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Hélio José Coelho-Júnior,1,2 Maria-Cláudia Irigoyen,3 Samuel da Silva Aguiar,2,4 Ivan de Oliveira Gonçalves,2,5 Niels Olsen Saraiva Câmara,6 Marco Antonio Cenedeze,7 Ricardo Yukio Asano,2,8 Bruno Rodrigues,1 Marco Carlos Uchida1 1Applied Kinesiology Laboratory–LCA, School of Physical Education, University of Campinas, Barão Geraldo, Campinas, São Paulo, 2Center of Health Sciences, University of Mogi das Cruzes, Mogi das Cruzes, 3Hypertension Unit, Heart Institute (InCor), Medical School of University of São Paulo, 4School of Physical Education, Catholic University of Brasília, Águas Claras, Brasília, 5Community Center for Older People of Poá, Poá, 6Department of Immunobiology, Laboratory of Transplantation Immunology, Institute of Biomedical Sciences, University of São Paulo, 7Nephrology Division, Federal University of São Paulo, 8School of Arts, Sciences and Humanities, University of São Paulo, São Paulo, Brazil Purpose: The purpose of this study was to compare the acute effects of resistance training (RT) and power training (PT) on the hemodynamic parameters and nitric oxide (NO) bioavailability of older women. Materials and methods: A randomized experimental design was used in this study. Twenty-one older women (age: 67.1±4.6 years; body mass index: 28.03±4.9 kg/m2; systolic blood pressure: 135.1±21.1 mmHg) were recruited to participate in this study. Volunteers were randomly allocated into PT, RT, and control session (CS) groups. The PT and RT groups underwent a single session of physical exercise equalized by training volume, characterized by 3 sets of 8–10 repetitions in 8 different exercises. However, RT group performed exercise at a higher intensity (difficult) than PT (moderate) group. On the other hand, concentric contractions were faster in PT group than in RT group. Hemodynamic parameters and saliva samples (for NO quantification) were collected before and during an hour after exercise completion. Results: Results demonstrated post-exercise hypotension during 35 minutes in the PT when compared to rest period (P=0.001). In turn, RT showed decreased heart rate and double product (P

Introduction: The objective of this study was to investigate the role of fenofibrate, a peroxisome proliferator-activated receptor-α agonist, in obesity-induced kidney damage (lipotoxicity) in mice with uninephrectomy., Methods: C57BL/6 mice underwent uninephrectomy and sham surgeries and were fed normocaloric or high-fat diets. After 10 weeks, obese mice were administered 0.02% fenofibrate for 10 weeks. Kidney function and morphology were evaluated, as well as levels of inflammatory and fibrotic mediators and lipid metabolism markers., Results: High-fat diet-fed mice developed characteristic obesity and hyperlipidemia, with subsequent renal lipid accumulation and damage, including mesangial expansion, interstitial fibrosis, inflammation, and proteinuria. These changes were greater in obese uninephrectomy mice than in obese sham mice. Fenofibrate treatment prevented hyperlipidemia and glomerular lesions, lowered lipid accumulation, ameliorated renal dysfunction, and attenuated inflammation and renal fibrosis. Furthermore, fenofibrate treatment downregulated renal tissue expression of plasminogen activator inhibitor-1, monocyte chemoattractant protein-1, and local expression of fibroblast growth factor-21., Conclusion: Peroxisome proliferator-activated receptor-α activation by fenofibrate, with subsequent lipolysis, attenuated glomerular and tubulointerstitial lesions induced by renal lipotoxicity, thus protecting the kidneys of uninephrectomy mice from obesity-induced lesions. The study findings suggest a pathway in the pharmacological action of fenofibrate, providing insight into the mechanisms involved in kidney damage caused by obesity in kidney donors.

Purpose: This study aimed to characterize the inflammatory mediators present in the tear film of patients with keratoconus (KC). It also aimed to investigate the gene expression of these mediators in corneal epithelial cells and their immune activity in conjunctival epithelial cells in patients with KC compared to a control group., Methods: This transversal study included 30 patients with KC and 23 control group participants. Tear samples were collected by washing the ocular surface with 60 μL of sterile buffered saline solution. The levels of interleukin IL-5, IL-13, IL-2, IL-6, IL-10, interferon-gamma, tumor necrosis factor-alpha, and IL-4 were measured using a LEGEND plex HU Th1/Th2 panel kit and analyzed using flow cytometry. Corneal epithelial samples were obtained via manual keratectomy from KC patients scheduled for corneal crosslinking and from individuals scheduled for photorefractive keratectomy (control group). These samples were immediately stored at -70 °C for mRNA extraction and subsequent reverse transcription polymerase chain reaction analysis to measure IL-5 and IL-6 gene expression. Conjunctival epithelium samples were collected using impression cytology and analyzed using immunohistochemistry and confocal microscopy to detect IL-5 and IL-6 immunoreactions., Results: Our study found no statistically significant differences in the tear film cytokine concentrations between the two groups. In addition, the gene expression of IL-5 and IL-6 in the corneal epithelium was higher in the KC group than in the control group, with IL-5 showing a 50% increase and IL-6 showing a 20% increase. Immunohistochemical analysis revealed a greater immunostaining of IL-5 and IL-6 in the conjunctival epithelium of patients with KC compared to the control group., Conclusions: In this study, despite higher levels of IL-5 and IL-6 in the tear film of patients with KC, there was no statistically significant difference compared to the control group. However, there was heightened immune activity in the corneal and conjunctival epithelial cells of patients with KC based on IL-5 and IL-6 gene expression and their immunodetection, respectively., (Copyright © 2024 Molecular Vision.)

Aim: The angiotensin-converting enzyme 2 (ACE2) and its homolog, the angiotensin converting enzyme 1 (ACE), are involved in COVID-19 physiopathology. Alterations in the enzymatic structure, expression, and/or activity may influence the risk of infection and severity of disease. For this reason, we aimed to identify different allelic forms of ACE2 G8790A and ACE I/D polymorphisms in a Brazilian cohort and evaluate their impact on ACE and ACE2 activities and their association with COVID-19 susceptibility and severity., Main Methods: A total of 549 COVID-19-negative and 270 COVID-19-positive participants from Ipaussu, Sao Paulo, Brazil, were recruited. ACE2 and ACE activities were measured by fluorogenic assays using MCA-Ala-Pro-Lys(Dnp) as the substrate for ACE2 and Z-Phe-His-Leu-OH (Z-FHL) and Hippuryl-His-Leu-OH (h-HL) as substrates for ACE. Genomic DNA was extracted from EDTA-peripheral blood, and the regions of the genes containing ACE2 G8790A and ACE I/D polymorphisms were amplified by PCR-restriction fragment length polymorphism and real-time PCR, respectively., Key Findings: The G allele of ACE2 G8790A polymorphism and D allele of ACE I/D polymorphism are associated with increased ACE and ACE2 activities. ACE activity ratio (Z-FHL/h-HL), an inflammatory marker, is increased in women with GG genotype and COVID-19-positive diagnosis., Significance: For the first time, it was demonstrated that in females, the GG genotype is associated with increased ACE activity ratio (Z-FHL/h-HL) in the COVID-19-positive group. Elevated ACE activity ratio (Z-FHL/h-HL) is highly linked to inflammation and may justify the associations between the G genotype and COVID-19 severity of symptoms and outcomes., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: This study aimed to characterize the tear film immunologic profile in keratoconus (KC) patients compared with healthy individuals (control group) and to investigate the correlation between the tear film immunologic profile and atopy, disease severity, and disease status over time., Methods: The study involved 30 KC patients and 18 healthy individuals. Tear collection was obtained using microcapillary tubes. Tear film levels of fractalkine, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon (IFN)-γ, interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-17A, IL-21, IL-23, interferon-inducible T-cell alpha chemoattractant (ITAC), macrophage inflammatory protein-1 alpha (MIP-1α), MIP-1β, MIP-3α, and tumor necrosis factor (TNF)-α were detected. Keratometric measurements and topographic patterns were used to diagnose and define disease progression. Tear immunologic profiles were compared, emphasizing the presence or absence of ocular allergy. Correlations between the cytokine profile, disease severity, and disease status were also analyzed longitudinally in the KC patients., Results: Lacrimal cytokine concentrations were higher in the KC patients than they were in the controls in 14 of 21 cytokines analyzed. IL-6 was the most relevant cytokine found in KC patients, especially when associated with ocular allergy. There was no correlation between KC progression and the level of inflammatory cytokines when analyzed longitudinally. KC severity correlated with IL-6 concentration, where the more severe KC presented a higher IL-6 concentration in tears., Conclusions: Inflammatory activity seems to be involved in the pathogenesis of KC. Out of 21 cytokines, 14 were more concentrated in the tears of KC patients than healthy subjects. IL-6 was significantly higher in KC patients' tears and was related to disease severity. Disease progression did not correlate with cytokine levels when analyzed longitudinally., (Copyright © 2021 Molecular Vision.)

Increasing evidence shows the essential participation of gut microbiota in human health and diseases by shaping local and systemic immunity. Despite an accumulating body of studies showing that chronic kidney disease (CKD) is closely associated with disturbances in the composition of gut microbiota, it remains unclear the importance of gut microbiota in the onset and development of CKD. For the purpose of untangling the role of gut microbiota in CKD, gut microbiota was depleted with a pool of broad-spectrum antibiotics in mice submitted to unilateral ureteral obstruction (UUO). Depletion of gut microbiota significantly decreased levels of proinflammatory cytokines and fibrosis markers, attenuating renal injury. Additionally, to study whether the pathogenic role of gut microbiota is dependent of microbial-host crosstalk, we generated mice lacking Myd88 (myeloid differentiation primary response gene 8) expression in intestinal epithelial cells (IECs) and performed UUO. The absence of Myd88 in IECs prevented a bacterial burden in mesenteric lymph nodes as observed in WT mice after UUO and led to lower expression of proinflammatory cytokines and chemokines, reducing deposition of type I collagen and, ultimately, attenuating renal damage. Therefore, our results suggest that the presence of gut microbiota is crucial for the development of CKD and may be dependent of Myd88 signaling in IECs, which appears to be essential to maturation of immune cells intimately involved in aggravation of inflammatory scenarios., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Watanabe, Andrade-Silva, Foresto-Neto, Felizardo, Matheus, Silva, Cenedeze, Honda, Perandini, Volpini, Pacheco-Silva and Câmara.)

High glucose concentration can activate TLR4 and NF-κB, triggering the production of proinflammatory mediators. We investigated whether the NF-κB pathway is involved in the pathogenesis and progression of experimental diabetic kidney disease (DKD) in a model of long-term type 1 diabetes mellitus (DM). Adult male Munich-Wistar rats underwent DM by a single streptozotocin injection, and were kept moderately hyperglycemic by daily insulin injections. After 12 months, two subgroups - progressors and non-progressors - could be formed based on the degree of glomerulosclerosis. Only progressors exhibited renal TLR4, NF-κB and IL-6 activation. This scenario was already present in rats with short-term DM (2 months), at a time when no overt glomerulosclerosis can be detected. Chronic treatment with the NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), prevented activation of renal TLR4, NF-κB or IL-6, without interfering with blood glucose. PDTC prevented the development of glomerular injury/inflammation and oxidative stress in DM rats. In addition, the NF-κB p65 component was detected in sclerotic glomeruli and inflamed interstitial areas in biopsy material from patients with type 1 DM. These observations indicate that the renal NF-κB pathway plays a key role in the development and progression of experimental DKD, and can become an important therapeutic target in the quest to prevent the progression of human DKD., (Copyright © 2020 Foresto-Neto, Albino, Arias, Faustino, Zambom, Cenedeze, Elias, Malheiros, Camara, Fujihara and Zatz.)

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a -/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.Gpr109a -/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Chronic kidney disease (CKD) patients present L-arginine (L-arg) deficiency and L-arg supplementation has been used as a treatment. In addition, sarcopenia is another common problem in CKD population, resistance training (RT) is one of the conservative strategies developed to prevent CKD progression, and however there are no evidences of a combination of these two strategies to treat CKD outcomes. The aim of this study was to evaluate the effects of oral L-arg supplementation combined with RT in an experimental model of CKD. Twenty-five Munich-Wistar male rats, 8-week-old were divided in 5 groups: Sham (sedentary control), Nx (CKD sedentary), Nx L-arg (CKD sedentary supplemented with 2% of L-arg), Nx RT (CKD exercised) Nx RT + L-arg (CKD exercised and supplemented with 2% of L-arg). CKD model was obtained by a subtotal 5/6 nephrectomy. RT was performed on a ladder climbing, three weekly sessions on non-consecutive days, with an intensity of 70% maximum carrying capacity. They were submitted to RT and/or L-arg supplementation for 10 weeks. There was a significant improvement in muscle strength, renal function, anti-inflammatory cytokines, arginase metabolism and renal fibrosis after RT. However, the combination of RT and L-arg impaired all the improvements promoted by RT alone. The L-arg supplementation alone did not impair renal fibrosis and renal function. In conclusion, RT improved inflammatory balance, muscle strength, renal function and consequently decreased renal fibrosis. Nevertheless, the association with L-arg supplementation prevented all these effects promoted by RT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The present study aimed at investigating the effects of a daily undulating periodization (DUP) and non-periodized (NP) resistance training programs on hemodynamic parameters of older women. Forty-two older women were randomized into one of the three experimental groups: NP, DUP, and control group (CG). Evaluations of the hemodynamic parameters occurred before, during and after the intervention. The exercise programs were performed twice a week over 22 weeks. NP and DUP groups were based on 3 sets of 8-10 repetitions in 9 exercises. In NP, the two exercise sessions were based on traditional strength training, which was performed at a Difficult intensity according to the rating of perceived exertion (RPE) method. In DUP, the first session was based on power resistance exercise, in which the concentric muscle contraction was performed as fast as possible at a moderate intensity based on RPE, while the second session was the same that was performed by NP. The findings demonstrated that diastolic blood pressure (90.4 vs. 76.2 mmHg) and mean arterial pressure (108.6 vs. 92.7 mmHg) were significantly reduced after NP, while no significant alterations were observed in DUP. Nevertheless, both training groups seem to have a cardio protective effect, since both training modes prevented the increase in HR reported in the experimental period in CG. In conclusion, our findings indicate that a 22-week NP resistance training program causes beneficial effects on hemodynamic parameters of older women. Trial Registration: NCT03443375.

Introduction: Chronic kidney disease (CKD) is considered a significant world health problem with elevated mortality rates. Patients with CKD are restricted to mild physical activity, present chronic inflammatory state and loss of muscle strength. Currently, the influence of resistance exercise (RE) on the progression of renal disease has not being fully elucidated., Purpose: To evaluate the effects of RE on the progression of CKD in a remnant kidney model (5/6Nx) in rats., Methods: Eight-week-old Wistar rats were submitted to 5/6 nephrectomy and were divided into four groups: Sham sedentary (Sham SD); Sham RE (Sham RE); 5/6Nx SD and 5/6Nx RE. The animals were trained for 8 weeks in a vertical climbing ladder for 3 days per week, on non-consecutive days., Results: As expected, 5/6Nx SD group presented a markedly loss of renal function, increased plasma inflammatory cytokines and increased oxidative stress with a reduced activity of nitric oxide. The higher macrophage infiltration and fibrosis confirmed these conditions. RE attenuated systolic blood pressure and renal function decrease and also improved serum lipid parameters in 5/6 Nx animals. It was evident the increase of muscle strength and mass in the trained groups while the sedentary group showed reduced muscle weight and strength compared to Sham SD., Conclusions: RE implemented following 5/6Nx retard the progression of chronic kidney injury while simultaneously allowed the maintenance of skeletal muscle strength., (Copyright © 2018. Published by Elsevier Inc.)

Acute kidney injury (AKI) is considered an inflammatory disease in which toll-like receptors (TLRs) signaling pathways play an important role. The activation of TLRs results in production of several inflammatory cytokines leading to further renal damage. In contrast, TLRs are key players on autophagy induction, which is associated with a protective function on cisplatin-induced AKI. Hence, the present study aimed to evaluate the specific participation of TLR2 and TLR4 molecules on the development of cisplatin-induced AKI. Complementarily, we also investigated the link between TLRs and heme oxygenase-1 (HO-1), a promisor cytoprotective molecule. First, we observed that only the absence of TLR2 but not TLR4 in mice exacerbated the renal dysfunction, tissue injury and mortality rate, even under an immunologically privileged microenvironment. Second, we demonstrated that TLR2 knockout (KO) mice presented lower expression of autophagy-associated markers when compared with TLR4 KO animals. Similar parameter was confirmed in vitro , using tubular epithelial cells derived from both KO mice. To test the cross-talking between HO-1 and TLRs, hemin (an HO-1 internal inducer) was administrated in cisplatin-treated TLR2 and TLR4 KO mice and it was detected an improvement in the global renal tissue parameters. However, this protection was less evident at TLR2 KO mice. In summary, we documented that TLR2 plays a protective role in cisplatin-induced AKI progression, in part, by a mechanism associated with autophagy up-regulation, considering that its interplay with HO-1 can promote renal tissue recover., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Patients with chronic kidney disease (CKD) have progressive renal fibrosis, inflammation, and reduced muscle mass and strength. Resistance training (RT) has been suggested to mitigate the loss of muscle mass, of strength and the inflammation in CKD, but the mechanisms are unknown. The aim of this study was to evaluate the influence of RT on renal fibrosis, renal cytokine expression, creatine kinase levels, and muscle mass and strength in CKD rats. A CKD model was obtained by 5/6 nephrectomy (Nx). Fifteen 8-week-old male rats were divided into 3 groups: Sham (control), Nx SED (CKD sedentary) and Nx RT (CKD trained). The RT consisted of ladder climbing at 70% of the animal's maximal carrying capacity for 10 weeks. Muscle strength, creatine kinase levels, renal fibrosis and mRNA interleukin (IL)-4, IL-6 and IL-10 were analyzed after the RT protocol. There was significant improvement in the muscle strength and creatine kinase levels in the Nx RT group. Moreover, renal fibrosis and inflammation were attenuated, with increased IL-4 and IL-10 expression and reduced IL-6 expression in the Nx RT group compared with that in the Nx SED group. No difference in muscle mass was observed among the groups. In conclusion, RT was effective in reducing fibrosis and inflammation, in addition to increasing muscle strength and creatine kinase levels, in rats with CKD, independent of muscle mass., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Mesenchymal stromal cells (MSCs) are multipotent cells with abilities to exert immunosuppressive response promoting tissue repair. Studies have shown that MSCs can secrete extracellular vesicles (MVs-MSCs) with similar regulatory functions to the parental cells. Furthermore, strong evidence suggesting that MVs-MSCs can modulate several immune cells (i.e., Th1, Th17, and Foxp3 + T cells). However, their precise effect on macrophages (Mϕs) remains unexplored. We investigated the immunoregulatory effect of MVs-MSCs on activated M1-Mϕs in vitro and in vivo using differentiated bone marrow Mϕs and an acute experimental model of thioglycollate-induced peritonitis, respectively. We observed that MVs-MSCs shared surface molecules with MSCs (CD44, CD105, CD90, CD73) and expressed classical microvesicle markers (Annexin V and CD9). The in vitro treatment with MVs-MSCs exerted a regulatory-like phenotype in M1-Mϕs, which showed higher CD206 level and reduced CCR7 expression. This was associated with decreased levels of inflammatory molecules (IL-1β, IL-6, nitric oxide) and increased immunoregulatory markers (IL-10 and Arginase) in M1-Mϕs. In addition, we detected that MVs-MSCs promoted the downregulation of inflammatory miRNAs (miR-155 and miR-21), as well as, upregulated its predicted target gene SOCS3 in activated M1-Mϕs. In vivo MVs-MSCs treatment reduced the Mϕs infiltrate in the peritoneal cavity inducing a M2-like regulatory phenotype in peritoneal Mϕs (higher arginase activity and reduced expression of CD86, iNOS, IFN-γ, IL-1β, TNF-α, IL-1α, and IL-6 molecules). This in vivo immunomodulatory effect of MVs-MSCs on M1-Mϕs was partially associated with the upregulation of CX3CR1 in F4/80 + /Ly6C + /CCR2 + Mϕs subsets. In summary, our findings indicate that MVs-MSCs can modulate an internal program in activated Mϕs establishing an alternative regulatory-like phenotype.

Mesenchymal stromal cells (MSCs) orchestrate tissue repair by releasing cell-derived microvesicles (MVs), which, presumably by small RNA species, modulate global gene expression. The knowledge of miRNA/mRNA signatures linked to a reparative status may elucidate some of the molecular events associated with MSC protection. Here, we used a model of cisplatin-induced kidney injury (acute kidney injury) to assess how MSCs or MVs could restore tissue function. MSCs and MVs presented similar protective effects, which were evidenced in vivo and in vitro by modulating apoptosis, inflammation, oxidative stress, and a set of prosurvival molecules. In addition, we observed that miRNAs (i.e., miR-880, miR-141, miR-377, and miR-21) were modulated, thereby showing active participation on regenerative process. Subsequently, we identified that MSC regulates a particular miRNA subset which mRNA targets are associated with Wnt/TGF-β, fibrosis, and epithelial-mesenchymal transition signaling pathways. Our results suggest that MSCs release MVs that transcriptionally reprogram injured cells, thereby modulating a specific miRNA-mRNA network.

Background: Proximal tubular dysfunction (PTD) is associated with a decreased long-term graft survival in renal transplant patients and can be detected by the elevation of urinary tubular proteins. This study investigated transcriptional changes in biopsies from renal transplant patients with PTD to disclose molecular mechanisms underlying graft injury and functional recovery., Methods: Thirty-three renal transplant patients with high urinary levels of retinol-binding protein, a biomarker of PTD, were enrolled in the study. The initial immunosuppressive scheme included azathioprine, cyclosporine, and steroids. After randomization, 18 patients (group 2) had their treatment modified by reducing cyclosporine dosage and substituting azathioprine for mycophenolate mofetil, while the other 15 patients (group 1) remained under the initial scheme. Patients were biopsied at enrollment and after 12 months of follow-up, and paired comparisons were performed between their intragraft gene expression profiles. The differential transcriptome profiles were analyzed by constructing gene co-expression networks and identifying enriched functions and central nodes in each network., Results: Only the alternative immunosuppressive scheme used in group 2 ameliorated renal function and tubular proteinuria after 12 months of follow-up. Intragraft molecular changes observed in group 2 were linked to autophagy, extracellular matrix, and adaptive immunity. Conversely, gene expression changes in group 1 were related to fibrosis, endocytosis, ubiquitination, and endoplasmic reticulum stress., Conclusion: These results suggest that molecular networks associated with the control of endocytosis, autophagy, protein overload, fibrosis, and adaptive immunity may be involved in improvement of graft function.

Unlabelled: Systemic lupus erythematosus (SLE) is an autoimmune disease with a persistent systemic inflammation. Exercise induced inflammatory response in SLE remains to be fully elucidated. The aim of this study was to assess the effects of acuteexercise on leukocyte gene expression in active (SLEACTIVE) and inactive SLE (SLEINACTIVE) patients and healthy controls(HC)., Methods: All subjects (n = 4 per group) performed a 30-min single bout of acute aerobic exercise (~70% of VO2peak) on a treadmill, and blood samples were collected for RNA extraction from circulating leukocyte at baseline, at the end of exercise, and after three hours of recovery. The expression of a panel of immune-related genes was evaluated by a quantitative PCR array assay. Moreover, network-based analyses were performed to interpret transcriptional changes occurring after the exercise challenge., Results: In all groups, a single bout of acute exercise led to the down-regulation of the gene expression of innate and adaptive immunity at the end of exercise (e.g., TLR3, IFNG, GATA3, FOXP3, STAT4) with a subsequent up-regulation occurring upon recovery. Exercise regulated the expression of inflammatory genes in the blood leukocytes of the SLE patients and HC, although the SLE groups exhibited fewer modulated genes and less densely connected networks (number of nodes: 29, 40 and 58; number of edges: 29, 60 and 195; network density: 0.07, 0.08 and 0.12, for SLEACTIVE, SLEINACTIVE and HC, respectively)., Conclusion: The leukocytes from the SLE patients, irrespective of disease activity, showed a down-regulated inflammatory geneexpression immediately after acute aerobic exercise, followed by an up-regulation at recovery. Furthermore, less organized gene networks were observed in the SLE patients, suggesting that they may be deficient in triggering a normal exercised-induced immune transcriptional response., (Copyright © 2015 International Society of Exercise and Immunology. All rights reserved.)

Aim: Experimental autoimmune encephalomyelitis (EAE) is a CD4(+)-mediated autoimmune pathology of the central nervous system (CNS) that is used as a model for the study of the human neuroinflammatory disease, multiple sclerosis. During the development of EAE, auto-reactive Th1 and Th17 CD4(+) T cells infiltrate the CNS promoting inflammatory cells recruitment, focal inflammation and tissue destruction. In this sense, statins, agents used to lower lipid levels, have recently shown to exert interesting immunomodulatory function. In fact, statins promote a bias towards a Th2 response, which ameliorates the clinical outcome of EAE. Additionally, simvastatin can inhibit Th17 differentiation. However, many other effects exerted on the immune system by statins have yet to be clarified, in particular during neuroinflammation. Thus, the aim of this study was to investigate the effects of simvastatin on the development of experimental autoimmune encephalomyelitis., Methods: Mice were immunized with MOG(35-55) and EAE severity was assessed daily and scored using a clinical scale. Cytokine secretion by mononuclear cells infiltrating the CNS was evaluated by flow cytometry., Results: Simvastatin (5 mg/kg/day) improved clinical outcome, induced an increase in TGF-β mRNA expression and inhibited IL-6, IL-12p40, IL-12p70, RANTES and MIP-1β secretion (p < 0.05). This was accompanied by a significant decrease in CNS inflammatory mononuclear cell infiltration, with reduced frequencies of both Th1 and Th17 cells. Simvastatin inhibited the proliferation of T lymphocytes co-cultured with primary microglial cells., Conclusions: Simvastatin treatment promotes EAE clinical amelioration by inhibiting T cell proliferation and CNS infiltration by pathogenic Th1 and Th17 cells.

Acute and chronic kidney injuries (AKI and CKI) constitute syndromes responsible for a large part of renal failures, and are today still associated with high mortality rates. Given the lack of more effective therapies, there has been intense focus on the use stem cells for organ protective and regenerative effects. Mesenchymal stem cells (MSCs) have shown great potential in the treatment of various diseases of immune character, although there is still debate on its mechanism of action. Thus, for a greater understanding of the role of MSCs, we evaluated the effect of adipose tissue-derived stem cells (AdSCs) in an experimental model of nephrotoxicity induced by folic acid (FA) in FVB mice. AdSC-treated animals displayed kidney functional improvement 24h after therapy, represented by reduced serum urea after FA. These data correlated with cell cycle regulation and immune response modulation via reduced chemokine expression and reduced neutrophil infiltrate. Long-term analyses, 4 weeks after FA, indicated that AdSC treatment reduced kidney fibrosis and chronic inflammation. These were demonstrated by reduced interstitial collagen deposition and tissue chemokine and cytokine expression. Thus, we concluded that AdSC treatment played a protective role in the framework of nephrotoxic injury via modulation of inflammation and cell cycle regulation, resulting in reduced kidney damage and functional improvement, inhibiting organ fibrosis and providing long-term immune regulation.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

Aim: To investigate a potential protective role of the kinin B2 receptor in a glycerol-induced rhabdomyolysis mouse model., Methods: We separated 28 C57Bl/6 male mice into 4 groups: untreated WT animals, untreated B2 knockout mice, glycerol-treated WT and glycerol-treated B2 knockout mice. Glycerol-treated animals received one intramuscular injections of glycerol solution (50% v/v, 7 mL/kg). After 48 h, urine and blood samples were collected to measure creatinine and urea levels. Additionally, kidney samples were extracted for histological evaluation, and the mRNA expression levels of kinin B1 and B2 receptors and inflammatory mediators were measured by real-time polymerase chain reaction., Results: Serum creatinine and urea levels showed differences between untreated wild-type and glycerol-treated wild-type mice (0.66 ± 0.04 vs 2.61 ± 0.53 mg/dL, P < 0.01; and 33.51 ± 2.08 vs 330.2 ± 77.7 mg/dL, P < 0.005), and between untreated B2 knockout mice and glycerol-treated knockout mice (0.56 ± 0.03 vs 2.23 ± 0.87 mg/dL, P < 0.05; and 42.49 ± 3.2 vs 327.2 ± 58.4 mg/dL, P < 0.01), but there was no difference between the glycerol-treated wild-type and glycerol-treated knockout mice. Glycerol was able to induce a striking increase in kinin B2 receptor expression (> 30 times, 31.34 ± 8.9) in kidney. Animals injected with glycerol had a higher degree of tubular injury than untreated animals. Wild-type and knockout mice treated with glycerol intramuscularly present kidney injury, with impairment in renal function. However, B2 knockout mice treated with glycerol did not show a different phenotype regarding kidney injury markers, when compared to the wild-type glycerol-treated group., Conclusion: We conclude that the kinin B2 receptor does not have a protective role in renal injury.

Focal and segmental glomerulosclerosis (FSGS) is one of the most important renal diseases related to end-stage renal failure. Bradykinin has been implicated in the pathogenesis of renal inflammation, whereas the role of its receptor 2 (B2RBK; also known as BDKRB2) in FSGS has not been studied. FSGS was induced in wild-type and B2RBK-knockout mice by a single intravenous injection of Adriamycin (ADM). In order to further modulate the kinin receptors, the animals were also treated with the B2RBK antagonist HOE-140 and the B1RBK antagonist DALBK. Here, we show that the blockage of B2RBK with HOE-140 protects mice from the development of FSGS, including podocyte foot process effacement and the re-establishment of slit-diaphragm-related proteins. However, B2RBK-knockout mice were not protected from FSGS. These opposite results were due to B1RBK expression. B1RBK was upregulated after the injection of ADM and this upregulation was exacerbated in B2RBK-knockout animals. Furthermore, treatment with HOE-140 downregulated the B1RBK receptor. The blockage of B1RBK in B2RBK-knockout animals promoted FSGS regression, with a less-inflammatory phenotype. These results indicate a deleterious role of both kinin receptors in an FSGS model and suggest a possible cross-talk between them in the progression of disease., (© 2014. Published by The Company of Biologists Ltd.)

Introduction: There is great interest in the use of animal models in the study of renal pathophysiology requires standardization of parameters., Objective: Standardize assessment of renal function in rats from in the Center for Reproductive Biology of Federal University of Juiz de Fora's colony., Methods: Thirty Wistar rats were used and performed measurements of creatinine (serum and urine), serum urea and proteinuria. Were evaluated: the urine collection interval in metabolic cages (24 hours or 12 hours), the need for 12-hour fast, the need of urine and serum deproteinization for creatinine measurement, need of serum deproteinization in animals with acute kidney injury to a spectrophotometer and ELISA, and the comparison of 24-hour proteinuria (PT 24 hours) with the protein/creatinine ratio (rP/C). Means were compared by the Student's t test, Pearson correlation, Bland-Altman plot for agreement and linear regression model to estimate PT 24 hours from rP/C., Results: The 24 hours urine output was greater than 12 hours, interfering with the creatinine clearance calculation. In the fasting group showed less water intake and lower urinary creatinine. There was great variability for the deproteinized whey and readings performed in the two devices were similar. There was a strong correlation between PT 24 hours and rP/C and the equation was generated: PT 24 hours = (8.6113 x rP/C) + 1.0869., Conclusion: Was standardized: 24-hour urine collection without fasting. The deproteinization showed no benefit. The measurements were performed with spectrophotometer reliability. It generated a practical formula for estimating PT 24 hours through rP/C.

There is a consensus in the scientific literature that supports the importance of the kallikrein kinin and renin angiotensin systems in renal physiology, but few studies have investigated their importance after renal transplantation. The aim of this study was to investigate the clinical effects of the insertion/deletion polymorphism in the angiotensin I-converting enzyme (ACE) gene and the +9/-9 polymorphism in the kinin B2 receptor (B2R) gene in kidney-transplanted patients (n=215 ACE, n=203 B2R) compared with 443 healthy individuals. Demographic results showed that there is a higher frequency of the D allele (high plasma ACE activity) and +9 allele (lower B2R expression) in transplant patients compared with control individuals. We also observed a higher frequency of these alleles in patients who had an elevated level of plasma creatinine. At day 7 post-transplantation, we found a higher prevalence of individuals with the DD genotype with elevated plasma creatinine level. Furthermore, individuals with the DD genotype had a higher chronic allograft dysfunction and graft loss compared with the II patient genotype, which showed no loss of graft. Taken together, our data suggest that the DD genotype is an indicator of an unfavorable prognosis following renal transplantation and could be related to kinin modulation.

Background/aims: Renal ischaemia-reperfusion injury (IRI) is a systemic inflammatory process in which Th1 responses predominate affecting other organs including the lungs. The present study explored the phagocytic and microbicidal capacity of macrophages in rats with lung inflammation that underwent IRI., Methods: The alveolar macrophages of rats sensitised to OVA were evaluated for phagocytosis and bacterial killing 24h after antigen challenge in animals with or without prior submission to 60 min of renal ischaemia., Results: Bronchoalveolar lavage had a high level of cellular infiltrate in immunised animals (420%) compared with control animals; IRI significantly reduced this infiltration (52%). Macrophages from animals immunised and challenged with OVA presented a 10x increase in phagocytic capacity compared to the control group, whereas immunised animals subjected to IRI showed a reduction in the phagocytic index of 68%. The killing of Klebsiella pneumoniae by macrophages from immunised animals was higher (56%) compared with the control group but reduced in animals submitted to IRI (45%). Immunised and challenged group showed an increase in gene expression levels of IL-10(450%), HO-1 (259%), INF-γ (460%) and MCP-1 (370%) compared to the immunised group subjected to IRI., Conclusions: Renal ischaemia and reperfusion injury apparently alters the phagocytic and microbicidal capacity of macrophages, reducing lung inflammation to OVA., (Copyright © 2013 S. Karger AG, Basel.)

Objective: the purpose of this study was to investigate the effect of low-level laser therapy (LLLT) on chronic kidney disease (CKD) in a model of unilateral ureteral obstruction (UUO)., Background Data: Regardless of the etiology, CKD involves progressive widespread tissue fibrosis, tubular atrophy, and loss of kidney function. This process also occurs in kidney allograft. At present, effective therapies for this condition are lacking. We investigated the effects of LLLT on the interstitial fibrosis that occurs after experimental UUO in rats., Methods: The occluded kidney of half of the 32 Wistar rats that underwent UUO received a single intraoperative dose of LLLT (AlGaAs laser, 780 nm, 22.5 J/cm(2), 30 mW, 0.75 W/cm(2), 30 sec on each of nine points). After 14 days, renal fibrosis was assessed by Sirius red staining under polarized light. Immunohistochemical analyses quantitated the renal tissue cells that expressed fibroblast (FSP-1) and myofibroblast (α-SMA) markers. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed to determine the mRNA expression of interleukin (IL)-6, monocyte chemotactic protein-1 (MCP-1), transforming growth factor (TGF)-β1 and Smad3., Results: The UUO and LLLT animals had less fibrosis than the UUO animals, as well having decreased expression inflammatory and pro-fibrotic markers., Conclusions: For the first time, we showed that LLLT had a protective effect regarding renal interstitial fibrosis. It is conceivable that by attenuating inflammation, LLLT can prevent tubular activation and transdifferentiation, which are the two processes that mainly drive the renal fibrosis of the UUO model.

Inflammation contributes to the pathogenesis of chronic kidney disease (CKD). Molecules released by the inflamed injured tissue can activate toll-like receptors (TLRs), thereby modulating macrophage and CD4(+) T-cell activity. We propose that in renal fibrogenesis, M2 macrophages are recruited and activated in a T helper subset 2 cell (T(H)2)-prone inflammatory milieu in a MyD88-dependent manner. Mice submitted to unilateral ureteral ligation (UUO) demonstrated an increase in macrophage infiltration with collagen deposition after 7 d. Conversely, TLR2, TLR4 and MyD88 knockout (KO) mice had an improved renal function together with diminished T(H)2 cytokine production and decreased fibrosis formation. Moreover, TLR2, TLR4 and MyD88 KO animals exhibited less M2 macrophage infiltration, namely interleukin (IL)-10(+) and CD206(+) CD11b(high) cells, at 7 d after surgery. We evaluated the role of a T(H)2 cytokine in this context, and observed that the absence of IL-4 was associated with better renal function, decreased IL-13 and TGF-β levels, reduced arginase activity and a decrease in fibrosis formation when compared with IL-12 KO and wild-type (WT) animals. Indeed, the better renal outcomes and the decreased fibrosis formation were restricted to the deficiency of IL-4 in the hematopoietic compartment. Finally, macrophage depletion, rather than the absence of T cells, led to reduced lesions of the glomerular filtration barrier and decreased collagen deposition. These results provide evidence that future therapeutic strategies against renal fibrosis should be accompanied by the modulation of the M1:M2 and T(H)1:T(H)2 balance, as T(H)2 and M2 cells are predictive of fibrosis toward mechanisms that are sensed by innate immune response and triggered in a MyD88-dependent pathway.

The pathogenesis of focal segmental glomerulosclerosis (FSGS) appears to be associated with type-2 cytokines and podocyte dysfunction. In this study, we tested the hypothesis that immunization with the polysaccharide fraction of Propionibacterium acnes (PS), a pro-Th1 agonist, may subvert the type-2 profile and protect podocytes from adriamycin-induced glomerulosclerosis. Adriamycin injection resulted in albuminuria and increased serum creatinine in association with loss of glomerular podocin and podoplanin expression, which is consistent with podocyte dysfunction. Renal tissue analysis revealed the expression of transcripts for GATA3 and fibrogenic-related proteins, such as TGF-β, tissue inhibitor of metalloproteinase-1 (TIMP-1) and metalloproteinase 9 (MMP9). In association with the expression of fibrogenic transcripts, we observed peri-glomerular expression of α-smooth muscle actin (α-SMA), indicating epithelial-to-mesenchymal transition, and increased expression of proliferating cell nuclear antigen (PCNA) in tubular cells, suggesting intense proliferative activity. Previous immunization with PS inhibited albuminuria and serum creatinine in association with the preservation of podocyte proteins and inhibition of fibrogenic transcripts and the expression of α-SMA and PCNA proteins. Tissue analysis also revealed that PS treatment induced expression of mRNA for GD3 synthase, which is a glycosiltransferase related to the synthesis of GD3, a ganglioside associated with podocyte physiology. In addition, PS treatment inhibited the influx of inflammatory CD8(pos) and CD11b(pos) cells to kidney tissue. Finally, PS treatment on day 4 post-ADM, a period when proteinuria was already established, was able to improve renal function. Thus, we demonstrate that the PS fraction of P. acnes can inhibit FSGS pathogenesis, suggesting that immunomodulation can represent an alternative approach for disease management., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Adipose tissue-derived stem cells (ASCs) are an attractive source of stem cells with regenerative properties that are similar to those of bone marrow stem cells. Here, we analyze the role of ASCs in reducing the progression of kidney fibrosis. Progressive renal fibrosis was achieved by unilateral clamping of the renal pedicle in mice for 1 h; after that, the kidney was reperfused immediately. Four hours after the surgery, 2 × 10(5) ASCs were intraperitoneally administered, and mice were followed for 24 h posttreatment and then at some other time interval for the next 6 weeks. Also, animals were treated with 2 × 10(5) ASCs at 6 weeks after reperfusion and sacrificed 4 weeks later to study their effect when interstitial fibrosis is already present. At 24 h after reperfusion, ASC-treated animals showed reduced renal dysfunction and enhanced regenerative tubular processes. Renal mRNA expression of IL-6 and TNF was decreased in ASC-treated animals, whereas IL-4, IL-10, and HO-1 expression increased despite a lack of ASCs in the kidneys as determined by SRY analysis. As expected, untreated kidneys shrank at 6 weeks, whereas the kidneys of ASC-treated animals remained normal in size, showed less collagen deposition, and decreased staining for FSP-1, type I collagen, and Hypoxyprobe. The renal protection seen in ASC-treated animals was followed by reduced serum levels of TNF-α, KC, RANTES, and IL-1α. Surprisingly, treatment with ASCs at 6 weeks, when animals already showed installed fibrosis, demonstrated amelioration of functional parameters, with less tissue fibrosis observed and reduced mRNA expression of type I collagen and vimentin. ASC therapy can improve functional parameters and reduce progression of renal fibrosis at early and later times after injury, mostly due to early modulation of the inflammatory response and to less hypoxia, thereby reducing the epithelial-mesenchymal transition.

Ischemia/reperfusion injury (IRI) is a leading cause of acute renal failure. The definition of the molecular mechanisms involved in renal IRI and counter protection promoted by ischemic pre-conditioning (IPC) or Hemin treatment is an important milestone that needs to be accomplished in this research area. We examined, through an oligonucleotide microarray protocol, the renal differential transcriptome profiles of mice submitted to IRI, IPC and Hemin treatment. After identifying the profiles of differentially expressed genes observed for each comparison, we carried out functional enrichment analysis to reveal transcripts putatively involved in potential relevant biological processes and signaling pathways. The most relevant processes found in these comparisons were stress, apoptosis, cell differentiation, angiogenesis, focal adhesion, ECM-receptor interaction, ion transport, angiogenesis, mitosis and cell cycle, inflammatory response, olfactory transduction and regulation of actin cytoskeleton. In addition, the most important overrepresented pathways were MAPK, ErbB, JAK/STAT, Toll and Nod like receptors, Angiotensin II, Arachidonic acid metabolism, Wnt and coagulation cascade. Also, new insights were gained about the underlying protection mechanisms against renal IRI promoted by IPC and Hemin treatment. Venn diagram analysis allowed us to uncover common and exclusively differentially expressed genes between these two protective maneuvers, underscoring potential common and exclusive biological functions regulated in each case. In summary, IPC exclusively regulated the expression of genes belonging to stress, protein modification and apoptosis, highlighting the role of IPC in controlling exacerbated stress response. Treatment with the Hmox1 inducer Hemin, in turn, exclusively regulated the expression of genes associated with cell differentiation, metabolic pathways, cell cycle, mitosis, development, regulation of actin cytoskeleton and arachidonic acid metabolism, suggesting a pleiotropic effect for Hemin. These findings improve the biological understanding of how the kidney behaves after IRI. They also illustrate some possible underlying molecular mechanisms involved in kidney protection observed with IPC or Hemin treatment maneuvers.

The Th1/Th2 balance represents an important factor in the pathogenesis of renal ischemia-reperfusion injury (IRI). In addition, IRI causes a systemic inflammation that can affect other tissues, such as the lungs. To investigate the ability of renal IRI to modulate pulmonary function in a specific model of allergic inflammation, C57Bl/6 mice were immunized with ovalbumin/albumen on days 0 and 7 and challenged with an ovalbumin (OA) aerosol on days 14 and 21. After 24 h of the second antigen challenge, the animals were subjected to 45 minutes of ischemia. After 24 h of reperfusion, the bronchoalveolar lavage (BAL) fluid, blood and lung tissue were collected for analysis. Serum creatinine levels increased in both allergic and non-immunized animals subjected to IRI. However, BAL analysis showed a reduction in the total cells (46%) and neutrophils (58%) compared with control allergic animals not submitted to IRI. In addition, OA challenge induced the phosphorylation of ERK and Akt and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lung homogenates. After renal IRI, the phosphorylation of ERK and expression of COX-2 and iNOS were markedly reduced; however, there was no difference in the phosphorylation of Akt between sham and ischemic OA-challenged animals. Mucus production was also reduced in allergic mice after renal IRI. IL-4, IL-5 and IL-13 were markedly down-regulated in immunized/challenged mice subjected to IRI. These results suggest that renal IRI can modulate lung allergic inflammation, probably by altering the Th1/Th2 balance and, at least in part, by changing cellular signal transduction factors., (Copyright © 2012 S. Karger AG, Basel.)

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.

A growing body of evidence demonstrates a correlation between Th2 cytokines and the development of focal and segmental glomerulosclerosis (FSGS). Therefore, we hypothesized that GSL-1, a monoglycosylceramide from Sphingomonas ssp. with pro-Th1 activity on invariant Natural Killer T (iNKT) lymphocytes, could counterbalance the Th2 profile and modulate glomerulosclerosis. Using an adriamycin(ADM)-based model of FSGS, we found that BALB/c mice presented albuminuria and glomerular degeneration in association with a Th2-like pro-fibrogenic profile; these mice also expressed a combination of inflammatory cytokines, such as IL-4, IL-1α, IL-1β, IL-17, TNF-α, and chemokines, such as RANTES and eotaxin. In addition, we observed a decrease in the mRNA levels of GD3 synthase, the enzyme responsible for GD3 metabolism, a glycolipid associated with podocyte physiology. GSL-1 treatment inhibited ADM-induced renal dysfunction and preserved kidney architecture, a phenomenon associated with the induction of a Th1-like response, increased levels of GD3 synthase transcripts and inhibition of pro-fibrotic transcripts and inflammatory cytokines. TGF-β analysis revealed increased levels of circulating protein and tissue transcripts in both ADM- and GSL-1-treated mice, suggesting that TGF-β could be associated with both FSGS pathology and iNKT-mediated immunosuppression; therefore, we analyzed the kidney expression of phosphorylated SMAD2/3 and SMAD7 proteins, molecules associated with the deleterious and protective effects of TGF-β, respectively. We found high levels of phosphoSMAD2/3 in ADM mice in contrast to the GSL-1 treated group in which SMAD7 expression increased. These data suggest that GSL-1 treatment modulates the downstream signaling of TGF-β through a renoprotective pathway. Finally, GSL-1 treatment at day 4, a period when proteinuria was already established, was still able to improve renal function, preserve renal structure and inhibit fibrogenic transcripts. In conclusion, our work demonstrates that the iNKT agonist GSL-1 modulates the pathogenesis of ADM-induced glomerulosclerosis and may provide an alternative approach to disease management.

Malaria associated-acute kidney injury (AKI) is associated with 45% of mortality in adult patients hospitalized with severe form of the disease. However, the causes that lead to a framework of malaria-associated AKI are still poorly characterized. Some clinical studies speculate that oxidative stress products, a characteristic of Plasmodium infection, as well as proinflammatory response induced by the parasite are involved in its pathophysiology. Therefore, we aimed to investigate the development of malaria-associated AKI during infection by P. berghei ANKA, with special attention to the role played by the inflammatory response and the involvement of oxidative stress. For that, we took advantage of an experimental model of severe malaria that showed significant changes in the renal pathophysiology to investigate the role of malaria infection in the renal microvascular permeability and tissue injury. Therefore, BALB/c mice were infected with P. berghei ANKA. To assess renal function, creatinine, blood urea nitrogen, and ratio of proteinuria and creatininuria were evaluated. The products of oxidative stress, as well as cytokine profile were quantified in plasma and renal tissue. The change of renal microvascular permeability, tissue hypoxia and cellular apoptosis were also evaluated. Parasite infection resulted in renal dysfunction. Furthermore, we observed increased expression of adhesion molecule, proinflammatory cytokines and products of oxidative stress, associated with a decrease mRNA expression of HO-1 in kidney tissue of infected mice. The measurement of lipoprotein oxidizability also showed a significant increase in plasma of infected animals. Together, our findings support the idea that products of oxidative stress, as well as the immune response against the parasite are crucial to changes in kidney architecture and microvascular endothelial permeability of BALB/c mice infected with P. berghei ANKA.

GENTA nephrotoxicity is likely to be caused, among other factors, by an increase of vasoconstrictors or a decrease of vasodilators such as NO. Few days after discontinuing GENTA treatment, the renal function is recovered, but if risk factors like advanced age, previous renal dysfunction, simultaneous use of other nephrotoxic drugs or dehydration are present, severe and progressive renal disease occurs. The aim of this study was to evaluate the renal function in rats during GENTA treatment and after its suspension as well as its relationship with NO. Rats were treated with water (vehicle, CTL) or GENTA (100 mg/kg BW) intraperitonially during 10 days; both n=24. Twelve animals of each group were sacrificed after blood and 24 h urine were collected, and their kidneys were removed for histology. In another rats this procedure underwent after 20 or 30 days of GENTA suspension. GENTA treated group developed a marked decrease in renal function, characterized by an increased serum urea and decreased creatinine clearance; NO was increased in the serum and decreased in the urine; all P < 0.01 vs CTL. Acute tubular necrosis was confirmed in GENTA treated group. After GENTA suspension we observed a normalization of urea, creatinine clearance and serum and urinary NO; the histological lesions were also attenuated. We suggest that NO could play a role in GENTA induced nephrotoxicity and recovery. The understanding of this physiopathology could be an useful tool to prevent or blunt the nephrotoxicity progression, mainly when risk factors are present., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: To analyze the role of adipose tissue-derived stem cells in reducing the progression of renal fibrosis., Methods: adipose tissue-derived stem cells were isolated from C57Bl/6 mice and characterized by cytometry and differentiation. Renal fibrosis was established after unilateral clamping of the renal pedicle for 1 hour. Four hours after reperfusion, 2.105 adipose tissue-derived stem cells were administered intraperitoneally and the animals were followed for 24 hours during 6 weeks. In another experimental group, 2.105adipose tissue-derived stem cells were administered only after 6 weeks of reperfusion, and they were euthanized and studied 4 weeks later. Twenty-four hours after reperfusion, the animals treated with adipose tissue-derived stem cells displayed reduced renal and tubular dysfunction and an increase of the regenerative process. Renal expression of IL-6 and TNF mRNA were decreased in the animals treated with adipose tissue-derived stem cells, while the levels of IL-4, IL-10, and HO-1 were increased, despite the fact that adipose tissue-derived stem cells were not observed in the kidneys via SRY analysis., Results: In 6 weeks, the kidneys of non-treated animals decreased in size, and the kidneys of the animals treated with adipose tissue-derived stem cells remained at normal size and display less deposition of type 1 collagen and FSP-1. The renal protection observed in animals treated with adipose tissue-derived stem cells was followed by a drop in serum levels of TNF-α, KC, RANTES, and IL-1a. Treatment with adipose tissue-derived stem cells after 6 weeks, when the animals already displayed established fibrosis, demonstrated an improvement in functional parameters and less fibrosis analyzed by Picrosirius stain, as well as a reduction of the expression of type 1 collagen and vimentin mRNA., Conclusion: Treatment with adipose tissue-derived stem cells may deter the progression of renal fibrosis by modulation of the early inflammatory response, likely via reduction of the epithelial-mesenchymal transition.

Background: The tubule-interstitial fibrosis is the hallmark of progressive renal disease and is strongly associated with inflammation of this compartment. Heme-oxygenase-1 (HO-1) is a cytoprotective molecule that has been shown to be beneficial in various models of renal injury. However, the role of HO-1 in reversing an established renal scar has not yet been addressed., Aim: We explored the ability of HO-1 to halt and reverse the establishment of fibrosis in an experimental model of chronic renal disease., Methods: Sprague-Dawley male rats were subjected to unilateral ureteral obstruction (UUO) and divided into two groups: non-treated and Hemin-treated. To study the prevention of fibrosis, animals were pre-treated with Hemin at days -2 and -1 prior to UUO. To investigate whether HO-1 could reverse established fibrosis, Hemin therapy was given at days 6 and 7 post-surgery. After 7 and/or 14 days, animals were sacrificed and blood, urine and kidney tissue samples were collected for analyses. Renal function was determined by assessing the serum creatinine, inulin clearance, proteinuria/creatininuria ratio and extent of albuminuria. Arterial blood pressure was measured and fibrosis was quantified by Picrosirius staining. Gene and protein expression of pro-inflammatory and pro-fibrotic molecules, as well as HO-1 were performed., Results: Pre-treatment with Hemin upregulated HO-1 expression and significantly reduced proteinuria, albuminuria, inflammation and pro-fibrotic protein and gene expressions in animals subjected to UUO. Interestingly, the delayed treatment with Hemin was also able to reduce renal dysfunction and to decrease the expression of pro-inflammatory molecules, all in association with significantly reduced levels of fibrosis-related molecules and collagen deposition. Finally, TGF-β protein production was significantly lower in Hemin-treated animals., Conclusion: Treatment with Hemin was able both to prevent the progression of fibrosis and to reverse an established renal scar. Modulation of inflammation appears to be the major mechanism behind HO-1 cytoprotection.

Introduction: TLR-4 has also been identified as a receptor for endogenous alarmins, which are increased post transplantation. TLR-4 has also been associated with a polymorphism that could impact graft outcome., Objective: To assess the expression of TLR-4 in kidney transplant patients carrying or not a polymorphism., Methods: TLR-4 polymorphism (A299G/T399I) was studied in 200 renal transplant patients. Healthy volunteers were also enrolled as control group. The polymorphism analysis was performed using restriction enzymes technique (RFLP). Functionality of TLR-4 polymorphism was assessed in samples from controls by quantification of TNF-α after LPS stimulus. TLR-4 and -2 expressions were also analyzed by flow cytometry., Results: TLR-4 polymorphism was present in 8.5% of renal transplant patients. This polymorphism was associated with impairment in TNF-α secretion. In general, in renal transplant patients, TLR-4 expression in monocytes and in neutrophils was lower than in health volunteers. TLR-2 and TLR-4 expressions in healthy volunteers with A299G/T399I TLR-4 polymorphism was higher than in wild-type genotype healthy volunteers (p<0.01 and p<0.05, respectively), and also higher than A299G/T399I TLR-4 polymorphism renal transplant patients (p<0.05). TLR-2 expression on neutrophils in wild-type genotype renal transplant patients was higher compared to wild-type genotype healthy volunteers, and was also higher in relation to A299G/T399I kidney transplanted patients (p<0.01)., Conclusion: Stable renal transplant patients with TLR-4 polymorphism have a lower expression of TLR-4 and TLR-2 receptors in peripheral mononuclear cells, which ultimately indicate a less responsiveness for alarmins., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Introduction: Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs., Aim: The objective was to study the pulmonary inflammatory systemic response after renal IRI., Methods: Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE(2) concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR., Results: Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 +/- 0.16 vs. 0.43 +/- 0.07, p < 0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 x 10(4) +/- 15.63 vs. 18.1 x 10(4) +/- 10.5, p < 0.05) 24 h (124 x 10(4) +/- 8.94 vs. 23.2 x 10(4) +/- 3.5, p < 0.05) and 48 h (79 x 10(4) +/- 15.72 vs. 22.2 x 10(4) +/- 4.2, p < 0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-alpha, IL-1beta, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-alpha, IL-1beta and MCP-1 and BALF PGE(2) concentrations were increased 24 h after renal IRI., Conclusion: Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal 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 1h. BMMCs were isolated from femurs and tibia, and after 6h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24h 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.

Objective and Design: This work explored the role of inhibition of cyclooxygenases (COXs) in modulating the inflammatory response triggered by acute kidney injury., Material: C57Bl/6 mice were used., Treatment: Animals were treated or not with indomethacin (IMT) prior to injury (days -1 and 0)., Methods: Animals were subjected to 45 min of renal pedicle occlusion and sacrificed at 24 h after reperfusion. Serum creatinine and blood urea nitrogen, reactive oxygen species (ROS), kidney myeloperoxidase (MPO) activity, and prostaglandin E2 (PGE(2)) levels were analyzed. Tumor necrosis factor (TNF)-alpha, t-bet, interleukin (IL)-10, IL-1beta, heme oxygenase (HO)-1, and prostaglandin E synthase (PGES) messenger RNA (mRNA) were studied. Cytokines were quantified in serum., Results: IMT-treated animals presented better renal function with less acute tubular necrosis and reduced ROS and MPO production. Moreover, the treatment was associated with lower expression of TNF-alpha, PGE(2), PGES, and t-bet and upregulation of HO-1 and IL-10. This profile was mirrored in serum, where inhibition of COXs significantly decreased interferon (IFN)-gamma, TNF-alpha, and IL-12 p70 and upregulated IL-10., Conclusions: COXs seem to play an important role in renal ischemia and reperfusion injury, involving the secretion of pro-inflammatory cytokines, activation of neutrophils, and ROS production. Inhibition of COX pathway is intrinsically involved with cytoprotection.

Purpose: To assess FasL mRNA levels in ejaculated sperm from adolescent patients with and without varicocele., Methods: Semen was obtained by masturbation following 2-4 days of ejaculatory abstinence, from 14 adolescents with varicocele grades II and III (study group), and 20 adolescents without varicocele (control group). Seminal analysis was done according to World Health Organization guidelines and morphology using Kruger's strict criteria. The Fas-ligand (FasL) gene expression was performed using reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis, according to the expression level of the housekeeping cyclophilin A gene. A Student's t-test was applied to compare the groups, and Spearman's rank test in order to verify possible correlations (p < 0.05)., Results: Quantitative RQ-PCR demonstrated that the expression of FasL mRNA in sperm from the varicocele group was higher than in the control group. Also, sperm concentration was higher in the controls, when compared to the varicocele group. When submitted to correlation analysis, adolescents with varicocele presented a correlation between sperm concentration and FasL gene expression levels (r = -0.470), not observed in controls., Conclusion: Our results allow us to conclude that, in adolescents with varicocele presenting lower sperm concentration, FasL mRNA levels are higher than in adolescents without varicocele.

Acute renal failure (ARF) is a frequent complication of Gram-negative sepsis, with a high risk of mortality. Lipopolysaccharide (LPS)-induced ARF is associated with hemodynamic changes that are strongly influenced by the overproduction of nitric oxide (NO) through the cytokine-mediated up-regulation of inducible NO synthase. LPS-induced reductions in systemic vascular resistance paradoxically culminate in renal vasoconstriction. Collagen XVIII is an important component of the extracellular matrix expressed in basement membranes. Its degradation by matrix metalloproteases, cathepsins and elastases results in the formation of endostatin, claimed to have antiangiogenic activity and to be a prominent vasorelaxing agent. We evaluated the expression of endostatin/collagen XVIII in an endotoxemic ARF model. ARF was induced in C57BL/6 mice by intraperitoneal injection of LPS (10 mg/kg) followed by sacrifice 4 and 12 h later. Kidney tissue was the source of RNA and protein and the subject of histological analysis. As early as 4 h after LPS administration, blood urea, creatinine and NO levels were significantly increased compared to control. Endostatin/collagen XVIII mRNA levels were 0.71 times lower than sham-inoculated mice 4 h after LPS inoculation, returning to normal levels 12 h after LPS inoculation. Immunohistological examination revealed that acute injury caused by LPS leads to an increase of endostatin basement membrane staining in association with the decrease of CD31 endothelial basement membrane staining. These results indicate that in the early phase of endotoxemic ARF the endostatin levels were not regulated by gene expression, but by the metabolism of collagen XVIII.

B lymphocyte infiltration in renal acute allograft rejection has been associated with steroid resistance and poor outcomes. We aimed to measure CD20 mRNA in urine of renal transplant patients with graft dysfunction and correlate with the histological diagnosis and immunohistochemical (IH) staining for CD20. A total of 48 urine samples were analyzed (21 with acute rejection, 10 with chronic allograft nephropathy, 11 with unspecific tubular lesions, 3 with acute pyelonephritis and 3 with polyomavirus nephropathy). Higher urinary CD20 levels associated with a positive IH staining for CD20 (>50 positive cells/HPF) in renal tissue (p=0.04), with a sensitivity of 83.3% and a specificity of 51.6%. Within the acute rejection group, a positive staining for CD20 was not associated with graft loss, steroid resistance or lack of return to basal creatinine after treatment, but was associated with higher serum creatinine at 3 and 6 months, 1 and 2 years after the acute episode (p<0.05). In conclusion, we showed that urinary levels of CD20 detected by RT-PCR had a high sensitivity for CD20+ staining in the corresponding renal tissue, but with a low specificity. Patients with clusters of CD20+ cells >50/HPF had higher serum creatinine after 2 years of follow up.