Urinary mitochondrial DNA associates with delayed graft function following renal transplantation.

BACKGROUND: Ischaemia-reperfusion (IR) injury is an important determinant of delayed graft function (DGF) affecting allograft function. Mitochondrial DNA (mtDNA) is released upon cell death and platelet activation into the extracellular environment and has been suggested to be a biomarker in several diseases. Whether extracellular mtDNA accumulates in plasma and/or urine upon renal IR and predisposes DGF is unknown. METHODS: C57BL/6J wild-type mice were subjected to renal IR. In addition, an observational case-control study was set up enrolling 43 patients who underwent kidney transplantation. One day post-IR in mice and a few days following renal transplantation in human, blood and urine were collected. Patients were stratified into DGF and non-DGF groups. RESULTS: mtDNA-encoded genes accumulate in urine and plasma in both mice subjected to renal IR injury and in humans following renal transplantation. In human renal transplant recipients, cold ischaemia time and renal function correlate with urinary mtDNA levels. Urinary mtDNA levels but not urinary nuclear DNA levels were significantly higher in the DGF group compared with the non-DGF group. Multiple receiver operating characteristic curves revealed significant diagnostic performance for mtDNA-encoded genes cytochrome c oxidase III (COXIII); nicotinamide adenine dinucleotide hydrogen subunit 1 (NADH-deh); mitochondrially encoded, mitochondrially encoded nicotinamide adenine dinucleotide dehydrogenase 2 (MT-ND2) with an area under the curve of, respectively, 0.71 [P = 0.03; 95% confidence interval (CI) 0.54-0.89], 0.75 (P = 0.01; 95% CI 0.58-0.91) and 0.74 (P = 0.02; 95% CI 0.58-0.89). CONCLUSIONS: These data suggest that renal ischaemia time determines the level of mtDNA accumulation in urine, which associates with renal allograft function and the diagnosis of DGF following renal transplantation.

Matrix metalloproteinase 2 is associated with stable and matrix metalloproteinases 8 and 9 with vulnerable carotid atherosclerotic lesions: a study in human endarterectomy specimen pointing to a role for different extracellular matrix metalloproteinase inducer glycosylation forms.

BACKGROUND AND PURPOSE: We studied matrix metalloproteinases (MMP) 2, 8, and 9 and extracellular matrix metalloproteinase inducer (EMMPRIN) levels in relation to carotid atherosclerotic plaque characteristics. METHODS: Carotid atherosclerotic plaques (n=150) were stained and analyzed for the presence of collagen, smooth muscle cell (SMC), and macrophages. Adjacent segments were used to isolate total protein to assess MMP-2 and MMP-9 activities and gelatin breakdown, MMP-8 activity, and EMMPRIN levels. RESULTS: Macrophage-rich lesions revealed higher MMP-8 and MMP-9 activities, whereas SMC-rich lesions showed higher MMP-2 activity. The levels of less glycosylated EMMPRIN-45kD were higher in SMC-rich lesions and lower in macrophage-rich plaques. EMMPRIN-45kD was associated with MMP-2 levels, whereas EMMPRIN-58kD was related to MMP-9 levels. CONCLUSIONS: MMP-2, MMP-8, and MMP-9 activities differed among carotid plaque phenotypes. Different EMMPRIN glycosylation forms are associated with either MMP-2 or MMP-9 activity, which suggests that EMMPRIN glycosylation may play a role in MMP regulation and plaque destabilization.

Involvement of furin-like proprotein convertases in the arterial response to injury.

BACKGROUND: Furin-like proprotein convertases (PCs) are proteolytic activators of proproteins, like membrane type 1-matrix metalloproteinase (MT1-MMP) and transforming growth factor beta (TGF-beta), that are described in the arterial response to injury. However, the involvement of furin-like PCs in the arterial response to injury has not been studied yet. We studied furin, MT1-MMP, MMP levels and TGF-beta signaling after arterial injury. We also investigated the effect of an inhibitor of furin-like PCs, alpha1-antitrypsin Portland (alpha1-PDX), on arterial injury following balloon dilation. METHODS AND RESULTS: NZW rabbit femoral and iliac arteries (N=42) were balloon dilated unilaterally and harvested after 2, 7, 14, 28 or 42 days. Furin mRNA levels were increased after 2 and 7 days. MMP-2 and MT1-MMP levels were increased after day 7 and TGF-beta signaling, by phosphorylating Smad 1/5 and 2/3, was increased at all time points. Inhibition of furin-like PCs, by adenoviral over-expression of alpha1-PDX, blocked proTGF-beta activation and Smad phosphorylation, and reduced MT1-MMP and MMP-2 activation (N=5). In vivo adventitial inhibition of furin-like PCs (N=9) resulted in a reduction of 13.1+/-5.2% in advential and 23.6+/-7.9% in intimal areas (P<0.05), but had no effect on lumen size due to decreased vessel areas. CONCLUSIONS: This study demonstrates that furin-like PCs are involved in the arterial response to injury possibly through activation of the TGF-beta-Smad signaling pathway and identifies furin-like PCs as a possible target to inhibit intimal hyperplasia.

The role of Toll-like receptor 2 in inflammation and fibrosis during progressive renal injury.

Tissue fibrosis and chronic inflammation are common causes of progressive organ damage, including progressive renal disease, leading to loss of physiological functions. Recently, it was shown that Toll-like receptor 2 (TLR2) is expressed in the kidney and activated by endogenous danger signals. The expression and function of TLR2 during renal fibrosis and chronic inflammation has however not yet been elucidated. Therefore, we studied TLR2 expression in human and murine progressive renal diseases and explored its role by inducing obstructive nephropathy in TLR2(-/-) or TLR2(+/+) mice. We found that TLR2 is markedly upregulated on tubular and tubulointerstitial cells in patients with chronic renal injury. In mice with obstructive nephropathy, renal injury was associated with a marked upregulation and change in distribution of TLR2 and upregulation of murine TLR2 danger ligands Gp96, biglycan, and HMGB1. Notably, TLR2 enhanced inflammation as reflected by a significantly reduced influx of neutrophils and production of chemokines and TGF-beta in kidneys of TLR2(-/-) mice compared with TLR2(+/+) animals. Although, the obstructed kidneys of TLR2(-/-) mice had less interstitial myofibroblasts in the later phase of obstructive nephropathy, tubular injury and renal matrix accumulation was similar in both mouse strains. Together, these data demonstrate that TLR2 can initiate renal inflammation during progressive renal injury and that the absence of TLR2 does not affect the development of chronic renal injury and fibrosis.