progresses spontaneously toward tubulointerstitial fibrosis and renal insufficiency. cGS was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, animals were randomly assigned to the following groups: cGS, cGS plus MMF (20 mg·kg body wt–1·day–1), cGS plus high-dose enalapril (12 mg·kg body wt–1·day–1), and cGS plus both. At week 16 after disease induction, MMF or enalapril alone reduced signs of chronic renal disease significantly and similarly compared with the untreated cGS group. Variables measured included proteinuria, blood pressure, tubulointerstitial and glomerular matrix accumulation, expression of transforming growth factor-1, fibronectin, and plasminogen activator inhibitor-1, infiltration of lymphocytes and macrophages, plasma creatinine and urea levels, and glomerular filtration rate. Combined MMF and enalapril treatment was not superior to single therapy. In conclusion, MMF slows the progression of chronic renal fibrosis and renal insufficiency as effectively as high-dose enalapril in the anti-thy1-induced chronic-progressive glomerulosclerosis model. The dual anti-inflammatory/anti-fibrotic intervention does not yield additive renoprotective effects, indicating that MMF and enalapril interfere with similar or very closely related pathways involved in progression of renal disease.
angiotensin-converting enzyme inhibition; transforming growth factor-1
THE HISTOLOGICAL PICTURE OF human and experimental chronic renal diseases is uniformly characterized by advancing fibrosis and inflammation in the tubulointerstitial compartment (10, 23, 33). Tubulointerstitial injury correlates closely with the progressive loss of function in chronic kidney disorders. The tubulointerstitial cell infiltrate consists of macrophages and various subsets of lymphocytes, whereas renal fibrosis is linked to overproduction of the key profibrotic cytokine transforming growth factor (TGF)- (1). TGF- overexpression, induced by various kinds of tissue injury, leads to renal matrix protein accumulation by increasing production of matrix proteins including fibronectin, biglycan, and collagens and decreasing of matrix protein degradation by increasing production of protease inhibitors including plasminogen activator inhibitor (PAI)-1 (1). Use of neutralizing antibodies to TGF- ameliorates pathological renal matrix expansion in both acute and chronic experimental renal disease (3, 35).
It is generally assumed that tubulointerstitial fibrosis and inflammation represent complementary pathways leading to end-stage renal disease, although some recent studies in experimental nonimmune kidney disorders have documented a relevant functional cross talk between tubulointerstitial cell infiltration and matrix protein expansion (8, 21, 32). To further characterize this functional interaction, we performed side-by-side comparison of interventions that were anticipated as being anti-inflammatory, anti-fibrotic, and combined anti-inflammatory/anti-fibrotic on the course of anti-thy1-induced chronic glomerulosclerosis in the rat. This is a model of progressive renal fibrosis and failure and is induced by a single injection of anti-thy1 antibody into uninephrectomized rats. A short-term acute mesangioproliferative glomerulonephritis is followed by a slow primarily nonimmune-mediated progression of the disease over several months toward glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency (12, 15). In our experimental approach, mycophenolate mofetil (MMF) was used as the intended primarily anti-inflammatory intervention. MMF serves conventionally as a relatively side effect-free immunosuppressant and is well established in solid organ transplantation (9, 33). The angiotensin-converting enzyme (ACE) inhibitor enalapril was used for the anticipated mainly anti-fibrotic intervention and conventionally serves as an antihypertensive drug. Beyond this, enalapril directly limits angiotensin II's key paracrine profibrotic actions in renal disease, including a direct induction of TGF-1 and its receptors (2, 17). Enalapril was given in a high dose to address the question of whether different pathways are targeted by the two drugs (17). Both drugs had previously shown beneficial effects in the acute and reversible model of anti-thy1 glomerulonephritis (17, 34). Treatments with MMF, enalapril, or both interventions together were started 1 wk after injection of anti-thy1 antibody when the glomerular disease was already established. At week 16 after disease induction, proteinuria, blood pressure, renal function, glomerular and tubulointerstitial fibrosis, and cell infiltration were used as measures of disease severity and therapeutic efficacy.
METHODS
Materials. Unless otherwise indicated, materials, chemicals, or culture media were purchased from Sigma (Taufkirchen, Germany).
Animals. Male Wistar rats (150–180 g) were obtained from Charles River (Sulzfeld, Germany). The animals were fed a normal protein diet (22.5% protein, Altromin, Lage, Germany) for at least 3 days before the experiment to allow equilibration. Animals were housed in a constant-temperature room with a 12:12-h dark-light cycle. Body weight was determined weekly. Food and water intakes were monitored daily. Animal care and treatment were in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by local authorities [Ministry of the government of Berlin (LaGetSi), Germany].
Model of anti-thy1-induced chronic-progressive glomerulosclerosis. Chronic-progressive glomerulosclerosis (cGS) was produced by surgically removing one kidney and intravenously injecting the monoclonal antibody mAb 1–22-3 [5 mg/kg body wt in phosphate-buffered saline (PBS), pH = 7.4] 3 days later (12, 15). The antibody was produced from hybridoma cell lines as previously described (15). mAb 1–22-3 antibody binds to a thy1-like antigen on the surface of mesangial cells of the kidney and causes a fast complement-dependent and nitric oxide-dependent mesangial cell lysis within the next 24 h (12, 15). The progression in cGS is linked to the uninephrectomy that is performed before anti-thy1 antibody injection, because the glomerular disease resolves over 4 wk in animals with two kidneys (12, 28). Control animals with and without a uninephrectomy were injected with equal volumes of PBS only.
Treatment with MMF and enalapril. MMF was given with the food in a daily dose of 20 mg/kg body wt. MMF is endogenously converted into mycophenolic acid (MPA), which preferentially antagonizes lymphocyte purine synthesis via inhibition of the inosine monophoshate dehydrogenase (IMPDH) enzyme (9). IMPDH plays an essential role in the de novo pathway of lymphocyte purine synthesis, and thus for lymphocyte proliferation. The daily dose of 20 mg/kg body wt has been chosen on the basis of previous reports in other renal disease models (23, 33). In dose-finding studies before the main experiments, we found that higher doses of MMF led to diarrhea in a relevant number of animals. The MMF-containing food was produced in our laboratory by using the flour of the standard rat chow (22.5% protein, A1311, Altromin). The drug was mixed into the dry food flour in appropriate amounts, water was added to form pellets, and the air-dried pellets were subsequently given to the MMF-treated animals.
Enalapril was given with the drinking water at a daily dose of 12 mg/kg body wt. Enalapril is endogenously converted into enalapril maleate, which inhibits the angiotensin-converting enzyme and thereby the generation of angiotensin II. The dose was chosen on the basis of a previous dose-response experiment, showing that this amount maximally limits TGF- overexpression in an acute model of anti-thy1 glomerulonephritis (17).
Experimental groups and design. Urine (24 h) was collected 1 wk after anti-thy1 antibody injection. On the basis of the actual 24-h proteinuria achieved, the diseased animals were randomly assigned to the treatment groups. The following groups were formed: 1) nonnephrectomized, PBS-injected controls (2-K Control; n = 6); 2) uninephrectomized, PBS-injected controls (1-K Control; n = 6); 3) uninephrectomized, anti-thy1-injected animals, no treatment (cGS; n = 12); 4) uninephrectomized, anti-thy1-injected animals treated with MMF (cGS+MMF; n = 8); 5) uninephrectomized, anti-thy1-injected animals treated with enalapril (cGS+Ena; n = 8); and 6) uninephrectomized, anti-thy1-injected animals treated with MMF and enalapril (cGS+MMF+Ena; n = 12).
In week 16, i.e., after 15 wk of MMF or/and enalapril administration, the effects of single and dual treatments on proteinuria, blood pressure, renal function, as well as glomerular and tubulointerstitial fibrosis and cell infiltration were determined. Blood creatinine and urea concentrations and calculated creatinine clearance served as markers of renal function. Glomerular and tubulointerstitial changes were analyzed separately. Glomeruli were isolated by a graded sieving technique. Because the renal cortex consists mainly of tubulointerstitial tissue (>95%), it was used as representative of the tubulointerstitium. Analysis of glomerular and tubulointerstitial fibrosis involved histological scoring of matrix accumulation, molecular analysis of TGF-1 as mediator of fibrosis, fibronectin as marker of matrix protein synthesis, and the protease inhibitor PAI-1 as marker of matrix protein degradation. Tubulointerstitial and glomerular cell infiltrations were analyzed by immunohistochemistry using an anti-ED1 antibody for macrophages and anti-CD4 and anti-CD8 antibodies for lymphocytes.
Measurement of systolic blood pressure and proteinuria. Systolic blood pressure was assessed before death in trained conscious animals by a tail-cuff method as previously described (15). The day before euthanasia, the animals were housed individually in metabolic cages for a 24-h urine collection. Urinary protein was measured using a pyrogallol red microtiter plate technique as previously described (15). Proteinuria is expressed as milligrams protein per 24 hours.
Euthanasia. The animals were anesthetized with 0.1 mg ketanest-0.01 mg xylazin/100 g body wt (ketamin 10%, WDT, Garbsen, Germany; Rompun 2%, Bayer Vital, Leverkusen, Germany). Following a midline abdominal incision, 5–10 ml of heparinized blood were drawn from the abdominal aorta and the kidneys were subsequently perfused with 30 ml of ice-cold PBS. Materials and tissues were subsequently processed as described in the following sections.
Blood analysis. Red and white blood cell counts were determined in an automated cell counter (XE 2,100, Sysmex, Norderstedt, Germany) using a standard fluorescent flow cytometry technology (15). Plasma and urine creatinine and plasma urea were measured spectrometrically in enzyme-based assays (16). The glomerular filtration rate was calculated on the basis of serum and urinary creatinine concentrations and the corresponding urine volume and is expressed as milliliters per minute per 100 grams body wt.
Light and immunohistochemical microscopy. For histological examination, cortical tissue was fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen. All histological studies were performed in a blinded fashion. Three-micrometer paraffin sections were stained with periodic acid-Schiff reaction to analyze tubulointerstitial and glomerular fibrosis by computer-based morphometric analysis. Renal sections were examined on a Leica DM LB2 light microscope (Leica Microsystems, Wetzlar, Germany) connected to a PL-A662 video camera and the Axiovision 2.05 image-analysis system (both Karl Zeiss Vision, Munich, Germany) using a 10 x 10 orthographic grid overlaid on digital images. The relative degree of tubulointerstitial fibrotic lesions, i.e., matrix deposition, tubular atrophy and dilation, was calculated in 15 randomly selected cortical areas per animal observed at x200 magnification graded on the following scale: 0 = normal, 1 = lesions involving <10% of cortical area, 2 = lesions involving 11–30% of cortical area, 3 = lesions involving 31–50% of cortical area, and 4 = lesions involving >50% of cortical area, respectively (15). The relative degree of glomerular matrix expansion was evaluated by rating the mesangial matrix occupying the area of 15 glomeruli/animal, using the following scoring system: 0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100% (15).
For analysis of cell infiltration, renal tissue was immunohistologically stained for CD4- and ED1 (macrophages)-positive cells. Paraffin-embedded tissues were incubated with a primary mouse anti-CD8 and mouse anti-ED1 antibody and cryoconserved material with a primary mouse anti-CD4 antibody, respectively (all from Serotec, Oxford, UK). Further staining was performed using a standard alkaline phosphatase anti-alkaline phosphatase technique (DakoCytomation, Hamburg, Germany), as previously described (15). Cell infiltration was evaluated by counting infiltrating cells in at least 15 glomerular sections (glomerular cell infiltration) and at least 15 randomly selected cortical areas (tubulointerstitial cell infiltration) from each rat. Tissue sections were observed at x200 magnification.
Cortical and glomerular TGF-1, fibronectin, and PAI-1 protein expression. Glomeruli from individual rats were isolated by a graded sieving technique (160-, 125-, and 71-μm mesh metal sieves), as described previously (17). For cultures of renal cortical tissue, a piece of cortical tissue was weighed and minced extensively with a razor blade (16). Glomeruli or cortical tissues were suspended in DMEM supplemented with 0.1 U/ml insulin, 100 U/ml penicillin, and 100 μg/ml streptomycin. Glomeruli were cultured at a density of 2,000 glomeruli/ml for 48 h and minced cortical tissue at a density of 10 mg/ml, respectively. After 48-h incubation at 37°C/5% CO2, supernatants were harvested and stored at –70°C until analysis of TGF-1, fibronectin, or PAI-1 content. TGF-1 content of the culture supernatant was measured after acid activation, using a commercially available ELISA kit (TGF-1 Duoset, R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. Fibronectin and PAI-1 levels were measured with modified inhibitory ELISA, according to published methods (22). Three samples from each rat were analyzed.
Cortical TGF-1, fibronectin, and PAI-1 mRNA expression. Cortical total RNA was extracted with TRIzol reagent (GIBCO BRL, Berlin, Germany) according to the manufacturer's instructions. The mRNA expression of TGF-1, fibronectin, PAI-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by a "two-step" RT-PCR (18). A cDNA copy was created with reverse transcriptase from an RNA PCR Core kit (Roche, Applied Biosystems, Branchburg, NJ). Real-time PCR was performed using the LightCycler System and SYBR Green I as the dsDNA binding dye (Roche Diagnostics, Mannheim, Germany). The following primer pairs were used (annealing temperature in parentheses): TGF-1: sense 5'-GGTGGCAGGCGAGAGCGCTGA-3' and antisense 5'-GGCATGGTAGCCCTTGGGCT-3' (64°C); fibronectin: sense 5'-GGTCCAAATCGGTCATGTTCCCA-3', antisense 5'-CGTAATGGGAAACCGTGTAAGGG-3'(64°C); PAI-1: sense 5'-CAGCATGTGGTCCAGGCCTCCAAA-3', antisense 5'-TGTGCCGCTCTCGTTCACCTCGATCT-3' (64°C); and GAPDH: sense 5'-CCATCTTCCAGGAGCGAGAT-3', antisense 5'-GATGACCTTGCCCACAGCCT-3' (59°C). For analysis, a relative quantification method was used as previously described (18, 19, 29). Briefly, amplification is described as N = N0·ECP, where N is the number of amplified molecules, N0 is the initial number of molecules, E is amplification efficiency, and CP is the crossing-point deviation, expressed as CP = CPtarget – CPGAPDH. In this quantification method, measured CP is defined as the point at which the fluorescence rises above the background fluorescence. Finally, N0 of the target gene was calculated and compared relative to expression of GAPDH mRNA as the housekeeping gene.
Statistical analysis. The results are expressed as means ± SE. Statistical analysis between groups was performed by ANOVA and subsequent Mann-Whitney U-testing. A P value <0.05 was considered significant.
RESULTS
Body weight, food intake, and blood cell analysis. At the end of the experiment, body weight was 588 ± 10 g in the normal (2-K Control) and 541 ± 12 g in the uninephrectomized control group (1-K Control), respectively. In comparison, final body weight was significantly lower in all nephritic groups (cGS: 492 ± 18 g, cGS+MMF: 469 ± 8 g, cGS+Ena: 509 ± 12 g, cGS+MMF+Ena: 476 ± 12 g, P < 0.05 vs. 2-K Control), indicating chronic renal disease and insufficiency. The differences between the nephritic groups were not significant. Food and water intakes did not differ between the groups throughout the experiment. Blood leukocyte, lymphocyte, and platelet numbers were not significantly different between any groups (data not shown). Compared with the uninephrectomized control animals, blood hemoglobin concentration was significantly lower in the untreated sGC animals (13.0 ± 0.5 vs. 14.7 ± 0.4 g/dl, P < 0.05). Treatment with enalapril partially prevented the decrease in hemoglobin levels (13.9 ± 0.1 g/dl, P < 0.05 vs. cGS), whereas MMF administration further lowered hemoglobin concentrations (cGS+MMF: 10.7 ± 0.6 g/dl, cGS+MMF+Ena: 10.9 ± 0.3 g/dl, P < 0.05 vs. cGS).
Disease severity in anti-thy1-induced chronic glomerulosclerosis 16 wk after disease induction. Compared with the normal controls, anti-thy1-induced chronic glomerulosclerosis 16 wk after disease induction was characterized by significant increases in proteinuria (149 ± 14 mg/day, Fig. 1A); blood pressure (145 ± 7 mmHg, Fig. 1B); histological tubulointerstitial matrix accumulation (matrix score 2.5 ± 0.3, Figs. 2 and 3A); protein expression of TGF-1 (189 ± 31 pg/ml); fibronectin (2,921 ± 605 ng/ml); and PAI-1 (294 ± 58 ng/ml) (Fig. 3, B–D; all P < 0.05 vs. 1-K Control and 2-K Control). TGF-1 mRNA was increased 2.9-fold, fibronectin 3.9-fold, and PAI-1 2.6-fold (all P < 0.05 vs. 1-K Control and 2-K Control). At the glomerular level, cGS animals showed increased histological matrix accumulation (matrix score 2.2 ± 0.1, Figs. 2 and 4A) and protein expression of TGF-1 (171 ± 15 pg/ml) and fibronectin (3,927 ± 239 ng/ml) (Fig. 4, B and C) (all P < 0.05 vs. 1-K Control and 2-K Control). Glomerular PAI-1 expression did not differ among all groups (Fig. 4D).
Renal matrix accumulation was paralleled by increases in infiltration with ED1-, CD4-, and CD8-positive cells into the tubulointerstitium (ED1: 186 ± 24 cells/section, CD4: 112 ± 14 cells/section, and CD8: 83 ± 11 cells/section) (Fig. 5, A–C) and the glomeruli (ED1: 3.1 ± 0.4 cells/cross section, CD4: 3.6 ± 0.4 cells/cross section and CD8: 0.4 ± 0.1 cells/cross section) (Fig. 6, A–C; all P < 0.05 vs. 1-K Control and 2-K Control). Moreover, untreated cGS animals showed significantly impaired renal function as documented by increased blood creatinine (1.2 ± 0.3 mg/dl) and urea concentrations (157 ± 53 mg/dl) and decreased glomerular filtration rate (0.32 ± 0.06 ml·min–1·100 g body wt–1) (Fig. 7, A–C; all P < 0.05 vs. 1-K Control and 2-K Control).
Effect of single treatment with MMF or enalapril on anti-thy1-induced chronic glomerulosclerosis. Compared with the untreated cGS animals, both the single MMF and enalapril treatments significantly limited disease progression (Figs. 1–7, P < 0.05 for all parameters vs. cGS, expect cortical PAI-1 protein) and overall to a very similar relative degree (P = not significant for all parameters cGS+MMF vs. cGS+Ena). This was documented on the basis of proteinuria blood pressure, histological tubulointerstitial fibrosis, protein expression of TGF-1 and fibronectin; tubulointerstitial mRNA expression of TGF-1, fibronectin, and PAI-1; glomerular histological matrix accumulation and protein expression of TGF-1 and fibronectin; immunohistological tubulointerstitial and glomerular cell infiltration with ED1-, CD4-, and CD8-positive cells; blood creatinine and urea concentrations; and glomerular filtration rate (Figs. 1–7). The relative changes with each intervention are summarized in Table 1.
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Effect of combined MMF and enalapril treatment on anti-thy1-induced chronic glomerulosclerosis. Compared with the single treatment groups, combined therapy with MMF and enalapril yielded very similar relative changes in the disease severity of anti-thy1-induced chronic glomerulonephritis (Figs. 1–7, P < 0.05 vs. cGS for all parameters, but P = not significant for all parameters vs. cGS+MMF and cGS+Ena). As shown in Table 1, in none of the multiple measures indicating renal function, tubulointerstitial and glomerular matrix accumulation, and cell infiltration the combined MMF and enalapril intervention was superior to single treatment with MMF or enalapril.
Taken together, the results show that the primarily anti-inflammatory approach using MMF is as effective as the anticipated mainly anti-fibrotic approach using a high dose of enalapril on renal matrix protein expression and accumulation, cell infiltration, and kidney function in experimental anti-thy1-induced chronic-progressive glomerulosclerosis. The dual anti-inflammatory/anti-fibrotic intervention does not yield additive renoprotective effects on renal fibrosis, cell infiltration, and insufficiency in this model of renal progression.
DISCUSSION
Progression to end-stage organ failure is a common feature of both experimental and human chronic renal diseases (10, 15, 23, 33). Ongoing loss of organ function occurs independently of whether the underlying disorder is glomerulonephritis, diabetes, or hypertension, suggesting that a common intrarenally determined program is operating. The model used here, anti-thy1-induced chronic glomerulosclerosis in the rat, is an experimental paradigm for a "one-hit, self-progressing kidney disease" as it is seen in patients who progress after a single episode of glomerulonephritis (10, 15). This contrasts with diabetic and hypertensive nephropathies, where continual or repeated extrarenal injurious stimuli drive the decline in renal function.
Using pharmacological interventions intended to target primarily tissue inflammation or fibrosis, our results suggest that tubulointerstitial cell infiltration and matrix protein expansion mediate renal progression by similar pathways. Although one would expect that anti-inflammatory MMF would be superior in reducing renal lymphocyte and macrophage infiltration and anti-fibrotic intervention by enalapril would be superior in reducing renal matrix protein accumulation, tubulointerstitial inflammation and tubulointerstitial fibrosis were found to be reduced with almost equal efficiency by both drugs. In addition, if renal inflammation and fibrosis represent complementary pathways leading to end-stage renal disease, one would expect an additive renoprotective effect with combination therapy. However, dual therapy did not produce additional beneficial effects on the progressive course of anti-thy1-induced glomerulosclerosis based on disease measures including proteinuria, blood pressure, tubulointerstitial and glomerular TGF- overexpression, matrix protein accumulation, and infiltration of lymphocytes and macrophages. In addition, similar therapeutic reductions in plasma creatinine and urea levels and increases in glomerular filtration rate were seen with mono- or dual therapy.
In the current understanding of renal disease, we separate inflammatory from fibrotic renal disease relatively clearly, although we know that inflammatory kidney disorders such as lupus nephritis are also characterized by marked matrix protein expansion (14), and fibrotic kidney diseases such as hypertensive and diabetic nephropathies show relevant infiltrations with lymphocytes and macrophages (23, 33). In addition to organ transplantation, MMF is used to treat inflammatory disease and the antihypertensive enalapril is used as an anti-fibrotic drug in organ protection (13, 17, 23, 33). However, research has revealed that MMF has important anti-fibrotic and enalapril has notable anti-inflammatory properties. MMF has been shown to inhibit directly basal and TGF--induced matrix protein expression of renal cells in vitro (5). Another important anti-fibrotic action of MMF is direct inhibition of the proliferation of renal cell types (23, 33). In many kidney diseases, renal cell proliferation precedes extracellular matrix protein expansion (7). Inhibition of lymphocyte and macrophage infiltration by MMF appears to prevent these cells from releasing a number of factors within the kidney that would amplify renal TGF- overexpression and matrix protein accumulation (15, 23, 33). On the other hand, enalapril, in addition to blocking angiotensin II's profibrotic effects, blocks proinflammatory actions of this octapeptid, including direct induction of reactive oxygen species, chemokines, and the transcription factor nuclear factor (NF)-B (11, 23, 31). The similarity of therapeutic effects with MMF and enalapril alone seen in this study supports the idea that these two drugs are neither solely "anti-inflammatory" nor solely "anti-fibrotic" and that renal tissue cell infiltration and matrix protein expansion represent functionally very similar pathways.
We also addressed the question of whether a combined anti-inflammatory/anti-fibrotic approach yields an additive therapeutic effect. Mechanistic additivity occurs when one pathway is maximally blocked by one intervention and a second pathway is blocked by a second intervention, leading to enhanced disease reduction. This contrasts with therapeutic additivity whereby combining two drugs that inhibit the same system, both at suboptimal doses, enhances efficacy. In the present study, enalapril was given at a dose shown to be maximally therapeutic in acute anti-thy1 glomerulonephritis (2, 17). While MMF may not have been given at maximal doses, addition of MMF to enalapril still should have yielded an enhanced therapeutic effect if these therapies target different pathways. By inquiring into a mechanistic additivity, our investigation goes beyond previous studies documenting additive renoprotection by MMF and RAS inhibition in nonimmune-mediated models of renal disease (8, 21, 32). To date, these studies have included interventions with the ACE inhibitor lisinopril or the AT1 antagonist losartan and MMF in models of renal mass reduction (8, 21) as well as losartan and MMF in a model of chronic cyclosporine nephropathy (32). However, lisinopril and losartan were used in low or moderate doses in these experiments compared with the doses needed to more effectively block the renin-angiotensin system as shown by others (17, 30). Therefore, the additivity seen in 5/6 nephrectomy and cyclosporine nephropathy may be therapeutic rather than mechanistic. While we feel it is likely that the additivity seen in these models was due to use of less than maximal doses of lisinopril and losartan, it remains possible that the results are due to differences in the animal models used (8, 21, 32) or timing of the pharmacological intervention (8).
This study provides in vivo data suggesting that inflammatory and fibrotic pathways are closely linked and thereby expands findings of previous, mostly in vitro studies. At the cellular or molecular level, there have been identified a number of functional crossing points between the two systems and it is likely that their interaction takes place continuously and through several pathways. At the extracellular level, it has been shown that the inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)- can directly induce renal cell TGF- expression and matrix protein production, renal cell proliferation, and even epithelial-myofibroblast transdifferentiation, an important feature in tubulointerstitial fibrosis (6, 20, 27). On the other hand, profibrotic growth factors such as angiotensin II have been found to enhance renal macrophage infiltration via induction of the chemokine RANTES and to directly induce the key proinflammatory transcription factor NF-B (11, 23, 31). Furthermore, IL-1 and TNF- production by cultured monocytes is amplified in the presence of extracellular matrix proteins, particularly fibronectin (4). Adding more complexity and redundancy, proinflammatory and profibrotic factors share intracellular signaling pathways involving protein tyrosine kinases, NF-B, and the mitogen-activating protein kinases (MAPK) c-Jun and p38 MAPK (11, 24–26).
In conclusion, MMF slows the progressive course toward chronic renal fibrosis and insufficiency as effectively as a high dose of enalapril in the model of anti-thy1-induced chronic glomerulosclerosis. Combining the intended primarily anti-inflammatory and anti-fibrotic interventions does not yield additive effects on renal function, matrix protein expansion, and cell infiltration. The findings suggest that tubulointerstitial inflammation and fibrosis represent functionally very similar or closely related pathways mediating the progression of renal disease.
GRANTS
This study was supported in part by grants from Hoffmann-La Roche (Grenzach-Whylen, Germany) and the Deutsche Forschungsgemeinschaft (PE 558/2–1 and PE 558/2–3, Bonn, Germany).
ACKNOWLEDGMENTS
We thank Nancy Noble for valuable comments on the manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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Department of Cell Biology and Institute of Nephrology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
ABSTRACT
Tubulointerstitial inflammation and fibrosis are hallmarks of chronic progressive renal diseases. To characterize the functional interaction between cell infiltration and matrix expansion, this study compared the immunosuppressant mycophenolate mofetil (MMF), intended as primarily anti-inflammatory intervention, the angiotensin-converting enzyme inhibitor enalapril, intended as primarily an anti-fibrotic drug, and a combination of both as anticipated anti-inflammatory/anti-fibrotic intervention. The model used was anti-thy1-induced chronic-progressive glomerulosclerosis (cGS) in the rat, where a brief anti-thy1-induced glomerular injury progresses spontaneously toward tubulointerstitial fibrosis and renal insufficiency. cGS was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, animals were randomly assigned to the following groups: cGS, cGS plus MMF (20 mg·kg body wt–1·day–1), cGS plus high-dose enalapril (12 mg·kg body wt–1·day–1), and cGS plus both. At week 16 after disease induction, MMF or enalapril alone reduced signs of chronic renal disease significantly and similarly compared with the untreated cGS group. Variables measured included proteinuria, blood pressure, tubulointerstitial and glomerular matrix accumulation, expression of transforming growth factor-1, fibronectin, and plasminogen activator inhibitor-1, infiltration of lymphocytes and macrophages, plasma creatinine and urea levels, and glomerular filtration rate. Combined MMF and enalapril treatment was not superior to single therapy. In conclusion, MMF slows the progression of chronic renal fibrosis and renal insufficiency as effectively as high-dose enalapril in the anti-thy1-induced chronic-progressive glomerulosclerosis model. The dual anti-inflammatory/anti-fibrotic intervention does not yield additive renoprotective effects, indicating that MMF and enalapril interfere with similar or very closely related pathways involved in progression of renal disease.
angiotensin-converting enzyme inhibition; transforming growth factor-1
THE HISTOLOGICAL PICTURE OF human and experimental chronic renal diseases is uniformly characterized by advancing fibrosis and inflammation in the tubulointerstitial compartment (10, 23, 33). Tubulointerstitial injury correlates closely with the progressive loss of function in chronic kidney disorders. The tubulointerstitial cell infiltrate consists of macrophages and various subsets of lymphocytes, whereas renal fibrosis is linked to overproduction of the key profibrotic cytokine transforming growth factor (TGF)- (1). TGF- overexpression, induced by various kinds of tissue injury, leads to renal matrix protein accumulation by increasing production of matrix proteins including fibronectin, biglycan, and collagens and decreasing of matrix protein degradation by increasing production of protease inhibitors including plasminogen activator inhibitor (PAI)-1 (1). Use of neutralizing antibodies to TGF- ameliorates pathological renal matrix expansion in both acute and chronic experimental renal disease (3, 35).
It is generally assumed that tubulointerstitial fibrosis and inflammation represent complementary pathways leading to end-stage renal disease, although some recent studies in experimental nonimmune kidney disorders have documented a relevant functional cross talk between tubulointerstitial cell infiltration and matrix protein expansion (8, 21, 32). To further characterize this functional interaction, we performed side-by-side comparison of interventions that were anticipated as being anti-inflammatory, anti-fibrotic, and combined anti-inflammatory/anti-fibrotic on the course of anti-thy1-induced chronic glomerulosclerosis in the rat. This is a model of progressive renal fibrosis and failure and is induced by a single injection of anti-thy1 antibody into uninephrectomized rats. A short-term acute mesangioproliferative glomerulonephritis is followed by a slow primarily nonimmune-mediated progression of the disease over several months toward glomerulosclerosis, tubulointerstitial fibrosis, and renal insufficiency (12, 15). In our experimental approach, mycophenolate mofetil (MMF) was used as the intended primarily anti-inflammatory intervention. MMF serves conventionally as a relatively side effect-free immunosuppressant and is well established in solid organ transplantation (9, 33). The angiotensin-converting enzyme (ACE) inhibitor enalapril was used for the anticipated mainly anti-fibrotic intervention and conventionally serves as an antihypertensive drug. Beyond this, enalapril directly limits angiotensin II's key paracrine profibrotic actions in renal disease, including a direct induction of TGF-1 and its receptors (2, 17). Enalapril was given in a high dose to address the question of whether different pathways are targeted by the two drugs (17). Both drugs had previously shown beneficial effects in the acute and reversible model of anti-thy1 glomerulonephritis (17, 34). Treatments with MMF, enalapril, or both interventions together were started 1 wk after injection of anti-thy1 antibody when the glomerular disease was already established. At week 16 after disease induction, proteinuria, blood pressure, renal function, glomerular and tubulointerstitial fibrosis, and cell infiltration were used as measures of disease severity and therapeutic efficacy.
METHODS
Materials. Unless otherwise indicated, materials, chemicals, or culture media were purchased from Sigma (Taufkirchen, Germany).
Animals. Male Wistar rats (150–180 g) were obtained from Charles River (Sulzfeld, Germany). The animals were fed a normal protein diet (22.5% protein, Altromin, Lage, Germany) for at least 3 days before the experiment to allow equilibration. Animals were housed in a constant-temperature room with a 12:12-h dark-light cycle. Body weight was determined weekly. Food and water intakes were monitored daily. Animal care and treatment were in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by local authorities [Ministry of the government of Berlin (LaGetSi), Germany].
Model of anti-thy1-induced chronic-progressive glomerulosclerosis. Chronic-progressive glomerulosclerosis (cGS) was produced by surgically removing one kidney and intravenously injecting the monoclonal antibody mAb 1–22-3 [5 mg/kg body wt in phosphate-buffered saline (PBS), pH = 7.4] 3 days later (12, 15). The antibody was produced from hybridoma cell lines as previously described (15). mAb 1–22-3 antibody binds to a thy1-like antigen on the surface of mesangial cells of the kidney and causes a fast complement-dependent and nitric oxide-dependent mesangial cell lysis within the next 24 h (12, 15). The progression in cGS is linked to the uninephrectomy that is performed before anti-thy1 antibody injection, because the glomerular disease resolves over 4 wk in animals with two kidneys (12, 28). Control animals with and without a uninephrectomy were injected with equal volumes of PBS only.
Treatment with MMF and enalapril. MMF was given with the food in a daily dose of 20 mg/kg body wt. MMF is endogenously converted into mycophenolic acid (MPA), which preferentially antagonizes lymphocyte purine synthesis via inhibition of the inosine monophoshate dehydrogenase (IMPDH) enzyme (9). IMPDH plays an essential role in the de novo pathway of lymphocyte purine synthesis, and thus for lymphocyte proliferation. The daily dose of 20 mg/kg body wt has been chosen on the basis of previous reports in other renal disease models (23, 33). In dose-finding studies before the main experiments, we found that higher doses of MMF led to diarrhea in a relevant number of animals. The MMF-containing food was produced in our laboratory by using the flour of the standard rat chow (22.5% protein, A1311, Altromin). The drug was mixed into the dry food flour in appropriate amounts, water was added to form pellets, and the air-dried pellets were subsequently given to the MMF-treated animals.
Enalapril was given with the drinking water at a daily dose of 12 mg/kg body wt. Enalapril is endogenously converted into enalapril maleate, which inhibits the angiotensin-converting enzyme and thereby the generation of angiotensin II. The dose was chosen on the basis of a previous dose-response experiment, showing that this amount maximally limits TGF- overexpression in an acute model of anti-thy1 glomerulonephritis (17).
Experimental groups and design. Urine (24 h) was collected 1 wk after anti-thy1 antibody injection. On the basis of the actual 24-h proteinuria achieved, the diseased animals were randomly assigned to the treatment groups. The following groups were formed: 1) nonnephrectomized, PBS-injected controls (2-K Control; n = 6); 2) uninephrectomized, PBS-injected controls (1-K Control; n = 6); 3) uninephrectomized, anti-thy1-injected animals, no treatment (cGS; n = 12); 4) uninephrectomized, anti-thy1-injected animals treated with MMF (cGS+MMF; n = 8); 5) uninephrectomized, anti-thy1-injected animals treated with enalapril (cGS+Ena; n = 8); and 6) uninephrectomized, anti-thy1-injected animals treated with MMF and enalapril (cGS+MMF+Ena; n = 12).
In week 16, i.e., after 15 wk of MMF or/and enalapril administration, the effects of single and dual treatments on proteinuria, blood pressure, renal function, as well as glomerular and tubulointerstitial fibrosis and cell infiltration were determined. Blood creatinine and urea concentrations and calculated creatinine clearance served as markers of renal function. Glomerular and tubulointerstitial changes were analyzed separately. Glomeruli were isolated by a graded sieving technique. Because the renal cortex consists mainly of tubulointerstitial tissue (>95%), it was used as representative of the tubulointerstitium. Analysis of glomerular and tubulointerstitial fibrosis involved histological scoring of matrix accumulation, molecular analysis of TGF-1 as mediator of fibrosis, fibronectin as marker of matrix protein synthesis, and the protease inhibitor PAI-1 as marker of matrix protein degradation. Tubulointerstitial and glomerular cell infiltrations were analyzed by immunohistochemistry using an anti-ED1 antibody for macrophages and anti-CD4 and anti-CD8 antibodies for lymphocytes.
Measurement of systolic blood pressure and proteinuria. Systolic blood pressure was assessed before death in trained conscious animals by a tail-cuff method as previously described (15). The day before euthanasia, the animals were housed individually in metabolic cages for a 24-h urine collection. Urinary protein was measured using a pyrogallol red microtiter plate technique as previously described (15). Proteinuria is expressed as milligrams protein per 24 hours.
Euthanasia. The animals were anesthetized with 0.1 mg ketanest-0.01 mg xylazin/100 g body wt (ketamin 10%, WDT, Garbsen, Germany; Rompun 2%, Bayer Vital, Leverkusen, Germany). Following a midline abdominal incision, 5–10 ml of heparinized blood were drawn from the abdominal aorta and the kidneys were subsequently perfused with 30 ml of ice-cold PBS. Materials and tissues were subsequently processed as described in the following sections.
Blood analysis. Red and white blood cell counts were determined in an automated cell counter (XE 2,100, Sysmex, Norderstedt, Germany) using a standard fluorescent flow cytometry technology (15). Plasma and urine creatinine and plasma urea were measured spectrometrically in enzyme-based assays (16). The glomerular filtration rate was calculated on the basis of serum and urinary creatinine concentrations and the corresponding urine volume and is expressed as milliliters per minute per 100 grams body wt.
Light and immunohistochemical microscopy. For histological examination, cortical tissue was fixed in 10% neutral buffered formalin or snap-frozen in liquid nitrogen. All histological studies were performed in a blinded fashion. Three-micrometer paraffin sections were stained with periodic acid-Schiff reaction to analyze tubulointerstitial and glomerular fibrosis by computer-based morphometric analysis. Renal sections were examined on a Leica DM LB2 light microscope (Leica Microsystems, Wetzlar, Germany) connected to a PL-A662 video camera and the Axiovision 2.05 image-analysis system (both Karl Zeiss Vision, Munich, Germany) using a 10 x 10 orthographic grid overlaid on digital images. The relative degree of tubulointerstitial fibrotic lesions, i.e., matrix deposition, tubular atrophy and dilation, was calculated in 15 randomly selected cortical areas per animal observed at x200 magnification graded on the following scale: 0 = normal, 1 = lesions involving <10% of cortical area, 2 = lesions involving 11–30% of cortical area, 3 = lesions involving 31–50% of cortical area, and 4 = lesions involving >50% of cortical area, respectively (15). The relative degree of glomerular matrix expansion was evaluated by rating the mesangial matrix occupying the area of 15 glomeruli/animal, using the following scoring system: 0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100% (15).
For analysis of cell infiltration, renal tissue was immunohistologically stained for CD4- and ED1 (macrophages)-positive cells. Paraffin-embedded tissues were incubated with a primary mouse anti-CD8 and mouse anti-ED1 antibody and cryoconserved material with a primary mouse anti-CD4 antibody, respectively (all from Serotec, Oxford, UK). Further staining was performed using a standard alkaline phosphatase anti-alkaline phosphatase technique (DakoCytomation, Hamburg, Germany), as previously described (15). Cell infiltration was evaluated by counting infiltrating cells in at least 15 glomerular sections (glomerular cell infiltration) and at least 15 randomly selected cortical areas (tubulointerstitial cell infiltration) from each rat. Tissue sections were observed at x200 magnification.
Cortical and glomerular TGF-1, fibronectin, and PAI-1 protein expression. Glomeruli from individual rats were isolated by a graded sieving technique (160-, 125-, and 71-μm mesh metal sieves), as described previously (17). For cultures of renal cortical tissue, a piece of cortical tissue was weighed and minced extensively with a razor blade (16). Glomeruli or cortical tissues were suspended in DMEM supplemented with 0.1 U/ml insulin, 100 U/ml penicillin, and 100 μg/ml streptomycin. Glomeruli were cultured at a density of 2,000 glomeruli/ml for 48 h and minced cortical tissue at a density of 10 mg/ml, respectively. After 48-h incubation at 37°C/5% CO2, supernatants were harvested and stored at –70°C until analysis of TGF-1, fibronectin, or PAI-1 content. TGF-1 content of the culture supernatant was measured after acid activation, using a commercially available ELISA kit (TGF-1 Duoset, R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. Fibronectin and PAI-1 levels were measured with modified inhibitory ELISA, according to published methods (22). Three samples from each rat were analyzed.
Cortical TGF-1, fibronectin, and PAI-1 mRNA expression. Cortical total RNA was extracted with TRIzol reagent (GIBCO BRL, Berlin, Germany) according to the manufacturer's instructions. The mRNA expression of TGF-1, fibronectin, PAI-1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by a "two-step" RT-PCR (18). A cDNA copy was created with reverse transcriptase from an RNA PCR Core kit (Roche, Applied Biosystems, Branchburg, NJ). Real-time PCR was performed using the LightCycler System and SYBR Green I as the dsDNA binding dye (Roche Diagnostics, Mannheim, Germany). The following primer pairs were used (annealing temperature in parentheses): TGF-1: sense 5'-GGTGGCAGGCGAGAGCGCTGA-3' and antisense 5'-GGCATGGTAGCCCTTGGGCT-3' (64°C); fibronectin: sense 5'-GGTCCAAATCGGTCATGTTCCCA-3', antisense 5'-CGTAATGGGAAACCGTGTAAGGG-3'(64°C); PAI-1: sense 5'-CAGCATGTGGTCCAGGCCTCCAAA-3', antisense 5'-TGTGCCGCTCTCGTTCACCTCGATCT-3' (64°C); and GAPDH: sense 5'-CCATCTTCCAGGAGCGAGAT-3', antisense 5'-GATGACCTTGCCCACAGCCT-3' (59°C). For analysis, a relative quantification method was used as previously described (18, 19, 29). Briefly, amplification is described as N = N0·ECP, where N is the number of amplified molecules, N0 is the initial number of molecules, E is amplification efficiency, and CP is the crossing-point deviation, expressed as CP = CPtarget – CPGAPDH. In this quantification method, measured CP is defined as the point at which the fluorescence rises above the background fluorescence. Finally, N0 of the target gene was calculated and compared relative to expression of GAPDH mRNA as the housekeeping gene.
Statistical analysis. The results are expressed as means ± SE. Statistical analysis between groups was performed by ANOVA and subsequent Mann-Whitney U-testing. A P value <0.05 was considered significant.
RESULTS
Body weight, food intake, and blood cell analysis. At the end of the experiment, body weight was 588 ± 10 g in the normal (2-K Control) and 541 ± 12 g in the uninephrectomized control group (1-K Control), respectively. In comparison, final body weight was significantly lower in all nephritic groups (cGS: 492 ± 18 g, cGS+MMF: 469 ± 8 g, cGS+Ena: 509 ± 12 g, cGS+MMF+Ena: 476 ± 12 g, P < 0.05 vs. 2-K Control), indicating chronic renal disease and insufficiency. The differences between the nephritic groups were not significant. Food and water intakes did not differ between the groups throughout the experiment. Blood leukocyte, lymphocyte, and platelet numbers were not significantly different between any groups (data not shown). Compared with the uninephrectomized control animals, blood hemoglobin concentration was significantly lower in the untreated sGC animals (13.0 ± 0.5 vs. 14.7 ± 0.4 g/dl, P < 0.05). Treatment with enalapril partially prevented the decrease in hemoglobin levels (13.9 ± 0.1 g/dl, P < 0.05 vs. cGS), whereas MMF administration further lowered hemoglobin concentrations (cGS+MMF: 10.7 ± 0.6 g/dl, cGS+MMF+Ena: 10.9 ± 0.3 g/dl, P < 0.05 vs. cGS).
Disease severity in anti-thy1-induced chronic glomerulosclerosis 16 wk after disease induction. Compared with the normal controls, anti-thy1-induced chronic glomerulosclerosis 16 wk after disease induction was characterized by significant increases in proteinuria (149 ± 14 mg/day, Fig. 1A); blood pressure (145 ± 7 mmHg, Fig. 1B); histological tubulointerstitial matrix accumulation (matrix score 2.5 ± 0.3, Figs. 2 and 3A); protein expression of TGF-1 (189 ± 31 pg/ml); fibronectin (2,921 ± 605 ng/ml); and PAI-1 (294 ± 58 ng/ml) (Fig. 3, B–D; all P < 0.05 vs. 1-K Control and 2-K Control). TGF-1 mRNA was increased 2.9-fold, fibronectin 3.9-fold, and PAI-1 2.6-fold (all P < 0.05 vs. 1-K Control and 2-K Control). At the glomerular level, cGS animals showed increased histological matrix accumulation (matrix score 2.2 ± 0.1, Figs. 2 and 4A) and protein expression of TGF-1 (171 ± 15 pg/ml) and fibronectin (3,927 ± 239 ng/ml) (Fig. 4, B and C) (all P < 0.05 vs. 1-K Control and 2-K Control). Glomerular PAI-1 expression did not differ among all groups (Fig. 4D).
Renal matrix accumulation was paralleled by increases in infiltration with ED1-, CD4-, and CD8-positive cells into the tubulointerstitium (ED1: 186 ± 24 cells/section, CD4: 112 ± 14 cells/section, and CD8: 83 ± 11 cells/section) (Fig. 5, A–C) and the glomeruli (ED1: 3.1 ± 0.4 cells/cross section, CD4: 3.6 ± 0.4 cells/cross section and CD8: 0.4 ± 0.1 cells/cross section) (Fig. 6, A–C; all P < 0.05 vs. 1-K Control and 2-K Control). Moreover, untreated cGS animals showed significantly impaired renal function as documented by increased blood creatinine (1.2 ± 0.3 mg/dl) and urea concentrations (157 ± 53 mg/dl) and decreased glomerular filtration rate (0.32 ± 0.06 ml·min–1·100 g body wt–1) (Fig. 7, A–C; all P < 0.05 vs. 1-K Control and 2-K Control).
Effect of single treatment with MMF or enalapril on anti-thy1-induced chronic glomerulosclerosis. Compared with the untreated cGS animals, both the single MMF and enalapril treatments significantly limited disease progression (Figs. 1–7, P < 0.05 for all parameters vs. cGS, expect cortical PAI-1 protein) and overall to a very similar relative degree (P = not significant for all parameters cGS+MMF vs. cGS+Ena). This was documented on the basis of proteinuria blood pressure, histological tubulointerstitial fibrosis, protein expression of TGF-1 and fibronectin; tubulointerstitial mRNA expression of TGF-1, fibronectin, and PAI-1; glomerular histological matrix accumulation and protein expression of TGF-1 and fibronectin; immunohistological tubulointerstitial and glomerular cell infiltration with ED1-, CD4-, and CD8-positive cells; blood creatinine and urea concentrations; and glomerular filtration rate (Figs. 1–7). The relative changes with each intervention are summarized in Table 1.
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Effect of combined MMF and enalapril treatment on anti-thy1-induced chronic glomerulosclerosis. Compared with the single treatment groups, combined therapy with MMF and enalapril yielded very similar relative changes in the disease severity of anti-thy1-induced chronic glomerulonephritis (Figs. 1–7, P < 0.05 vs. cGS for all parameters, but P = not significant for all parameters vs. cGS+MMF and cGS+Ena). As shown in Table 1, in none of the multiple measures indicating renal function, tubulointerstitial and glomerular matrix accumulation, and cell infiltration the combined MMF and enalapril intervention was superior to single treatment with MMF or enalapril.
Taken together, the results show that the primarily anti-inflammatory approach using MMF is as effective as the anticipated mainly anti-fibrotic approach using a high dose of enalapril on renal matrix protein expression and accumulation, cell infiltration, and kidney function in experimental anti-thy1-induced chronic-progressive glomerulosclerosis. The dual anti-inflammatory/anti-fibrotic intervention does not yield additive renoprotective effects on renal fibrosis, cell infiltration, and insufficiency in this model of renal progression.
DISCUSSION
Progression to end-stage organ failure is a common feature of both experimental and human chronic renal diseases (10, 15, 23, 33). Ongoing loss of organ function occurs independently of whether the underlying disorder is glomerulonephritis, diabetes, or hypertension, suggesting that a common intrarenally determined program is operating. The model used here, anti-thy1-induced chronic glomerulosclerosis in the rat, is an experimental paradigm for a "one-hit, self-progressing kidney disease" as it is seen in patients who progress after a single episode of glomerulonephritis (10, 15). This contrasts with diabetic and hypertensive nephropathies, where continual or repeated extrarenal injurious stimuli drive the decline in renal function.
Using pharmacological interventions intended to target primarily tissue inflammation or fibrosis, our results suggest that tubulointerstitial cell infiltration and matrix protein expansion mediate renal progression by similar pathways. Although one would expect that anti-inflammatory MMF would be superior in reducing renal lymphocyte and macrophage infiltration and anti-fibrotic intervention by enalapril would be superior in reducing renal matrix protein accumulation, tubulointerstitial inflammation and tubulointerstitial fibrosis were found to be reduced with almost equal efficiency by both drugs. In addition, if renal inflammation and fibrosis represent complementary pathways leading to end-stage renal disease, one would expect an additive renoprotective effect with combination therapy. However, dual therapy did not produce additional beneficial effects on the progressive course of anti-thy1-induced glomerulosclerosis based on disease measures including proteinuria, blood pressure, tubulointerstitial and glomerular TGF- overexpression, matrix protein accumulation, and infiltration of lymphocytes and macrophages. In addition, similar therapeutic reductions in plasma creatinine and urea levels and increases in glomerular filtration rate were seen with mono- or dual therapy.
In the current understanding of renal disease, we separate inflammatory from fibrotic renal disease relatively clearly, although we know that inflammatory kidney disorders such as lupus nephritis are also characterized by marked matrix protein expansion (14), and fibrotic kidney diseases such as hypertensive and diabetic nephropathies show relevant infiltrations with lymphocytes and macrophages (23, 33). In addition to organ transplantation, MMF is used to treat inflammatory disease and the antihypertensive enalapril is used as an anti-fibrotic drug in organ protection (13, 17, 23, 33). However, research has revealed that MMF has important anti-fibrotic and enalapril has notable anti-inflammatory properties. MMF has been shown to inhibit directly basal and TGF--induced matrix protein expression of renal cells in vitro (5). Another important anti-fibrotic action of MMF is direct inhibition of the proliferation of renal cell types (23, 33). In many kidney diseases, renal cell proliferation precedes extracellular matrix protein expansion (7). Inhibition of lymphocyte and macrophage infiltration by MMF appears to prevent these cells from releasing a number of factors within the kidney that would amplify renal TGF- overexpression and matrix protein accumulation (15, 23, 33). On the other hand, enalapril, in addition to blocking angiotensin II's profibrotic effects, blocks proinflammatory actions of this octapeptid, including direct induction of reactive oxygen species, chemokines, and the transcription factor nuclear factor (NF)-B (11, 23, 31). The similarity of therapeutic effects with MMF and enalapril alone seen in this study supports the idea that these two drugs are neither solely "anti-inflammatory" nor solely "anti-fibrotic" and that renal tissue cell infiltration and matrix protein expansion represent functionally very similar pathways.
We also addressed the question of whether a combined anti-inflammatory/anti-fibrotic approach yields an additive therapeutic effect. Mechanistic additivity occurs when one pathway is maximally blocked by one intervention and a second pathway is blocked by a second intervention, leading to enhanced disease reduction. This contrasts with therapeutic additivity whereby combining two drugs that inhibit the same system, both at suboptimal doses, enhances efficacy. In the present study, enalapril was given at a dose shown to be maximally therapeutic in acute anti-thy1 glomerulonephritis (2, 17). While MMF may not have been given at maximal doses, addition of MMF to enalapril still should have yielded an enhanced therapeutic effect if these therapies target different pathways. By inquiring into a mechanistic additivity, our investigation goes beyond previous studies documenting additive renoprotection by MMF and RAS inhibition in nonimmune-mediated models of renal disease (8, 21, 32). To date, these studies have included interventions with the ACE inhibitor lisinopril or the AT1 antagonist losartan and MMF in models of renal mass reduction (8, 21) as well as losartan and MMF in a model of chronic cyclosporine nephropathy (32). However, lisinopril and losartan were used in low or moderate doses in these experiments compared with the doses needed to more effectively block the renin-angiotensin system as shown by others (17, 30). Therefore, the additivity seen in 5/6 nephrectomy and cyclosporine nephropathy may be therapeutic rather than mechanistic. While we feel it is likely that the additivity seen in these models was due to use of less than maximal doses of lisinopril and losartan, it remains possible that the results are due to differences in the animal models used (8, 21, 32) or timing of the pharmacological intervention (8).
This study provides in vivo data suggesting that inflammatory and fibrotic pathways are closely linked and thereby expands findings of previous, mostly in vitro studies. At the cellular or molecular level, there have been identified a number of functional crossing points between the two systems and it is likely that their interaction takes place continuously and through several pathways. At the extracellular level, it has been shown that the inflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)- can directly induce renal cell TGF- expression and matrix protein production, renal cell proliferation, and even epithelial-myofibroblast transdifferentiation, an important feature in tubulointerstitial fibrosis (6, 20, 27). On the other hand, profibrotic growth factors such as angiotensin II have been found to enhance renal macrophage infiltration via induction of the chemokine RANTES and to directly induce the key proinflammatory transcription factor NF-B (11, 23, 31). Furthermore, IL-1 and TNF- production by cultured monocytes is amplified in the presence of extracellular matrix proteins, particularly fibronectin (4). Adding more complexity and redundancy, proinflammatory and profibrotic factors share intracellular signaling pathways involving protein tyrosine kinases, NF-B, and the mitogen-activating protein kinases (MAPK) c-Jun and p38 MAPK (11, 24–26).
In conclusion, MMF slows the progressive course toward chronic renal fibrosis and insufficiency as effectively as a high dose of enalapril in the model of anti-thy1-induced chronic glomerulosclerosis. Combining the intended primarily anti-inflammatory and anti-fibrotic interventions does not yield additive effects on renal function, matrix protein expansion, and cell infiltration. The findings suggest that tubulointerstitial inflammation and fibrosis represent functionally very similar or closely related pathways mediating the progression of renal disease.
GRANTS
This study was supported in part by grants from Hoffmann-La Roche (Grenzach-Whylen, Germany) and the Deutsche Forschungsgemeinschaft (PE 558/2–1 and PE 558/2–3, Bonn, Germany).
ACKNOWLEDGMENTS
We thank Nancy Noble for valuable comments on the manuscript.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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