Effect of corticosteroid withdrawal on tacrolimus and mycophenolate mofetil exposure in a randomized multicenter study.

As corticosteroid-sparing protocols are increasingly utilized in kidney transplant recipients, it is crucial to understand potential drug interactions between tacrolimus (TAC) and the effect of corticosteroid withdrawal as well as to characterize dose adjustments of mycophenolate mofetil (MMF) in this setting. This prospective, multicenter, randomized, double-blind study included 397 patients who were randomized on posttransplant day 8 to receive either placebo (CSWD) or corticosteroid continuance (CCS). TAC trough levels at week two posttransplant were significantly greater in the CSWD group whereas TAC doses were comparable to the CCS group. This interaction was not observed in the African American subgroup. Higher serum creatinine and potassium levels were also observed in the CSWD group. MMF dose was significantly reduced in the CSWD group by the investigators because of decreased WBC counts, mostly outside of study protocol criteria, despite similar incidence of neutropenia and reported cytomegalovirus infection. Understanding TAC and MMF exposure in the context of corticosteroid-sparing protocols should allow for improved dosing of immunosuppressants and better management of posttransplant patients.

Cytomegalovirus incidence between everolimus versus mycophenolate in de novo renal transplants: pooled analysis of three clinical trials.

Everolimus (EVR) in heart and renal transplant (RTx) recipients may be associated with a decreased incidence of cytomegalovirus (CMV). A detailed analysis of the association between EVR versus mycophenolic acid (MPA) and CMV events has not been reported. CMV data from 2004 de novo RTx recipients from three-randomized, prospective, EVR studies A2309 (N = 833), B201 (N = 588) and B251 (N = 583) were retrospectively analyzed to identify differences between two EVR dosing groups and MPA. EVR groups received 1.5 mg/day, or 3 mg/day with either standard (SD-CsA) or reduced dose cyclosporine (RD-CsA). Controls received MPA with SD-CsA. CMV prophylaxis was as per center practice. CMV incidence (infection/syndrome, disease, viremia) was captured per local center evaluations. Kaplan-Meier analyses demonstrated that freedom from CMV viremia and infection/syndrome was significantly greater for EVR versus MPA for recipients without CMV prophylaxis. Among recipients who received prophylaxis, freedom from viremia was greater for EVR 3.0 mg; freedom from infection/syndrome was greater for EVR 3.0 and 1.5 mg. Although freedom from organ involvement was numerically greater for EVR, it was not statistically significant. This analysis documents significant reductions in the incidence of CMV infection/syndrome and viremia in EVR-treated de novo RTx recipients, especially those who did not receive CMV prophylaxis versus MPA.

Preconditioning: from experimental findings to novel therapies in acute kidney injury.

Improving the ability of the kidney to tolerate injury through preconditioning is likely to have important clinical implications. Although a number of preconditioning strategies have been studied, ischemic preconditioning (IP) has been studied the most experimentally. The information gathered has helped us shed more light into the mechanisms responsible for this tissue adaptation that confers to tissues a more resistant status. IP is effective within minutes, suggesting that preformed mediators are involved. This is followed by delayed preconditioning, a phenomenon that is less potent but longer acting. Remote preconditioning occurs also in non-affected tissues and can be transferable. A number of mediators and transcription factors have been implicated including kinases, heat shock proteins, nitric oxide and neurogenic pathways, all of which help change the cell into a more resistant phenotype. There is evidence that IP also occurs in the human environment with lessons learned from myocardial ischemia, hepatic resection and cerebral ischemia. Because of the ethical impediment with intentionally applying organ ischemia, there has been an interest in pharmacological preconditioning lately. Exogenously administered erythropoietin was shown to benefit kidneys subjected to different insults. In addition, mesenchymal stem cells-based approaches for the prevention and treatment of acute kidney injury (AKI) are being studied. Calcineurin inhibitors may represent a viable way to reduce ischemia reperfusion injury in transplantation. Translating the experimental findings to the clinical arena remains a challenge. The discovery of new biomarkers for AKI should help initiate therapy early, when therapy could make a difference.

Lipid-based amphotericin in pulmonary zygomycosis: safety and efficacy of high exposure in a renal allograft recipient.

Zygomycosis is associated with a high mortality in immunosuppressed patients. Treatment typically includes surgical resection and administration of intravenous amphotericin B. Success of treatment may require withdrawal of immunosuppression, with risk of graft loss. We report the successful treatment of invasive pulmonary zygomycosis, following initial surgical resection, using very high doses of lipid-based amphotericin B without withdrawal of immunosuppression. The patient received daily doses up to 10 mg/kg/day (51 g cumulatively) of lipid-based amphotericin B along with a brief course of intrapleural amphotericin. Despite immunosuppression not being withdrawn, the patient's kidney allograft function remained stable. We conclude that high doses of lipid-based amphotericin B can be safe and effective as part of the treatment regimen for pulmonary zygomycosis.

Expression of vascular endothelial growth factor and its receptors Flt-1 and KDR/Flk-1 in chronic cyclosporine nephrotoxicity.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen involved in angiogenesis, wound healing, and inflammation. METHODS: Rats placed on low salt diet (LSD) or normal salt diet (NSD) were treated with cyclosporine (CsA) or vehicle (VH) and killed at 7 or 28 days. We studied the expression of VEGF and its receptors Flt-1 and KDR/Flk-1 mRNA by Northern and that of VEGF protein by Western blot. RESULTS: CsA induced VEGF mRNA and protein expressions at 7 and 28 days in LSD rats. At 7 days, CsA up-regulated the expression of Flt-1 and KDR/Flk-1 receptors; however, at 28 days, Flt-1 remained unchanged whereas KDR/Flk-1 expression declined. In NSD rats, in which the lesion did not develop, the expression of VEGF and its receptors remained similar to control. CONCLUSIONS: What causes VEGF to be up-regulated remains unclear. Further studies are needed to study the role of hypoxia and other cytokines in relation to VEGF in this model.

De novo fibrillary glomerulopathy in the renal allograft of a patient with systemic lupus erythematosus.

Lupus glomerulonephritis is a complication of systemic lupus erythematosus, with 10% of the patients developing end-stage renal disease. It is accepted that lupus patients are good candidates for kidney transplantation and that the disease activity is subdued after transplantation due to rigorous immunosuppression, with a low rate of graft loss due to recurrent glomerulonephritis. While recurrent fibrillary glomerulopathy has been reported in renal allografts, de novo disease has not. We report a patient with systemic lupus who underwent a renal transplantation and subsequently lost her allograft due to de novo fibrillary glomerulopathy. Four years after her first kidney transplant, the patient presented with acute deterioration of her renal function. A renal biopsy was performed, and it revealed a focal mesangioproliferative pattern with positive amorphous mesangial immunofluorescence staining for IgG and C3. Congo red staining was negative. Electron microscopy demonstrated the presence of randomly oriented nonamyloid fibrils in the mesangiun. The diagnosis of de novo fibrillary glomerulopathy was made. The patient lost her allograft and received a second cadaveric renal transplant 1 year later. She has had a stable renal function since then.

Effect of nitric oxide modulation on TGF-beta1 and matrix proteins in chronic cyclosporine nephrotoxicity.

BACKGROUND: Chronic cyclosporine (CsA) nephrotoxicity is characterized by interstitial fibrosis and afferent arteriolar hyalinosis. L-arginine (L-Arg), the substrate for nitric oxide (NO) synthase and N-nitro-L-arginine-methyl ester (L-NAME), the NO synthase inhibitor, were shown to modulate acute CsA nephrotoxicity. However, the mechanism of fibrosis in chronic CsA nephrotoxicity remains unclear. Thus, we examined the effect of NO modulation on fibrosis and the expression of transforming growth factor-beta1 (TGF-beta1) and matrix proteins in chronic CsA nephrotoxicity. METHODS: Rats were administered CsA (7.5 mg/kg), CsA + L-Arg (1.7 g/kg), CsA + L-NAME (3.5 mg/kg), vehicle (VH), VH + L-Arg, and VH + L-NAME, and were sacrificed at 7 or 28 days. NO production, physiologic parameters, and histology were studied in addition to the mRNA expression of TGF-beta1, plasminogen activator inhibitor-1 (PAI-1) and the matrix proteins biglycan and collagens type I and IV by Northern and the protein expression of PAI-1 and fibronectin by enzyme-linked immunosorbent assay. RESULTS: While L-NAME strikingly reduced NO biosynthesis and worsened the glomerular filtration rate and CsA-induced fibrosis, L-Arg had the opposite beneficial effect. In addition, the CsA-induced up-regulated expression of TGF-beta1, PAI-1, and the matrix proteins biglycan, fibronectin, and collagen I was significantly increased with L-NAME and strikingly improved with L-Arg. Collagen IV expression was not affected. Also, NO modulation did not affect VH-treated rats. CONCLUSIONS: Chronic CsA nephrotoxicity can be aggravated by NO blockade and ameliorated by NO enhancement, suggesting that NO maintains a protective function. NO modulation was associated with a change in TGF-beta1 expression, which, in turn, was associated with alterations in matrix deposition and matrix degradation through its effect on PAI-1.

Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis.

UNLABELLED: Expression of apoptosis regulatory genes in chronic cyclosporine nephrotoxicity favors apoptosis. BACKGROUND: Chronic cyclosporine (CsA) nephrotoxicity is characterized by interstitial fibrosis, tubular dropout, and loss of cellularity in areas of fibrosis. Apoptosis was found to play a role in CsA-induced fibrosis. We evaluated the role of the death genes p53, Bax, and Fas-L (ligand), survival gene Bcl-2, interleukin-converting enzyme (ICE), and caspase-3. METHODS: Salt-depleted rats were administered CsA 15 mg/kg/day or vehicle (VH) and were sacrificed at 7 or 28 days. Apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling assay. p53 and Bax expressions were evaluated by Northern and Western blot analysis. Fas-L and Bcl-2 expressions were evaluated by immunofluorescence. In addition to ICE mRNA, caspase-3 enzymatic activity was assayed. RESULTS: Although no differences were seen at one week, apoptosis-positive cells increased with CsA at four weeks (P < 0.05) and correlated with tubular atrophy and interstitial fibrosis (r = 0.8, P < 0.05). CsA induced the expression of p53 (P < 0.05) and Bax (P < 0.01) and decreased that of Bcl-2 (P < 0.05). CsA up-regulated Fas-L expression (P < 0.001). ICE mRNA and caspase-3 activity were also increased (P < 0.01). The changes occurred as early as one week and remained statistically significant at four weeks. CONCLUSIONS: Specific apoptotic genes are increased in chronic CsA nephrotoxicity. The balance favors the induction of apoptosis. Increased apoptosis could explain the tubular dropout and loss of cellularity with fibrosis. This then may impair the ability of the tubulointerstitium to remodel. Apoptosis could also contribute to some of CsA immunosuppressive effects on activated lymphocytes.

Angiotensin II blockade decreases TGF-beta1 and matrix proteins in cyclosporine nephropathy.

Angiotensin II (Ang II) is implicated in fibrosis but the precise mechanism of this effect remains unclear. In a model of chronic cyclosporine (CsA) nephropathy, we previously showed that TGF-beta1 plays a role in CsA-induced tubulointerstitial fibrosis and arteriolopathy by stimulating extracellular matrix (ECM) protein synthesis and inhibiting ECM degradation through increasing the synthesis of plasminogen activator inhibitor (PAI)-1. We hypothesized that Ang II contributes to fibrosis by inducing TGF-beta1. Salt-depleted rats were given placebo, CsA alone, CsA + nilvadipine, CsA + hydralazine/hydrochlorthiazide, CsA + losartan (AT1 receptor antagonist) or CsA + enalapril (Ang converting enzyme inhibitor) and were sacrificed at 7 and 28 days. All treated groups achieved similar blood pressures and glomerular filtration rates. The lesion of chronic CsA nephropathy was ameliorated by concomitant therapy with losartan or enalapril at 28 days, a phenomenon not observed in the other treatment groups. Similarly, Ang II blockade resulted in decreased expression of TGF-beta1 and PAI-1 by Northern and ELISA. Similarly, the expression of ECM proteins directly influenced by TGF-beta decreased with Ang II blockade. These results suggest that CsA-induced fibrosis in this model is independent of renal hemodynamics and is mediated, at least partly, through Ang II induction of TGF-beta1 expression.

Sodium depletion enhances fibrosis and the expression of TGF-beta1 and matrix proteins in experimental chronic cyclosporine nephropathy.

The major limitation to the clinical use of cyclosporine (CsA) is renal toxicity. In the past, the lack of an animal model of chronic CsA nephropathy has hampered the study of its pathogenesis. Rats given CsA and placed on a low sodium diet (LSD) develop a histology similar to human lesions of chronic CsA nephropathy, a phenomenon not observed in animals on a normal sodium diet (NSD). We have previously shown that transforming growth factor-beta1 (TGF-beta1) is involved in the CsA-induced renal fibrosis in rats on a LSD. We hypothesized that sodium depletion is critical to the increase in TGF-beta1 expression, which, in turn, results in excessive matrix accumulation. Pair-fed rats were placed on a NSD or LSD, treated with CsA or vehicle, and killed at 7 or 28 days (N = 4 to 6 in each group). All rats achieved similar blood pressure control, and all CsA-treated rats achieved similar CsA blood levels. However, while CsA did not affect creatinine clearance in rats on a NSD, it lowered creatinine clearance in rats on a LSD (P < 0.01). Cyclosporine-induced tubulointerstitial fibrosis and arteriolopathy was observed at 28 days only in the rats on a LSD (P < 0.05). In addition, peripheral renin activity was increased only in the rats on a LSD (P < 0.01), while it remained normal in the rats on a NSD. In addition, CsA-treated rats on a LSD developed a progressive increase in the mRNA expression of TGF-beta1 and the matrix proteins biglycan and type I collagen at 7 and 28 days. Most of the changes were seen at 28 days (P < 0.001 for TGF-beta1, P < 0.01 for biglycan and type I collagen). On the other hand, CsA treatment in rats on a NSD did not affect the mRNA expression of TGF-beta1 and matrix proteins. Most of the changes in the immunofluorescence deposition of the glycoproteins tenascin and fibronectin EDA+ were in the tubulointerstitium and vessels of the kidneys of rats on a LSD and were mostly significant at 28 days, in accordance with the characteristic histology of chronic CsA nephropathy. The mRNA expression of plasminogen activator inhibitor-1, a protease inhibitor involved in matrix degradation and stimulated by TGF-beta1, was observed only in kidneys of rats on a LSD (P < 0.01). Since sodium depletion elevates peripheral renin activity, our experiments suggest a role for the renin-angiotensin system in the expression of TGF-beta1 and matrix proteins in CsA-induced renal fibrosis of rats on a LSD.

Identification of promoter activity and differential expression of transcripts encoding the murine stromelysin-1 gene in renal cells.

Stromelysin-1, matrix metalloproteinase-3 (MMP-3), is an important endopeptidase selectively expressed by somatic cells in organ tissues. The renal tubulointerstitium, for example, comprises tubular epithelium and interstitial fibroblasts forming the principal mass of the kidney. We observed that mRNA encoding stromelysin-1 is detectable in murine renal fibroblasts, but not in proximal tubular epithelium. Transcripts measured by RNase protection assay in renal fibroblasts increase following exposure to phorbol ester, and thereafter, activated stromelysin-1 protein can be detected in culture media by Western blotting. A 6.4 Kb genomic clone containing the putative stromelysin-1 promoter was isolated and a relevant 2.1 Kb PstI restriction fragment including 2.1 Kb of the immediate 5'-flanking region was sequenced on both strands. Two transcriptional start sites were identified by primer extension; the major start site corresponded to a previously established position in the rat promoter, and a second undescribed minor transcriptional start site was located 16 bp upstream of the primary site. A HiNF-A chromatin-activating element at -106 bp was found in the early promoter region of pR336 and an active AP-1 site at -72 bp with an Ets/PEA-3 motif at -203 bp was suggested by transient transfection of luciferase minigenes into renal fibroblasts responsive to phorbol ester. This Ets element was identical to a site in the early promoter of the fibroblast-specific gene FSP1. A baseline enhancement in activity of pR336 in fibroblasts was further observed with the addition of 5' flanking sequence out to -1980 bp. This additional region of flanking sequence contains two modular regions: one of multiple PEA-3 elements between -684 bp and -1955 bp and a second region between -1929 bp and -1980 bps containing a second AP-1 site at -1929 bp, a MBF-1/ MEP-1 metal binding site, and a PPAR peroxisome proliferator element at -1950 bp. Our findings implicate a gene structure with expected activity in a mesenchymal phenotype. The PKC-dependent regulation of the stromelysin-1 gene supports the notion that it may be modulated during inflammation or tissue remodeling.

Mechanism of fibrosis in experimental tacrolimus nephrotoxicity.

The clinical use of tacrolimus (FK506) is limited by nephrotoxicity. The pathogenesis of fibrosis in chronic FK506 nephrotoxicity remains unknown. Because transforming growth factor (TGF)-beta plays a key role in the fibrogenesis of many diseases, including cyclosporine nephrotoxicity, we studied a salt-depleted rat model of chronic FK506 nephropathy in which clinically relevant FK506 blood levels are obtained and which shows similarities to the lesions described in patients receiving FK506. Pair-fed rats were treated with either FK506 (1 mg/kg/day s.c.) or an equivalent dose of vehicle and were killed at 7 or 28 days. Characteristic histologic changes of tubular injury, interstitial fibrosis, and arteriolopathy developed in FK506-treated rats at 28 days and were accompanied by worsening kidney function, decreased concentrating ability, and enzymuria. FK506-treated kidneys had a progressive increase in the expression of TGF-beta1 and matrix proteins (biglycan, tenascin, fibronectin, and type I collagen). This effect seems to be specific because the expression of type IV collagen, a basement membrane collagen, was not affected. Matrix deposition was present mostly in the tubulointerstitium and vessels in accordance with the FK506 chronic lesion. The expression of plasminogen activator inhibitor-1, a protease inhibitor influenced by TGF-beta, followed TGF-beta1 and matrix proteins, suggesting that the fibrosis of chronic FK506 nephropathy likely involves the dual action of TGF-beta1 on matrix deposition and degradation. Since both peripheral and tissue renin expression were elevated with FK506, the renin-angiotensin system may play a role in the pathogenesis of this condition.

Expression of TGF-beta 1 and matrix proteins is elevated in rats with chronic rejection.

The pathogenesis of fibrosis in chronic renal allograft rejection remains unknown. Since TGF-beta 1 plays a key role in fibrogenesis, we studied a rat model of chronic allograft rejection that shows similarities to the structural lesion described in patients. We previously demonstrated an increased expression of TGF-beta 1 in human kidney biopsies with acute and chronic rejection. Recipients of renal allografts (F344-Lewis) and isografts (Lewis-Lewis) were sacrificed at 4, 8, 24 and 52 weeks. Characteristic histologic changes of chronic rejection developed in the allografts as early as four weeks and were accompanied by progressive albuminuria significant by eight weeks. Allografts showed a progressive increase in mRNA expression of TGF-beta 1 and matrix proteins during the 52 week course. Increased matrix deposition by immunofluorescence was mostly present in the interstitium and vessels early and in all kidney compartments later. The mRNA expression of plasminogen activator inhibitor, a protease inhibitor stimulated by TGF-beta 1, increased along with TGF-beta 1 and matrix proteins. These results suggest that the fibrosis of chronic renal allograft rejection is mediated, at least partly, by the dual action of TGF-beta 1 on matrix deposition and degradation.

Cyclosporine nephropathy: pathophysiology and clinical impact.

Cyclosporine (CsA) has markedly improved the results of transplantation, and its use has been extended to include autoimmune and primary renal diseases. However, the major limitation to its nephrotoxicity. Acutely, a reversible form of renal vasoconstriction can occur in all patients receiving CsA. In addition, progressive renal insufficiency owing to CsA has also been well documented. Clinically, long-term CsA nephrotoxicity is characterized by slowly progressive azotemia, proteinuria, and hypertension. Histologically, there is striped tubulointerstitial fibrosis, tubular atrophy, and afferent arteriolopathy. New evidence suggests that the histological changes in long-term CsA nephropathy are independent of the functional changes. Recently transforming growth factor (TGF)-beta was shown to be upregulated along with matrix proteins in a model of CsA nephrotoxicity. Because TGF-beta has immunosuppressive activities, it was suggested that it may contribute not only to CsA nephrotoxicity, but also to the mechanism of action of CsA. A number of strategies have been devised to try to minimize CsA nephrotoxicity, but remain inconclusive. Presently, it is not clear what role CsA therapy plays in the chronic failure of renal allografts. Continued research should give new insights into measures to overcome CsA-induced scarring in the kidney.

Hyperadrenergic state following acute withdrawal from clonidine used at supratherapeutic doses.

Abrupt cessation of clonidine treatment precipitates a physiological withdrawal syndrome, thought to be due to a hyperactive state of central autonomic and cognitive adrenergic neuronal systems dependent on presynaptic alpha 2-adrenoceptors and/or imidazoline receptors. We hereby describe a 36-year-old male with history of end-stage renal disease, hypertension and medication non-compliance, who presented with severe hypertension and remarkable agitation. His daily clonidine intake was estimated to be 10 mg. The patient had abruptly discontinued his clonidine five days prior to admission. The following indices of adrenergic activity were measured in plasma (normal control values in parentheses): noradrenaline (NA) 8.59 nmol/l (1.32-4.56 nmol/l), adrenaline (Adr) 1.86 nmol/l (0.83-4.20) nmol/l), total 3-methoxy-4-hydroxyphenylglycol (MHPG) 152.8 nmol/l (45.1-111.5 nmol/l), and free MHPG 33.0 nmol/l (12.2-31.4 nmol/l). Plasma clonidine level was 3.53 ng/ml (15.9 nmol/l) with the usual therapeutic level being < 2.0 ng/ml (8.9 nmol/l). Initially, the patient received sedatives and was started on clonidine for the first 24 hours only, after which time period prazosin was started, with good response of his blood pressure and reversal of his mental status changes. At that point, the plasma values of indices of adrenergic activity had decreased compared with their corresponding initial values by the following percentages: NA 60.6%, Adr 22.6%, total MHPG 42.2% and free MHPG 11.5%. Plasma clonidine level had decreased now by 43.6% to an absolute value of 1.99 ng/ml (8.85 nmol/l). We emphasize that physicians should be aware of clonidine's abuse potential and caution should be taken, as well as the appropriate route chosen, when prescribing clonidine in patients who show features of poor compliance to medications and especially in patients with psychoses, suicide potential or personality disorders.

Role of transforming growth factor-beta 1 in experimental chronic cyclosporine nephropathy.

The pathogenesis of fibrosis in chronic cyclosporine (CsA) nephropathy remains unknown. Since TGF-beta 1 plays a key role in the fibrogenesis of a number of renal diseases, we studied a salt-depleted rat model of chronic CsA nephropathy which shows similarity to the structural and functional lesions described in patients. Pair fed rats were treated with either CsA (15 mg/kg/day s.c.) or an equivalent dose of olive oil and sacrificed at 7 and 28 days. Characteristic histologic changes of proximal tubular injury, tubulointerstitial fibrosis and arteriolopathy developed in CsA-treated rats at day 28. They were accompanied by physiologic changes of increased serum creatinine, decreased creatinine clearance, increased enzymuria and decreased concentrating ability. CsA-treated rats showed a progressive increase in mRNA expression of TGF-beta 1 and matrix proteins at days 7 and 28. Most of the changes were in the tubulointerstitial and vascular compartments by immunofluorescence with a predominant involvement of the medulla as compared to cortex. The mRNA expression of plasminogen activator inhibitor, a protease inhibitor stimulated by TGF-beta 1, followed TGF-beta 1 and matrix proteins, suggesting that the fibrosis of chronic CsA nephropathy likely involves the dual action of TGF-beta on matrix deposition and degradation.

Transforming growth factor-beta and matrix protein expression in acute and chronic rejection of human renal allografts.

Because the increased tissue expression of TGF-beta underlies fibrosis in many diseases, it was hypothesized that sustained elevated transforming growth factor (TGF)-beta overexpression might be responsible for fibrosis in chronic rejection of the renal allograft. To test this hypothesis, biopsies were obtained from 5 patients with acute rejection, 5 patients with chronic rejection, 10 normal individuals, and 10 patients with kidney disease. The tissues were examined by immunofluorescence for the three TGF-beta isoforms (1, 2, and 3) and the two matrix proteins induced by TGF-beta that serve as markers of fibrosis: fibronectin extradomain A positive (EDA+) and plasminogen activator inhibitor-1 (PAI-1). The tubulointerstitium from all cases of acute rejection and chronic rejection showed highly significant increases in immunostaining for the three TGF-beta isoforms (P < 0.001), fibronectin EDA+ (P < 0.005), and PAI-1 (P < 0.001). In the glomeruli, only TGF-beta 1 expression achieved statistical significance (P < 0.005) in acute rejection, whereas in chronic rejection, all three TGF-beta isoforms (p < 0.001) in addition to fibronectin EDA+ (p < 0.001) and PAI-1 (p < 0.001) were elevated. There was both cellular and matrix staining of the TGF-beta isoforms. In striking contrast, control kidney tissues were negative or only weakly positive. Because TGF-beta was present both in acute and in chronic rejection but not in control tissues and because acute rejection episodes are a good predictor for chronic rejection, these results suggest that TGF-beta may play a role in the pathogenesis of fibrosis in chronic rejection.