Viscoelastic properties of human bladder tumours.

The urinary bladder is an organ which facilitates the storage and release of urine. The bladder can develop tumours and bladder cancer is a common malignancy throughout the world. There is a consensus that there are differences in the mechanical properties of normal and malignant tissues. However, the viscoelastic properties of human bladder tumours at the macro-scale have not been previously studied. This study investigated the viscoelastic properties of ten bladder tumours, which were tested using dynamic mechanical analysis at frequencies up to 30Hz. The storage modulus ranged between 0.052MPa and 0.085MPa while the loss modulus ranged between 0.019MPa and 0.043MPa. Both storage and loss moduli showed frequency dependent behaviour and the storage modulus was higher than the loss modulus for every frequency tested. Viscoelastic properties may be useful for the development of surgical trainers, surgical devices, computational models and diagnostic equipment.

Prograf produces a molecular environment favoring antifibrosis, an effect reversed by the addition of rapamune.

Rapamune, an inhibitor of the mammalian target of rapamycin, exhibits antiproliferative actions and is increasingly used as adjuvant therapy with calcineurin inhibitors. This study investigated the effect of Rapamune on functional and molecular markers in a rat model of calcineurin inhibitor-induced graft dysfunction. Prograf (6 mg), with or without addition of Rapamune (1 mg), was administered to salt-depleted male rats (n = 6/group). Urinary protein excretion and serum creatinine were measured. Rats were culled at 28 days, and messenger RNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was evaluated with reverse transcriptase polymerase chain reaction. Serum creatinine increased with Prograf (P = .01), but not Rapamune (P = .69) treatment, compared to controls at 28 days. The combination of Rapamune and Prograf produced a rise in serum creatinine at 7 (P = .007) and 14 (P = .01) days, but this was not observed at later time points. Urinary protein excretion was unaltered by any drug or combination. While confirming a synergistic effect of Rapamune and calcineurin inhibitors on renal function, these results suggest that sole therapy with Prograf produces inhibition of fibrotic gene expression. Rapamune alone has no deleterious effect on gene expression but addition of Rapamune cancels out the beneficial effects of Prograf.

The novel antifibrotic agent pirfenidone attenuates the profibrotic environment generated by calcineurin inhibitors in the rat salt-depletion model.

Calcineurin inhibitors (CNIs) promote fibrosis in renal allografts by altering the dynamics of extracellular matrix (ECM) turnover. Graft structure is changed and functional disturbance may follow. This study examined the effects of an antifibrotic agent, pirfenidone, on functional, structural, and molecular markers of fibrosis in a rat model. Cyclosporine or tacrolimus were administered to salt-depleted rats, with or without varying doses of pirfenidone. Both CNIs increased serum creatinine, and pirfenidone attenuated this functional disturbance. No changes in urinary protein excretion or graft histology were observed, suggesting structural and functional alterations can be dissociated. Messenger RNA expression of pro- and antifibrotic genes affecting ECM was estimated with semiquantitative RT-PCR. Cyclosporine-induced increases in collagen III mRNA expression were attenuated by pirfenidone (500 mg/kg/d), and increases in TIMP-1 expression were reversed by all doses of pirfenidone. Matrix metalloproteinase-2 expression was decreased by cyclosporine; all doses of pirfenidone significantly reversed this effect. Tacrolimus alone decreased TGF-beta and TIMP-1 expression, suggesting some antifibrotic action; addition of pirfenidone had no further effect. The mechanism of action of pirfenidone is reversal of some CNI-induced changes in fibrotic gene expression. It also attenuates creatinine rise in salt-depleted rats in this model of CNI-induced nephrotoxicity.

Mycophenolate mofetil inhibits intimal hyperplasia and attenuates the expression of genes favouring smooth muscle cell proliferation and migration.

AIM: Intimal hyperplasia remains the leading cause of late graft failure following heart transplantation. The immunosuppressive drug mycophenolate mofetil has been shown to inhibit the development of intimal hyperplasia. This study aimed to assess the efficacy of a combination of mycophenolate mofetil, calcineurin inhibition, and sirolimus on the development of intimal hyperplasia. METHODS: Male Sprague-Dawley rats received mycophenolate mofetil (30 mg/kg per day) and either tacrolimus (0.1 mg/kg per day), cyclosporine (5 mg/kg per day), or sirolimus (0.05 mg/kg per day) and were compared to an untreated control group. All animals underwent left common carotid artery balloon angioplasty. Morphometric analysis was performed on representative transverse sections, and intima medial ratios calculated at 2 weeks. Profibrotic gene expression was assessed with competitive RT-PCR at 2 weeks for metalloproteinase-2, metalloproteinase-9, TIMP-1, collagen III, and TGF-beta. Sections were stained with sirius red, and extracellular matrix deposition was quantified. RESULTS: Mycophenolate mofetil in combination with rapamycin was associated with the greatest reduction in intimal thickening (intima medial ratio 0.79; range 0.45-0.86), compared to its combination with either cyclosporine (1.41; range 1.06-1.68, P < .02) or tacrolimus (0.93; range 0.81-1.37, P < .05) and controls (1.47; range 1.02-2.04, P < .005). Mycophenolate mofetil and rapamycin significantly inhibited all profibrotic genes studied compared to controls (P < .01) but there were no differences between tacrolimus and cyclosporine. Mycophenolate mofetil and sirolimus significantly attenuated extracellular matrix deposition compared to tacrolimus and cyclosporin (P < .023). CONCLUSION: The benefits of mycophenolate mofetil in combination with sirolimus are preferential over those with cyclosporine or tacrolimus. Randomised trials are warranted to assess if mycophenolate mofetil should be an alternative agent to calcineurin-inhibitors when used in combination with sirolimus.

Cyclosporine and rapamycin act in a synergistic and dose-dependent manner in a model of immunosuppressant-induced kidney damage.

The combination of cyclosporine (CSA) and rapamycin (RAPA) is a potent and commonly used approach to immunosuppression following solid-organ transplantation. By applying varying doses of CSA and RAPA to the rat salt-depleted model, we aimed to find a dose combination that favored antiproliferation/antifibrosis rather than toxicity. Male Sprague-Dawley rats (350 to 500 g) were salt-depleted for 7 days prior to commencing CSA and RAPA treatment. Serum creatinine and urinary protein/creatinine ratios were measured. Fibrosis was estimated with Sirius red staining of extracellular collagen. mRNA expression of TGF-beta, MMP-2, MMP-9, TIMP-1, and collagen III was assessed with reverse transcriptase PCR. A rise in serum creatinine at 7 and 28 days was observed for CSA 15 mg/kg/d (P = .002) but not CSA 7.5 mg (P = .06) or RAPA 1 mg (P = .69) compared to controls. Twenty-four-hour urinary protein excretion was unchanged compared to controls for all drug doses and combinations. Of the dose combinations, CSA 7.5 mg/d + RAPA 0.5 mg/d produced the lowest serum creatinine for all time points, and inhibited profibrotic TIMP-1 (P = .017), while increasing antifibrotic MMP-2 (P = .009) mRNA expression, compared to CSA treatment alone. Expression of TGF-beta and collagen III was unaltered between groups. CSA treatment produced molecular and biochemical changes indicating renal damage. Addition of RAPA can attenuate this damage, but only with a dose reduction of both agents. The most favorable results were for the dose combination CSA 7.5 mg/kg/d plus RAPA 0.5 mg/kg/d.

Fibrosis-associated gene expression in renal transplant glomeruli after acute renal allograft rejection.

BACKGROUND: Acute allograft rejection is thought to be a risk factor for chronic allograft nephropathy, the cardinal features of which are vasculopathy, interstitial fibrosis and glomerulosclerosis. Fibrosis-associated genes might act as ad interim surrogate markers for chronic allograft nephropathy. The aim of this study was to determine mRNA expression of fibrosis-associated genes in glomeruli plucked from protocol renal transplant biopsies, in patients with or without a history of acute rejection. METHODS: A consecutive series of 52 patients (31 male, 21 female) was assessed. Donor categories were cadaveric, living related or asystolic. Transplant recipients received either cyclosporin- or tacrolimus-based immunosuppression. Patients routinely underwent percutaneous needle-core renal transplant biopsy at 1 week, and 3 and 6 months. Acute rejection episodes were confirmed histologically and treated with intravenous methylprednisolone, or antithymocyte globulin if steroid resistant. Individual glomeruli were plucked and total mRNA was extracted. Fibrosis-associated genes were amplified by reverse transcriptase-polymerase chain reaction (PCR) and quantified by enzyme-linked immunosorbent assay. RESULTS: The expression of both collagen type III (mean 0.42 versus 0.31 arbitrary units of PCR products corrected for a housekeeping gene) and collagen IV (mean 0.46 versus 0.42 arbitrary units) at 6 months did not differ between recipients who experienced acute rejection episodes and those who were free from rejection. There was also no significant difference between groups in terms of mRNA expression of collagen IValpha2, matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinases 1 and 2, transforming growth factor beta or tenascin. CONCLUSION: These results suggest that acute rejection episodes do not increase the expression of fibrosis-associated genes in glomeruli from renal transplant biopsies.

Rapamycin inhibits vascular remodeling in an experimental model of allograft vasculopathy and attenuates associated changes in fibrosis-associated gene expression.

BACKGROUND: Rapamycin inhibits extracellular matrix (ECM) accumulation (fibrosis) and vascular remodeling in experimental models of chronic allograft dysfunction (CAD) by poorly understood mechanisms. The aim of this study was to assess the effect of rapamycin on the expression of fibrosis-associated genes and correlate this with observed changes in ECM remodeling in an experimental of model allograft vasculopathy. METHODS: Vascular remodeling and ECM accumulation (picrosirius red) were measured by computerized histomorphometry of F344-to-Lewis rat aortic allograft sections harvested at serial timepoints. Expression of fibrosis associated genes was studied by means of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Rapamycin (0.5 mg/kg/day) inhibited intimal hyperplasia, medial ECM accumulation and expansive vascular remodeling (increasing vessel circumference) in rat aortic allografts. This was associated with attenuation of the graft inflammatory infiltrate and a reduction in intragraft gelatinase, collagen III and tissue inhibitor of metalloproteinase 1 (TIMP 1) mRNA levels. At a lower dosage (0.25 mg/kg/day), rapamycin inhibited intimal hyperplasia and medial ECM accumulation, but there was a lesser effect on vascular remodeling. Lower dose allografts were also seen to have a more severe inflammatory infiltrate and larger amounts of intragraft matrix metalloproteinase 9 (MMP 9) mRNA than those treated with the higher dose. CONCLUSIONS: These data suggest that, in addition to the tissue response to injury, the alloimmune injury itself may contribute directly to the vascular remodeling that occurs in allograft vasculopathy. Rapamycin at higher but not lower doses inhibited both of these pathologic processes.

The impact of cyclosporine dose reduction with or without the addition of rapamycin on functional, molecular, and histological markers of chronic allograft nephropathy.

BACKGROUND: Overexposure to cyclosporine is a risk factor for chronic allograft nephropathy (CAN) and dose reduction has been advocated. The purpose of this study was to determine the impact of adding the non-nephrotoxic immunosuppressant, rapamycin, after cyclosporine dose reduction in renal-allograft recipients with CAN. METHODS: Thirty-one patients with biopsy-confirmed CAN were prospectively randomized to receive a 40% cyclosporine dose reduction with (rapamycin, n=16) or without (control, n=15) the addition of rapamycin 2 mg/day. Renal function and side-effect parameters were assessed. Patients had renal allograft biopsies taken at recruitment and after 6 months. Glomeruli were isolated from these and underwent total mRNA extraction followed by RT-PCR-ELISA to assess transforming growth factor-beta1, collagen III, TIMP-1, TIMP-2, and matrix metalloproteinase-2 expression. Samples were also stained with Sirius red and the percentage interstitial volume fraction quantified by computerized histomorphometric analysis. Data are presented as mean (+/-SD). RESULTS: Patient characteristics and cyclosporine trough levels after dose reduction (rapamycin 68 [+/-21] vs. control 56 [+/-19] ng/mL, P=NS) were similar in both groups. Rapamycin patients had a significant fall in Cr-51 radioisotope glomerular filtration rate (31.6 [+/-8.9] to 27.3 [+/-8.6] mL/min, P<0.01) that was not significant in controls (29.5 [+/-10.4] to 27.0 [+/-8.0] mL/min, P=NS). Transforming growth factor-beta1 expression fell over time in control but remained constant in rapamycin patients. Conversely collagen III expression increased over the 6-month follow-up in rapamycin patients but not in controls. Both had comparable increases in TIMP-1 and matrix metalloproteinase-2 but only rapamycin patients developed a significant increase in TIMP-2. Sirius red-stained interstitial volume fraction fell over the study in controls (15.3-11.2%, P=0.06) but not in rapamycin patients (16.2-16.3%, P=NS). CONCLUSION: Rapamycin (2 mg/day) did not improve functional, molecular, or histological outcome in patients with CAN after cyclosporine dose reduction. Further studies involving larger numbers of patients are necessary to confirm these findings.

Effects of the combination of rapamycin with tacrolimus or cyclosporin on experimental intimal hyperplasia.

BACKGROUND: Allograft vasculopathy remains the leading cause of late allograft failure following transplantation and can be inhibited by the antiproliferative drug rapamycin. This study assessed the efficacy of combining rapamycin therapy with calcineurin inhibition. METHODS: Male Sprague-Dawley rats received rapamycin 0.05 mg/kg daily and either tacrolimus 0.1 mg/kg or cyclosporin 5 mg/kg daily, and findings were compared with those in an untreated control group. Animals underwent left common carotid artery balloon angioplasty; the artery was explanted after 2 weeks. Morphometric analysis was performed on transverse sections and the intima : media ratio was calculated. Profibrotic gene expression was measured with competitive reverse transcriptase-polymerase chain reaction at 14 and 28 days. Proliferation was determined with proliferating cell nuclear antigen at 14 and 28 days. Extracellular matrix deposition was quantified with Sirius red. RESULTS: The combination of rapamycin and tacrolimus was associated with the greatest reduction in intimal thickening. Furthermore, treatment with rapamycin and tacrolimus significantly attenuated extracellular matrix deposition compared with rapamycin and cyclosporin (P < 0.02). CONCLUSION: The effects of rapamycin in combination with tacrolimus were better than those observed with rapamycin and cyclosporin.

Microemulsion cyclosporin inhibits vascular remodelling and attenuates associated changes in profibrotic gene expression in an experimental model of allograft vasculopathy.

BACKGROUND: Chronic allograft dysfunction (CAD), the leading cause of solid organ transplant failure, is characterized by histological evidence of extracellular matrix (ECM) accumulation (fibrosis). The aim of this study was to characterize the changes in fibrosis-associated gene expression in an experimental model of CAD and to measure the effect of the immunosuppressant cyclosporin on these changes. METHODS: Lewis recipients of F344 rat thoracic to abdominal transplants were administered cyclosporin or no treatment. Vascular remodelling and ECM accumulation (picrosirius red) were measured using computerized histomorphometry. Fibrosis-associated gene expression was studied by semiquantitative reverse transcription-polymerase chain reaction. RESULTS: Cyclosporin inhibited medial ECM accumulation and vascular remodelling in allografts. This was associated with an attenuation of the graft inflammatory infiltrate and a reduction in intragraft matrix metalloproteinase (MMP) 2 and MMP-9 messenger RNA (mRNA) levels. There was a significant negative correlation between neoadventitial ECM density and MMP-9 expression, as well as with vessel circumference. Neoadventitial ECM density was significantly higher in the cyclosporin-treated group than in animals with untreated allografts, as were mRNA levels of collagen 3 and tissue inhibitor of metalloproteinase 1. CONCLUSION: The alloimmune injury itself may contribute directly to vascular remodelling and fibrosis in allograft vasculopathy. Cyclosporin attenuated this component of the pathophysiology of CAD effectively.

Renal transplant fibrosis correlates with intragraft expression of tissue inhibitor of metalloproteinase messenger RNA.

BACKGROUND: Chronic renal allograft nephropathy is characterized by an abnormal accumulation of extracellular matrix proteins in the glomeruli and tubulo-interstitium. The aim of this study was to determine the relationship between intragraft expression of the genes controlling the accumulation of extracellular matrix and the development of chronic renal allograft nephropathy in human renal transplants. METHODS: Forty renal allografts with stable renal function were biopsied 6 months after transplantation. Single glomeruli were plucked from the surface of these protocol biopsies and total messenger RNA (mRNA) was extracted. Reverse transcriptase-polymerase chain reaction was used to study the intragraft expression of several fibrosis-associated genes (collagen III, collagen IValpha2, matrix metalloproteinase (MMP) 2, tissue inhibitors of metalloproteinases (TIMPs) 1 and 2, tenascin and transforming growth factor (TGF) beta1). The level of tubulo-interstitial fibrosis was measured by quantitative immunostaining of collagen III. RESULTS: There were positive correlations between the level of tubulo-interstitial collagen III immunostaining and intragraft expression of the genes for TIMP-1 (rs= 0.70, P < 0.02) and TIMP-2 (rs = 0.59, P < 0.02). Interstitial fibrosis was also strongly correlated with the levels of TGF-beta mRNA (rs = 0.67, P < 0.002). Finally, TIMP-1 expression increased with TGF-beta expression (rs = 0.77, P < 0.002). CONCLUSION: Failure of extracellular matrix degradation may be an important molecular mechanism in the pathogenesis of chronic renal allograft damage.

Pirfenidone inhibits early myointimal proliferation but has no effect on late lesion size in rats.

AIMS: intimal hyperplasia is mediated by smooth muscle cell proliferation, migration and deposition of extracellular matrix. The anti-fibrotic agent pirfenidone has been shown to inhibit pro-fibrotic growth factors in non-vascular inflammatory models. This study investigated the effect of the novel anti-fibrotic agent pirfenidone on the development of neointima. METHODS: male Sprague-Dawley rats received either standard diet or diet supplemented with pirfenidone (250, 500, 1000 mg/kg/day). Animals underwent left common carotid balloon angioplasty and were explanted at 4, 8, 14 and 28 days and analysed for intimal thickening, pro-fibrotic gene expression, extracellular matrix deposition and metalloproteinase activity. RESULTS: neointimal thickness was significantly reduced in a dose-dependent manner at 14 days; pirfenidone 250 mg/kg (p<0.005), pirfenidone 500 mg/kg (p<0.001), pirfenidone 1 g/kg ( p<0.001). There were no significant differences in intimal thickening at 28 days. Expression of MMP-2, MMP-9, TIMP-1, collagen III and TGF-beta were all significantly inhibited at 14 days. Both collagen III expression and ECM deposition were reduced at 28 days ( p<0.05 and <0.002 respectively). CONCLUSION: pirfenidone reduces expression of MMPs governing smooth muscle cell proliferation and migration (MMP-2 and 9), and genes favouring ECM accumulation (TIMP-1 and collagen III). This study shows that inhibition of MMP activity is not sufficient to inhibit late lesion size.

Tacrolimus has less fibrogenic potential than cyclosporin A in a model of renal ischaemia-reperfusion injury.

BACKGROUND: Cyclosporin is associated with significant chronic nephrotoxicity, manifest in the long term mainly as renal fibrosis. There have been claims that tacrolimus is a less fibrotic drug than cyclosporin, and this study was designed to determine the effect of the two drugs on the expression of fibrosis-associated genes. METHODS: Male Wistar rats underwent clamping of the right renal pedicle for 45 min together with left nephrectomy; this model has previously been shown to be associated with upregulation of fibrosis-associated genes. Experimental groups (six animals per group) received cyclosporin A 10 mg/kg daily, tacrolimus 0.2 mg/kg daily or no treatment. Animals were killed at 16 weeks, and the renal cortical expression of fibrosis-associated genes was studied by means of quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Tacrolimus-treated animals developed significantly less proteinuria and had lower serum creatinine levels than those receiving cyclosporin. Tacrolimus administration also significantly reduced the expression of transforming growth factor beta and tissue inhibitor of metalloproteinases 1, both the products of genes associated with fibrosis. Although cyclosporin treatment reduced levels of the matrix-degrading enzymes, matrix metalloproteinase (MMP) 2 and MMP-9, this was not statistically significant. CONCLUSION: Tacrolimus has less nephrotoxicity than cyclosporin in this model. It also appears to have less fibrogenic potential, and this may have implications for the choice of long-term immunosuppressant in renal transplantation.

Differential effects of cyclosporin and tacrolimus on the expression of fibrosis-associated genes in isolated glomeruli from renal transplants.

BACKGROUND: Chronic allograft nephropathy is characterized by an excessive accumulation of extracellular matrix proteins leading to glomerular and interstitial fibrosis. The aim of this study was to determine the effects of two different immunosuppressive agents (cyclosporin and tacrolimus) on the expression of the genes controlling extracellular matrix deposition in renal transplant glomeruli. METHODS: Fifty-one renal transplant recipients were randomized to receive immunosuppression with either microemulsion cyclosporin or tacrolimus. Isolated glomeruli were plucked from protocol transplant biopsies performed 1 week, 3 months and 6 months after transplantation. Expression of the genes for collagen IValpha2, collagen III, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases (TIMP) 1 and TIMP-2, tenascin and transforming growth factor (TGF) beta1 was studied by quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The expression of messenger RNA (mRNA) for collagen III and TIMP-1 was significantly higher in patients receiving cyclosporin therapy than in those having tacrolimus (P < 0.01); this finding was accounted for by differences in the biopsy material at 1 week. A significant difference in collagen III, TIMP-1 and TIMP-2 mRNA expression was also detected between patients depending on the source of renal donor (cadaveric or living). There were no significant differences in the level of glomerular TGF-beta1. CONCLUSION: The data provide new in vivo evidence that tacrolimus may exert a less fibrogenic influence on transplant glomeruli than cyclosporin.

Molecular changes in extracellular matrix turnover after renal ischaemia-reperfusion injury.

BACKGROUND: Renal ischaemia-reperfusion (IR) injury is an inevitable consequence of transplantation and contributes to later graft fibrosis. This study aimed to elucidate the possible mechanisms by studying the expression of genes associated with extracellular matrix (ECM) turnover. METHODS: Male Wistar rats underwent laparotomy, clamping of the right renal pedicle for 45 min, and left nephrectomy. Control animals underwent left nephrectomy only, or had no operation. Animals were killed at 8, 16 and 24 weeks and messenger RNA was extracted from renal tissue. Genes of interest were amplified and then quantified in an enzyme-linked immunosorbent assay system with levels expressed as a ratio to a known housekeeping gene (GAPDH). RESULTS: Experimental animals developed progressive proteinuria from 16 weeks onwards. At 8 weeks after IR injury, gene levels of matrix metalloproteinase (MMP) 2, an ECM-degrading enzyme, were significantly increased. Levels then fell progressively. This was associated with increasing expression of tissue inhibitor of metalloproteinases (TIMP) 1, an inhibitor of MMP-2, and of transforming growth factor (TGF) beta, a profibrotic cytokine, by 24 weeks following injury. CONCLUSION: These results suggest that, after an initial phase of increased ECM turnover following IR injury, the balance turns towards one of reduced degradation. This is likely to be an important mechanism in the subsequent development of fibrosis.