Luciana Aparecida Reis

Bone Marrow-Derived Mesenchymal Stem Cells Repaired but Did Not Prevent Gentamicin-Induced Acute Kidney Injury through Paracrine Effects in Rats

This study evaluated the effects of bone marrow-derived mesenchymal stem cells (BMSCs) or their conditioned medium (CM) on the repair and prevention of Acute Kidney Injury (AKI) induced by gentamicin (G). Animals received daily injections of G up to 20 days. On the 10th day, injections of BMSCs, CM, CM+trypsin, CM+RNase or exosome-like microvesicles extracted from the CM were administered. In the prevention groups, the animals received the BMSCs 24 h before or on the 5th day of G treatment. Creatinine (Cr), urea (U), FENa and cytokines were quantified. The kidneys were evaluated using hematoxylin/eosin staining and immunohystochemistry. The levels of Cr, U and FENa increased during all the periods of G treatment. The BMSC transplantation, its CM or exosome injections inhibited the increase in Cr, U, FENa, necrosis, apoptosis and also increased cell proliferation. The pro-inflammatory cytokines decreased while the anti-inflammatory cytokines increased compared to G. When the CM or its exosomes were incubated with RNase (but not trypsin), these effects were blunted. The Y chromosome was not observed in the 24-h prevention group, but it persisted in the kidney for all of the periods analyzed, suggesting that the injury is necessary for the docking and maintenance of BMSCs in the kidney. In conclusion, the BMSCs and CM minimized the G-induced renal damage through paracrine effects, most likely through the RNA carried by the exosome-like microvesicles. The use of the CM from BMSCs can be a potential therapeutic tool for this type of nephrotoxicity, allowing for the avoidance of cell transplantations.

Transplantation of bone marrow mesenchymal stem cells decreases oxidative stress, apoptosis, and hippocampal damage in brain of a spontaneous stroke model

Stroke is the most common cause of motor disabilities and is a major cause of mortality worldwide. Adult stem cells have been shown to be effective against neuronal degeneration through mechanisms that include both the recovery of neurotransmitter activity and a decrease in apoptosis and oxidative stress. We chose the lineage stroke-prone spontaneously hypertensive rat (SHRSP) as a model for stem cell therapy. SHRSP rats can develop such severe hypertension that they generally suffer a stroke at approximately 1 year of age. The aim of this study was to evaluate whether mesenchymal stem cells (MSCs) decrease apoptotic death and oxidative stress in existing SHRSP brain tissue. The results of qRT-PCR assays showed higher levels of the antiapoptotic Bcl-2 gene in the MSC-treated animals, compared with untreated. Our study also showed that superoxide, apoptotic cells, and by-products of lipid peroxidation decreased in MSC-treated SHRSP to levels similar those found in the animal controls, Wistar Kyoto rats. In addition, we saw a repair of morphological damage at the hippocampal region after MSC transplantation. These data suggest that MSCs have neuroprotective and antioxidant potential in stroke-prone spontaneously hypertensive rats.

Receptor-Induced Dilatation in the Systemic and Intrarenal Adaptation to Pregnancy in Rats

Normal pregnancy is associated with systemic and intrarenal vasodilatation resulting in an increased glomerular filtration rate. This adaptive response occurs in spite of elevated circulating levels of angiotensin II (Ang II). In the present study, we evaluated the potential mechanisms responsible for this adaptation. The reactivity of the mesangial cells (MCs) cultured from 14-day-pregnant rats to Ang II was measured through changes in the intracellular calcium concentration ([Cai]). The expression levels of inducible nitric oxide synthase (iNOS), the Ang II-induced vasodilatation receptor AT2, and the relaxin (LGR7) receptor were evaluated in cultured MCs and in the aorta, renal artery and kidney cortex by real time-PCR. The intrarenal distribution of LGR7 was further analyzed by immunohistochemistry. The MCs displayed a relative insensitivity to Ang II, which was paralleled by an impressive increase in the expression level of iNOS, AT2 and LGR7. These results suggest that the MCs also adapt to the pregnancy, thereby contributing to the maintenance of the glomerular surface area even in the presence of high levels of Ang II. The mRNA expression levels of AT2 and LGR7 also increased in the aorta, renal artery and kidney of the pregnant animals, whereas the expression of the AT1 did not significantly change. This further suggests a role of these vasodilatation-induced receptors in the systemic and intrarenal adaptation during pregnancy. LGR7 was localized in the glomeruli and on the apical membrane of the tubular cells, with stronger labeling in the kidneys of pregnant rats. These results suggest a role of iNOS, AT2, and LGR7 in the systemic vasodilatation and intrarenal adaptation to pregnancy and also suggest a pivotal role for relaxin in the tubular function during gestation.

Heme oxygenase 1 improves glucoses metabolism and kidney histological alterations in diabetic rats

One important concern in the treatment of diabetes is the maintenance of glycemic levels and the prevention of diabetic nephropathy. Inducible heme oxygenase 1 (HO-1) is a rate-limiting enzyme thought to have antioxidant and cytoprotective roles. The goal of the present study was to analyze the effect of HO-1 induction in chronically hyperglycemic rats. The hyperglycemic rats were divided into two groups: one group, called STZ, was given a single injection of streptozotocin; and the other group was given a single streptozotocin injection as well as daily injections of hemin, an HO-1 inducer, over 60 days (STZ + HEME). A group of normoglycemic, untreated rats was used as the control (CTL).Body weight, diuresis, serum glucose levels, microalbuminuria, creatinine clearance rate, urea levels, sodium excretion, and lipid peroxidation were analyzed. Histological alterations and immunohistochemistry for HO-1 and inducible nitric oxide synthase (iNOS) were assessed. After 60 days, the STZ group exhibited an increase in blood glucose, diuresis, urea, microalbuminuria, and sodium excretion. There was no weight gain, and there was a decrease in creatinine clearance in comparison to the CTL group. In the STZ + HEME group there was an improvement in the metabolic parameters and kidney function, a decrease in blood glucose, serum urea, and microalbuminuria, and an increase of creatinine clearance, in comparison to the STZ group.There was glomerulosclerosis, collagen deposition in the STZ rats and increase in iNOS and HO-1 expression. In the STZ + HEME group, the glomerulosclerosis and fibrosis was prevented and there was an increase in the expression of HO-1, but decrease in iNOS expression and lipid peroxidation. In conclusion, our data suggest that chronic induction of HO-1 reduces hyperglycemia, improves glucose metabolism and, at least in part, protects the renal tissue from hyperglycemic injury, possibly through the antioxidant activity of HO-1.

Synergistic effect of apoptosis and necroptosis inhibitors in cisplatin-induced nephrotoxicity

Abstract Necroptosis is a nonapoptotic cell death pathway. We aim to study the effect of necrostatin-1 (a specific necroptosis inhibitor) in cisplatin-induced injury. We analyzed the effect of the combined use of inhibitors of apoptosis (z-vad) and necroptosis (necrostatin-1) in acute kidney injury by cisplatin in human proximal tubule cells. Our results showed moderate effectiveness in cytoprotection after treatment with z-vad. But the concomitant use of inhibitors (z-vad and necrostatin-1) presented synergistic and additive protection. The present study analyzed the caspase-3 activity and we observed a significant decrease in the group treated with z-vad and cisplatin. However we did not observe changes in the group treated with both inhibitors (z-vad and necrostatin-1) and cisplatin. Thus, demonstrating that necroptosis is a caspase-independent mechanism. We also analyzed the effect of necrostatin-1 in vivo model. C57BL/6 mice were treated with cisplatin and/or inhibitors. The concomitant use of inhibitors (z-vad and necrostatin-1) recovered renal function and decreased levels of urinary Ngal. Additionally, we analyzed the expression of RIP-1, a specific marker for necroptosis. In animals treated with cisplatin and z-VAD levels of RIP-1 were higher. This result reinforces that necroptosis occurs only in conditions where apoptosis was blocked. However, the use of both inhibitors (z-vad and necrostatin-1) provided additional protection. In conclusion, our study has a significant potential to show in vitro and in vivo protection obtained by necrostatin-1. Therefore, our results suggest that necroptosis may be an important mechanism of cell death after kidney injury.

Bone Marrow-Derived Mesenchymal Stem Cells and Their Conditioned Medium Attenuate Fibrosis in an Irreversible Model of Unilateral Ureteral Obstruction.

The therapeutic potential of mesenchymal stem cells (MSCs) and their conditioned medium (MSC-CM) has been extensively studied. MSCs can repair tissue, reduce local inflammation, and modulate the immune response. Persistent renal tubular interstitial inflammation results in fibrosis and leads to chronic kidney disease (CKD). Unilateral ureteral obstruction (UUO) is a very well-accepted renal fibrosis model. In this study, we evaluated factors influenced by the administration of MSCs or MSC-CM in the UUO model. MSCs extracted from rat bone marrow were cultivated in vitro and characterized by flow cytometry and cellular differentiation. Eight groups of female rats were used in experiments (n = 7, each), including Sham, UUO, UUO + MSC (obstruction + MSC), and UUO + CM (obstruction + MSC-CM) for 7 days of obstruction and Sham, UUO, UUO + MSC, and UUO + CM for 14 days of obstruction. The MSCs or MSC-CM was administered via the abdominal vena cava after total ligation of the left ureter. After 7 or 14 days, rats were euthanized, and serum and obstructed kidney samples were collected. MSCs or MSC-CM decreased the expression of molecules, such as Collai, α-SMA, and TNF-α. We also observed reductions in the levels of caspase 3, α-SMA, and PCNA in treated animals by immunohistochemistry. Our results suggest that the intravenous administration of MSCs or MSC-CM improves fibrosis progression and factors involved in apoptosis, inflammation, cell proliferation, and epithelial–mesenchymal transition in Wistar rats subjected to UUO, indicating a potential tool for preventing CKD.

Preconditioning induced by gentamicin protects against acute kidney injury: The role of prostaglandins but not nitric oxide

Nephrotoxicity is the main side effect of gentamicin (GENTA). Preconditioning (PC) refers to a situation in which an organ subjected to an injury responds less intensely when exposed to another injury. The aim of this study was to evaluate the effect of PC with GENTA on nephrotoxic acute kidney injury (AKI). GENTA group rats were injected daily with GENTA (40 mg/kg/BW) for 10 days. PC animals were injected with GENTA for 3 days (40 mg/kg/BW/daily) and, after one rest week, were injected daily with GENTA for 10 days. Animals of the L-NAME and DICLO groups were preconditioned for 3 days and then received daily injections of GENTA for 10 days; they were concomitantly treated with L-NAME (10 mg/kg/BW) and diclofenac (DICLO, 5 mg/kg/BW) for 13 days. Blood and urine were collected for measurement of serum creatinine, urea, urine sodium, protein, hydroperoxides, lipid peroxidation and nitric oxide (NO). The animals were killed; kidneys were removed for histology and immunohistochemistry for apoptosis and cell proliferation. GENTA group rats showed an increase in plasma creatinine, urea, urine sodium, hydroperoxides, lipid peroxidation, proteinuria, necrosis and apoptosis, characterizing nephrotoxic AKI. PC animals showed a decrease in these parameters and increased proliferation. The blockade of NO synthesis by L-NAME potentiated the protective effect, suggesting that NO contributed to the injury caused by GENTA. The blockade of prostaglandin synthesis with DICLO increased serum and urinary parameters, blunting the protective effect of PC. Our data suggest that PC could be a useful tool to protect against nephrotoxic AKI.

Priming prevent nephrotoxic acute renal failure through stimulation of antioxidant defense mechanism.

INTRODUCTION Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. OBJECTIVE To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). METHODS Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. RESULTS Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. CONCLUSION Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.Introduction: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. Objective: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). Methods: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. Results: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. Conclusion: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.

Effects of Exosomes (EXOs) Derived by Renal Pluripotent Stem Cells (rPSCs) on the Cisplatin (Cis) Nephrotoxicity in Mice

The acute kidney injury (AKI) is characterized by Cis that induces hypoxia and generates free radicals, inflammation and apoptosis of renal tubules [1]. For these reasons it becomes essential to use strategies to prevent this acute damage. Therefore EXOs are potential tools [2]. In this study, we evaluated the EXOs derived by rPSCs on the nephrotoxicity induced by Cis. For in vitro methods, rPSCs mice (C57BL6-J) were used to obtain cultured using standard techniques following glomerular isolation by differential sieving and rPSCs. The rPSCs were characterized by immunofluorescence and FACS utilizing positive antibodies (Wnt1, CD24, PAX2 and ZO1) and negative antibodies (CD45, Thy-1 and pan cytokeratin). MSCs Mice (C57BL6-J) were used to obtained cultured using standard techniques following bone marrow isolation by differential sieving and MSCs. EXOs: rPSCs were incubed for 24 hours. Then, the EXOs were obtained by ultracentrifugation technique and characterized by Western blot utilizing CD9 and CD63. For in vivo methods, mice were treated with Cis (10mg/BW) or PBS (vehicle CTL group) during 5 days. At the 3th day of this treatment, animals were treated with EXOs (35μg/ml) derived from rPSCs in only one injection. At the end of these treatment, urine 24 hours and blood were collected by biochemical and cytokines (IL2, IL6 and IL10) analysis and kidney to evaluated for HE, caspase 3 and KI67. Results: It was observed that the rPSCs positive for the markers scored (Wnt1, CD24, PAX2 and ZO1); EXOs marked to CD9 and 63. The creatinine, urea, IL2 and IL6 increased in Cis group when compared to CTL; Cr:(2.5±0.9 vs. 1.4±0.4 mg/dl; p<0.05); U: (300±20.6 vs. 100±22.8 mg/dl; p<0.05); IL2: (1.2±0.3 vs. 0.2±0.02 pg/ml; p<0.05); IL6: (1.8±0.4 vs. 1.1±0.07pg/ml; p<0.05); Therefore, t`s not observed difference in IL10 (0.2±0.01 vs. 0.2±0.03pg/ml; p<0.05). When these animals received EXOs it was observed a decrease in Cr, U, IL2, IL6 and a significant increase of anti-inflammatory cytokines IL10 when compared to Cis group; Cr:(1.4±0.7 vs. 1.4±0.4 mg/dl; p<0.05); U: (208±32.7 vs. 100±22.8 mg/dl; p<0.05); IL2: (0.4±0.01 vs. 0.2±0.02 pg/ml; p<0.05); IL6: (0.7±0.5 vs. 1.1±0.07pg/ml; p<0.05); Therefore, ť s not observed difference in IL10 (1.1±0.02 vs. 0.2±0.03pg/ml; p<0.05). In Cis-group, the kidneys showed a small marked KI67 and intensive caspase 3 and acute tubular necrosis (ATN) expression but differently, it was highly marked for KI67 and lower expression for caspase 3 in Cis+EXOs groups and no histological ATN lesions were observed. These results strongly suggest that EXOs derived from rPSCs can minimize AKI induced by Cis. These therapeutics EXOs effects have a significant impact on renal function and holds substantial potential use especially by avoiding transplanting cells with potential adverse effects, in this experimental model.

Comparative study of intravenous and topical administration of mesenchymal stem cells in experimental colitis

Adult marrow-derived mesenchymal stem cells (MSCs) have anti-inflammatory properties in patients with Inflammatory Bowel Disease (IBD), but systemic delivery is associated with safety concerns. Whether topical delivery of MSCs would provide similar efficacy to systemic administration is unknown. To compare topicallydelivered MSCs to systemic asministration, and non-MSC therapy, in animal model of colitis. Trinitrobenzenesulfonic acid (TNBS) colitis was induced in Wistar rats. Topical MSCs were compared to intravenous MSCs, adalimumab, infliximab and control in this model. Serial measurements of clinical criteria were used (i.e., weight, stool characteristics), and serum interleukin 6 (IL-6) and TNF measurements and macroscopic and microscopic scores were used to evaluate treatment efficacy. Topical and intravenous stem cell treatments, significantly prevented weight loss in TNBS mice on day 3 of colitis. There was greater evidence of a difference mainly on the third day (p<0.001). IV or PR stem cells also reduced serum IL-6 and TNF levels to similar levels to those of anti-TNF treated animals. In the intestinal tract, stem-cell treatment ameliorated the microscopic and macroscopic damage caused by TNBS. Rectal-delivered stem cells produced similar results to IV-delivered cells. This study demonstrates that rectal stem cells can treat colitis in an animal model to a similar extent to IV stem cells and systemic anti-TNF therapies. The mechanisms of this effect warrants further study. Introduction Inflammatory bowel diseases have pathophysiologies based on genetic inheritance associated with environmental factors related either to the intestinal lumen or not, and produce an exacerbated inflammatory response, resulting in intestinal or extraintestinal clinical manifestations [1-4]. In recent years, experimental models have significantly contributed to the understanding of these diseases, allowing the development of drugs with high specificity that act directly on target inflammatory mediators, such as Tumor Necrosis Factor alpha (TNF-alpha) and adhesion molecules, among others.This advancement has enabled treatment providers to change the natural history of these diseases, increasing the quality of life of affected patients and decreasing hospitalization and surgical intervention rates [5]. Although these drugs have modified the treatment scenario, primary drug failure due to loss of response has been observed over the years [6]. Primary failure has corresponded to more than 40% of cases [7], and serial clinical studies indicate a primary failure rate of 10 to 20%. During one year, this loss of response may vary between 23 to 43% of cases [6]. Genetic factors, disease duration, smoking and even the production of antidrug antibodies have been associated with primary failure [6,8]. Conversely, recent studies indicate the possibility of treatment using an immune path (cell therapy) different from drug therapy in patients who are intolerant to conventional drug treatments or when such treatments fail [9]. Cell therapy could represent the optimization of intestinal factors produced by mesenchymal stem cells, which have demonstrated the capacity to inhibit TNF-alpha and Interleukin 6 (IL-6) by regulating the incorrectly exacerbated immune response and inducing intestinal homeostasis, producing an important clinical response for healing the colonic mucosa in both experimental models and humans [9-11]. One of the limiting factors of this treatment is associated with opportunistic infections and uncontrolled cell development, which lead to neoplasms [12,13]. In addition to these data, these cells have the capacity to migrate to the lesion site, even when applied far from the lesion [1,14]. Such lines of evidence have encouraged the studies regarding the topical application of these cells in the colon, which has potential as a new drug strategy and may decrease the side effects of this therapy [1]. Objectives This study aims to assess the applicability and the results of mesenchymal stem cell implantation in animals submitted to Correspondence to: Didia Bismara Cury, Director, Center of Inflammatory Bowel Disease, Clínica Scope, Campo Grande, MS 79002212, Brazil, Tel: +55673-3256040 Fax: +55-673-3256040; E-mail: didia_cury@uol.com.br