Metallothionein I and II gene expression as a response to metal contamination in bank vole Clethrionomys glareolus

The expression of two metallothionein genes (Mt-I and Mt-II) in the liver, kidney, and gonad of bank voles collected at four metal-contaminated sites (Cd, Zn, Pb, and Fe) were measured using the quantitative real-time PCR method (QPCR). Relative Mt gene expression was calculated by applying a normalization factor (NF) using the expression of two housekeeping genes, ribosomal 18S and beta-actin. Relative Mt expression in tissues of animals from contaminated sites was up to 54.8-fold higher than those from the reference site for Mt-I and up to 91.6-fold higher for Mt-II. Mt-II gene expression in the livers of bank voles from contaminated sites was higher than Mt-I gene expression. Inversely, Mt-II expression in the kidneys of voles was lower than Mt-I expression. Positive correlations between cadmium levels in the tissues and Mt-I were obtained in all studied tissues. Zinc, which undergoes homeostatic regulation, correlated positively with both Mt-I and Mt-II gene expression only in the kidney. Results showed that animals living in chronically contaminated environments intensively activate detoxifying mechanisms such as metallothionein expression. This is the first time that QPCR techniques to measure MT gene expression have been applied to assess the impact of environmental metal pollution on field collected bank voles.

Systemic multicompartmental effects of the gut microbiome on mouse metabolic phenotypes

To characterize the impact of gut microbiota on host metabolism, we investigated the multicompartmental metabolic profiles of a conventional mouse strain (C3H/HeJ) (n=5) and its germ-free (GF) equivalent (n=5). We confirm that the microbiome strongly impacts on the metabolism of bile acids through the enterohepatic cycle and gut metabolism (higher levels of phosphocholine and glycine in GF liver and marked higher levels of bile acids in three gut compartments). Furthermore we demonstrate that (1) well-defined metabolic differences exist in all examined compartments between the metabotypes of GF and conventional mice: bacterial co-metabolic products such as hippurate (urine) and 5-aminovalerate (colon epithelium) were found at reduced concentrations, whereas raffinose was only detected in GF colonic profiles. (2) The microbiome also influences kidney homeostasis with elevated levels of key cell volume regulators (betaine, choline, myo-inositol and so on) observed in GF kidneys. (3) Gut microbiota modulate metabotype expression at both local (gut) and global (biofluids, kidney, liver) system levels and hence influence the responses to a variety of dietary modulation and drug exposures relevant to personalized health-care investigations.

Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies

Small interfering RNA (siRNA), antisense oligonucleotides (ODNs), ribozymes and DNAzymes have emerged as sequence-specific inhibitors of gene expression that may have therapeutic potential in the treatment of a wide range of diseases. Due to their rapid degradation in vivo, the efficacy of naked gene silencing nucleic acids is relatively short lived. The entrapment of these nucleic acids within biodegradable sustained-release delivery systems may improve their stability and reduce the doses required for efficacy. In this study, we have evaluated the potential in vitro and in vivo use of biodegradable poly (d,l-lactide-co-glycolide) copolymer (PLGA) microspheres as sustained delivery devices for ODNs, ribozyme, siRNA and DNA enzymes. In addition, we investigated the release of ODN conjugates bearing 5′-end lipophilic groups. The in vitro sustained release profiles of microsphere-entrapped nucleic acids were dependent on variables such as the type of nucleic acid used, the nature of the lipophilic group, and whether the nucleic acid used was single or double stranded. For in vivo studies, whole body autoradiography was used to monitor the bio-distribution of either free tritium-labelled ODN or that entrapped within PLGA microspheres following subcutaneous administration in Balb-c mice. The majority of the radioactivity associated with free ODN was eliminated within 24 h whereas polymer-released ODN persisted in organs and at the site of administration even after seven days post-administration. Polymer microsphere released ODN exhibited a similar tissue and cellular tropism to the free ODN. Micro-autoradiography analyses of the liver and kidneys showed similar bio-distribution for polymer-released and free ODNs with the majority of radioactivity being concentrated in the proximal convoluted tubules of the kidney and in the Kupffer cells of the liver. These findings suggest that biodegradable PLGA microspheres offer a method for improving the in vivo sustained delivery of gene silencing nucleic acids, and hence are worthy of further investigation as delivery systems for these macromolecules.

The development of a real-time PCR to detect pathogenic Leptospira species in kidney tissue

A LightCycler(R) real-time PCR hybridization probe-based assay that detects a conserved region of the 16S rRNA gene of pathogenic but not saprophytic Leptospira species was developed for the rapid detection of pathogenic leptospires directly from processed tissue samples. In addition, a differential PCR specific for saprophytic leptospires and a control PCR targeting the porcine beta-actin gene were developed. To assess the suitability of these PCR methods for diagnosis, a trial was performed on kidneys taken from adult pigs with evidence of leptospiral infection, primarily a history of reproductive disease and serological evidence of exposure to pathogenic leptospires (n = 180) and aborted pig foetuses (n = 24). Leptospire DNA was detected by the 'pathogenic' specific PCR in 25 tissues (14%) and the control beta-actin PCR was positive in all 204 samples confirming DNA was extracted from all samples. No leptospires were isolated from these samples by culture and no positives were detected with the 'saprophytic' PCR. In a subsidiary experiment, the 'pathogenic' PCR was used to analyse kidney samples from rodents (n = 7) collected as part of vermin control in a zoo, with show animals with high microagglutination titres to Leptospira species, and five were positive. Fifteen PCR amplicons from 1 mouse, 2 rat and 14 pig kidney samples, were selected at random from positive PCRs (n = 30) and sequenced. Sequence data indicated L. interrogans DNA in the pig and rat samples and L. inadai DNA, which is considered of intermediate pathogenicity, in the mouse sample. The only successful culture was from this mouse kidney and the isolate was confirmed to be L. inadai by classical serology. These data suggest this suite of PCRs is suitable for testing for the presence of pathogenic leptospires in pig herds where abortions and infertility occur and potentially in other animals such as rodents. Crown Copyright (C) 2007 Published by Elsevier Ltd. All rights reserved.

Characteristics of the equine embryo and fetus from days 15 to 107 of pregnancy

In spite of numerous, substantial advances in equine reproduction, many stages of embryonic and fetal morphological development are poorly understood, with no apparent single source of comprehensive information. Hence, the objective of the present study was to provide a complete macroscopic and microscopic description of the equine embryo/fetus at various gestational ages. Thirty-four embryos/fetuses were aged based on their crown rump length (CRL), and submitted to macroscopic description, biometry, light and scanning microscopy, as well as the alizarin technique. All observed developmental changes were chronologically ordered and described. As examples of the main observed features, an accentuated cervical curvature was observed upon macroscopic examination in all specimens. In the nervous system, the encephalic fourth ventricle and the encephalic vesicles forebrain, midbrain, and hindbrain, were visualized from Day 19 (ovulation = Day 0). The thoracic and pelvic limbs were also visualized; their extremities gave rise to the hoof during development from Day 27. Development of other structures such as pigmented optical vesicle, liver, tail, cardiac area, lungs, and dermal vascularization started on Days 25, 25, 19, 19, 34, and 35, respectively. Light and scanning microscopy facilitated detailed examinations of several organs, e.g., heart, kidneys, lungs, and intestine, whereas the alizarin technique enabled visualization of ossification. Observations in this study contributed to the knowledge regarding equine embryogenesis, and included much detailed data from many specimens collected over a long developmental interval. (C) 2011 Elsevier Inc. All rights reserved.

L-NAME-treatment alters ectonucleotidase activities in kidney membranes of rats

Aims: To investigate the effect of N omega-Nitro-L-arginine methyl ester CL-NAME) treatment, known to induce a sustained elevation of blood pressure, on ectonucleotidase activities in kidney membranes of rats. Main methods: L-NAME (30 mg/kg/day) was administered to Wistar rats for 14 days in the drinking water. Enzyme activities were determined colorimetrically and their gene expression patterns were analyzed by semi-quantitative RT-PCR. The metabolism of ATP and the accumulation of adenosine were evaluated by HPLC in kidney membranes from control and hypertensive rats. PKC phosphorylation state was investigated by Western blot. Key findings: We observed an increase in systolic blood pressure from 115 +/- 12 mmHg (control group) to 152 18 mmHg (L-NAME-treated group). Furthermore, the hydrolysis of ATP, ADP, AMP, and p-Nph-5`TMP was also increased (17%, 35%, 27%, 20%, respectively) as was the gene expression of NTPDase2, NTPDase3 and NPP3 in kidneys of hypertensive animals. Phospho-PKC was increased in hypertensive rats. Significance: The general increase in ATP hydrolysis and in ecto-5`-nucleotidase activity suggests a rise in renal adenosine levels and in renal autoregulatory responses in order to protect the kidney against the threat presented by hypertension. (C) 2010 Elsevier Inc. All rights reserved.

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Local and cardiorenal effects of periodontitis in nitric oxide-deficient hypertensive rats

Objective: In this study we have assessed the renal and cardiac consequences of ligature-induced periodontitis in both normotensive and nitric oxide (NO)-deficient (L-NAME-treated) hypertensive rats. Materials and methods: Oral L-NAME (or water) treatment was started two weeks prior to induction of periodontitis. Rats were sacrificed 3, 7 or 14 days after ligature placement, and alveolar bone loss was evaluated radiographically. Thiobarbituric reactive species (TBARS; a lipid peroxidation index), protein nitrotyrosine (NT; a marker of protein nitration) and myeloperoxidase activity (MPO; a neutrophil marker) were determined in the heart and kidney. Results: In NO-deficient hypertensive rats, periodontitis-induced alveolar bone loss was significantly diminished. In addition, periodontitis-induced cardiac NT elevation was completely prevented by L-NAME treatment. On the other hand L-NAME treatment enhanced MPO production in both heart and kidneys of rats with periodontitis. No changes due to periodontitis were observed in cardiac or renal TBARS content. Conclusions: In addition to mediating alveolar bone loss, NO contributes to systemic effects of periodontitis in the heart and kidney. (C) 2010 Elsevier Ltd. All rights reserved.

THE ROLE OF INNATE IMMUNITY IN SEPTIC ACUTE KIDNEY INJURIES

Acute kidney injury (AKI) is an important clinical syndrome characterized by abnormalities in the hydroelectrolytic balance. Because of high rates of morbidity and mortality (from 15% to 60%) associated with AKI, the study of its pathophysiology is critical in searching for clinical targets and therapeutic strategies. Severe sepsis is the major cause of AKI. The host response to sepsis involves an inflammatory response, whereby the pathogen is initially sensed by innate immune receptors (pattern recognition receptors [PRRs]). When it persists, this immune response leads to secretion of proinflammatory products that induce organ dysfunction such as renal failure and consequently increased mortality. Moreover, the injured tissue releases molecules resulting from extracellular matrix degradation or dying cells that function as alarmines, which are recognized by PRR in the absence of pathogens in a second wave of injury. Toll-like receptors (TLRs) and NOD-like receptors (NLRs) are the best characterized PRRs. They are expressed in many cell types and throughout the nephron. Their activation leads to translocation of nuclear factors and synthesis of proinflammatory cytokines and chemokines. TLRs` signaling primes the cells for a robust inflammatory response dependent on NLRs; the interaction of TLRs and NLRs gives rise to the multiprotein complex known as the inflammasome, which in turn activates secretion of mature interleukin 1 beta and interleukin 18. Experimental data show that innate immune receptors, the inflammasome components, and proinflammatory cytokines play crucial roles not only in sepsis, but also in organ-induced dysfunction, especially in the kidneys. In this review, we discuss the significance of the innate immune receptors in the development of acute renal injury secondary to sepsis.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

A role for regulatory T cells in renal acute kidney injury

Ischemia reperfusion injury (IRI) is a potential contributor for the development of chronic allograft nephropathy. T cells are important mediators of injury, even in the absence of alloantigens. We performed a depletion of TCD4(+)CTLA4(+)Foxp3(+) cells with anti-CD25(PC61), a treatment with anti-GITR (DTA-1) and rat-IgG, followed by 45 min of ischemia and 24/72 h of reperfusion, and then analyzed blood urea, kidney histopathology and gene expression in kidneys by QReal Time PCR. After 24 h of reperfusion, depletion of TCD4(+)CTLA4(+)Foxp3(+) cells reached 30.3%(spleen) and 67.8%(lymph nodes). 72 h after reperfusion depletion reached 43.1%(spleen) and 90.22%(lymph nodes) and depleted animals presented with significantly poorer renal function, while DTA-1 (anti-GITR)-treated ones showed a significant protection, all compared to serum urea from control group (IgG: 150.10 +/- 50.04; PC61: 187.23 +/- 31.38; DTA-1: 64.53 +/- 25.65, mg/dL, p<0.05). These data were corroborated by histopathology. We observed an increase of HO-1 expression in animals treated with DTA-1 at 72 h of reperfusion with significant differences. Thus, our results suggest that PC61 (anti-CD25) mAb treatment is deleterious, while DTA-1 (anti-GITR) mAb treatment presents a protective role in the renal IRI, indicating that some regulatory populations of T cells might have a role in IRI. (C) 2009 Elsevier B.V. All rights reserved.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Improved renal function after kidney transplantation is associated with heme oxygenase-1 polymorphism

Heme oxygenase-1 (HO-1) has a microsatellite polymorphism based on the number of guanosine-thymidine nucleotide repeats (GT) repeats that regulates expression levels and could have an impact on organ survival post-injury. We correlated HO-1 polymorphism with renal graft function. The HO-1 gene was sequenced (N = 181), and the allelic repeats were divided into subclasses: short repeats (S) (< 27 repeats) and long repeats (L) (>= 27 repeats). A total of 47.5% of the donors carried the S allele. The allograft function was statistically improved six months, two and three yr after transplantation in patients receiving kidneys from donors with an S allele. For the recipients carrying the S allele (50.3%), the allograft function was also better throughout the follow-up, but reached statistical significance only three yr after transplantation (p = 0.04). Considering only those patients who had chronic allograft nephropathy (CAN; 74 of 181), allograft function was also better in donors and in recipients carrying the S allele, two and three yr after transplantation (p = 0.03). Recipients of kidney transplantation from donors carrying the S allele presented better function even in the presence of CAN.