Severe acute renal failure (ARF) remains a common, largely treatment-resistant clinical problem with disturbingly high mortality rates. Therefore, we tested whether administration of multipotent mesenchymal stem cells (MSC) to anesthetized rats with ischemia-reperfusion-induced ARF (40-min bilateral renal pedicle clamping) could improve the outcome through amelioration of inflammatory, vascular, and apoptotic/necrotic manifestations of ischemic kidney injury. Accordingly, intracarotid administration of MSC (∼ 106/animal) either immediately or 24 h after renal ischemia resulted in significantly improved renal function, higher proliferative and lower apoptotic indexes, as well as lower renal injury and unchanged leukocyte infiltration scores. Such renoprotection was not obtained with syngeneic fibroblasts. Using in vivo two-photon laser confocal microscopy, fluorescence-labeled MSC were detected early after injection in glomeruli, and low numbers attached at microvasculature sites. However, within 3 days of administration, none of the administered MSC had differentiated into a tubular or endothelial cell phenotype. At 24 h after injury, expression of proinflammatory cytokines IL-1β, TNF-α, IFN-γ, and inducible nitric oxide synthase was significantly reduced and that of anti-inflammatory IL-10 and bFGF, TGF-α, and Bcl-2 was highly upregulated in treated kidneys. We conclude that the early, highly significant renoprotection obtained with MSC is of considerable therapeutic promise for the cell-based management of clinical ARF. The beneficial effects of MSC are primarily mediated via complex paracrine actions and not by their differentiation into target cells, which, as such, appears to be a more protracted response that may become important in late-stage organ repair.

Acute kidney injury (AKI) is a major clinical problem in which a critical vascular, pathophysiological component is recognized. We demonstrated previously that mesenchymal stem cells (MSC), unlike fibroblasts, are significantly renoprotective after ischemia-reperfusion injury and concluded that this renoprotection is mediated primarily by paracrine mechanisms. In this study, we investigated whether MSC possess vasculoprotective activity that may contribute, at least in part, to an improved outcome after ischemia-reperfusion AKI. MSC-conditioned medium contains VEGF, HGF, and IGF-1 and augments aortic endothelial cell (EC) growth and survival, a response not observed with fibroblast-conditioned medium. MSC and EC share vasculotropic gene expression profiles, as both form capillary tubes in vitro on Matrigel alone or in cooperation without fusion. MSC undergo differentiation into an endothelial-like cell phenotype in culture and develop into vascular structures in vivo. Infused MSC were readily detected in the kidney early after reflow but were only rarely engrafted at 1 wk post-AKI. MSC attached in the renal microvascular circulation significantly decreased apoptosis of adjacent cells. Infusion of MSC immediately after reflow in severe ischemia-reperfusion AKI did not improve renal blood flow, renovascular resistance, or outer cortical blood flow. These data demonstrate that the unique vasculotropic, paracrine actions elicited by MSC play a significant renoprotective role after AKI, further demonstrating that cell therapy has promise as a novel intervention in AKI.

Our data show that renal SDF-1 is a currently unrecognized mediator of homing to and migration of CXCR4 expressing cells in the injured kidney. Because certain cells that express CXCR4 may have renoprotective effects, our results suggest that SDF-1 may be a major signal involved in kidney repair.

A split intein capable of protein transsplicing is identified in a DnaE protein of the cyanobacterium Synechocystis sp. strain PCC6803. The N-and C-terminal halves of DnaE (catalytic subunit ␣ of DNA polymerase III) are encoded by two separate genes, dnaE-n and dnaE-c, respectively. These two genes are located 745,226 bp apart in the genome and on opposite DNA strands. The dnaE-n product consists of a N-extein sequence followed by a 123-aa intein sequence, whereas the dnaE-c product consists of a 36-aa intein sequence followed by a C-extein sequence. The N-and C-extein sequences together reconstitute a complete DnaE sequence that is interrupted by the intein sequences inside the ␤-and -binding domains. The two intein sequences together reconstitute a split mini-intein that not only has intein-like sequence features but also exhibited protein trans-splicing activity when tested in Escherichia coli cells.Inteins have been defined as protein sequences embedded in-frame within a precursor protein sequence and excised during a maturation process termed protein splicing (1, 2). Protein splicing is a post-translational event involving precise excision of the intein sequence and concomitant ligation of the flanking sequences (N-and C-exteins) by a normal peptide bond (3-5). Most reported inteins are thought to be bifunctional elements, possessing a protein splicing activity and an endonuclease activity (6-9). Crystal structure of the Sce VMA1 intein revealed a two-domain structure, with domain I consisting of the N-and C-terminal regions of the intein sequence and domain II formed by the middle part of the intein sequence (10). Domain I (or a part of it) was suggested to be the splicing domain, whereas domain II corresponded to the endonuclease domain. Such a bipartite structure may be applicable to many other inteins, as has been suggested by studies including mutagenesis (11, 12) and sequence statistical modeling (7-9). Functional studies of mini-inteins, either found in nature or engineered in vitro, also confirmed such a two-domain model (13-15), further suggesting that the N-and C-terminal regions of an intein make up a functional splicing domain. Molecular mechanisms of protein splicing involve an N3S (or N3O) acyl shift at the N-terminal splice site (16-18), formation of a branched intermediate (19,20), and cyclization of an invariant Asn residue at the C terminus of intein to form succinimide (21), leading to excision of the intein. The ligated exteins undergo an S3N (or O3N) acyl shift to form a native peptide bond (21). Amino acid residues that are implicated in the splicing mechanism include a nucleophilic amino acid (Cys, Ser, or Thr) both at the beginning of the intein sequence and at the beginning of the C-extein sequence, an internal His, and a His-Asn dipeptide at the end of the intein sequence. In crystal structures of two inteins, these amino acids are indeed positioned at or near the active site of protein splicing (10,22).Approximately 50 intein-coding sequences have been found in Ͼ20 differe...

Little is known about the cell biology or the biologic roles of polymorphonuclear cell (PMN)-derived matrix metalloproteinase-8 (MMP-8). When activated with proinflammatory mediators, human PMN release only ∼15–20% of their content of MMP-8 (∼60 ng/106 cells) exclusively as latent pro-MMP-8. However, activated PMN incubated on type I collagen are associated with pericellular collagenase activity even when bathed in serum. PMN pericellular collagenase activity is attributable to membrane-bound MMP-8 because: 1) MMP-8 is expressed in an inducible manner in both pro- and active forms on the surface of human PMN; 2) studies of activated PMN from mice genetically deficient in MMP-8 (MMP-8−/−) vs wild-type (WT) mice show that membrane-bound MMP-8 accounts for 92% of the MMP-mediated, PMN surface type I collagenase activity; and 3) human membrane-bound MMP-8 on PMN cleaves types I and II collagens, and α1-proteinase inhibitor, but is substantially resistant to inhibition by tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2. Binding of MMP-8 to the PMN surface promotes its stability because soluble MMP-8 has t1/2 = 7.5 h at 37°C, but membrane-bound MMP-8 retains >80% of its activity after incubation at 37°C for 18 h. Studies of MMP-8−/− vs WT mice given intratracheal LPS demonstrate that 24 h after intratracheal LPS, MMP-8−/− mice have 2-fold greater accumulation of PMN in the alveolar space than WT mice. Thus, MMP-8 has an unexpected, anti-inflammatory role during acute lung injury in mice. TIMP-resistant, active MMP-8 expressed on the surface of activated PMN is likely to be an important form of MMP-8, regulating lung inflammation and collagen turnover in vivo.