Karine S. Verdoorn

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.

Bone Marrow Mononuclear Cells Attenuate Interstitial Fibrosis and Stimulate the Repair of Tubular Epithelial Cells after Unilateral Ureteral Obstruction

The growing number of patients suffering from chronic renal disease is a challenge for the development of innovative therapies. Benefits of cell therapy in acute renal diseases in animal models have been reported but seldom for chronic lesions. We present evidence for the improvement of renal morphology in a model of tubulointerstitial fibrosis. Wistar rats were submitted to unilateral ureteral obstruction (UUO), treated with bone-marrow mononuclear cells (UUO+BMMC) infused via the cava vein, and killed on day 14. Labeled BMMC were seen in renal tissue after 7 days in the group UUO+BMMC. UUO+BMMC also showed a reduction in ED1+ cells and tubular apoptotic cells together with enhanced tubular proliferation. Myofibroblasts were also reduced after BMMC which is consistent with a decrease in collagen deposition (picro Sirius staining) and RT-PCR data showing lower levels of procollagen-I mRNA. Simultaneously, nestin+ cells increased in the interstitium and decreased in the tubules. Double stained nestin+/α-SMA+ cells were present only in the interstitium, and their levels did not change after BMMC infusion. These data indicate a renoprotective effect of BMMC through increased tubular cell regeneration, inhibition of tubular cell apoptosis and partially blocking of the inflammatory and fibrotic events that occur after unilateral ureteral obstruction.

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.

Delta Opioid Receptors: The Link between Exercise and Cardioprotection

This study investigated the role of opioid receptor (OR) subtypes as a mechanism by which endurance exercise promotes cardioprotection against myocardial ischemia-reperfusion (IR) injury. Wistar rats were randomly divided into one of seven experimental groups: 1) control; 2) exercise-trained; 3) exercise-trained plus a non-selective OR antagonist; 4) control sham; 5) exercise-trained plus a kappa OR antagonist; 6) exercise-trained plus a delta OR antagonist; and 7) exercise-trained plus a mu OR antagonist. The exercised animals underwent 4 consecutive days of treadmill training (60 min/day at ∼70% of maximal oxygen consumption). All groups except the sham group were exposed to an in vivo myocardial IR insult, and the myocardial infarct size (IS) was determined histologically. Myocardial capillary density, OR subtype expression, heat shock protein 72 (HSP72) expression, and antioxidant enzyme activity were measured in the hearts of both the exercised and control groups. Exercise training significantly reduced the myocardial IS by approximately 34%. Pharmacological blockade of the kappa or mu OR subtypes did not blunt exercise-induced cardioprotection against IR-mediated infarction, whereas treatment of animals with a non-selective OR antagonist or a delta OR antagonist abolished exercise-induced cardioprotection. Exercise training enhanced the activities of myocardial superoxide dismutase (SOD) and catalase but did not increase the left ventricular capillary density or the mRNA levels of HSP72, SOD, and catalase. In addition, exercise significantly reduced the protein expression of kappa and delta ORs in the heart by 44% and 37%, respectively. Together, these results indicate that ORs contribute to the cardioprotection conferred by endurance exercise, with the delta OR subtype playing a key role in this response.

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.

A comparative proteomic analysis of Vibrio cholerae O1 wild-type cells versus a phoB mutant showed that the PhoB/PhoR system is required for full growth and rpoS expression under inorganic phosphate abundance.

UNLABELLED PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae. BIOLOGICAL SIGNIFICANCE Certain V. cholerae strains are pathogenic to humans, causing cholera, an acute dehydrating diarrhoeal disease endemic in Southern Asia, parts of Africa and Latin America, where it has been responsible for significant mortality and economical damage. Its ability to grow within distinct niches is dependent on gene expression regulation. PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. However, Pho regulon genes also play roles in virulence, motility and biofilm formation, among others. In this paper we report that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways, in Pi abundance. Moreover, we showed, for the first time, that the interrelationship between PhoB-RpoS-(p)ppGpp-poly(P) in V. cholerae, is somewhat diverse from the model of inter-regulation between those systems, described in Escherichia coli. The V. cholerae dependence on PhoB/PhoR for the RpoS mediated stress response and cellular growth under Pi abundance, suggests that this systems roles are broader than previously thought.

Biphasic regulation of type II phosphatidylinositol-4 kinase by sphingosine: Cross talk between glycero- and sphingolipids in the kidney

Phosphatidylinositol-4 kinase (PI-4K) is responsible for the generation of phosphatidylinositol-4 phosphate (PtdIns(4)P), a bioactive signaling molecule involved in several biological functions. In this study, we show that sphingosine modulates the activity of the PI-4K isoform associated with the basolateral membranes (BLM) from kidney proximal tubules. Immunoblotting with an anti-α subunit PI-4K polyclonal antibody revealed the presence of two bands of 57 and 62kDa in the BLM. BLM-PI-4K activity retains noteworthy biochemical properties; it is adenosine-sensitive, not altered by wortmanin, and significantly inhibited by Ca(2+) at the μM range. Together, these observations indicate the presence of a type II PI-4K. Endogenous phosphatidylinositol (PI) alone reaches PI-4K half-maximal activity, revealing that even slight modifications in PI levels at the membrane environment promote significant variations in BLM-associated-PI-4K activity. ATP-dependence assays suggested that the Mg.ATP(2-) complex is the true substrate of the enzyme and that free Mg(2+) is an essential cofactor. Another observation indicated that higher concentrations of free ATP are inhibitory. BLM-associated-PI-4K activity was ~3-fold stimulated in the presence of increasing concentration of sphingosine, while in concentrations higher than 0.4mM, in which S1P is pronouncedly formed, there was an inhibitory effect on PtdIns(4)P formation. We propose that a tightly coupled regulatory network involving phosphoinositides and sphingolipids participate in the regulation of key physiological processes in renal BLM carried out by PI-4K.

Transplantation of bone marrow-derived MSCs improves renal function and Na + +K + -ATPase activity in rats with renovascular hypertension

Renovascular hypertension (RVH) is a progressive disease, leading to chronic kidney disease when untreated and no specific treatment is available. Therefore, development of new therapeutic modalities is imperative. RVH is triggered by renal artery stenosis and subsequent renin–angiotensin–aldosterone system activation; it can be experimentally induced by the 2 Kidneys–1 Clip (2K1C) model. This study investigates the therapeutic potential of renal subcapsular mesenchymal stem cell (MSC) infusion in 2K1C rats. Renal morphological and functional changes were analyzed, including Na++K+-ATPase activity and expression, renin angiotensin-converting enzyme (ACE) and angiotensin-II type 1 (AT1R) and type 2 (AT2R) receptors expression. 2K1C rats developed hypertension accompanied by renin upregulation (clipped kidney) and renal Na++K+-ATPase activity and expression reduction. MSC therapy decreased systolic blood pressure, renin, ACE, and AT1R, upregulated AT2R and podocin expression and restored renal Na++K+-ATPase activity and expression. In addition, MSC improved renal morphology, reduced fibrosis and TGF-β expression in the clipped kidney, decreased proteinuria and restored protein plasma levels. In conclusion, transplantation into a renal subcapsule is an efficient route and MSC is a good candidate for cell therapy, which may represent an interesting approach for chronic kidney disease treatment.

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.