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Abstract: Type 2 Diabetes Mellitus (DM) is the most common form of diabetes. The initial treatment of type 2 DM consists of the adoption of healthy lifestyle habits together with several classes of hypoglycemic agents. However, these medications are not always able to reduce the blood glucose levels in all patients. Therefore, creatine supplementation has emerged as a new putative candidate for type 2 DM treatment. This systematic review aimed to investigate the effects (benefits and harms) of creatine supplementation in patients with type 2 diabetes through a systematic review. The studies were searched in MEDLINE, EMBASE, LILACS, CENTRAL, SPORTDiscus, and CINAHL databases, without date or language restrictions. Methodological quality was assessed using the Cochrane risk-of-bias table. The certainty of the evidence was classified using the Grading of Recommendations Assessment, Development and Evaluation approach. Three randomized controlled trials (RCTs) were included (87 participants). Overall, the methodological quality was classified as unclear to a high risk of bias. Each trial compared creatine supplementation with a different control group (placebo, metformin, and glibenclamide). Creatine supplementation seems to be effective in decreasing glycemic levels and glycosylated hemoglobin concentrations compared to placebo. No difference was observed compared to metformin or glibenclamide with creatine, and all treatments were able to reduce blood glucose levels. No major adverse effects were observed. Based on the low certainty of evidence, creatine supplementation was shown to be a hypoglycemic intervention for patients with type 2 diabetes, without major adverse events reported. However, well- designed RCTs with larger sample sizes and long-term outcomes are needed to support this evidence.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) is the gold standard technique for gene expression analysis, but the choice of quantitative reference genes (housekeeping genes, HKG) remains challenging. Identification of the best HKG is essential for estimating the expression level of target genes. The aim of this study was to identify the best HKG for an in vitro model with mouse mesangial cells (MMCs) stimulated with 5 ng/mL of TGF-β. Candidate five HKG were selected: Actb, Hprt, Gapdh, 18S and Ppia. After quantitative expression, the best combination of these genes was analyzed in silico using six software programs. To validate the results, the best genes were used to normalize the gene expression of fibronectin, vimentin and α-SMA. In silico analysis revealed that Ppia, Gapdh and 18S were the most stable genes between the groups. GenEX software and Spearman's correlation determined that combination of Ppia and Gapdh is the best HKG pair. Validation of the HKG by normalizing fibronectin, vimentin and α-SMA were consistent with results from the literature. This study established the combination of Ppia and Gapdh as the best HKG pair for gene expression analysis by RT-PCR in this in vitro model using MMCs treated with TGF-β.

In chronic kidney disease (CKD), evidence suggests that soluble αKlotho (sKlotho) has cardioprotective effects. Contrariwise, high circulating levels of fibroblast growth factor 23 (FGF23) are related to uremic cardiomyopathy development. Recently, it has been demonstrated that sKlotho can act as a soluble FGF23 co-receptor, allowing sKlotho to modulate FGF23 actions in the myocardium, leading to the activation of cardioprotective pathways. Fibroblast growth factor 21 (FGF21) is a cardiomyokine with sKlotho-like protective actions and has never been evaluated in uremic cardiomyopathy. Here, we aimed to evaluate whether recombinant αKlotho (rKlotho) replacement can attenuate cardiac remodeling in an established uremic cardiomyopathy, and to explore its impact on myocardial FGF21 expression. Forty-six male Wistar rats were divided into three groups: control, CKD-untreated, and CKD treated with rKlotho (CKD + KL). CKD was induced by 5/6 nephrectomy. From weeks 4–8, the control and CKD-untreated groups received vehicle, whereas the CKD + KL group received subcutaneous rKlotho replacement (0.01 mg/kg) every 48 h. Myocardial remodeling was evaluated by heart weight/tibia length (HW/TL) ratio, echocardiographic parameters, myocardial histomorphometry, and myocardial expression of β-myosin heavy chain (MHCβ), alpha smooth muscle actin (αSMA), transient receptor potential cation channel 6 (TRPC6), and FGF21. As expected, CKD animals had reduced levels of sKlotho and increased serum FGF23 levels. Compared to the control group, manifest myocardial remodeling was present in the CKD-untreated group, while it was attenuated in the CKD + KL group. Furthermore, cardiomyocyte diameter and interstitial fibrotic area were reduced in the CKD + KL group compared to the CKD-untreated group. Similarly, rKlotho replacement was associated with reduced myocardial expression of TRPC6, MHCβ, and αSMA and a higher expression of FGF21. rKlotho showed cardioprotective effects by attenuating myocardial remodeling and reducing TRPC6 expression. Interestingly, rKlotho replacement was also associated with increased myocardial FGF21 expression, suggesting that an interaction between the two cardioprotective pathways needs to be further explored. Impact statement This study aimed to evaluate whether rKlotho replacement can attenuate cardiac remodeling in a post-disease onset therapeutic reasoning and explore the impact on myocardial FGF21 expression. This study contributes significantly to the literature, as the therapeutic effects of rKlotho replacement and FGF21 myocardial expression have not been widely evaluated in a setting of uremic cardiomyopathy. For the first time, it has been demonstrated that subcutaneous rKlotho replacement may attenuate cardiac remodeling in established uremic cardiomyopathy and increase myocardial expression of FGF21, suggesting a correlation between αKlotho and myocardial FGF21 expression. The possibility of interaction between the αKlotho and FGF21 cardioprotective pathways needs to be further explored, but, if confirmed, would point to a therapeutic potential of FGF21 in uremic cardiomyopathy.

Introdução: O Rio Pouca Saúde como é popularmente conhecido e está localizado próximo a Hospital, Indústrias, palafitas e Terminais no Porto de Santos/SP e classificase como gamboa. Objetivo: Realizar o controle toxicológico do rio com utilização de bioensaios durante o verão e inverno. Material e métodos: Foram coletadas amostras de águas superficiais das margens do rio em dos dois pontos, durante o verão e o inverno: ponto 1 (comunidade ribeirinha) e ponto 2 (atividades industriais), esses resultados foram comparados com o controle com água potável. Foi escolhido o bioindicador Allium cepa tipo pirulito (Cebolas) padronizadas, feitas em triplicatas e imersas diretamente em controle e amostras sem diluição, por 72h. Posteriormente foi realizada a análise macroscópica das raízes. No teste genotoxico foi contado 400 células e calculado o índice mitótico (IM%= nº de células em mitose/nº de células analisadas x100). Resultados: Em análise macroscópica, observou-se no verão um crescimento de 18  1,1 raízes com 2,5  0,4 cm no ponto 1, no ponto 2 cresceram 18  1,5 raízes com comprimento de 3,2  0,3 cm, entretanto, amostras controles (40  10 raízes de 2,5  0,37 cm). No inverno, as amostras do ponto 1 não germinaram e no ponto 2 cresceram 18  1,52 raízes com 0,6  0,1 cm. Em ambas estações, as amostras apresentaram raízes amarronzadas e com deficiência no crescimento. Foi observado no verão IM 20% no ponto 1 e IM 35% no ponto 2, muito inferior ao comparar com o controle de IM 95%. No inverno, o ponto 1 não germinou e no ponto 2 foi observado IM 23%, reduzido comparando com o controle IM 94%. Conclusão: Nossos resultados sugerem que o Rio Pouca Saúde pode estar presentes poluentes que estão interferindo no crescimento das plantas e consequentemente podendo interferir na saúde da população ribeirinha.

Background Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis. Methods Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy donor (HC) was used as a comparative control to normalize the many technical steps for reprogramming EPs and for the organoids generation. EPs were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. Results iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. Conclusion The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney development model, which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

Background. Kidney organoids have been broadly obtained from commercially available induced pluripotent stem cells (iPSCs); however, it has been a great challenge to efficiently produce renal organoid models from patients with autosomal dominant polycystic kidney disease (ADPKD) that recapitulate both embryogenesis and the mechanisms of cystogenesis.Methods. Blood erythroid progenitors (EPs) from two ADPKD patients and one healthy control (HC) donor were reprogrammed by an episomal vector into iPSCs, which were differentiated into renal tubular organoids and then stimulated by forskolin to induce cysts formation. Results. iPSCs derived from EPs exhibited all characteristics of pluripotency and were able to differentiate into all three germ layers. 3D tubular organoids were generated from single cells after 28 days in Matrigel. HC and ADPKD organoids did not spontaneously form cysts, but upon forskolin stimulation, cysts-like structures were observed in the ADPKD organoids but not in the HC-derived organoids. Conclusion. The findings of this study showed that kidney organoids were successfully generated from the blood EP cells of ADPKD patients and a healthy control donor. This approach should contribute as a powerful tool for embryonic kidney developing model which is able to recapitulate the very early pathophysiological mechanisms involved in cytogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Background. Chronic renal artery stenosis is considered one of the most common causes of renovascular hypertension (RH). Chronic hypoxia can lead to irreversible damage to renal tissue and to a progressive deterioration of renal function. We have previously shown that bone marrow-derived mesenchymal stem cells (BMSCs) improved renal parenchyma and function in a model of RH (2 kidneys, 1 clip model (2K-1C) in rats. Microvesicles (MVs) and exosomes (EXs) released by MSCs have been shown to induce effects similar to those induced by whole cells but with fewer side effects. In this study, we compared the effects of adipose-derived MSCs (ASCs) with those of the MVs and EXs released by ASCs on tissue inflammation and renal function in 2 K-1C rats. Results. Flow cytometry analysis showed that even after 15 days, ASCs were still detected in both kidneys. The expression of a stem cell homing marker (SDF1-α) was increased in ASC-treated animals in both the stenotic and contralateral kidneys. Interestingly, SDF1-α expression was also increased in MV- and EX-treated animals. A hypoxia marker (HIF1-α) was upregulated in the stenotic kidney, and treatments with ASCs, MVs, and EXs were effective in reducing the expression of this marker. Stenotic animals showed a progressive increase in systolic blood pressure (SBP), while animals treated with ASCs, MVs, and EXs showed a stabilization of SBP, and this stabilization was similar among the different treatments. Stenotic animals developed significant proteinuria, which was reduced by ASCs and MVs but not by EXs. The increased expression of Col I and TGFβ in both kidneys was reduced by all the treatments, and these treatments also effectively increased the expression of the anti-inflammatory cytokine IL-10 in both kidneys; however, only ASCs were able to reduce the overexpression of the proinflammatory cytokine IL-1β in both kidneys of 2K-1C animals. Conclusion. The results of this study demonstrated that the EVs released by ASCs produced beneficial results but with lower efficacy than whole cells. ASCs produced stronger effects in this model of renal chronic hypoxia, and the use of EVs instead of whole cells should be evaluated depending on the parameter to be corrected.

Background: Mesenchymal stem cells (MSC) improve renal function and renovascular hypertension in the 2-kidney 1-clip model (2K-1C). While MSC play an immunomodulatory role, induce neoangiogenesis, and reduce fibrosis, they do not correct sodium loss by the contra­lateral kidney. Objectives: We investigated the tubular function of both stenotic and contralateral kidneys and the effect of MSC treatment by evaluating diuresis, natriuresis, and the expression of the main water and sodium transporters. Method: Adult Wistar rats were allocated into four groups: control (CT), CT+MSC, 2K-1C, and 2K-1C+MSC. MSC (2 × 105) were infused through the tail vein 3 and 5 weeks after clipping. Systolic blood pressure (SBP) was monitored weekly by plethysmography. Six weeks after clipping, 24-hour urine and blood samples were collected for biochemical analysis. Gene expression of the Na/H exchanger-3, epithelial sodium channel, Na/K-ATPase, Na/K/2Cl cotransporter, and aquaporins 1 and 2 (AQP1 and AQP2) were analyzed by RT-PCR. Intrarenal distribution of AQP1 and AQP2 was analyzed by immunohistochemistry. Results: In hypertensive 2K-1C animals, MSC prevented additional increases in BP. AQP1, but not AQP2, was suppressed in the contralateral kidney, resulting in significant increase in urinary flow rate and sodium excretion. Gene expressions of sodium transporters were similar in both kidneys, suggesting that the high perfusing pressure in the contralateral kidney was responsible for increased natriuresis. Contralateral hypertensive kidney showed signs of renal deterioration with lower GFR in spite of normal RPF levels. Conclusions: MSC treatment improved renal function and enhanced the ability of the contralateral kidney to excrete sodium through a tubular independent mechanism contributing to reduce SBP.

Mesenchymal stem cells (MSC) induced neovascularization and improved renal morphology of the stenotic kidney in 2 kidneys-1 clip (2K-1C) model of renovascular hypertension. The present study evaluated the effects of MSC in the contralateral hypertensive kidney. Three weeks after left renal artery occlusion, MSC were injected into the tail vein of the 2K-1C rats. Renal function and morphology were analyzed in both kidneys. Labeled MSC were found in stenotic and contralateral kidneys. Hypertensive 2K-1C animals presented increased circulating levels of Angiotensin II (Ang II) and renin. MSC prevented the progressive increase of blood pressure and reduced circulating Ang II and renin levels. Stenotic kidney showed reduced renal plasma flow (RPF) and glomerular filtration rate (GFR), whereas the contralateral kidney had a tendency (p > 0.5) of reduction in GFR in spite of unchanged RPF. MSC treatment caused an improvement in GFR with no effect of on RPF in the stenotic kidney. Contralateral kidney showed increased diuresis and natriuresis that were even higher in MSC-treated animals, indicating that cell treatment improved the capacity of the contralateral kidney to excrete sodium. Contralateral kidney expressed higher levels of inflammatory cytokines (IL-6, TNF-α) and signs of fibrosis, which were attenuated by MSC treatment. MSC treatment improved the stenotic kidney function, and it was also beneficial to the contralateral hypertensive kidney because it improved the morphology and preserved its capacity to excrete sodium.

Renin angiotensin system (RAS) blockade reduces the progression of chronic kidney disease (CKD) independently of its antihypertensive effect. Ang II-induced fibrosis can be mediated by molecules such as klotho, peroxisome proliferator-activate receptor γ (PPAR-γ), and the Wnt/β-catenin pathway; however, the interaction among these molecules and RAS activation is not completely known. The aim of this study was to investigate a possible link between RAS, PPAR-γ, and Klotho in the 5/6 nephrectomy (NX) animals. NX rats presented hypertension that was blunted by both losartan and propranolol, however, only losartan was able to reduce the expression levels of fibronectin FSP1 and TGF-β in the remnant kidney. The anti-fibrotic Klotho and PPAR-γ were reduced in the remnant kidney, and losartan, but not propranolol, restored their levels. In contrast, the profibrotic Wnt 7a and Wnt 3 were upregulated and losartan prevented the increase in Wnts. In vitro, Ang II induced a decrease in both klotho and in PPAR-γ in Madin-Darby canine kidney (MDCK) cells, and this effect was blunted by losartan. However, klotho expression was increased by pioglitazone, an agonist of PPAR-γ, and suppressed by BADGE, an antagonist of PPAR-γ, suggesting that the effect of Ang II downregulating klotho is mediated by PPAR-γ. These data suggest that activation of the Wnt pathway together with downregulation of PPAR-γ that in turn suppresses klotho contribute to potentiating the profibrotic effect of Ang II.

Mesangial cells stimulated with high glucose (HG) exhibit increased intracellular angiotensin II (AngII) synthesis that is correlated with the upregulation of AngII target genes, such as profibrotic cytokines. The intracrine effects of AngII can be mediated by several molecules transferred to other cells via exosomes (Exos), which play a key role in cellular communication under many physiological and pathological conditions. The aim of this study was to investigate the effects of exosomes derived from HG-stimulated human mesangial cells (HG-HMCs) on normal unstimulated HMCs. Exosomes from HMCs (C-Exos) and HG-HMCs (HG-Exos) were obtained from cell culture supernatants. HMCs were incubated with C-Exos or HG-Exos. HG stimulus induced a change in the amount but not the size of Exos. Both C-Exos and HG-Exos contained angiotensinogen and renin, but no angiotensin converting enzyme was detected. Compared with HMCs treated with C-Exos, HMCs treated with HG-Exos presented higher levels of fibronectin, angiotensinogen, renin, AT1 and AT2 receptors, indicating that HG-Exos modified the function of normal HMCs. These results suggest that the intercellular communication through Exos may have pathophysiological implications in the diabetic kidney.

Acute kidney injury is mostly reversible, and hepatocyte growth factor (HGF) has a relevant role in the tissue repair. MicroRNA (miR)-26a is an endogenous modulator of HGF. The role of miR-26a in the kidney repair process was evaluated in Wistar rats submitted to an acute kidney injury model of rhabdomyolysis induced by glycerol (6 mL/kg). Animals were evaluated 3, 12, 48, 96, and 120 hours after glycerol injection. Serum creatinine (SCr) and gene expression of HGF, c-met, signal transducer and activator of transcription 3 (STAT3), and miR-26a were estimated. Also, tubular NK52E cells were transfected with anti-miR26a and stimulated with Fe3+ for 24 hours to mimic the effects of myoglobin in vitro. SCr was highest after 48 hours. After 96 hours, SCr started to decrease, characterizing the recovery phase, with normalization after 120 hours. HGF expression increased during the onset phase (3 hours), with a low relationship with miR-26a. In contrast, in the recovery phase, the increase in miR-26a was coincident with HGF messenger RNA suppression, suggesting that in the recovery phase, miR-26a may have a role in HGF modulation. Fe3+ induced cellular death after 3 hours and proliferation after 24 hours. There was no correlation between miR-26a and STAT3 during the death phase; however, during the proliferation phase, an increase in STAT3 was paralleled with a decrease in miR-26a. miR-26a silencing induced increases in cell viability and the phosphorylated form of STAT3 protein expression in cells receiving Fe3+ . In conclusion, miR-26a may have a key role in modulating HGF levels after its proliferative effects have been triggered.

Proteinuria is critical in the tubulointerstitial changes that ultimately lead to renal insufficiency. Increased protein filtration has direct toxic effects on tubular epithelial cells, leading to epithelial mesenchymal transition (EMT) to a myofibroblast phenotype. Angiotensin II and transforming growth factor (TGF)-β1 are the main mediators of EMT. Calcitriol may exert a potential renoprotective effect by reducing the activity of the renin angiotensin system by suppressing renin gene expression and also by inhibiting the proinflammatory nuclear factor-κB pathway. The present study investigated the benefits of calcitriol treatment in a puromycin-induced protein-uric nephropathy model. Uninephrectomized adult male Wistar rats received intraperitoneal administration of a single dose of puromycin (100 mg/kg) or vehicle. After eight weeks, the animals were divided into two groups and received vehicle or calcitriol (0.5 μg/kg) for four weeks. The vehicle-treated, proteinuric rats developed progressive proteinuria and tubulointerstitial fibrosis after 12 weeks. Increased collagen deposition and fibrosis were significantly ameliorated by calcitriol treatment. Calcitriol was effective in preventing an increase in the EMT markers, α-smooth muscle actin and fibroblast-specific protein 1, reducing macrophage infiltration as evidenced by levels of ED-1. In addition, calcitriol increased the anti-inflammatory cytokine interleukin-10 and reduced the pro-oxidant p47 phox enzyme. These effects were paralleled by a reduction in TGF-β/Smad3 expression. Calcitriol may have therapeutic potential in the proteinuric nephropathy model used in the present study by inhibiting the TGF-β1 axis.

Obesity and its consequences can damage the kidney over time. However, less is known about the impact of developing overweight/obesity during childhood on the kidney in adulthood and the renal impact of a superimposed acute kidney injury (AKI). This study evaluated the effect of obesity induced by a high-fat diet initiated soon after weaning on the adult life of mice and their response to superimposed nephrotoxic effects of cisplatin. C57BL/6 post-weaning mice (3 weeks old) were divided into a control group (CT, n = 12) and a high-fat diet group (HF, n = 12). After 9 weeks, animals were further divided into the following groups: CT, CT treated with a single dose of cisplatin (CTCis, 20 mg/kg, i.p.), HF and HF treated with cisplatin (HFCis). The HF group exhibited higher body weight gain compatible with a moderate obesity. Obese mice presented increased visceral adiposity, hyperkalemia, sodium retention, glomerular hyperfiltration and proteinuria, without any significant changes in blood pressure and glycemia. AKI induced by cisplatin was exacerbated in obese animals with a 92% reduction in the GFR versus a 31% decrease in the CTCis group; this sharp decline resulted in severely elevated serum creatinine and urea levels. Acute tubular necrosis induced by cisplatin was worsened in obese mice. The HFCis group exhibited robust systemic and intrarenal inflammation that was significantly higher than that in the CTCis group; the HFCis group also showed a higher degree of renal oxidative stress. In conclusion, the moderate degree of obesity induced shortly after weaning resulted in mild early renal alterations, however, obese young animals were prone to develop a much more severe AKI induced by cisplatin.

Bone disease as a consequence of diabetes mellitus (DM) is not fully understood. The effects of high glucose (30 mM), high insulin (50 nM), or mannitol (30 mM; osmotic control) were evaluated on MC3T3-E1 cells (osteoblasts) in vitro. The mRNA and protein levels of parathyroid hormone (PTH) receptor (PTH1R), collagen I, RANKL, osteoprotegerin (OPG), alkaline phosphatase (ALP), and glucose transporter (GLUT1) were estimated by real-time polymerase chain reaction or Western blotting. The mineralization capacity was analyzed by von Kossa staining. High glucose induced overexpression of RANKL (2×) and OPG (30×), suggesting that RANKL-induced osteoclast activity might not be a dominant mechanism of bone disease in DM, since this increase was followed by increased OPG. Collagen I increased by 12×, indicating an excess of organic matrix production. The expression of ALP decreased by 50%, indicating a deficit in mineralization capacity, confirmed by von Kossa staining. Mannitol induced similar effects as glucose suggesting that extracellular hyperosmolarity was able to stimulate organic matrix production. GLUT1 expression was not altered, and insulin did not reverse most of the effects of glucose, suggesting that glucose uptake by osteoblasts was not altered by high glucose. The data suggest that the bone fragility typical of DM is not a consequence of excessive bone reabsorption but is instead attributable to a defect in organic matrix mineralization. The heightened increase in OPG versus RANKL might cause a decrease in the bone-remodeling cycle. Osteoblasts appear to be more sensitive to extracellular hypertonicity than to the intracellular metabolic effects of hyperglycemia.

Hypothesis/Introduction: Transformer Growth Factor (TGF-β1) and angiotensin II (AngII) induce epithelial mesenchymal transition (EMT) and myofibroblastic transdifferentiation (MFT) contributing to renal fibrosis. The present study evaluated the capacity of an AT1 receptor blocker (losartan) to induce the regression of pre-existing fibrosis via interference with MFT and EMT in a rat model of type 2 diabetes, and in cultured mesangial cells (MCs) stimulated with high glucose and AngII. Materials and methods: After 12 weeks of diabetes induction (D12 group), animals showing evidence of nephropathy, were divided in groups untreated for additional 8 weeks (D20 group) and treated for additional 8 weeks with losartan (D20+los group). Results: D12 animals presented hyperglycemia, insulin resistance, hypertension, proteinuria, increased levels of TGF-β1 and MFT/EMT markers. Losartan stabilized all of these parameters and hindered the progression of fibrosis, but it did not reverse the pre-existing fibrotic manifestations. Losartan reduced TGF-β1 in the tubules, but not in the glomeruli. Stimulated MC exhibited myofibroblast phenotype and capacity for migration, which were completely reversed by losartan. Conclusions: Cellular transition may play a role in diabetes-inducing renal fibrogenesis in both AngII-TGF-β1 axis-dependent and independent manners. Losartan was efficient in preventing cells from undergoing further transdifferentiation, but this strategy was not sufficient to induce regression of the pre-existing tissue fibrosis.

Hyperuricemia is associated with increases in cardiovascular risk and renal disease. Mesangial cells regulate glomerular filtration rates through the release of hormones and vasoactive substances. This study evaluates the signaling pathway of uric acid (UA) in immortalized human mesangial cells (ihMCs). To evaluate cell proliferation, ihMCs were exposed to UA (6–10 mg/dL) for 24–144 h. In further experiments, ihMCs were treated with UA (6–10 mg/dL) for 12 and 24 h simultaneously with losartan (10−7 mmol/L). Angiotensin II (AII) and endothelin-1 (ET-1) were assessed using the enzyme-linked immunosorbent assay (ELISA) technique. Pre-pro-ET mRNA was evaluated by the real-time PCR technique. It was observed that soluble UA (8 and 10 mg/dL) stimulated cellular proliferation. UA (10 mg/dL) for 12 h significantly increased AII protein synthesis and ET-1 expression and protein production was increased after 24 h. Furthermore, UA increased [Ca2+]i, and this effect was significantly blocked when ihMCs were preincubated with losartan. Our results suggested that UA triggers reactions including AII and ET-1 production in mesangial cells. In addition, UA can potentially affect glomerular function due to UA-induced proliferation and contraction of mesangial cells. The latter mechanism could be related to the long-term effects of UA on renal function and chronic kidney disease.

Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca2+]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin–angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 μg/mL). AngII-induced [Ca2+]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca2+]i and an impaired response to AngII-induced rise in [Ca2+]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca2+]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.

Mesangial cells (MCs) play a central role in the pathogenesis of diabetic nephropathy (DN). MC dysfunction arises from excessive glucose uptake through insulin-independent glucose transporter (GLUT1). The role of the insulin-dependent transporter (GLUT4) remains unknown. This study evaluated the effect of high glucose on GLUT1, GLUT4, and fibronectin expression levels. Glucose uptake was determined in the absence and presence of insulin. Angiotensin II has been implicated as a mediator of MC abnormalities in DN, and its effects on the GLUTs expression were evaluated in the presence of losartan. MCs were exposed to normal (NG, 10 m M) or high (HG, 30 m M) glucose for 1, 4, 12, 24, and 72 hrs. Glucose uptake was elevated from 1 hr up to 24 hrs of HG, but returned to NG levels after 72 hrs. HG induced an early (1-, 4-, and 12-hrs) rise in GLUT1 expression, returning to NG levels after 72 hrs, whereas GLUT4 was overexpressed at later timepoints (24 and 72 hrs). HG during 4 hrs induced a 40% rise in glucose uptake, which was unaffected by insulin. In contrast, after 72 hrs, glucose uptake was increased by 50%, only under insulin stimulus. Losartan blunted the effects of HG on GLUT1, GLUT4, and fibronectin expression and on glucose uptake. Results suggest that MCs can be highly susceptible to the HG environment since they uptake glucose in both an insulin-independent and insulin-dependent manner. The beneficial effects of angiotensin II inhibition in DN may also involve a decrease in the rate of glucose uptake by MCs.

Renovascular hypertension induced by 2 Kidney-1 Clip (2K-1C) is a renin-angiotensin-system (RAS)-dependent model, leading to renal vascular rarefaction and renal failure. RAS inhibitors are not able to reduce arterial pressure (AP) and/or preserve the renal function, and thus, alternative therapies are needed. Three weeks after left renal artery occlusion, fluorescently tagged mesenchymal stem cells (MSC) (2×10(5) cells/animal) were injected weekly into the tail vein in 2K-1C hypertensive rats. Flow cytometry showed labeled MSC in the cortex and medulla of the clipped kidney. MSC prevented a further increase in the AP, significantly reduced proteinuria and decreased sympathetic hyperactivity in 2K-1C rats. Renal function parameters were unchanged, except for an increase in urinary volume observed in 2K-1C rats, which was not corrected by MSC. The treatment improved the morphology and decreased the fibrotic areas in the clipped kidney and also significantly reduced renal vascular rarefaction typical of 2K-1C model. Expression levels of IL-1β, TNF-α angiotensinogen, ACE, and Ang II receptor AT1 were elevated, whereas AT2 levels were decreased in the medulla of the clipped kidney. MSC normalized these expression levels. In conclusion, MSC therapy in the 2K-1C model (i) prevented the progressive increase of AP, (ii) improved renal morphology and microvascular rarefaction, (iii) reduced fibrosis, proteinuria and inflammatory cytokines, (iv) suppressed the intrarenal RAS, iv) decreased sympathetic hyperactivity in anesthetized animals and v) MSC were detected at the CNS suggesting that the cells crossed the blood-brain barrier. This therapy may be a promising strategy to treat renovascular hypertension and its renal consequences in the near future.

Pregnancy is characterized by maternal systemic and intrarenal vasodilation, leading to increases in the renal plasma flow (RPF) and glomerular filtration rate (GFR). These responses are mainly mediated by nitric oxide (NO) and relaxin. The impact of cigarette smoking on the maternal adaptations to pregnancy is unclear. Here we evaluated the effects of chronic exposure to nicotine on systemic and intrarenal parameters in virgin (V) and 14-day pregnant (P) Wistar rats. V and P groups received saline or nicotine (6 mg·kg(-1)·day(-1)) respectively, via osmotic minipumps for 28 days, starting 14 days before pregnancy induction. Nicotine induced a 10% increase in blood pressure in the V group and minimized the characteristic pregnancy-induced hypotension. Renal sympathetic nerve activity (rSNA) and baroreflex sensitivity were impaired by nicotine mainly in the P group, indicating that the effect of nicotine on blood pressure was not mediated by nervous system stimulation. Nicotine had no effect on GFR in the V rats but reduced GFR of the P group by 30%. Renal expression of sodium and water transporters was downregulated by nicotine, resulting in increased fractional sodium excretion mainly in the P group, suggesting that nicotine compromised the sodium and water retention required for normal gestation. There was a reduction in the expression of inducible NO synthase (iNOS) in both the kidney tissue and renal artery, as well as in the expression of the relaxin receptor (LGR7). These results clearly show that nicotine induced deleterious effects in both virgin and pregnant animals, and abolished the maternal capacity to adapt to pregnancy.

The prorenin receptor [(P)RR] is upregulated in the diabetic kidney and has been implicated in the high glucose (HG)-induced overproduction of profibrotic molecules by mesangial cells (MCs), which is mediated by ERK1/2 phosphorylation. The regulation of (P)RR gene transcription and the mechanisms by which HG increases (P)RR gene expression are not fully understood. Because intracellular levels of angiotensin II (AngII) are increased in MCs stimulated with HG, we used this in vitro system to evaluate the possible role of AngII in (P)RR gene expression and function by comparing the effects of AT1 receptor blockers (losartan or candesartan) and (P)RR mRNA silencing (siRNA) in human MCs (HMCs) stimulated with HG. HG induced an increase in (P)RR and fibronectin expression and in ERK1/2 phosphorylation. These effects were completely reversed by (P)RR siRNA and losartan but not by candesartan (an angiotensin receptor blocker that, in contrast to losartan, blocks AT1 receptor internalization). These results suggest that (P)RR gene activity may be controlled by intracellular AngII and that HG-induced ERK1/2 phosphorylation and fibronectin overproduction are primarily induced by (P)RR activation. This relationship between AngII and (P)RR may constitute an additional pathway of MC dysfunction in response to HG stimulation.

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

The effects of long-term diabetes in the presence of established nephropathy on tubular function remains poorly understood. We evaluated the levels of the main sodium and water transport proteins expressed in the kidney after long-term (8 weeks) of streptozotocin (STZ)-induced type 1 diabetes mellitus (DM) in untreated (D) and insulin (4 U/s.c./day)-treated (D+I) rats. D animals presented upregulation ( approximately 4.5-fold) of Na/glucose cotransporter (SGLT1), whereas the alpha-subunit of the epithelial sodium channel (alpha-ENaC) and aquaporin 1 (AQP1) were downregulated ( approximately 20 and 30% respectively) with no change in the Na/H exchanger (NHE3), Na/Cl cotransporter (TSC) and AQP2. Insulin replacement partially prevented these alterations and caused increases in the expression of alpha-ENaC and AQP2. These effects suggest an action of insulin in the tubular transport properties. The upregulation of SGLT1 may constitute a mechanism to prevent greater glucose losses in the urine but it may result in glucotoxicity to the proximal epithelial cells contributing to the diabetic nephropathy. The decrease of alpha-ENaC in D animals may compensate for the increased sodium reabsorption via SGLT1 resulting in discrete natriuresis. DM-induced polyuria was not due to changes in AQP2 expression.