Clinical and histopathologic examination of renal allografts treated with tacrolimus (FK506) for at least one year

BACKGROUND: We clinically and pathologically analyzed renal allografts from 1 9 renal transplant patients treated with tacrolimus (FK506) for more than 1 year. METHODS: Twenty-six renal allograft biopsy specimens from 1 9 renal transplant patients who underwent transplantations between 1991 and 1993 were evaluated. Thirteen biopsies were performed from stable functioning renal allografts with informed consent (nonepisode biopsy) and the other 13 were from dysfunctional renal allografts with a clinical indication for biopsy (episode biopsy). RESULTS: The main pathologic diagnoses (some overlap) were acute rejection (AR; n = 4), chronic rejection (CR; n=5), AR+CR (n =4), recurrent IgA nephropathy (n =5), normal findings (n =2), minimal-type chronic FK506 nephropathy (n = 9), and mild-type FK506 nephropathy (n = 11). Of the nonepisode biopsies, 7 and 4 biopsies showed minimal-type and mild-type chronic FK506 nephropathy, respectively. Chronic FK506 nephropathy consisted of rough and foamy tubular vacuolization (5 biopsies), arteriolopathy (angiodegeneration of the arteriolar wall; 20 biopsies), focal segmental glomerulosclerosis (4 biopsies) and the striped form of interstitial fibrosis (11 biopsies). The serum creatinine levels of patients in the mild-type chronic FK506 nephropathy group, which included 7 episode biopsies, were statistically higher than those in the minimum-type chronic FK506-nephropathy group (P< 0.001). CONCLUSIONS: This study demonstrates that chronic FK506 nephropathy consists primarily of arteriolopathy manifesting as insudative hyalinosis of the arteriolar wall, and suggests that mild-type chronic FK506 nephropathy is a condition which may lead to deterioration of renal allograft function.     

Probing residual structure and backbone dynamics on the milli- to picosecond timescale in a urea-denatured fibronectin type III domain

BACKGROUND: Human immunodeficiency virus-associated nephropathy (HIVAN) is a renal disease of unknown pathogenesis. Recent evidence suggests that the fibrogenic cytokine transforming growth factor-beta (TGF-beta) might be involved. We hypothesized that overproduction of TGF-beta in the kidney might be involved in the pathogenesis of HIVAN. METHODS: The mRNA and protein expression of TGF-beta isoforms, TGF-beta 1, TGF-beta 2, and TGF beta 3, deposition of matrix proteins induced by TGF-beta, and levels of HIV Tat protein were studied in HIVAN. Controls included normal and diseased kidneys from HIV-positive and -negative patients. The ability of Tat to induce production of TGF-beta and matrix proteins was also studied in human mesangial cells. RESULTS: Normal kidneys, thin basement membrane nephropathy, and minimal change disease were negative for the three TGF-beta isoforms and Tat. In HIVAN, levels of TGF-beta isoforms and Tat were significantly increased, along with the expression of TGF-beta mRNA and deposition of matrix proteins stimulated by TGF-beta. Increased levels of TGF-beta isoforms, but not Tat, were also found in other glomerular diseases characterized by matrix accumulation. HIV infection, in the absence of HIVAN, was not associated with an increase in TGF-beta or Tat expression. Tat stimulated the expression and production of TGF-beta 1 and matrix proteins by human mesangial cells. CONCLUSIONS: Our findings suggest that overproduction of TGF-beta is involved in the pathogenesis of HIVAN.     

Comparison of the stability of technetium-labeled peptides to challenge with cysteine

-Tacrolimus (FK 506) is a powerful, widely used immunosuppressant. The clinical utility of FK 506 is complicated by substantial hypertension and nephrotoxicity. To clarify the mechanisms of FK 506-induced hypertension, we studied the chronic effects of FK 506 on the synthesis of endothelin-1 (ET-1), the expression of mRNA of ET-1 and endothelin-converting enzyme-1 (ECE-1), the endothelial nitric oxide synthase (eNOS) activity, and the expression of mRNA of eNOS and C-type natriuretic peptide (CNP) in rat blood vessels. In addition, the effect of the specific endothelin type A receptor antagonist FR 139317 on FK 506-induced hypertension in rats was studied. FK 506, 5 mg. kg-1. d-1 given for 4 weeks, elevated blood pressure from 102+/-13 to 152+/-15 mm Hg and increased the synthesis of ET-1 and the levels of ET-1 mRNA in the mesenteric artery (240% and 230%, respectively). Little change was observed in the expression of ECE-1 mRNA and CNP mRNA. FK 506 decreased eNOS activity and the levels of eNOS mRNA in the aorta (48% and 55%, respectively). The administration of FR 139317 (10 mg. kg-1. d-1) prevented FK 506-induced hypertension in rats. These results indicate that FK 506 may increase blood pressure not only by increasing ET-1 production but also by decreasing NO synthesis in the vasculature.     

Helicobacter pylori infection and gastric secretion

We examined whether an immunosuppressant, FK506, inhibits delayed neuronal death in the gerbil hippocampal CA1 sector after 5-min forebrain ischemia. After reperfusion, gerbils were injected intravenously with FK506. Gerbils in the early injection group were injected with FK506 immediately after reperfusion, and gerbils in the delayed injection group were injected with FK506 1 or 2 h postischemia. The body temperature of the FK506-treated gerbils in the normothermic group was maintained at 37.5-38.0 degrees C for 2 h postischemia. In the chronic survival group, neuroprotection was assessed after recovery for 45 days. Seven or 45 days after reperfusion, neuronal density in the CA1 was assessed following perfusion fixation. FK506 ameliorated cell death in the CA1 in a dose-dependent manner in every group, although it showed a hypothermic effect. FK506 is neuroprotective against forebrain ischemia in gerbils.     

An animal model of Peyronie's-like condition associated with an increase of transforming growth factor beta mRNA and protein expression

PURPOSE: Transforming growth factor beta (TGF-beta) is involved in numerous vital processes including tissue fibrosis. Our objective was to study the role of TGF-beta in the induction of a Peyronie's-like condition and to produce an animal model for the further study of Peyronie's disease. MATERIALS AND METHODS: Twenty-four adult male Sprague-Dawley rats were divided into two groups. Different concentrations of cytomodulin, a synthetic heptopeptide with TGF-beta-like activity, were injected into the tunica of each rat from the first group (n = 18). Rats in the second group (n = 6) received saline injections as a control. The tunical tissues were taken after 3 days, 2 weeks, and 6 weeks and were examined using Hart and Trichrome stains. In the same tissue samples, TGF-beta mRNA and protein expression were studied. RESULTS: Histological alterations were observed in 15 out of 18 cytomodulin-injected rats, especially in tissue examined after 6 weeks. The most prominent changes were chronic cellular infiltration, focal and diffuse elastosis, thickening, disorganization and clumping of the collagen bundles. Results from immunoblot revealed remarkable TGF-beta1 protein expression in all the cytomodulin-injected rats only after 2 and 6 weeks. No remarkable TGF-beta2 or TGF-beta3 protein expression was observed. TGF-beta1 mRNA expression in the cytomodulin-injected rats was noticed in rats injected with higher concentrations after 3 days, while it was expressed in all rats after 2 weeks. There was no expression in the control group after either 3 days or 2 weeks. CONCLUSIONS: Cytomodulin can induce Peyronie's-like condition in the rat penis, which may explain the role of TGF-beta in the pathogenesis of Peyronie's disease.     

FK506 promotes liver regeneration by suppressing natural killer cell activity

We examined the mechanism of promotion of liver regeneration by tacrolimus hydrate (FK506), a potent immunosuppressant, after partial hepatectomy. The administration of FK506 significantly increased the bromodeoxyuridine (BrdU) labelling index at 36 and 48 h after 70% hepatectomy compared with the placebo group. Using the same model, we examined the effect of FK506 on the expression of hepatocyte growth factor (HGF) and transforming growth factor-beta 1 (TGF-beta 1) and found no changes in HGF and TGF-beta 1 mRNA expression in the liver or in the HGF protein concentration in plasma. We found that pretreatment with FK506 markedly reduced the activity and number of liver-resident natural killer (NK) cells at the time of partial hepatectomy. Our observations suggest that the promotion of liver regeneration by FK506 may be attributable to a reduction in the number of liver-resident NK cells and to inhibition of their activity.     

A constitutive law for mitral valve tissue

BACKGROUND: Obstructive nephropathy is a primary cause of renal failure in infancy. Chronic unilateral ureteral obstruction (UUO) in the neonatal rat results in reduced renal expression of epidermal growth factor (EGF), renal tubular epithelial (RTE) cell apoptosis and interstitial fibrosis. We wished to determine whether these changes could be prevented by exogenous administration of EGF. METHODS: Thirty-three Sprague-Dawley rats underwent UUO within the first 48 hours of life, and received daily injections of either EGF (0.1 mg/kg/day) or saline (control) for the following seven days, after which obstructed and intact opposite kidneys were removed for study. These were compared to 11 sham-operated rats that received either no injections, EGF injections, or saline injections. Renal cell proliferation was determined by proliferating cell nuclear antigen, apoptosis was measured by the TUNEL technique, and the distribution of vimentin, clusterin, transforming growth factor-beta 1 (TGF-beta 1), and alpha-smooth muscle actin were determined by immunohistochemistry. Tubular dilation, tubular atrophy, and interstitial collagen deposition were quantitated by histomorphometry. RESULTS: Compared to controls, EGF treatment increased RTE cell proliferation in the obstructed kidney by 76%, decreased apoptosis by 80%, and reduced vimentin, clusterin and TGF-beta 1 immunostaining (all P < 0.05). EGF treatment reduced tubular dilation by 50%, atrophic tubules by 30%, and interstitial fibrosis by 50% (all P < 0.05). There was no significant effect of EGF on renal alpha smooth muscle actin distribution. There was no effect of saline or EGF injections on kidneys from sham-operated rats for any of the parameters studied. CONCLUSIONS: We conclude that EGF stimulates RTE cell proliferation and maturation and reduces apoptosis in the neonatal rat kidney subjected to chronic UUO. These effects may contribute to the reduction in tubular dilation, tubular atrophy, and interstitial fibrosis. By preserving renal development, administration of EGF attenuates the renal injury resulting from chronic UUO.     

Functional and structural characteristics of experimental FK 506 nephrotoxicity

1. FK 506 (Tacrolimus, Prograf) is a novel immunosuppressant which is effective in solid organ transplantation and autoimmune diseases. The lack of a suitable animal model has hindered the study of the nephrotoxicity of the drug which has emerged as a common adverse effect in clinical trials. We report both acute and chronic nephrotoxicity with tacrolimus (FK) in which renal structure and function are worsened by sodium depletion. 2. Pair fed male Sprague-Dawley rats were given FK (3 or 6 mg/kg, p.o.) or vehicle for 7, 21 and 42 days on low salt or normal diet. The FK whole blood trough levels achieved (3-10 ng/mL) were similar to those observed in FK treated transplant patients. 3. In salt depleted animals treated for 7 days, FK (6 mg/kg) decreased renal blood flow and glomerular filtration rate (1.8 +/- 0.1 and 0.2 +/- 0.1 mL/min per 100 g vs 2.9 +/- 0.2 and 1.1 +/- 0.1 mL/min per 100 g in the vehicle group, P < 0.01). 4. After 21 days of treatment of FK on low salt diet but not normal salt, FK induced focal collapse and vacuolization in proximal tubules and discrete or confluent zones of tubulointerstitial oedema and mononuclear cell infiltration. 5. After 42 days in salt depleted rats, there was significant tubulointerstitial scarring that was associated with an increased plasma renin activity (PRA) (64 +/- 10 vs 30 +/- 4 ng AI/mL per h in the vehicle group, P < 0.05). Animals given normal salt diets did not develop significant histological lesions even up to 42 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substance use among adolescents with juvenile rheumatoid arthritis

BACKGROUND: Delayed graft function is a common and severe complication after cadaveric kidney transplantation. Besides a more complicated postoperative course, DGF can worsen the overall graft survival. In particular, DGF enhances the nephrotoxicity of mainstream immunosuppressants cyclosporine and FK506. This study evaluates a new therapeutical approach to the treatment of DGF related nephrotoxicity, based on the administration of IGF-I. METHODS: Sixty inbred Lewis rats underwent a bilateral clamping of the renal pedicles (20') as standard damage. The animals were stratified in six groups, according to the postoperative treatment. Group 1 served as control and received only the standard ischemic injury. Cyclosporine and FK506 were added in groups 3 and 5. Groups 2, 4 and 6 had the same treatment of groups 1, 3, 5 respectively, plus the administration of IGF-I. Blood samples were drawn daily to evaluate creatinine and BUN for 7 days. RESULTS: The rats treated with IGF-I had significantly better values compared to the respective controls (2-way ANOVA, p < 0.05). CONCLUSIONS: In conclusion, IGF-I improves the nephrotoxicity of mainstream immunosuppressants in this model. Its use is potentially beneficial for transplantation.     

Comparative dose-dependence study of FK506 and cyclosporin A on the rate of axonal regeneration in the rat sciatic nerve

The new immunosuppressant drug FK506 (Tacrolimus) increases the rate of nerve regeneration in vivo (Gold et al., 1994; Gold et al., 1995). In the present study, we have examined the dose-dependence of FK506's ability to enhance nerve regeneration. In the first set of experiments, rats received daily s.c. injections of FK506 (2 mg/kg, 5 mg/kg or 10 mg/kg) for 18 days after a sciatic nerve crush injury. Signs of functional recovery in the hind feet appeared earlier than in saline-treated control rats at all three FK506 dosage; recovery was maximally accelerated in the 5-mg/kg group. Light microscopy at 18 days after nerve crush revealed more regenerating myelinated fibers in FK506-treated rats than in controls; this was most apparent in the 5-mg/kg group. Morphometric analysis of axonal areas in the soleus nerve confirmed that axonal calibers were maximally increased in the 5-mg/kg group. In the second set of experiments, the rate of axonal regeneration was determined by radiolabeling the L5 dorsal root ganglion. Regeneration rate for sensory axons was maximally increased (by 34%) in the 5-mg/kg group. In contrast, cyclosporin A (10 or 50 mg/kg; dosages were selected on the basis of the 1/10 lower potency of cyclosporin A) did not significantly alter the rate of axonal regeneration. Cyclosporin A (50 mg/kg) also failed to increase functional recovery or axonal calibers in the soleus nerve. Because the two drugs share a common mechanism for producing immunosuppression (i.e., calcineurin inhibition), these results indicate that FK506's nerve regenerative property involves a distinct, calcineurin-independent mechanism.     

Postoperative plasma concentrations of procalcitonin after different types of surgery

Transforming growth factor (TGF)-beta 1 is an important cytokine involved in the pathobiology of tissue fibrosis through its stimulation of the production of, and inhibition of the degradation of, extracellular matrix proteins. We examined the clinical usefulness of plasma TGF-beta 1 concentration as a marker of fibrogenesis in patients with chronic viral hepatitis. Thirty-five patients, 11 with minimal chronic hepatitis, 14 with mild chronic hepatitis and 10 with moderate chronic hepatitis and 20 healthy subjects were studied. Transforming growth factor-beta 1 concentrations in platelet-poor plasma were measured with a TGF-beta 1 enzyme-linked immunosorbent assay system kit after acid-ethanol extraction. Plasma TGF-beta 1 levels were significantly elevated in patients with mild and moderate chronic hepatitis, but not in those with minimal chronic hepatitis, compared with the levels in the controls. Plasma TGF-beta 1 levels were increased in parallel with the histological degree of necroinflammation and of liver fibrosis. Plasma TGF-beta 1 levels were positively correlated with blood levels of procollagen type III N-peptide, and 7S fragment and central triple-helix of type IV collagen. These results suggest that plasma TGF-beta 1 level is a useful marker in assessing the situation of liver active fibrogenesis in patients with chronic viral hepatitis.     

Three-dimensional triple-quantum-filtered 23Na imaging of the dog head in vivo

Uninephrectomized rats with diet-induced hypercholesterolemia develop interstitial inflammation and fibrosis after 8 to 12 weeks. Fibrosis has been associated with the accumulation of lipid peroxidation products within the tubulointerstitium, along with increased renal mRNA levels for transforming growth factor beta-1 (TCF-beta 1), some matrix proteins, and the tissue inhibitor of metalloproteinases (TIMP-1). However, mRNA levels for urokinase-type plasminogen activator (uPA) have been found to be decreased. The purpose of the present study was to determine whether antioxidant therapy could attenuate interstitial fibrosis in hypercholesterolemic rats and to determine changes in the pattern of renal gene expression induced by antioxidant therapy. Three groups of uninephrectomized rats were studied after 12 weeks of feeding standard rat chow, an atherogenic diet (standard chow plus 4% cholesterol/1% cholic acid), or an atherogenic diet supplemented with high doses of the antioxidants probucol and vitamin E. Rats fed the atherogenic diet developed hypercholesterolemia and a 56% increase in total kidney collagen compared with rats fed standard chow. In comparison, the hypercholesterolemic rats treated with antioxidants had normal levels of renal lipid peroxidation products and a normal kidney collagen content. In contrast, there were no significant differences in urinary albumin excretion rates or the number of interstitial macrophages between the two hypercholesterolemic groups. Compared with the untreated hypercholesterolemic group, antioxidant therapy induced significant reductions in renal mRNA levels for procollagen III (to 60% of untreated levels), collagen IV (60%), and TIMP-1 (20%), while uPA levels were significantly increased (to 210%). Paradoxically, antioxidant therapy was associated with a significant increase in renal TGF-beta 1 mRNA levels (to 150%), although TGF-beta 1 protein expression shifted from interstitial to tubular epithelial cells in predominance. The results of the present study demonstrate the efficiency of antioxidant therapy in preventing renal interstitial fibrosis in hypercholesterolemic rats with a single kidney. Based on changes in renal gene expression at the mRNA level, impaired matrix protein synthesis and increased intrarenal activity of the metalloproteinases and uPA/plasmin may play a role in the attenuation of fibrosis.     

Nephrotoxicity studies of the immunosuppressants tacrolimus (FK506) and ascomycin in rat models

The nephrotoxic potential of ascomycin, the C21-ethyl analogue of FK506, was defined and ways explored to enhance its detection. After 14-day dosing in the Fischer-344 rat, FK506 and ascomycin reduced creatinine clearance by >50% at doses of 1 and 3 mg/kg, i.p., respectively. Ascomycin also had a 3-fold lower immunosuppressive potency in a popliteal lymph node hyperplasia assay, resulting in an equivalent therapeutic index consistent with a common mechanistic dependence on calcineurin inhibition. Renal impairment with different routes of administration was correlated with pharmacokinetics. Sensitivity of detection was not adequate with shorter dosing durations in rats with unilateral nephrectomy or in mice using a cytochrome P-450 inhibitor, SKF-525A. In 14-day studies, nephrotoxicity was not induced by continuous i.p. infusion of ascomycin at 10 mg/kg/day or daily oral administration (up to 50 mg/kg/day) in rats on a normal diet, nor by continuous i.v. infusion (up to 6 mg/kg/day) in rats on a low salt diet to enhance susceptibility. The lack of toxicity at high oral doses of FK506 or ascomycin, and the finding of non-linear oral pharmacokinetics of ascomycin show that this drug class has an oral absorption ceiling. The negative results with continuous infusion suggest that ascomycin nephrotoxicity is governed by peak drug levels. In addition to defining ways to meaningfully compare the nephrotoxic potential of FK506 derivatives, these results have implications for overall safety assessment and improved clinical use.     

Renal expression of transforming growth factor-beta inducible gene-h3 (beta ig-h3) in normal and diabetic rats

BACKGROUND: Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of a number of kidney diseases characterized by glomerulosclerosis and tubulointerstitial fibrosis. TGF-beta is secreted in a latent form requiring extracellular modification to become biologically active. TGF-beta inducible gene-h3 (beta ig-h3) is a recently identified TGF-beta-induced gene product. The present study sought to examine beta ig-h3 expression in normal and diabetic rats. METHODS: Beta ig-h3, TGF-beta1 and alpha1 (IV) collagen gene expression were assessed by Northern blot analysis and in situ hybridization in 20 Sprague Dawley rats, randomly assigned to receive streptozotocin (diabetic, N = 11) or citrate buffer alone (control, N = 9) and sacrificed eight months later. The effect of exogenous TGF-beta1 on beta ig-h3 expression was also assessed in cultured proximal tubular cells. RESULTS: In situ hybridization localized beta ig-h3 gene expression to the juxtaglomerular apparatus and the pars recta (S3 segment) of proximal tubules in both control and diabetic animals. Kidney TGF-beta 1, beta ig-h3 and alpha1 (IV) collagen mRNA from diabetic rats were increased two- to threefold compared with controls (P < 0.01). There was a significant correlation between TGF-beta1 and beta ig-h3 gene expression in kidneys from diabetic rats (r = 0.73, P = 0.01). In addition, beta ig-h3 mRNA increased in response to exogenous TGF-beta1 in a dose-dependent fashion in cultured proximal tubular cells. CONCLUSION: These findings support the hypothesis that biologically active TGF-beta plays a pathogenetic role in diabetic kidney disease and suggest that beta ig-h3 may be a useful index of TGF-beta1 bioactivity in the kidney.