The microbiota of the intestinal tract of chickens plays an important role in inhibiting the establishment of intestinal pathogens. Earlier culturing and microscopic examinations indicated that only a fraction of the bacteria in the cecum of chickens could be grown in the laboratory. Therefore, a survey of cecal bacteria was done by retrieval of 16S rRNA gene sequences from DNA isolated from the cecal content and the cecal mucosa. The ribosomal gene sequences were amplified with universal primers and cloned or subjected to temporal temperature gradient gel electrophoresis (TTGE). Partial 16S rRNA gene sequences were determined from the clones and from the major bands in TTGE gels. A total of 1,656 partial 16S rRNA gene sequences were obtained and compared to sequences in the GenBank. The comparison indicated that 243 different sequences were present in the samples. Overall, sequences representing 50 phylogenetic groups or subgroups of bacteria were found, but approximately 89% of the sequences represented just four phylogenetic groups (Clostridium leptum, Sporomusa sp., Clostridium coccoides, and enterics). Sequences of members of the Bacteroides group, the Bifidobacterium infantis subgroup, and of Pseudomonas sp. each accounted for less than 2% of the total. Sequences related to those from the Escherichia sp. subgroup and from Lactobacillus, Pseudomonas, and Bifidobacterium spp. were generally between 98 and 100% identical to sequences already deposited in the GenBank. Sequences most closely related to those of the other bacteria were generally 97% or less identical to those in the databases and therefore might be from currently unknown species. TTGE and random cloning indicated that certain phylogenetic subgroups were common to all birds analyzed, but sequence data from random cloning also provided evidence for qualitative and quantitative differences among the cecal microbiota of individual birds reared under very similar conditions.

Mesenchymal stromal/stem cells (MSCs) are clinically useful for cell-based therapy, but concerns regarding their ability to replicate limit their human application. MSCs release extracellular vesicles (EVs) that mediate at least in part the paracrine effects of the parental cells. To understand the molecular basis of their biological properties, we characterized the RNA cargo of EVs from porcine adipose-tissue derived MSCs. Comprehensive characterization of mRNA and miRNA gene expression using high-throughput RNA sequencing (RNA-seq) revealed that EVs are selectively enriched for distinct classes of RNAs. For example, EVs preferentially express mRNA for transcription factors (e.g. MDFIC, POU3F1, NRIP1) and genes involved in angiogenesis (e.g. HGF, HES1, TCF4) and adipogenesis (e.g. CEBPA, KLF7). EVs also express Golgi apparatus genes (ARRB1, GOLGA4) and genes involved in TGF-β signaling. In contrast, mitochondrial, calcium signaling, and cytoskeleton genes are selectively excluded from EVs, possibly because these genes remain sequestered in organelles or intracellular compartments. RNA-seq generated reads for at least 386 annotated miRNAs, but only miR148a, miR532-5p, miR378, and let-7f were enriched in EVs compared to MSCs. Gene ontology analysis indicates that these miRNA target transcription factors and genes that participate in several cellular pathways, including angiogenesis, cellular transport, apoptosis, and proteolysis. Our data suggest that EVs transport gene regulatory information to modulate angiogenesis, adipogenesis, and other cell pathways in recipient cells. These observations may contribute to development of regenerative strategies using EVs to overcome potential complications of cell-based therapy.

Mesenchymal stem/stromal cells (MSCs) have distinct capability for renal repair, but may have safety concerns. MSC-derived extracellular vesicles emerged as a novel non-cellular alternative. Using a porcine model of metabolic syndrome and renal artery stenosis we tested whether extracellular vesicles attenuate renal inflammation, and if this capacity is mediated by their cargo of the anti-inflammatory cytokine interleukin (IL)10. Pigs with metabolic syndrome were studied after 16 weeks of renal artery stenosis untreated or treated four weeks earlier with a single intrarenal delivery of extracellular vesicles harvested from adipose tissue-derived autologous MSCs. Lean and sham metabolic syndrome animals served as controls (seven each). Five additional pigs with metabolic syndrome and renal artery stenosis received extracellular vesicles with pre-silenced IL10 (IL10 knock-down). Single-kidney renal blood flow, glomerular filtration rate, and oxygenation were studied in vivo and renal injury pathways ex vivo. Retention of extracellular vesicles in the stenotic-kidney peaked two days after delivery and decreased thereafter. Four weeks after injection, extracellular vesicle fragments co-localized with stenotic-kidney tubular cells and macrophages, indicating internalization or fusion. Extracellular vesicle delivery attenuated renal inflammation, improved medullary oxygenation and fibrosis. Renal blood flow and glomerular filtration rate fell in metabolic syndrome and renal artery stenosis compared to metabolic syndrome, but was restored in pigs treated with extracellular vesicles. These renoprotective effects were blunted in pigs treated with IL10-depleted extracellular vesicles. Thus, extracellular vesicle-based regenerative strategies might be useful for patients with metabolic syndrome and renal artery stenosis.

Background Reno-protective strategies are needed to improve renal outcomes in patients with atherosclerotic renal artery stenosis (ARAS). Adipose tissue-derived mesenchymal stem cells (MSCs) can promote renal regeneration, but their potential for attenuating cellular injury and restoring kidney repair in ARAS has not been explored. We hypothesized that replenishment of MSC as an adjunct to percutaneous transluminal renal angioplasty (PTRA) would restore renal cellular integrity and improve renal function in ARAS pigs. Methods and Results Four groups of pigs (n=7 each) were studied after 16 weeks of ARAS, ARAS 4 weeks after PTRA and stenting with or without adjunct intra-renal delivery of MSC (10×106 cells), and controls. Stenotic kidney blood flow (renal blood flow[RBF]) and glomerular filtration rate (GFR) were measured using multidetector computer tomography (CT). Renal microvascular architecture (micro-CT), fibrosis, inflammation, and oxidative stress were evaluated ex-vivo. Four weeks after successful PTRA, mean arterial pressure fell to a similar level in all revascularized groups. Stenotic kidney GFR and RBF remained decreased in ARAS (p=0.01 and p=0.02) and ARAS+PTRA (p=0.02 and p=0.03) compared to normal, but rose to normal levels in ARAS+PTRA+MSC (p=0.34 and p=0.46 vs. normal). Interstitial fibrosis, inflammation, microvascular rarefaction, and oxidative stress were attenuated only in PTRA+MSC-treated pigs. Conclusions A single intra-renal delivery of MSC in conjunction with renal revascularization restored renal hemodynamics and function, and decreased inflammation, apoptosis, oxidative stress, microvascular loss, and fibrosis. This study suggests a unique and novel therapeutic potential for MSC in restoring renal function when combined with PTRA in chronic experimental renovascular disease.

Objective-Mechanisms of renal injury distal to renal artery stenosis (RAS) remain unclear. We tested the hypothesis that it involves microvascular remodeling consequent to increased oxidative stress. Methods and Results-Three groups of pigs (nϭ6 each) were studied after 12 weeks of RAS, RASϩantioxidant supplementation (100 IU/kg vitamin E and 1 g vitamin C daily), or controls. The spatial density and tortuousity of renal microvessels (Ͻ500 m) were tomographically determined by 3D microcomputed tomography. The in situ production of superoxide anion and the expression of vascular endothelial growth factor (VEGF), its receptor VEGFR-2, hypoxia-inducible-factor (HIF)-1␣, von Hippel-Lindau (VHL) protein, and NAD(P)H oxidase (p47phox and p67phox subunits) were determined in cortical tissue. RAS and RASϩantioxidant groups had similar degrees of stenosis and hypertension. The RAS group showed a decrease in spatial density of cortical microvessels, which was normalized in the RASϩantioxidant group, as was arteriolar tortuousity. RAS kidneys also showed tissue fibrosis (by trichrome and Sirius red staining), increased superoxide anion abundance, NAD(P)H oxidase, VHL protein, and HIF-1␣ mRNA expression. In contrast, expression of HIF-1␣, VEGF, and VEGFR-2 protein was downregulated. These were all significantly improved by antioxidant intervention. Conclusions-Increased oxidative stress in the stenotic kidney alters growth factor activity and plays an important role in renal microvascular remodeling, which can be prevented by chronic antioxidant intervention. (Arterioscler Thromb