Kaija Inkinen

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.

Urine neutrophil gelatinase-associated lipocalin is a marker of graft recovery after kidney transplantation

Delayed graft function (DGF), especially long-lasting DGF, complicates kidney transplant outcome. Neutrophil gelatinase-associated lipocalin (NGAL) is an acute kidney injury marker; therefore, we tested whether urine NGAL could predict DGF, prolonged DGF (lasting over 14 days), or the quality of kidney function in transplant recipients without DGF (non-DGF). We collected urine samples from 176 recipients transplanted with deceased donor kidneys before and various days after transplantation. A total of 70 transplantations had DGF, of which 26 were prolonged. Patients who developed DGF had a significantly slower decrease in urinary NGAL compared with those without DGF, such that day 1 NGAL predicted DGF (area under the curve (AUC) 0.75) and predicted DGF in 15 of 112 cases with day 1 urine output over 1 l (AUC 0.70) and in 19 of 86 cases with a day 1 decrease in creatinine over 50 μmol/l (AUC 0.74). The urinary NGAL level on day 1 predicted prolonged DGF (AUC 0.75), which had significantly worse 1-year graft survival (73%), compared with shorter DGF (100%). In non-DGF, high day 3 NGAL (greater than the mean) was associated with significantly worse kidney function at 3 weeks compared with low NGAL, but not at 3 months and 1 year. NGAL did not correlate with long-term function in DGF. Hence, day 1 urinary NGAL predicted DGF even when it was not clinically expected early on, and importantly, it predicted prolonged DGF that led to worse graft survival.

Deceased donor neutrophil gelatinase-associated lipocalin and delayed graft function after kidney transplantation: a prospective study

IntroductionExpanding the criteria for deceased organ donors increases the risk of delayed graft function (DGF) and complicates kidney transplant outcome. We studied whether donor neutrophil gelatinase-associated lipocalin (NGAL), a novel biomarker for acute kidney injury, could predict DGF after transplantation.MethodsWe included 99 consecutive, deceased donors and their 176 kidney recipients. For NGAL detection, donor serum and urine samples were collected before the donor operation. The samples were analyzed using a commercial enzyme-linked immunosorbent assay kit (serum) and the ARCHITECT method (urine).ResultsMean donor serum NGAL (S-NGAL) concentration was 218 ng/mL (range 27 to 658, standard deviation (SD) 145.1) and mean donor urine NGAL (U-NGAL) concentration was 18 ng/mL (range 0 to 177, SD 27.1). Donor S-NGAL and U-NGAL concentrations correlated directly with donor plasma creatinine levels and indirectly with estimated glomerular filtration rate (eGFR) calculated using the modification of diet in renal disease equation for glomerular filtration rate. In transplantations with high (greater than the mean) donor U-NGAL concentrations, prolonged DGF lasting longer than 14 days occurred more often than in transplantations with low (less than the mean) U-NGAL concentration (23% vs. 11%, P = 0.028), and 1-year graft survival was worse (90.3% vs. 97.4%, P = 0.048). High U-NGAL concentration was also associated with significantly more histological changes in the donor kidney biopsies than the low U-NGAL concentration. In a multivariate analysis, U-NGAL, expanded criteria donor status and eGFR emerged as independent risk factors for prolonged DGF. U-NGAL concentration failed to predict DGF on the basis of receiver operating characteristic curve analysis.ConclusionsThis first report on S-NGAL and U-NGAL levels in deceased donors shows that donor U-NGAL, but not donor S-NGAL, measurements give added value when evaluating the suitability of a potential deceased kidney donor.

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.

Fibrosis and matrix metalloproteinases in rat renal allografts.

The temporal activity and gene expression of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinase (TIMP) were investigated in a rat model of chronic allograft nephropathy. Gelatinolytic activity of MMP‐2 and ‐9 were demonstrated by zymography, and MMP‐2,‐9 and TIMP‐3 mRNA by in situ hybridization. The generation of fibrosis was determined as total collagen content/DNA. Significantly more latent and active MMP‐2, as well as latent MMP‐9, were seen in allografts than in autografts. Intense MMP‐2 mRNA expression was demonstrated in the allografts during the first 20 days after transplantation, located mainly in the interstitium of the kidney. In addition, some tubular cells expressed MMP‐2 mRNA. After day 20, MMP‐2 gene expression was faint. MMP‐9 mRNA expression in allografts was located mainly in the glomerulus. TIMP‐3 mRNA expression was downregulated in allografts. MMP‐2, MMP‐9 and TIMP‐3 seem to play a critical role in the development of fibrosis in the renal allograft.

Connective tissue growth factor and its correlation to other growth factors in experimental granulation tissue

Connective tissue growth factor (CTGF) is upregulated in a variety of fibrotic disorders, probably secondary to the activation and production of transforming growth factor-beta (TGF- g ). We have studied the expression of CTGF in a rat wound-healing model using Northern blot, in situ hybridization, and immunohistochemistry. The expression of CTGF mRNA in Northern blot and immunohistochemistry were correlated to the expression of TGF- g 1 and platelet-derived growth factor (PDGF). Northern hybridization showed the maximum expression of CTGF mRNA on day 14, whereas TGF- g 1 expression was maximal on days 7 and 14 and the time-related changes were smaller than for CTGF. PDGF A and PDGF B mRNA expressions were at maximum on day 14 and on day 21, respectively. In situ hybridization showed that fibroblast-like cells expressed CTGF most intensively, expression declining rapidly after day 14. CTGF mRNA and protein were found in blood vessel cells during the first week. In immunohistochemistry, all growth factors were expressed by fibroblast-like cells, macrophage-like cells, and blood vessels but CTGF-positive cells were fewer and were more restricted on days 5 and 7. These results demonstrate that CTGF expression together with TGF- g and PDGF are upregulated in wound healing, and CTGF expression in blood vessels suggests that CTGF is involved in angiogenesis.

Cloning of cDNA for Rat Pro α1(V) Collagen mRNA. Expression Patterns of Type I, Type III and Type V Collagen Genes in Experimental Granulation Tissue

A cDNA clone for rat pro alpha1(V) collagen mRNA was constructed using PCR amplification, with primers based on human and hamster COL5A1 gene sequences. The clone pRCVA1 is 560 nucleotides long and it encodes for the carboxy propeptide of type V procollagen. Homology shared with type I collagen sequence was 64%, with type II collagen 65% and with type III collagen 61%. To evaluate the spatial and temporal expression of type V collagen mRNA in wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 5, 8, 14, 21, 30, 59 and 84. Specific cDNA probes to pro alpha1(I), pro alpha1(III) and pro alpha1(V) collagen mRNA were used in slot blot, Northern and in situ hybridization. Type I collagen gene expression was upregulated at the initial stage of wound healing, type III collagen gene expression was constant and from the day 14 onwards type I and III collagen gene expressions were at the same level. Type V collagen gene expression was seen at every time point studied but at a considerably lower level than type I and III collagens. In situ hybridization showed that type V collagen was expressed in two different cell types. In conclusion, type V collagen was expressed in the wound healing model from at least day 5 onwards and it was synthesized by fibroblast-like and rounded cells.

Cytomegalovirus infection, viral DNA, and immediate early-1 gene expression in rejecting rat liver allografts.

Background. Cytomegalovirus (CMV) infection has been linked to acute and chronic rejection. We have previously shown that concomitant rat cytomegalovirus (RCMV) infection increases portal inflammation and bile duct destruction in rejecting rat liver allografts. Many of the pro-inflammatory effects of CMV have been attributed to the immediate early (IE) proteins of CMV. We wanted to investigate whether RCMV and IE-1 gene expression persist in the liver graft in our model. Methods. Liver transplantations were performed from PVG (RT1c) into BN (RT1n) rats. One day after transplantation, the rats were infected with RCMV. No immunosuppression was given. The graft infection was studied by viral culture, immunofluorescence, DNA in situ hybridization and RT-PCR for the detection of IE-1 mRNA at various time points. Results. RCMV caused an active infection from 5 days to 2 weeks after transplantation, during which infectious virus was found in the graft. Thereafter the cultures were negative. RCMV antigens and DNA were found in hepatocytes, endothelial, inflammatory, and bile duct cells during the active infection. At 4 weeks, RCMV DNA positive hepatocytes, endothelial, inflammatory, and bile duct cells could still be found, but in much smaller quantities. IE-1 mRNA expression was, however, only detected during the active infection, not at 4 weeks postinfection. Conclusions. RCMV IE-1 expression does not persist in the graft after the active infection, although some viral DNA can be detected in the graft up to 4 weeks. In our model, the CMV-induced increase in graft damage does not seem to require the continued expression of IE-1.