Rafael S. Lindoso

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.

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-PK1 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.

Renal recovery after injury: the role of Pax-2

Tubular epithelial cells often suffer injury and damage caused by different factors such as ischaemia or toxicity (for review, see [1,2]). From the observations in human studies and animal models, it is clear that these acute insults can result in chronic kidney disease [3]. The structural and functional restoration of the kidney depends on a delicate balance of growth and transcription factors that guide gene expression [4]. The signalling pathways triggered during this process often resemble those observed during kidney development. Tissue regeneration comprises dedifferentiation, proliferation and transdifferentiation processes [5]. After injury, surviving cells suffer dedifferentiation assuming progenitor cell characteristics (epithelial-to-mesenchymal transition) [6]. Indeed, markers of undifferentiated cells are reexpressed such as vimentin, which occurs in mesenchymal cells and not in mature cells [7], and neural cell adhesion molecules expressed initially in the metanephric mesenchyme [8]. Many genes are reactivated during renal repair, such as c-Jun, c-Fos, c-Myc and EGR-1, leading these cells to resemble kidney organogenesis behaviour [9]. It is important to mention that not all genes involved in tissue repair are related to those activated during embryogenesis and vice-versa. These undifferentiated cells present a higher proliferation rate than that from normal adult kidney cells [10]. Data shown by Witzgall and coworkers [7] revealed that irreversible injured cells (expressing clusterin) do not express vimentin or proliferation cell nuclear antigen (PCNA), which are markers of undifferentiated and proliferating cells, respectively, supporting the idea that only surviving cells are capable of dedifferentiation and proliferation giving rise to cells that subsequently will suffer transdifferentiation. The new cells undergo mesenchymalto-epithelial differentiation functionally replacing the cells lost during injury. It seems to be a promising issue to explore these genes and transcriptional factors that are selectively expressed during embryogenesis and potentially re-expressed after tissue injury and thereby possibly modulate and enhance the regeneration process. Despite the well-established importance of growth factors in kidney organogenesis and regeneration [11,12], there is much to investigate about the downstream effector pathways they regulate. Transcription factors activated by these pathways may be interesting targets for regenerative treatment strategies, especially because some of them are tissue specific. In this review, we focused on Pax-2, an important transcription factor that regulates transition of mesenchymal cells to an epithelial phenotype, expressed in the kidney during development [13] and re-expressed after injury [6,14]. Here we resume the relationship between the transcription factor Pax-2 and renal recovery process after injury, and discuss the mechanisms related to the regulation of its expression.

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.

Renal regenerative potential of different extra-cellular vesicle populations derived from bone marrow mesenchymal stromal cells

Extracellular vesicles (EVs) derived from human bone marrow mesenchymal stromal cells (MSCs) promote the regeneration of kidneys in different animal models of acute kidney injury (AKI) in a manner comparable with the cells of origin. However, due to the heterogeneity observed in the EVs isolated from MSCs, it is unclear which population is responsible for the proregenerative effects. We therefore evaluated the effect of various EV populations separated by differential ultracentrifugation (10K population enriched with microvesicles and 100K population enriched with exosomes) on AKI recovery. Only the exosomal-enriched population induced an improvement of renal function and morphology comparable with that of the total EV population. Interestingly, the 100K EVs exerted a proproliferative effect on murine tubular epithelial cells, both in vitro and in vivo. Analysis of the molecular content from the different EV populations revealed a distinct profile. The 100K population, for instance, was enriched in specific mRNAs (CCNB1, CDK8, CDC6) reported to influence cell cycle entry and progression; miRNAs involved in regulating proliferative/antiapoptotic pathways and growth factors (hepatocyte growth factor and insulin-like growth factor-1) that could explain the effect of renal tubular cell proliferation. On the other hand, the EV population enriched in microvesicles (10K) was unable to induce renal regeneration and had a molecular profile with lower expression of proproliferative molecules. In conclusion, the different molecular composition of exosome- and microvesicle-enriched populations may explain the regenerative effect of EVs observed in AKI.

Extracellular vesicles as regulators of tumor fate: crosstalk among cancer stem cells, tumor cells and mesenchymal stem cells

The tumor microenvironment comprises a heterogeneous population of tumorigenic and non-tumorigenic cells. Cancer stem cells (CSCs) and mesenchymal stem cells (MSCs) are components of this microenvironment and have been described as key regulators of different aspects of tumor physiology. They act differently on the tumor: CSCs are described as tumor initiators and are associated with tumor growth, drug resistance and metastasis; MSCs can integrate the tumor microenvironment after recruitment and interact with cancer cells to promote tumor modifications. Extracellular vesicles (EVs) have emerged as an important mechanism of cell communication under the physiological and pathological conditions. In cancer, secretion of EVs seems to be one of the main mechanisms by which stem cells interact with other tumor and non-tumor cells. The transfer of bioactive molecules (lipids, proteins and RNAs) compartmentalized into EVs triggers different responses in the target cells, regulating several processes in the tumor as angiogenesis, tumor invasiveness and immune escape. This review focuses on the role of CSCs and MSCs in modulating the tumor microenvironment through secretion of EVs, addressing different aspects of the multidirectional interactions among stem cells, tumor and tumor-associated cells.

Resident Stem Cells in Kidney Tissue

Adult stem/progenitor cells have been identified in different segments of the kidney (papilla, cortex, tubules, and interstitium). Detection of these cells is based on classical stem-cell features, such as a slow cell cycle and multipotential capacity, as well as the expression of surface markers, like Sca-1, CD133, and CD24. The lack of kidney-specific stem/progenitor cell markers creates a challenge for their identification in the adult kidney. Although beneficial applications of these cells have been described, their role in renal tissue maintenance and repair is still unknown. Kidney regeneration after injury seems to involve different cell types and mechanisms, and renal stem/progenitor cells may play an important role in this process. It remains to be seen whether these cells can contribute to the establishment and progression of renal disorders, as well as playing a role in renal cancer development. Understanding these issues will help to develop new therapeutic strategies for kidney diseases.

Inside back cover: Proteomics of cell–cell interactions in health and disease

DOI: 10.1002/pmic.201500341 Understanding the cell to cell interactions by proteomic approaches. Venn diagram shows the proteomic methodologies that can be applied to analyze proteins differentially expressed in cell to cell interaction mechanisms. The background figures describe data from previous studies published in Proteomics. For details, see the review by Rafael S. Lindoso et al. on page 328.

miRNA Expression in Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) express defined patterns of miRNAs which have been shown to be involved in stemness and differentiation processes. Modulation of miRNA expression by MSCs has been shown to change their biological properties. Moreover, in light of the paracrine hypothesis of MSC action, increasing evidence indicates that miRNA transfer between MSCs and injured cells in tissues accounts for the healing properties exhibited by MSCs. Extracellular vesicles (EVs) are candidates for the trafficking of encapsulated miRNAs between cells, as they are able to cross easily biological barriers and protect nucleic acids from degradation. Moreover, EVs, by expressing the same surface receptors of MSCs, may be recruited to the site of injury as well as the cell of origin. These properties of EVs may be exploited for the generation of specially engineered EVs carrying specific miRNAs for therapeutic purposes.