Early Effects of Extracellular Vesicles Secreted by Adipose Tissue Mesenchymal Cells in Renal Ischemia Followed by Reperfusion: Mechanisms Rely on a Decrease in Mitochondrial Anion Superoxide Production.

Acute kidney injury (AKI) caused by ischemia followed by reperfusion (I/R) is characterized by intense anion superoxide (O2•-) production and oxidative damage. We investigated whether extracellular vesicles secreted by adipose tissue mesenchymal cells (EVs) administered during reperfusion can suppress the exacerbated mitochondrial O2•- formation after I/R. We used Wistar rats subjected to bilateral renal arterial clamping (30 min) followed by 24 h of reperfusion. The animals received EVs (I/R + EVs group) or saline (I/R group) in the kidney subcapsular space. The third group consisted of false-operated rats (SHAM). Mitochondria were isolated from proximal tubule cells and used immediately. Amplex Red™ was used to measure mitochondrial O2•- formation and MitoTracker™ Orange to evaluate inner mitochondrial membrane potential (Δψ). In vitro studies were carried out on human renal proximal tubular cells (HK-2) co-cultured or not with EVs under hypoxic conditions. Administration of EVs restored O2•- formation to SHAM levels in all mitochondrial functional conditions. The gene expression of catalase and superoxide dismutase-1 remained unmodified; transcription of heme oxygenase-1 (HO-1) was upregulated. The co-cultures of HK-2 cells with EVs revealed an intense decrease in apoptosis. We conclude that the mechanisms by which EVs favor long-term recovery of renal structures and functions after I/R rely on a decrease of mitochondrial O2•- formation with the aid of the upregulated antioxidant HO-1/Nuclear factor erythroid 2-related factor 2 system, thus opening new vistas for the treatment of AKI.

Type 2 diabetes mellitus alters cardiac mitochondrial content and function in a non-obese mice model.

Type 2 diabetes mellitus (T2DM) is associated with an increase of premature appearance of several disorders such as cardiac complications. Thus, we test the hypothesis that a combination of a high fat diet (HFD) and low doses of streptozotocin (STZ) recapitulate a suitable mice model of T2DM to study the cardiac mitochondrial disturbances induced by this disease. Animals were divided in 2 groups: the T2DM group was given a HFD and injected with 2 low doses of STZ, while the CNTRL group was given a standard chow and a buffer solution. The combination of HFD and STZ recapitulate the T2DM metabolic profile showing higher blood glucose levels in T2DM mice when compared to CNTRL, and also, insulin resistance. The kidney structure/function was preserved. Regarding cardiac mitochondrial function, in all phosphorylative states, the cardiac mitochondria from T2DM mice presented reduced oxygen fluxes when compared to CNTRL mice. Also, mitochondria from T2DM mice showed decreased citrate synthase activity and lower protein content of mitochondrial complexes. Our results show that in this non-obese T2DM model, which recapitulates the classical metabolic alterations, mitochondrial function is impaired and provides a useful model to deepen study the mechanisms underlying these alterations.

Proteomics of cell-cell interactions in health and disease.

The mechanisms of cell-cell communications are now under intense study by proteomic approaches. Proteomics has unraveled changes in protein profiling as the result of cell interactions mediated by ligand/receptor, hormones, soluble factors, and the content of extracellular vesicles. Besides being a brief overview of the main and profitable methodologies now available (evaluating theory behind the methods, their usefulness, and pitfalls), this review focuses on-from a proteome perspective-some signaling pathways and post-translational modifications (PTMs), which are essential for understanding ischemic lesions and their recovery in two vital organs in mammals, the heart, and the kidney. Knowledge of misdirection of the proteome during tissue recovery, such as represented by the convergence between fibrosis and cancer, emerges as an important tool in prognosis. Proteomics of cell-cell interaction is also especially useful for understanding how stem cells interact in injured tissues, anticipating clues for rational therapeutic interventions. In the effervescent field of induced pluripotency and cell reprogramming, proteomic studies have shown what proteins from specialized cells contribute to the recovery of infarcted tissues. Overall, we conclude that proteomics is at the forefront in helping us to understand the mechanisms that underpin prevalent pathological processes.

Mechanisms of kidney repair by human mesenchymal stromal cells after ischemia: a comprehensive view using label-free MS(E).

Acute kidney injury (AKI) is one of the more frequent and lethal pathological conditions seen in intensive care units. Currently available treatments are not totally effective but stem cell-based therapies are emerging as promising alternatives, especially the use of mesenchymal stromal cells (MSC), although the signaling pathways involved in their beneficial actions are not fully understood. The objective of this study was to identify signaling networks and key proteins involved in the repair of ischemia by MSC. Using an in vitro model of AKI to investigate paracrine interactions and label-free high definition 2D-NanoESI-MS(E) , differentially expressed proteins were identified in a human renal proximal tubule cell lineage (HK-2) exposed to human MSC (hMSC) after an ischemic insult. In silico analysis showed that hMSC stimulated antiapoptotic activity, normal ROS handling, energy production, cytoskeleton organization, protein synthesis, and cell proliferation. The proteomic data were validated by parallel experiments demonstrating reduced apoptosis in HK-2 cells and recovery of intracellular ATP levels. qRT-PCR for proteins implicated in the above processes revealed that hMSC exerted their effects by stimulating translation, not transcription. Western blotting of proteins associated with ROS and energy metabolism confirmed their higher abundance in HK-2 cells exposed to hMSC.

Paracrine interaction between bone marrow-derived stem cells and renal epithelial cells.

BACKGROUND/AIMS: Renal tubular cells are the main target of ischemic insult associated with acute renal injury. Low oxygen and nutrient supplies result in ATP depletion, leading to cell death and loss of renal function. A possible mechanism by which bone marrow-derived cells support renal tissue regeneration relies on the capacity of mononuclear cells (BMMC), particularly mesenchymal stem cells (MSC), to secrete paracrine factors that mediate support for kidney regeneration. METHODS: BMMC/MSC and renal cells (LLC-PK(1) from pig and IRPTC from rat) were co-cultured under stressful conditions (ATP depletion and/or serum free starvation), physically separated by a microporous membrane (0.4 µm), was used to determine whether bone marrow-derived cells can interact with renal cells in a paracrine manner. RESULTS: This interaction resulted in stimulation of renal cell proliferation and the arrest of cell death. MSC elicit effective responses in renal cells in terms of stimulating proliferation and protection. Such effects are observed in renal cells co-cultured with rat BMMC/MSC, an indication that paracrine mechanisms are not entirely species-specific. CONCLUSION: The paracrine action of BMMC/MSC was influenced by a renal cell stimulus released during stress, indicating that cross-talk with injured cells is required for renal regeneration supported by bone marrow-derived cells.

Bone marrow mononuclear cells shift bioactive lipid pattern in injured kidney towards tissue repair in rats with unilateral ureteral obstruction.

BACKGROUND: Bioactive lipids are important in tissue injury and regeneration. Ceramide (Cer) is known for its pro-apoptotic action and sphingosine-1-phosphate (S1P) for inducing proliferation and cell survival; diacylglycerol (DAG) and lysophosphatidic acid (LPA) are involved in various signalling pathways including modulation of ion transport. LPA signalling through its receptor LPA(1) is also related to the progression of fibrosis. This study investigated the modulation of lipid signalling pathways induced by administration of bone marrow-derived mononuclear cells (BMMC) in chronic kidney disease. METHODS: Unilateral ureteral obstruction (UUO) was followed by intravenous injection of ∼2 × 10(7) BMMC. Controls were UUO group treated with buffered solution and sham-operated group. Animals were killed 14 days after surgery, and lipid phosphorylation assays and immunoblotting were performed on the kidney homogenates. RESULTS: More DAG was available in the UUO rats (2.4 ± 0.4 and 2.4 ± 0.3 vs 1.0 ± 0.2 pmol (32)PA mg(-)(1) min(-)(1), in UUO and UUO + BMMC vs SHAM). Sphingosine kinase was 150 ± 12% more active in UUO + BMMC than in UUO and SHAM. Cer levels were 76 ± 7% lower in the UUO + BMMC than UUO. LPA receptor type 1 (LPA(1)) expression was 169 ± 7% higher in the UUO group than in UUO + BMMC and SHAM. BMMC maintain control levels of Ca(2+)-ATPase expression altered by UUO by 40%. CONCLUSIONS: BMMC infusion modulated diverse lipid signalling pathways and protein expression, shifted sphingolipid metabolism toward a regenerative pattern and favourably reduced the levels of a receptor involved in the progression of tissue fibrosis. These results strengthen the benefits of BMMC treatment and give insight into its paracrine mechanisms of action.

Acute Myocardial Infarction Reduces Respiration in Rat Cardiac Fibers, despite Adipose Tissue Mesenchymal Stromal Cell Transplant.

Adipose-derived mesenchymal stromal cell (AD-MSC) administration improves cardiac function after acute myocardial infarction (AMI). Although the mechanisms underlying this effect remain to be elucidated, the reversal of the mitochondrial dysfunction may be associated with AMI recovery. Here, we analyzed the alterations in the respiratory capacity of cardiomyocytes in the infarcted zone (IZ) and the border zone (BZ) and evaluated if mitochondrial function improved in cardiomyocytes after AD-MSC transplantation. Female rats were subjected to AMI by permanent left anterior descending coronary (LAD) ligation and were then treated with AD-MSCs or PBS in the border zone (BZ). Cardiac fibers were analyzed 24 hours (necrotic phase) and 8 days (fibrotic phase) after AMI for mitochondrial respiration, citrate synthase (CS) activity, F0F1-ATPase activity, and transmission electron microscopy (TEM). High-resolution respirometry of permeabilized cardiac fibers showed that AMI reduced numerous mitochondrial respiration parameters in cardiac tissue, including phosphorylating and nonphosphorylating conditions, respiration coupled to ATP synthesis, and maximal respiratory capacity. CS decreased in IZ and BZ at the necrotic phase, whereas it recovered in BZ and continued to drop in IZ over time when compared to Sham. Exogenous cytochrome c doubled respiration at the necrotic phase in IZ. F0F1-ATPase activity decreased in the BZ and, to more extent, in IZ in both phases. Transmission electron microscopy showed disorganized mitochondrial cristae structure, which was more accentuated in IZ but also important in BZ. All these alterations in mitochondrial respiration were still present in the group treated with AD-MSC. In conclusion, AMI led to mitochondrial dysfunction with oxidative phosphorylation disorders, and AD-MSC improved CS temporarily but was not able to avoid alterations in mitochondria function over time.

Extracellular vesicles released from mesenchymal stromal cells modulate miRNA in renal tubular cells and inhibit ATP depletion injury.

The mechanisms involved in renal repair by mesenchymal stromal cells (MSCs) are not entirely elucidated. The paracrine secretion of bioactive molecules has been implicated in the protective effects. Besides soluble mediators, MSCs have been shown to release extracellular vesicles (EVs), involved in renal repair process for different injury models. EVs have been shown to mediate communication between cells through the transference of several molecules, like protein, bioactive lipids, mRNA, and microRNAs (miRNAs). The miRNAs are noncoding RNAs that posttranscriptionally modulate gene expression and are involved in the regulation of several cellular processes, including those related to repair. The aim of the present study was to investigate the role of MSC-EVs in the modulation of miRNAs inside renal proximal tubular epithelial cells (PTECs) in an in vitro model of ischemia-reperfusion injury induced by ATP depletion. In this model we evaluated whether changes in miRNA expression were dependent on direct miRNA transfer or on transcription induction by MSC-EVs. The obtained results showed an enhanced incorporation of MSC-EVs in injured PTECs with protection from cell death. This biological effect was associated with EV-mediated miRNA transfer and with transcriptional modulation of miRNAs expressed by injured PTECs. Prediction of miRNA targets showed that miRNAs modulated in PTECs are involved in process of renal recovery with downregulation of coding-mRNAs associated with apoptosis, cytoskeleton reorganization, and hypoxia, such as CASP3 and 7, SHC1 and SMAD4. In conclusion, these results indicate that MSC-EVs may transfer and modulate the expression of several miRNAs involved in the repair and recovery process in PTECs.