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Chronic cyclosporine (CsA) nephrotoxicity is characterized by tubulointerstitial fibrosis. Pirfenidone (PFD) is a novel antifibrotic compound that was shown to prevent and even reverse fibrosis. The mechanism of action of PFD is unclear but involves inhibition of transforming growth factor-β (TGF-β). Salt-depleted rats were administered CsA, CsA + PFD, vehicle (VH) or VH + PFD and sacrificed at 28 days. Physiologic and histologic changes were studied in addition to TGF-β1, plasminogen activator inhibitor-1 (PAI-1) and biglycan mRNA expressions by Northern blot. TGF-β1 immunohistochemistry was also performed. Treatment with PFD ameliorated CsA-induced fibrosis by about 50% (p < 0.05). CsA-induced decrease in creatinine clearance improved with PFD but the difference was not significant. TGF-β1, PAI-1 and biglycan mRNA expressions increased with CsA (p < 0.05 vs. VH) but strikingly improved with PFD treatment (p < 0.05 vs. CsA), which brought the levels down to VH levels. PFD treatment also decreased TGF-β1 protein expression by 80%. These results demonstrate that PFD can attenuate renal fibrosis in this model. PFD was associated with a decrease in TGF-β1 expression, which, in turn, was associated with a decrease in matrix deposition. These experiments suggest that PFD can be clinically useful for preventing chronic CsA nephrotoxicity and may prove to be helpful in other progressive renal diseases.
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Cyclosporine (CsA) remains an important immunosuppressive drug used in transplantation and autoimmune diseases (1). However, the major dose-limiting adverse effect of long-term CsA administration is chronic nephrotoxicity, an irreversible renal lesion characterized by striped tubulointerstitial fibrosis, tubular atrophy and afferent arteriolar hyalinosis (2). The mechanisms leading to fibrosis in CsA nephrotoxicity remain poorly understood. Our previous studies using an experimental model of chronic CsA nephrotoxicity have shown that the fibrogenic cytokine transforming growth factor-β1 (TGF-β1) is involved in the fibrosis of this model by increasing extracellular matrix (ECM) synthesis and by decreasing ECM degradation through increasing the activity of plasminogen activator inhibitor-1 (PAI-1) (3). Apoptosis seems to play a role in this model and is directly correlated with tubulointerstitial fibrosis where the loss of cells may prevent the ability of the kidney to remodel effectively (4).
Attempts at preventing or decreasing the amount of fibrosis in chronic CsA nephrotoxicity and other models of fibrosis have been, at best, only partially effective. In this model, we have shown that the renin-angiotensin system (RAS) was up-regulated and that blocking the activity of the RAS resulted in a beneficial effect in reducing fibrosis by decreasing the expression of TGF-β1 and various ECM components (5). In addition, L-arginine treatment protected CsA-treated animals from impaired renal function and from the development of tubulointerstitial fibrosis (6). This beneficial effect seems to be mediated, at least partly, by decreasing the expression of TGF-β1 and ECM proteins. All this evidence point to a central role for TGF-β1 in the development of fibrosis in this model and, thus, attempts to decrease TGF-β1 expression should prove to be beneficial in chronic CsA nephrotoxicity. Moreover, strategies to prevent ECM accumulation in the interstitium, as well as in the glomeruli, have previously aimed at blocking the action or activation of TGF-β (7). The efficacy of this approach has been supported by a number of in vivo and in vitro studies (8–12).
Pirfenidone (PFD) is a recently developed antifibrotic molecule [5-methyl-1-phenyl-2(1H)-pyridone) that was shown to prevent and even reverse ECM accumulation in experimental pulmonary fibrosis (13) and peritoneal sclerosis (14). In patients with end-stage pulmonary fibrosis, PFD not only improved the survival rate, but also restored pulmonary function (15). More recently, the beneficial effect of PFD was demonstrated in experimental progressive renal diseases such as monoclonal anti-Thy-1 antibody glomerulonephritis (16), the subtotal nephrectomy remnant kidney model (17), and in unilateral ureteral obstruction (18), a well-characterized model of tubulointerstitial fibrosis. In these models, PFD not only prevented the progression of sclerotic glomerular and tubulointerstitial lesions, but also decreased collagen accumulation in the kidney.
At the moment, the mechanism underlying the antifibrotic action of PFD remains poorly understood (19). However, suppression of increased TGF-β and inhibition of its effect has been postulated as one possibility as shown in the bleomycin hamster model of lung fibrosis (20, 21) and in the remnant and postobstruction kidney models (18). Since the fibrosis of chronic CsA nephrotoxicity is associated with an up-regulated TGF-β expression and since the beneficial effect of PFD is, at least partly, mediated by TGF-β, we investigated the role of PFD on renal function and histology in an experimental model of chronic CsA nephrotoxicity. Our results suggest that PFD exerts a protective function in this model and that it decreases the expression of TGF-β1 and ECM proteins.
Materials and Methods
Thirty-two male Sprague-Dawley rats (Charles River, Wilmington, MA, USA) weighing 325–350 g were used. They were housed in individual cages in a temperature- and light-controlled environment, and received a low-salt diet (0.05% sodium, Teklad Premier, WI, USA) and were allowed free access to tap water. After 1 week on a low-salt diet, they were assigned to one of four groups of 8 animals. The experimental groups were: Group A, vehicle (VH) olive-oil control; Group B, VH and PFD; Group C, CsA only and Group D, CsA and PFD. The animals were pair fed to the amount of food consumed the day before by Group C (CsA only). On day 28, systolic blood pressure was measured by tail plethysmography (Natsume Seisakusho Co. Ltd, Tokyo, Japan) and 24-h urine samples were collected in metabolic cages (Nalge Company, Rochester, NY, USA). The following day, rats were anesthetized with intraperitoneal ketamine, the abdomen was opened through a midline incision and the aorta was cannulated retrogradely below the renal arteries with an 18-gauge needle. With the aorta occluded by ligation above the renal arteries and the renal veins opened by a small incision for outflow, the kidneys were perfused with 20 mL of cold heparinized saline. The left kidney was removed and processed for light microscopy. After removing the right kidney, the cortex was dissected from the medulla, and the cortex was processed for RNA analysis. After the experiment, the animals were euthanized by deep anesthesia with ketamine followed by exsanguination.
CsA (Novartis Research Institute, East Hanover, NJ, USA) was diluted in olive oil and administered subcutaneously (sc) at a dose of 7.5 mg/kg/day. The vehicle group received olive oil 1 mL/kg/day sc. PFD was generously supplied by Dr Solomon Margolin from Marnac Inc., Dallas, TX. PFD was added in powder form to a low-salt diet at 0.5% w/w (500 mg PFD/100 g diet) and administered to animals at a dose of 250 mg/kg/day.
Blood was collected from the jugular vein in plastic syringes transferred to metal-free tubes and chilled on ice. Plasma was harvested immediately by centrifugation at 4 °C and stored at − 70 °C until determined. Urinary and plasma creatinine was measured by a Cobas autoanalyzer (Roche Diagnostics, Division Hoffman-La Roche Inc., Nutley, NJ, USA). Urinary osmolality was measured by freezing point depression (Osmette A, Precision Systems Inc., Natick, MA, USA). Creatinine clearance (Ccr) was calculated using a standard formula. CsA blood level was measured by a monoclonal radioimmunoassay for CsA (Incstar Co., Stillwater, MN, USA).
Rats were orally given PFD at a dose of 250 mg/kg/day or water for 4 days (n= 4 for each group). After 1 h from the last administration with PFD or water, the animals were anesthetized with ketamine (50 mg/kg) intraperitoneally. Following that, polyethylene catheters (PE-50) were inserted into the jugular vein for blood-sample collections and into the femoral vein for CsA infusion. CsA was diluted with saline and administered intravenously through the femoral vein over 2 min at a dose of 7.5 mg/kg. Blood samples were drawn (0.2 mL sharp in EDTA tube) from the jugular vein at 15, 60, 180, 360 and 1440 min after the infusion of CsA. The animals were kept under light anesthesia up to 6 h after the injection.
Tissue was fixed in 10% buffered formalin and embedded in paraffin. Sections 2–4 μm thick were stained with periodic acid-Schiff's reagent and trichrome stain and examined by light microscopy. The histological findings were subdivided into three categories: tubular injury, nephrocalcinosis and tubulointerstitial fibrosis. Findings ascribed to tubular injury included cellular and intercellular vacuolization, tubular collapse (unassociated with interstitial fibrosis or tubular basement membrane thickening) and tubular distention. Nephrocalcinosis was defined as the presence of calcium crystals within the tubular lumen. A minimum of 30 fields at 200× magnification were assessed and graded in each biopsy using a 0–3 semiquantitative score by an observer masked to treatment groups using a color image analyzer (Nikon E400, Nikon Inc., Tokyo, Japan; Pixera Professional digital camera, Los Gatos, CA, USA; Macintosh Powerbook G3, NIH Image vs. 1.5). Tubulointerstitial fibrosis was estimated using the following score: 0 = normal interstitium; 0.5 = < 5% of areas injured: minimal interstitial fibrosis with slight disruption and mild interstitial thickening between tubules; 1 = 5–20% of areas injured: mild to moderate fibrosis with mild interstitial thickening between tubules; 1.5 = 21–35% of areas injured; 2 = 36–50% of areas injured: severe fibrosis with tubular dilation, significant interstitial thickening between tubules and thickening of the tubular basement membrane; 2.5 = 51–65% of areas injured and 3 = > 65% of areas injured. The extent of changes in cortical tubules was graded according to the following score: 0 = no tubular injury; 0.5 = < 5% of tubules injured; 1 = 5–20% of tubules injured; 1.5 = 21–35% of tubules injured; 2 = 36–50% of tubules injured; 2.5 = 51–65% of tubules injured and 3 = > 65% of tubules injured. Nephrocalcinosis was assessed using the following scoring system: 0 = no nephrocalcinosis present, 0.5 = < 5% of tubules found; 1 = 5–10% of tubules found; 1.5 = 11–15% of tubules found; 2 = 16–20% of tubules found; 2.5 = 21–25% of tubules found and 3 = > 25% of tubules found.
Northern blot analysis
Renal tissue was finely minced with a razor blade on ice, and then homogenized in TRIzol reagent (GibcoBrl, Grand Island, NY, USA). RNA extraction was performed according to the manufacturer's protocol. After resuspension in Tris-ethylenedinitrilo-tetracetic and (EDTA) buffer, RNA concentrations were determined using spectrophotometric readings at Absorbance260. Thirty micrograms of RNA from each individual rat kidney were electrophoresed in each lane in 0.9% agarose gels containing 2.2 m formaldehyde and 0.2 m Mops (pH 7.0) and transferred to a nylon membrane (ICN Biomedicals, Costa Mesa, CA, USA) overnight by capillary blotting. Nucleic acids were cross-linked by ultraviolet irradiation (Stratagene, La Jolla, CA, USA). The membranes were prehybridized for 2 h at 42 °C with 50% formamide, 10% Denhardt's solution, 0.1% sodium dodecyl sulphate (SDS), 5× standard saline citrate (SSC), and 200 μg/mL denatured salmon sperm DNA. They were then hybridized at 42 °C for 18 h with cDNA probes labeled with 32P-dCTP by random oligonucleotide priming (Boehringer Mannheim). The blots were washed in 2× SSC, 0.1% SDS at room temperature for 15 min and in 0.1× SSC, 0.1% SDS at 50 °C for 15 min. Films were exposed at − 70 °C for different time periods to ensure linearity of densitometric values and exposure time. Autoradiographs were scanned on an imaging densitometer (GS-700, Bio-Rad Laboratories, Hercules, CA, USA). RNA from each individual animal, and not pooled RNA, was analyzed. Each band represented data from an individual animal. The density of bands for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to control for differences in the total amount of RNA loaded onto each gel line. For quantitative purposes, the values were divided by the density of bands for GAPDH in the same lane. The cDNA probes used for Northern were: a mouse TGF-β1 cDNA probe (plasmid MUI5) (22); a rat PAI-1 cDNA probe (plasmid pBluescript SK(–)) (23); a human biglycan cDNA probe (plasmid P16) (24); and a rat GAPDH cDNA probe (plasmid pBluescript KS II) (25).
To examine the expression of TGF-β1, immunohistochemical study was performed using the avidin-biotin-peroxidase complex technique (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). The kidney tissues were embedded in OCT compound (Sakura, Tokyo, Japan) and snap-frozen in liquid nitrogen. Cryostat sections (4–8 μm thick) were washed with PBS and treated with 0.3% H2O2 in PBS for 20 min After wash and blocking with 1.5% normal goat serum, the specimens were incubated overnight at 4 °C with primary antibody diluted in 1% bovine serum albumin (BSA)/PBS: rabbit polyclonal anti-TGF-β1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After being rinsed with 1% BSA/PBS, the sections were incubated with biotinylated anti-rabbit IgG for 2 h, then with the avidin-biotin-peroxidase complex. Nonimmune rabbit serum was used as negative control and gave no significant staining. In these experiments, the slides were read in a blinded fashion and analyzed semi-quantitatively (0–100%) using photoimage analysis according to the degree of positive staining (area of TGF-β1 positive tubule and interstitium/area of examined section × 100).
Results are presented as mean ± standard error. Comparisons between groups were done by analysis of variance (Kruskal–Wallis test, followedby the Tukey test). The level of statistical significance was chosen asp < 0.05.
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The introduction of CsA into clinical practice has markedly improved short-term allograft survival and, as a result, CsA-based immunosuppressive regimens continue to be a mainstay in organ transplantation. Unfortunately, CsA use can lead to an irreversible renal lesion characterized by tubular atrophy, tubulointerstitial fibrosis and afferent arteriolopathy (1–3). In this experiment, we examined an animal model of chronic CsA nephrotoxicity with pathologic features similar to the human lesion (26). Although the mechanisms responsible for CsA-induced kidney damage are not well understood, the fibrogenic process certainly leads to the permanent loss of the normal integrity and function of the kidney. Furthermore, interstitial fibrosis represents a final common pathway associated with a variety of progressive kidney disorders (27). It is for this reason that pharmacologic interventions that can reverse or prevent the progression of interstitial fibrosis are needed. Thus, the search for effective antifibrotic agents is of great importance not only for elucidating the mechanism of CsA-induced fibrosis but also for ameliorating the renal fibrosis seen in various progressive renal diseases.
Fibrotic diseases are characterized by the accumulation of ECM components (27). The current state of our knowledge suggests that TGF-β overexpression is largely responsible for this accumulation through its actions to induce production of ECM, inhibit its degradation and increase integrin expression on cells, thereby enhancing ECM deposition (7). Although TGF-β is essential for normal tissue repair following injury, sustained or excessive expression of TGF-β is considered to be clearly causal and can be viewed as the final common element in the pathogenesis of human and experimental fibrotic diseases (7). In this experimental model of chronic CsA nephrotoxicity, we have previously shown an elevated expression of TGF-β1, the plasmin protease system inhibitor PAI-1 and various ECM components (3). Our group, in addition to others, has then suggested that TGF-β plays an important role in the pathogenesis of this disease (2–6, 28).
There is considerable interest in potential therapeutic applications to manipulate TGF-β actions either directly or indirectly. Some of the directly targeted therapies have included neutralizing antibodies to TGF-β (8), inhibition of TGF-β synthesis with antisense oligonucleotides (9), the use an inhibitory molecule against TGF-β such as the small proteoglycan decorin (10, 11), or gene therapy with a chimeric inhibitor molecule TGFβRII/Fc (12). Other therapies have included low protein intake, angiotensin II blockade, or L-arginine supplementation that were shown to decrease TGF-β overproduction and, as a result, were effective in decreasing the accumulation of ECM (5, 6, 29–31). In this experimental model of chronic CsA nephrotoxicity, we were able to show a beneficial effect with angiotensin II blockade and L-arginine supplementation on the expression of TGF-β1 and ECM proteins, in addition to an amelioration in the CsA-induced renal fibrosis (5, 6).
In the present study, we evaluated an antifibrotic agent, PFD, for its ability to block kidney fibrosis in chronic CsA nephrotoxicity. We were able to clearly document the significant ameliorating effect of PFD on CsA-induced renal fibrosis. The PFD-treated animals showed a striking attenuation of the CsA-induced histological changes with very little fibrosis in the interstitium. On the other hand, while PFD clearly improved the CsA-induced decline in GFR, the difference did not achieve statistical significance. The beneficial effect seen with PFD on CsA-induced renal fibrosis was independent of renal hemodynamics since PFD treatment did not result in significant changes in GFR or serum creatinine. The findings in this study are consistent with a recently published observation in streptozotocin-diabetic rats where PFD administration had a beneficial effect on renal fibrosis but failed to improve GFR or renal blood flow (32). This is also in accord with our previous findings in the same model where the beneficial effect of Ang II blockade on CsA-induced renal fibrosis was independent of its effect on renal hemodynamics (5).
The mechanisms for the ameliorating effect of PFD on CsA-induced fibrosis are not well understood. However, PFD administration did not alter CsA blood levels and AUC, thus excluding the possibility that PFD could have exerted its beneficial effect by altering CsA pharmacokinetics. Our data also indicate that treatment with PFD resulted in normalization of the mRNA levels of the ECM proteins biglycan and PAI-1 almost down to the VH control levels. Turning off the mRNA expression of the ECM proteins correlated very well and was coincident with the decrease in renal fibrosis, indicating that blocking renal fibrosis by PFD can prevent progressive renal disease. Our data also indicate that CsA-treated kidneys exhibit an increased TGF-β1 mRNA and protein levels, and that PFD treatment prevented that increase. Thus, it seems possible that the inhibition of ECM expression and the resultant amelioration in renal fibrosis and restoration of renal function by PFD may be mediated, at least partly, in part by the suppression of TGF-β1. However, it remains unclear whether the decreased TGF-β1 expression is due to a direct effect of PFD and/or through a secondary mechanism that results in the attenuation of fibrosis.
In other experimental models in which PFD treatment was applied such as the anti-Thy-1-induced glomerulonephritis, the model of subtotal 5/6 nephrectomy, and the unilateral ureteral obstruction model, the beneficial effect of PFD was also associated with a decrease in TGF-β expression (16–18). In the bleomycin hamster model of lung fibrosis, PFD treatment suppressed the overexpression of TGF-β production at the transcriptional level and subsequently down-regulated the bleomycin-induced overexpression of procollagen genes in the lung (20, 21). It is also possible that PFD treatment may act directly on interstitial cells responsible for TGF-β production such as macrophages, fibroblasts and epithelial cells and compromise their ability to synthesize and release TGF-β. Alternatively, PFD may be directly inhibiting the activity of serine proteases required to convert the latent form of TGF-β into the active form (33). Regardless of the mechanism, these data suggest that the beneficial effect of PFD is related, at least partly, to a down-regulated expression of TGF-β.
PFD has been shown to inhibit collagen accumulation when such accumulation is excessive (18–21). A study done in myometrial cells showed that PFD significantly inhibited steady-state mRNA levels for both collagen type I and III, which may reflect an inhibitory effect on gene transcription or an increase in mRNA turnover (34). In vivo studies using the hamster model of artificially induced lung fibrosis have shown that PFD causes a marked reduction in proline hydroxylase levels (13). This finding suggests that PFD may reduce the availability of the hydroxyproline required for collagen synthesis and therefore may inhibit collagen synthesis at the translational level as well. However, in contrast to other collagen modulator substances, PFD does not alter the basal levels of collagen content and thus, it may have the ability to normalize the overexpression of ECM components that contribute to fibrosis (35, 36). The accumulation of ECM results from a balance between production and degradation processes. However, in the postobstructed kidney model, PFD did not affect the expression of the matrix metalloproteinase MMP-2; thus, its beneficial effect in this model did not seem to be related to increased ECM degradation (18).
Macrophage infiltration has been implicated as being pathologically important in cellular alterations leading to tubulointerstitial fibrosis (27). Indeed, ED-1 positive cell infiltration was shown to be present in CsA-treated kidneys in the early phases of fibrosis (37). Several cytokines secreted by infiltrating macrophages act as chemoattractants and stimulate fibroblasts proliferation (27). Since TGF-β is known to be a strong chemoattractant for monocytes, this may represent another plausible explanation for the beneficial effect of PFD. Interstitial fibroblasts can then produce ECM proteins with ensuing fibrosis. PFD has been shown to inhibit TGF-β1-induced collagen synthesis by human fibroblasts (38). In a model of obliterative bronchiolitis, PFD treatment was effective in inhibiting fibroblast proliferation by controlling epithelial cell-regulated fibroblast proliferation (39). Other studies have shown that fibroblasts treated with PFD were unable to exit the G1 phase of the cell cycle, which suggests a postreceptor site of action for PFD (34).
Moreover, in in vitro culture of human synovial fibroblasts, PFD was shown to down-regulate the expression of the intercellular adhesion molecule-1 (ICAM-1), which is necessary for the infiltration of inflammatory cells (40). As such, PFD might prevent the progression of interstitial fibrosis by inhibiting cytokine production, such as TGF-β from the inflammatory fibroblasts, which results in the suppression of adhesion molecule expression. In addition, it was shown that PFD scavenges reactive oxygen species (ROS) in in vivo and in vitro conditions (13, 41). As a result, it is possible that PFD could be exerting its anti-inflammatory and antifibrotic effects by being able to scavenge the ROS generated by the influx of inflammatory cells.
Recently, the use of PFD was studied in patients with advanced idiopathic pulmonary fibrosis (15). The results of this study were very encouraging. Patients whose lung function had deteriorated prior to enrollment appeared to stabilize after beginning treatment; few patients had improved oxygenation. PFD also improved the 1- and 2-year survival rate in these patients with end-stage pulmonary fibrosis. The drug was well tolerated with minimal side-effects and no evidence of toxicity, which further suggests that this agent does not affect normal turnover of the extracellular matrix.
We conclude that PFD is a promising, safe, new antifibrotic agent for the prevention and treatment of chronic CsA nephrotoxicity. In this experiment, PFD treatment reduced interstitial fibrosis and was associated with a decrease in the expression of TGF-β1 and ECM proteins. Its beneficial effect has also been observed in other fibrotic diseases, which supports the concept that PFD treatment may offer a new and exciting possibility for retarding the progression of chronic renal diseases. Further studies are needed to determine the clinical efficacy and toxicity of PFD in human renal diseases.