Juhani Ahonen

Angiotensin II induces connective tissue growth factor gene expression via calcineurin-dependent pathways.

Connective tissue growth factor (CTGF) is a polypeptide implicated in the extracellular matrix synthesis. Previous studies have provided evidence that angiotensin II (Ang II) promotes collagen synthesis and regulates collagen degradation. We investigated whether or not CTGF mediates the profibrotic effects of Ang II in the heart and kidneys and the role of calcineurin-dependent pathways in CTGF gene regulation. In transgenic rats harboring human renin and angiotensinogen genes, Ang II induced an age-dependent increase in myocardial CTGF expression, which was 3.5-fold greater compared to normotensive Sprague Dawley (SD) rats. CTGF overexpression correlated closely with the Ang II-induced rise in blood pressure. CTGF mRNA and protein were located predominantly in areas with leukocyte infiltration, myocardial, and vascular lesions and co-localized with TGFbeta(1), collagen I, and collagen III mRNA expressions. Ang II induced CTGF mRNA and protein to a lesser extent in the kidneys, predominantly in glomeruli, arterioles, and in the interstitium with ample inflammation. However, no expression was found in the right ventricle or pulmonary arteries. Blockade of calcineurin activity by cyclosporine A completely normalized Ang II-induced CTGF overexpression in heart and kidney, suppressed the inflammatory response, and mitigated Ang II-induced cell proliferation and apoptosis. In contrast, blockade of mTOR (target of rapamycin) pathway by everolimus, further increased the expression of CTGF even though everolimus ameliorated cell proliferation and T-cell-mediated inflammation. Our findings provide evidence that CTGF mediates Ang II-induced fibrosis in the heart and kidneys via blood pressure and calcineurin-dependent pathways.

Effect of cytomegalovirus on an experimental model of chronic renal allograft rejection under triple-drug treatment in the rat.

BACKGROUNDnCytomegalovirus (CMV) infection is thought to be a risk factor of chronic rejection. In clinical studies and animal models, mainly concerning graft vasculopathy, CMV has been demonstrated to enhance allograft arteriosclerosis. In this study we have investigated the effect of CMV on the early inflammatory response and graft histology in an experimental model of renal transplantation in a rat strain combination that develops chronic rejection under triple-drug immunosuppression.nnnMETHODSnRenal transplantations were performed in a rat strain combination of DA-->BN receiving triple-drug treatment (2 mg/kg methylprednisolone, 2 mg/kg azathioprine, 5 mg/kg cyclosporine daily subcutaneously). One group of immunosuppressed animals was infected with rat CMV, the Maastricht strain (10(5) plaque-forming units intraperitoneally), and the other group was left uninfected. As a positive control for alloresponse, one group of recipients received neither immunosuppression nor virus. Syngenic transplantations with triple-drug treatment and CMV were used as negative controls. The grafts were monitored by frequent ultrasound-guided fine-needle aspiration biopsies, and the intragraft inflammation was quantified in detail by the increment method and expressed in corrected increment units (CIU). Graft histology was performed in parallel.nnnRESULTSnNonimmunosuppressed animals developed acute rejection with a high peak of inflammation (7.9+/-3.2 CIU), a typical blast response, and lymphocytosis followed by infiltration of macrophages and necrosis within 7 days. Triple drug-treated animals had a short, mild inflammatory response (3.3+/-1.4 CIU at the peak) in the graft 3-5 days after transplantation but ended up with histological changes characteristic of chronic rejection with vasculopathy and fibrosis 40-60 days later. Triple drug-treated animals with CMV demonstrated a significantly stronger inflammation (4.5+/-1.8 CIU, P<0.01) than those without, and lymphoid activation continued longer and was followed by infiltration of macrophages in the graft. CMV infection of the graft was demonstrated by viral culture and antigen detection. In histology, chronic rejection with intimal thickening of arteries and arterioles and medial necrosis of large arteries was seen at 14 days, ending up with remarkable graft fibrosis within 20 days after transplantation.nnnCONCLUSIONnCMV prolonged and increased graft inflammation and accelerated chronic rejection of renal allografts under triple-drug treatment.

Effects of Dietary Sodium and Magnesium on Cyclosporin A–Induced Hypertension and Nephrotoxicity in Spontaneously Hypertensive Rats

Arterial hypertension, nephrotoxicity, and magnesium loss are common side effects of the immunosuppressive agent cyclosporin A (CsA). In the present study, the effects of dietary sodium and magnesium on CsA toxicity were examined in spontaneously hypertensive rats. A 6-week treatment with CsA during a moderately low-sodium diet (Na 0.3%, Mg 0.2% of the dry weight of the chow) raised blood pressure only slightly, without evidence of nephrotoxicity. By contrast, CsA during a high-sodium diet (Na 2.6%) produced a pronounced rise in blood pressure as well as marked nephrotoxicity, comprising decreased creatinine clearance, increased levels of serum creatinine and urea, and increased urinary protein excretion. During the high-sodium diet, CsA decreased myocardial and bone magnesium concentration and increased myocardial and renal calcium concentration. Magnesium supplementation (Mg 0.6%) protected against the CsA-induced hypertension and nephrotoxicity during the high-sodium diet. Magnesium supplementation also completely prevented the CsA-induced myocardial magnesium depletion and calcium accumulation in the heart and kidney during the high-sodium diet. Our findings indicate a detrimental interaction between increased sodium intake and CsA treatment and a marked protection by concomitant oral magnesium supplementation.

Long-term graft outcome is not necessarily affected by delayed onset of graft function and early acute rejection.

BACKGROUNDnBoth acute rejection episodes and delayed graft function (DGF) have been shown to be associated with decreased 1-year renal allograft survival. In our center, the incidence and the intensity of acute rejection episodes have been reduced by cyclosporine-based triple-drug therapy. We have also shown that DGF alone is not a risk factor for long-term graft survival.nnnMETHODSnWe have now investigated whether an acute rejection episode together with DGF significantly effects long-term graft outcome. This study involved 862 first cadaveric renal allografts and 182 regrafts.nnnRESULTSnThe incidence of DGF was 33% after first transplants and 44% after retransplants. The overall incidence of acute rejection episodes was 23% in first grafts and 28% in regrafts. After first grafts, there were no statistically significant differences in graft survival rates and half-lives between the early graft function (EGF) and DGF groups with or without acute rejection. In regrafts, graft survival was significantly higher in the EGF group without acute rejection than in the DGF group with acute rejection. However, if all other causes except chronic rejection were censored, the half-life in the EGF group without acute rejection was 17.3 years in first grafts, and in the DGF group with acute rejection, that number was 11.5 years in first grafts; for regrafts, the half-life was 12.3 years and 6.1 years, respectively.nnnCONCLUSIONSnAcute rejection together with DGF could contribute to initial damage to the graft, and this might lead to later chronic allograft failure. In our study, this effect was evident only in the case of retransplants.

Long-term consequences of different immunosuppressive regimens for renal allografts.

The long-term effects of four different immunosuppressive regimens on renal allografts have been investigated up to four years. A total of 128 recipients of first cadaveric renal allograft were randomized, after an initial induction period, to receive either triple drug therapy—i.e., (low-dose) cyclosporine, azathioprine, and methylprednisolone, or any possible combination of two drugs—i.e., Aza plus CsA, Aza plus MP, and CsA plus MP. The actual four-year graft survival rates for the triple therapy, Aza plus CsA, Aza plus MP, and CsA plus MP groups were 72%, 69%, 75%, and 59%, and patient survival rates were 78%, 81%, 81%, and 84%, respectively, with no significant differences. The cumulative number of chronic rejections up to 4 years was 0.09, 0.29, 0.25, and 0.34 per patient per group (P=ns), respectively. At 2, 3, and 4 years posttransplantation, the graft function was significantly worse in the Aza plus MP group compared with the triple therapy group (JP<.05). Of the 98 patients who did not have type I or II diabetes at the time of transplantation, 17 developed posttransplantation diabetes mellitus or an abnormal glucose tolerance test within 2 years posttransplanta-tion. All these patients had received steroids and none of the patients without steroids had these abnormalities. At two years the mean cholesterol level was highest in the Aza plus MP group, 6.8 mmol/L and lowest in the group receiving triple therapy, 5.8 mmol/L (P=ns). The use of (low-dose) CsA was not associated with lipid abnormalities or with disturbances in glucose metabolism. A protocol graft biopsy was performed at two years on all functioning kidneys, and the histological changes were scored blindly. No CsA-specific changes, except isometric vacuolation in tubuli, were found. Histological alterations characteristic of chronic rejection were expressed as “chronic allograft damage index.” Chronic allograft damage index was lowest in the triple therapy group, 1.5, compared with the other groups, 3.2–4.3 (P=.01), indicating the least histopathological change in the triple therapy group. In conclusion, this long-term study did not show any serious cyclosporine-related side-effects when used in low dose in combination with two other drugs. Some side-effects, such as posttrans-plant diabetes mellitus and probably some lipid abnormalities, could rather be traced to a higher dose of steroids. Moreover, the triple drug therapy was more efficacious than any double drug regimen in the prevention of chronic histological changes in renal allografts.

Expression and activity of matrix metalloproteinase-2 and -9 in experimental granulation tissue

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix‐modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP‐2 and MMP‐9 and to study the regulation of MMP‐2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP‐2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP‐9 gene expression was low between days 8–21 and increased after 4 weeks of granulation tissue formation. Membrane‐type 1 MMP (MT1‐MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP‐2) mRNA later during wound healing. In in situ hybridization the expression of MMP‐2 mRNA was seen mostly in fibroblast‐like cells and MMP‐9 mRNA in macrophage‐like cells. MMP‐9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage‐like cells on days 3–8. MMP‐9 proteolytic activity was observed only on days 3–8. The active form of the MMP‐2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP‐2 did not show any apparent changes during the experimental period. We conclude that MMP‐2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP‐9 was demonstrated only in early wound healing, and the MMP‐9 gene is upregulated when the granulation tissue matures.

Cyclosporine induces myocardial connective tissue growth factor in spontaneously hypertensive rats on high-sodium diet

Background. The introduction of cyclosporine (CsA) has led to an improvement in the prognosis of solid organ transplantation. However, drug-induced hypertension and nephrotoxicity, associated with the development of atherosclerosis and coronary heart disease, still worsen the long-term outcome of CsA-treated patients. Whether the CsA-induced myocardial changes are associated with the induction of connective tissue growth factor (CTGF), a recently found polypeptide implicated in extracellular matrix synthesis, is not known. Methods. Spontaneously hypertensive rats (8–9 weeks old) were treated with CsA (5 mg·kg−1·d−1 subcutaneously) for 6 weeks. The influence of angiotensin-converting enzyme inhibition (enalapril 30 mg·kg−1·d−1 orally) and angiotensin-1 receptor blockade (valsartan 3 and 30 mg·kg−1·d−1 orally) on CsA toxicity was also investigated. Myocardial morphology was examined, and vascular lesions were scored. Localization and the quantitative expression of CTGF, as well as collagen I and collagen III, mRNA were evaluated by in situ hybridization and Northern blot. Results. CsA-induced hypertension and nephrotoxicity were associated with myocardial infarcts and vasculopathy of the coronary arteries. CsA increased myocardial CTGF, collagen I, and collagen III mRNA expressions by 91%, 198%, and 151%, respectively. CTGF mRNA expression colocalized with the myocardial lesions. Blockade of the renin-angiotensin system prevented vascular damage and the CsA-induced CTGF, collagen I, and collagen III mRNA overexpressions in the heart. Conclusions. CsA increases CTGF, collagen I, and collagen III mRNA expressions in the heart. The induction of CTGF gene is mediated, at least in part, by angiotensin II.

An experimental model of chronic renal allograft rejection in the rat using triple drug immunosuppression

BACKGROUNDnChronic rejection is a major problem in renal transplantation. Various experimental models have been developed to study vasculopathy of chronic rejection. However, animal models resembling the clinical situation of renal transplantation with combination therapy of basic immunosuppression are not available. The aim of this study was to find an experimental model of a donor-recipient rat strain combination that, under triple drug immunosuppressive treatment (methylprednisolone, cyclosporine, and azathioprine), would develop chronic rejection within a few weeks.nnnMETHODSnRenal transplantations were performed in strain combinations of DA-->AO, PVG-->BN, and DA-->BN. In each group, 5-8 animals received triple drug treatment of methylprednisolone (2 mg/kg), azathioprine (2 mg/kg), and cyclosporine (5 mg/kg) daily, 5-10 animals were left without treatment, and 6 syngenic transplantations were performed. The grafts were monitored with ultrasound-guided fine needle aspiration biopsies to quantify the inflammation in the graft. Graft histology was performed in parallel and quantified by using the chronic allograft damage index (CADI).nnnRESULTSnIn nonimmunosuppressed animals, irreversible acute rejection with a high peak of inflammation appeared in every strain combination within 5-8 days. In triple drug-treated rats, the DA-->AO combination demonstrated a prolonged acute rejection but no characteristic chronic changes, and the PVG-->BN combination showed practically no inflammation and did not develop any signs of chronic rejection within 60 days (CADI: 2.7+/-2.1), but the DA-->BN combination showed an early, mild inflammatory response 5-7 days after transplantation and developed chronic rejection within 40-60 days after transplantation (CADI: 7.9+/-3.1). Syngenic animals showed no inflammation or histological alterations (CADI: 1.7+/-2.0).nnnCONCLUSIONSnIn conclusion, in the DA-->BN combination with triple drug treatment, early mild inflammation was followed by the development of chronic rejection and can be used as an experimental model that resembles the clinical situation in renal transplantation.

SOLUBLE INTERCELLULAR ADHESION MOLECULE-1 (sICAM-1) AFTER KIDNEY TRANSPLANTATION: THE ORIGIN AND ROLE OF URINARY sICAM-1? 1

BACKGROUNDnIntercellular adhesion molecule-1 (ICAM-1) binds to leukocyte adhesion receptors LFA-1 and MAC-1, and mediates leukocyte adhesion to target structures. During acute rejection there is increased expression of ICAM-1 in vascular and tubulointestial cells, and consequently accumulation of inflammatory leukocytes. Soluble ICAM-1 (sICAM-1) is released from ICAM-1 expressing cells and excreted into the surrounding fluid. Increased serum sICAM-1 levels are found in patients with acute rejections of various allografts, and high urinary levels in steroid resistant acute kidney allograft rejection.nnnMETHODSnUrinary excretion of sICAM-1 was measured by EIA in 136 kidney allograft recipients during the first 1-6 post transplant weeks: 30 patients developed acute rejection, and 106 patients had stable graft function. The molecular weight, binding to hyaluronan, and the origin of urinary sICAM-1 were studied.nnnRESULTSnWe show that urinary sICAM-1 circulates as a monomer with a molecular weight between 50 and 100 kD. It binds to immobilized, but not to circulating hyaluronan. About one week after transplantation the mean sICAM-1/creatinine ratio (306 ng/mmol) in transplanted patients was higher than in the healthy controls (167 ng/mmol, P<0.01), and remained basically unchanged during the follow-up in patients with stable graft function, whereas it increased in patients developing rejection, being about 2.5-fold above the initial level a few days before rejection (P<0.01). Urinary sICAM-1 did not correlate with the urinary albumin, whereas in patients developing rejection it correlated with urinary IL-2R (r=0.5146, P<0.001), a marker of lymphocyte activation. In the urinary sediment of rejecting patients ICAM-1 was demonstrated in the tubular epithelial cells, and in the macrophages.nnnCONCLUSIONSnIncreased urinary excretion of sICAM-1 was demonstrated in kidney transplanted patients a few days before acute rejection. It seems to originate from activated macrophages and/or from the tubular epithelial cells. The fact that urinary sICAM-1 is not bound to hyaluronan or to leukocytes suggests that it is not able to compete with membrane-bound ICAM-1 for these bindings, but may do so for the binding of activated macrophages.

Fine-needle aspiration biopsy in the monitoring of liver allografts. II. Applications to human liver allografts.

Serial fine-needle aspiration biopsies (FNAB) were used for clinical monitoring of human liver allografts. Nine liver allograft recipients were monitored with FNAB at 1–3-day intervals. No complications were recorded. All patients underwent at least 1 inflammatory episode of acute rejection; altogether 11 episodes, all reversible, were recorded. The inflammatory infiltrate consisted mainly of lymphoid cells, including lymphoid blasts, with minor involvement of monocytes, monoblasts, and macrophages. Further analysis of lymphoid cell subpopulations by immunoperoxidase techniques demonstrated an increase of T cells during rejection, both the CD4 (T4) and CDS (T8) subsets were increased. A slight increase of B cells in the graft was also seen. The CD4/CD8 (T4/T8) ratio was first low, peaked at the onset, and decreased toward the end of the episode. No clear correlations to the intragraft cellular events were recorded in corresponding blood specimens. However, an episode of eosinophilia was seen in the blood at the beginning of rejection, correlating with fever in the recipient. Degenerative changes in the parenchymal cells and bile droplets in the hepatocytes, indicating cholestasis and hepatocyte damage, were seen during all episodes of rejection, and these changes persisted even 10 days after the inflammation had subsided. The FNAB-findings correlated well with biochemical laboratory parameters, but the diagnosis of rejection could be established by the FNAB already 1–5 days earlier than elevated serum values indicated liver dysfunction.