Folic Acid-induced acute kidney injury.

<p>Mice were submitted to folic acid-induced kidney injury (FA) or sodium bicarbonate (Bic) vehicle treatment and after 24 h, received adipose tissue-derived stem cells (AdSC). Kidney tissue and serum samples were extracted 24 h after AdSC therapy for kidney function and protein expression analyses; control (ctr) mice received no treatment, (Bic) mice received vehicle only (sodium bicarbonate, 0.15 M). <b>(A)</b> Urea biochemistry essays revealed that AdSC treatment conferred protection against acute kidney dysfunction as seen through lower serum urea accumulation (n = 2–6 for each group); <b>(B-D)</b> Immunohistochemistry of kidney tissue. Semi-quantification of proliferating cell nuclear antigen (PCNA) staining demonstrates normalized cell proliferation rates after AdSC treatment vs. FA mice (B,C) (n = 2–5 for each group). Immunohistochemistry also showed reduced neutrophil infiltrate seen through myeloperoxidase (MPO) staining after AdSC treatment vs. FA mice (B,D) (n = 2–3 for each group). Arrows indicate positive MPO staining (original magnification: 20x). <b>(E)</b> Kidney bioplex essays showed reduced inflammatory profiles in stem cell treated mice, (n = 3 for each group). Neutrophil chemoattractant protein chemokine (C-X-C motif) ligand 1 (CXCL1), and macrophage inflammatory protein 1 alpha (MIP-1<b>α</b>) levels n were increased in AdSC-treated mice. In parallel, granulocyte macrophage colony stimulating factor (GM-CSF) and CXCL-1 expression was reduced in the AdSC-treated group. A tendency towards lower monocyte chemoattractive protein-1 (MCP-1) and interleukine 6 (IL-6) expression was also observed for these animals (n = 2–3 for each group). Serum urea levels are expressed as (mg/dL ± s.d.). Immunohistochemistry values are expressed as the percentage of area positive for PCNA ± s.d. or the number of cells positively stained for MPO per mm² ± s.d.). Scale bar, 25 <b>μ</b>m. Bioplex values are expressed as (pg/mL) ± s.d. *<i>P</i><0.05, **<i>P</i><0.01 ***<i>P</i><0.001</p

Folic Acid-induced chronic kidney injury.

<p>Mice were submitted to folic acid-induced kidney injury (FA) or sodium bicarbonate (Bic) vehicle treatment and after 24 h, received adipose tissue-derived stem cells (AdSC). Kidney tissue and serum samples were extracted 4 weeks after FA for kidney function and protein expression analyses; control (Ctr) mice received no treatment, (Bic) mice received vehicle only (sodium bicarbonate, 0.15 M). <b>(A)</b> Urea biochemistry essays showed no significant difference of long term renal functional between groups (n = 2–7 for each group) <b>(B,C)</b> Kidney sections were viewed under both bright-field (a-e) and polarized light (f-j) microscopy. AdSC-treated mice kidneys displayed reduced deposit of tissue collagen in comparison to FA mice (n = 3–5 for each group) (original magnification: 20x). <b>(D)</b> Bioplex essays show stem cell-treated mice develop ameliorated chronic kidney inflammation (n = 3 for each group). FA-treated mice expressed higher levels of interferon-gamma (IFN-<b>γ</b>) vs. control mice, while in AdSC-treated mice, IFN-<b>γ</b> and eotaxin expression was shown to be reduced in kidney tissue vs. the latter. A tendency towards lower interleukine-2 (IL-2) and MIP-1<b>α</b> expression was also observed for these animals (n = 2–6 for each group). Immunohistochemistry values are expressed as the percentage of area of polarized light ± s.d. Scale bar, 50 <b>μ</b>m. Bioplex values are expressed as (μg/mL) ± s.d. *<i>P</i><0.05, **<i>P</i><0.01 ***<i>P</i><0.001</p

Additional file 2: of Intragraft transcriptional profiling of renal transplant patients with tubular dysfunction reveals mechanisms underlying graft injury and recovery

High-hub, hub, and bottleneck genes identified in each comparison. Hubs were defined as highly connected nodes according to node degree values. High-hubs are top-ranked hubs presenting also high betweenness centrality values. Bottleneck genes were classified as nodes with high betweenness centrality but low node degree values

Stem cell characterization.

<p>AdSC from FVB mice were maintained for a maximum of 5 passages and stem cell profile was evaluated through membrane receptor phenotyping and differentiation assays. <b>(A)</b> Representative image of AdSC displaying cell phenotype under bright-field microscopy (original magnification: 20x). <b>(B-D)</b> Stem cell pluripotency was evaluated by culture adipogenesis and osteogenesis under differentiation stimuli and posterior staining with (<b>B)</b> Oil Red and (<b>C)</b> Alizarin Red or (<b>D)</b> Von Kossa, respectively (original magnification: 20x). <b>(E)</b> Representative histograms for stem cell marker expression (CD34, CD45, CD73, CD90 and CD105) obtained by cytometric analysis.</p

ADSC confer increased graft survival upon adoptive transfer.

<p>Allogeneic donor CBA/J skin allografts were transplanted onto C57BL/6 recipients. On day +1 following surgery, recipients were separated into three experimental groups: (A) (Allo-ADSC) were injected intraperitoneally with 5×10⁵ ADSC (n = 8); (BMMC) were injected intraperitoneally with 5×10⁵ bone marrow mononuclear cells (n = 6); (B6-ADSC) were injected intraperitoneally with 5×10⁵ ADSC from syngeneic C57BL/6 donors; (Balb/c-ADSC) were injected intraperitoneally with 5×10⁵ ADSC from a third party strain; (Allo) were intraperitoneally with PBS (n = 10). Isogenic skin transplants were used as controls (n = 5). Morphometric analyses from skin histology at day 3 and 10 show: (B) collagen orientation (Sirius red), (C) neutrophil infiltration and (D) necrosis. (E) VEGF quantification was performed by RT-PCR. (F) Numbers represent the percentage of Foxp3⁺ cells within gated CD4⁺ T cells on day 3 from axillary draining lymph nodes. (G) RT-PCR data showing Foxp3, IFN-γ and IL-2 expression in axillary draining lymph nodes and skin on the day of rejection. Data are represented as mean ± SEM; <i>n</i> = 3 independent experiments. <i>*P</i><0.05.</p

ADSC are effective suppressors of CD4⁺ T cell proliferation in an <i>in vitro</i> MLR co-culture and inhibit Th-1/Th-17 polarization.

<p>(A) In an MLR culture, naïve CD4⁺ T cells from the spleens of C57BL/6 mice were stimulated with mature dendritic cells from CBA/J mice for 4 days in the presence or absence of different concentration of ADSC. CD4⁺ T cell proliferation in these cultures was analyzed by flow cytometry. Gates represent the CFSE dilution peaks using FlowJo. (B) Expansion of cell generations was determined using FlowJo. (C) Cell proliferation relative index was determined using FlowJo. (D) Cytokine levels for IL4, IL-10 and IFN-γ from the culture supernatants were analyzed by Bioplex. (E) Intracellular staining for IL-17 and IFN-γ was performed on cultured T cells and analyzed by flow cytometry in gated CD4⁺ T cells using FlowJo.</p

ADSC change the cytokine milieu <i>in vivo</i> and block Th-17 responses.

<p>C57BL/6 mice were grafted with full thickness allogeneic tail skin from CBA/J mice and treated or not with donor (CBA/J) ADSC. Tissues were analyzed on days 3 and 10 after transplantation. RNA was isolated from (A) draining axillary lymph nodes and (B) skin. Gene expression of Foxp3, TGF-β, IL-10, IFN-γ, IL-17 and IL-6 was assessed by quantitative RT-PCR. Samples were normalized by expression of an endogenous housekeeping gene (HPRT). Data are represented as mean ± SEM; <i>n</i> = 3 independent experiments done in triplicate, leading to a total of ≥10 independent values for each point. <i>*P</i><0,05.</p

Transcriptome Analysis of Renal Ischemia/Reperfusion Injury and Its Modulation by Ischemic Pre-Conditioning or Hemin Treatment

<div><p>Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.</p> </div

Oligonucleotides sequence.

<p>Oligonucleotides sequence.</p

Top ranked biological functions after renal ischemia/reperfusion injury in mice that received previous hemin treatment.

<p>A) KEGG categories showing significant enriched functions for differentially expressed genes at IRI+Hemin <i>vs</i> IRI comparison; B) Biological Process Ontology (GO) of the differentially expressed genes at IRI+Hemin <i>vs</i> IRI comparison. The bar plot represents the percentage of genes differentially expressed and functionally annotated in isolated kidney tissue. Bar plot colors represent up (red) and down (green) regulated genes.</p