Experimental Chronic Kidney Disease Research Articles

Impact of intravenous iron on cardiac and skeletal oxidative stress and cardiac mitochondrial function in experimental uraemia chronic kidney disease.

Introuction: Uraemia leads to changes in cardiac structure, metabolic remodeling and anaemia, key factors in the development of heart failure in patients with chronic kidney disease. Previous studies have identified abnormalities in mitochondrial function, potentially impairing energy provision and enhancing oxidative stress. This study characterised oxidant status and changes in mitochondrial function in uraemia and the impact of correcting anaemia via intravenous iron therapy. Methods: Experimental uraemia was induced in male Sprague-Dawley rats via a subtotal nephrectomy and parenteral iron administration given 6 weeks post-surgery. Oxidative stress from tissue samples was evaluated by measuring pro-oxidant activities and anti-oxidant capacities in both sham and uraemic animals with and without iron supplementation. Thiobarbituric acid-reactive substances (TBARS), aconitase activity and cardiolipin were measured. Mitochondrial function was assessed using the Seahorse XFp analyser on isolated mitochondria excised from cardiac tissue. Results: Oxidative stress in this uraemic model was increased in cardiac tissue (increased GSSG/GSH ratio, TBARS and increased activities of pro-oxidant enzymes). There was no impact on skeletal tissue. Parenteral iron ameliorated oxidative stress by enhancing the anti-oxidant defense system in cardiac tissue and skeletal tissue. Examination of respiratory reserve in cardiac mitochondria demonstrated that parenteral iron restored mitochondrial function. This experimental model of uraemia demonstrated a specific oxidative stress on the heart muscle without significant changes in skeletal oxidant status. Iron therapy improved anti-oxidant defence system, consequently reducing oxidative stress in the heart and skeletal tissue. There was an improvement in cardiac mitochondrial function. Conclusions: This experimental evidence indicates that iron therapy could reduce vulnerability to oxidative stress and potentially improve both cardiac and skeletal functional capacity from improvements in mitochondrial function.

Urinary DPP4 correlates with renal dysfunction, and DPP4 inhibition protects against the reduction in megalin and podocin expression in experimental CKD.

This study investigated the molecular mechanisms underlying the antiproteinuric effect of DPP4 inhibition in 5/6 renal ablation rats and tested the hypothesis that the urinary activity of DPP4 correlates with chronic kidney disease (CKD) progression. Experiments were conducted in male Wistar rats who underwent 5/6 nephrectomy (Nx) or sham operation followed by 8 wk of treatment with the DPP4 inhibitor (DPP4i) sitagliptin or vehicle. Proteinuria increased progressively in Nx rats throughout the observation period. This increase was remarkably mitigated by sitagliptin. Higher levels of proteinuria in Nx rats compared to control rats were accompanied by higher urinary excretion of retinol-binding protein 4, a marker of tubular proteinuria, as well as higher urinary levels of podocin, a marker of glomerular proteinuria. Retinol-binding protein 4 and podocin were not detected in the urine of Nx + DPP4i rats. Tubular and glomerular proteinuria was associated with the reduced expression of megalin and podocin in the renal cortex of Nx rats. Sitagliptin treatment partially prevented this decrease. Besides, the angiotensin II renal content was significantly reduced in the Nx rats that received sitagliptin compared to vehicle-treated Nx rats. Interestingly, both urinary DPP4 activity and abundance increased progressively in Nx rats. Additionally, urinary DPP4 activity correlated positively with serum creatinine levels, proteinuria, and blood pressure. Collectively, these results suggest that DPP4 inhibition ameliorated both tubular and glomerular proteinuria and prevented the reduction of megalin and podocin expression in CKD rats. Furthermore, these findings suggest that urinary DPP4 activity may serve as a biomarker of renal disease and progression.

Therapeutic potential of human induced pluripotent stem cells and renal progenitor cells in experimental chronic kidney disease

BackgroundChronic kidney disease (CKD) is a global public health problem. Cell therapy using pluripotent stem cells represents an attractive therapeutic approach for the treatment of CKD.MethodsWe transplanted mitomycin C (MMC)-treated human induced pluripotent stem cells (hiPSCs) and renal progenitor cells (RPCs) into a CKD rat model system. The RPC and hiPSC cells were characterized by immunofluorescence and qRT-PCR. Untreated 5/6 nephrectomized rats were compared to CKD animals receiving the same amount of MMC-treated hiPSCs or RPCs. Renal function, histology, and immunohistochemistry were evaluated 45 days post-surgery.ResultsWe successfully generated hiPSCs from peripheral blood and differentiated them into RPCs expressing renal progenitor genes (PAX2, WT1, SIX2, and SALL1) and podocyte-related genes (SYNPO, NPHS1). RPCs also exhibited reduced OCT4 expression, confirming the loss of pluripotency. After cell transplantation into CKD rats, the body weight change was significantly increased in both hiPSC and RPC groups, in comparison with the control group. Creatinine clearance (CCr) was preserved only in the hiPSC group. Similarly, the number of macrophages in the kidneys of the hiPSC group reached a statistically significant reduction, when compared to control rats. Both treatments reduced positive staining for the marker α-smooth muscle actin. Histological features showed decreased tubulointerstitial damage (interstitial fibrosis and tubular atrophy) as well as a reduction in glomerulosclerosis in both iPSC and RPC groups.ConclusionsIn conclusion, we describe that both MMC-treated hiPSCs and RPCs exert beneficial effects in attenuating CKD progression. Both cell types were equally efficient to reduce histological damage and weight loss caused by CKD. hiPSCs seem to be more efficient than RPCs, possibly due to a paracrine effect triggered by hiPSCs. These results demonstrate that the use of MMC-treated hiPSCs and RPCs improves clinical and histological CKD parameters, avoided tumor formation, and therefore may be a promising cell therapy strategy for CKD.Graphical abstract

Ameliorative effect of a nano chromium metal-organic framework on experimental diabetic chronic kidney disease.

Metal-Organic Frameworks (MOFs) are a new class of crystalline porous structures which can be used as a novel structure in diverse fields of medical science. Several studies have shown that chromium supplementation can be effective in amelioration of biochemical parameters of diabetes and its renal complications. Therefore, a chromium-containing MOF (DIFc) was synthetized by nanochelating technology in the present study and then its effect on biochemical indices in diabetic rats was evaluated. Diabetes was induced by high-fat diet consumption and streptozotocin (35 mg/kg) injection and then the treatment started 8 weeks after disease induction and continued for 8 weeks. The results showed that DIFc treatment decreased HOMA-IR index, blood urea nitrogen, uric acid and malondialdehyde in plasma samples. This nano MOF also reduced albumin, malondialdehyde and 8-isoprostane in urine specimen, while it increased creatinine clearance. In conclusion, DIFc MOF demonstrated promising results in the present study, indicating that it can be developed and evaluated in future investigations with the aim of designing a novel agent for management of diabetes and its renal complications.

Genetic Deletion of NOD1 Prevents Cardiac Ca2+ Mishandling Induced by Experimental Chronic Kidney Disease.

Risk of cardiovascular disease (CVD) increases considerably as renal function declines in chronic kidney disease (CKD). Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) has emerged as a novel innate immune receptor involved in both CVD and CKD. Following activation, NOD1 undergoes a conformational change that allows the activation of the receptor-interacting serine/threonine protein kinase 2 (RIP2), promoting an inflammatory response. We evaluated whether the genetic deficiency of Nod1 or Rip2 in mice could prevent cardiac Ca2+ mishandling induced by sixth nephrectomy (Nx), a model of CKD. We examined intracellular Ca2+ dynamics in cardiomyocytes from Wild-type (Wt), Nod1−/− and Rip2−/− sham-operated or nephrectomized mice. Compared with Wt cardiomyocytes, Wt-Nx cells showed an impairment in the properties and kinetics of the intracellular Ca2+ transients, a reduction in both cell shortening and sarcoplasmic reticulum Ca2+ load, together with an increase in diastolic Ca2+ leak. Cardiomyocytes from Nod1−/−-Nx and Rip2−/−-Nx mice showed a significant amelioration in Ca2+ mishandling without modifying the kidney impairment induced by Nx. In conclusion, Nod1 and Rip2 deficiency prevents the intracellular Ca2+ mishandling induced by experimental CKD, unveiling new innate immune targets for the development of innovative therapeutic strategies to reduce cardiac complications in patients with CKD.

The influence of the prebiotic gum acacia on the intestinal microbiome composition in rats with experimental chronic kidney disease

Chronic kidney disease (CKD) is a globally common and important disease and there are evidence for a bidirectional relationship between microbiota and CKD. The aim of the study was to examine the influence of prebiotic – gum acacia (GA) on the intestinal microbiota in rats with adenine-induced CKD. Animals were randomly distributed into four equal groups (n = 6): control, adenine, GA and adenine + GA groups. CKD was induced by adenine (0.75% w/w) given in the diet daily for four weeks, and GA was administered in drinking water at a concentration of 15% w/v. The 16s rRNA analysis was performed on Illumina Miseq targeting V3-V4 region to characterize microbial composition. The abundance of Actinobacteria, Proteobacteria, Tenericutes and Verrucomicrobia bacteria was increased in adenine-induced CKD, and GA treatment successfully reversed those levels. Interestingly, alpha and beta diversity index were both reduced with GA treatment in rats with CKD. Short chain fatty acids (SCFAs) measurement and PICRUSt analysis have shown that GA treatment completely restored the depleted butyrate level and various perturbated functional pathways, respectively, in CKD rats. Taking together, our results suggest that GA supplementation has a beneficial role in treating CKD, through an increased production of butyrate, as well as its anti-inflammatory, antioxidant capacity and anti-nitrosative properties.

Development of experimental chronic kidney disease and vascular calcification alters diurnal variation of phosphate and its hormonal regulators.

The mineral‐bone axis is tightly regulated and dependent on renal function. In chronic kidney disease (CKD) progressive loss of renal capacity disrupts this axis over‐time, with marked changes in circulating calcium, phosphate, PTH, and fibroblast growth factor‐23 (FGF‐23). These changes contribute to the development of cardiovascular disease, like vascular calcification (VC), which worsens morbidity and mortality in CKD. Although the chronic changes in these circulating factors and their relationships are well known, no experimental studies have examined how the progressive development of CKD and VC alter the circadian rhythms of these factors. An adenine‐induced experimental model of CKD in rats was used to establish (i) general circulating trends, (ii) if renal dysfunction affects these observed trends, and (iii) identify potential changes in these trends caused by VC. This study clearly discerned patterns of daily variations in circulating minerals and hormones, finding that both phosphate and PTH follow modelable diurnal variations whereas calcium and FGF‐23 maintain relative stability over 24‐hr. Surprisingly, the development of CKD was not sufficient to disrupt these patterns of diurnal variation and only altered the magnitude of change; however, it was found that the diurnal rhythms of circulating phosphate and daily stability of calcium were only significantly altered in the setting of CKD with established VC.

Jian-Pi-Yi-Shen Regulates EPO and Iron Recycling Protein Expressions in Anemic Rats with Chronic Kidney Disease: Accumulation of Hypoxia Inducible Factor-2α via ERK Signaling.

Jian-Pi-Yi-Shen (JPYS), the traditional Chinese medicine (TCM) decoction, has been commonly used to treat chronic kidney disease (CKD) and its complications such as anemia. JPYS has been previously found to induce erythropoietin (EPO) production in HEK293T cells and CKD rats. However, the mechanism of JPYS in treating anemia of CKD rats has remained largely unknown. Here, we further extend our effort to investigate the translational control of hypoxia inducible factor- (HIF-) α protein via ERK signaling and the effect on iron recycling-related protein expression by JPYS, thus revealing the mechanism of JPYS in correcting anemia in CKD. Experimental CKD rats with anemia were induced by 5/6 nephrectomy. Rats were administrated orally with high dose (6.0 g/kg/d) and low dose (1.5 g/kg/d) of JPYS for 90 days. Serum hepcidin level was determined to evaluate iron homeostasis. The protein expressions of HIF-2α, erythropoietin (EPO), ferritin, and ferroportin (FPN) and the phosphorylation level of extracellular signal-regulated kinase 1/2 (ERK1/2) were detected by Western blot. The results showed that JPYS treatment significantly ameliorated kidney function by reducing increased levels of blood urea nitrogen (BUN), serum creatinine (Scr), and urine protein (UPRO). Periodic acid-Schiff (PAS) and Masson staining observation showed that the renal pathological damage was restored in JPYS-treated CKD rats. In parallel, JPYS markedly improved CKD anemia through upregulation of red blood cell (RBC), hemoglobin (HGB), and hematocrit (HCT). JPYS stimulated EPO and HIF-2α protein expressions in both the kidney and liver of CKD rats. Furthermore, JPYS induced the phosphorylation of ERK1/2 protein. In addition, JPYS regulated protein expression of ferritin and FPN in both the liver and spleen of CKD rats and the serum level of hepcidin. In conclusion, JPYS induces the expression of EPO through ERK-mediated HIF-2α protein accumulation and regulates systemic iron recycling, supporting its role in promoting erythropoiesis and improvement of anemia in CKD.

The effect of energy restriction on development and progression of chronic kidney disease: review of the current evidence.

Energy restriction (ER) has anti-ageing effects and probably protects from a range of chronic diseases including cancer, diabetes and chronic kidney disease (CKD). Specifically, ER has a positive impact on experimental kidney ageing, CKD (diabetic nephropathy, polycystic kidney disease) and acute kidney injury (nephrotoxic, ischaemia-reperfusion injury) through such mechanisms as increased autophagy, mitochondrial biogenesis and DNA repair, and decreased inflammation and oxidative stress. Key molecules contributing to ER-mediated kidney protection include adenosine monophosphate-activated protein kinase, sirtuin-1 and PPAR-γ coactivator 1α. However, CKD is a complex condition, and ER may potentially worsen CKD complications such as protein-energy wasting, bone-mineral disorders and impaired wound healing. ER mimetics are drugs, such as metformin and Na-glucose co-transporter-2 which mimic the action of ER. This review aims to provide comprehensive data regarding the effect of ER on CKD progression and outcomes.

Abstract MP32: Effectiveness Of Investigational RNAi Therapeutics Targeting Liver Angiotensinogen In Experimental Chronic Kidney Disease

Background: RNAi therapeutics targeting angiotensinogen (AGT) lower blood pressure in animal models of essential hypertension, but whether this treatment is beneficial in hypertensive chronic kidney disease (CKD) is yet unknown. Our aim was to assess the effectiveness of a small interfering RNA (siRNA) targeting hepatic AGT in a rat model of hypertensive CKD. Methods: Sprague Dawley rats (n=7-12 per group) were subjected to 5/6th nephrectomy, allowed to recover for four weeks, and subsequently subjected to one of five treatments: (1) vehicle, (2) AGT siRNA (10-30 mg/kg, s.c. every 2 weeks), (3) AGT siRNA + losartan (30 mg/kg/d), (4) losartan (30 mg/kg/d), or (5) losartan + captopril (30 and 6 mg/kg/d). Mean arterial pressure (MAP) was measured by radiotelemetry for 4 weeks, glomerular filtration rate (GFR) was measured by transcutaneous FITC-sinistrin clearance. Results: AGT siRNA and AGT siRNA + losartan reduced plasma AGT by 96.5 ± 1.4% and 97.0 ± 2.3%, respectively. Over the course of 4 weeks, proteinuria increased with vehicle and this increase was abrogated by all treatments (Δ proteinuria at 4 weeks versus baseline: +0.17 [0.12, 0.23], +0.02 [-0.04, 0.05], -0.01 [-0.07, 0.06], -0.05 [-0.12, 0.00], -0.01 [-0.06, 0.11] for vehicle, AGT siRNA, AGT siRNA + losartan, losartan, losartan + captopril, P < 0.05 for all groups compared with vehicle). Similarly, heart weight was equally reduced by all treatments except vehicle. No intervention affected GFR. Over the course of 4 weeks, MAP increased (from a baseline of 154 ± 18 mmHg) during vehicle treatment and this rise was prevented by AGT siRNA (+13 ± 15 mmHg vs. -2 ± 14 mmHg, P > 0.05 for difference between groups). Combination treatment with AGT siRNA + losartan, losartan + captopril, and losartan alone caused a similar and marked reduction in MAP (-17 ± 14 mmHg, -21 ± 17 mmHg, and -33 ± 20 mmHg, P < 0.05 versus vehicle for all; differences between groups not significant). Conclusion: In experimental CKD, AGT siRNA monotherapy appears to reduce proteinuria and heart weight in a blood pressure-independent fashion, to a similar degree as losartan or combination treatment. Thus, an siRNA targeting hepatic AGT may be a potential therapeutic approach for providing renal and cardioprotection in CKD.

P0094KLOTHO DECLINE IS ASSOCIATED WITH PRO-HYPERTROPHIC SIGNALING IN MYOCARDIUM OF NEPHRECTOMIZED SPONTANEOUSELY HYPERTENSIVE RATS (PILOT SYUDY)

Abstract Background and Aims Klotho deficiency is suggested to be involved in cardiac complications in chronic kidney disease (CKD) through modulation of intracellular pro-hypertrophic signaling pathways. The aim of this study was to investigate the molecular mechanisms of myocardial remodeling in the settings of Klotho decline in experimental CKD. Method Systolic blood pressure (BP), myocardial mass index (MMI), serum and urinary creatinine (Cr), serum urea (Ur), inorganic phosphate (Pi), 25-hydroxyvitamin D (25OHD), parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho, myocardial Klotho, calcineurin/ NFAT and beta-catenin expression (IHC), quantitative morphometry of cardiomyocite (CM) thickness and nuclei area (hematoxylin and eosin), myocardial perivascular (PF) and interstitial fibrosis (IF), renal fibrosis (RF) (Masson’s trichrome) and Klotho expression (rKl, IHC) were analyzed in spontaneously hypertensive rats with 3/4 or 5/6 nephrectomy (Nx) and duration of experimental exposure 1 and 2 mo (Nx1, n = 9; Nx2, n = 8). Controls were sham operated (SO) animals (SO1, n = 9; SO2, n = 9). Results The experimental model was corresponded to the initial stages of CKD (Ur level was between 6.64 and 13.36 mmol/L, serum Cr increased less than 50% vs SO, RF did not exceed 22 % FOV). Serum concentration of Klotho and rKl expression decreased, while MMI, CM thickness increased in both Nx1 and Nx2 (Table). There were no differences in serum Pi, FGF23 and PTH levels in Nx1 vs SO (Table). In Nx2 Klotho decline was accompanied by the development of IF and PF, serum 25OHD decrease, Pi and FGF23 increase vs SO2 (Table). In multiple regression analyzes including SO and Nx animals rKl was independently associated with MMI (β = -0.38 ± 0.16, p = 0.026), and CM thickness (β = -0.64 ± 0.14, p <0.001). Myocardial fibrosis was associated with rKl level (IF: β = -0.87 ± 0.19, p = 0.001) and serum 25OHD (PF: β =-0.34 ± 0.15, p=0.032). According to pilot data of IHC, in Nx rat myocardium Klotho protein expression decreased, cytoplasmic calcineurin and nuclear NFAT in CM were observed (Figure), suggesting an increase in the activity of the calcineurin / NFAT signaling pathway. An intracellular redistribution of β-catenin from the cytoskeleton to the cytoplasm and nuclei were observed in CM of Nx rats (Figure), likely corresponding to increase in the activity of Wnt signaling. Conclusion Associations of CM hypertrophy, myocardial fibrosis and Klotho decline in CKD may be mediated by calcineurin / NFAT and Wnt signaling in myocardium.

P0871THE IMPACT OF ENDOGENOUS PTH/PTH1R/ATF4 AXIS ON TRABECULAR AND CORTICAL BONE REMODELING AND BONE GROWTH OF YOUNG RATS WITH EXPERIMENTAL CHRONIC KIDNEY DISEASES

Abstract Background and Aims Chronic kidney disease - mineral bone disorder (CKD–MBD) is one of the major clinical complications in patients with chronic kidney disease (CKD). Bone remodeling has been suggested to play a fundamental role in the maintenance of skeletal integrity via a balance between the bone formation/resorption process. The parathyroid hormone (PTH) is a key hormone controlling bone metabolism. PTH is known to affect bone with a net catabolic and anabolic effect but the mechanisms responsible for these differing effects are poorly understood. Previously, we demonstrated the distinct effect of mild degree of CKD on trabecular and cortical bone strength in rapidly growing rats (Pawlak et al. PlosOne, 2016). The aim of the present study was to evaluate the effect of endogenous PTH, its receptor PTH1R and activating transcription factor 4 (ATF4) – the major regulators of the anabolic PTH response in osteoblasts on bone remodeling and growth of young rats with experimental CKD. Method Four-week old Wistar male rats were divided into 2 groups: with CKD induced by surgical 5/6 subtotal nephrectomy, and sham-operated (CON). After one (CON-1; CKD-1) and three months (CON-3; CKD-3) of the surgery the femurs were collected and their lengths were measured. The activity of alkaline phosphatase (ALP), a bone formation marker, and tartrate-resistant acid phosphatase (TRACP5b) reflecting bone resorption were determined in homogenates from trabecular and cortical left femurs. The expression of PTH1R and ATF4 gene was determined by QRT-PCR in right femurs. Serum PTH was analyzed using ELISA kit. Computations were performed using Statistica ver.10 computer software. Results Serum PTH was increased in CKD-3 compared to CKD-1, moreover the slight increase in PTH levels was noted in CKD-3 compared to appropriate controls (both p<0.05). The activity of ALP and TRACP5b in trabecular bone tissue were significantly lower in CKD-3 compared to CKD-1 rats (p<0.01 and p<0.05; respectively). There was a strong positive correlation between ALP and TRACP5b in this bone region (R =0.624, p =0.002), whereas both ALP and TRACP5b were inversely related to serum PTH (R = -0.534, p =0.012 and R = -0.636, p =0.002; respectively). The activity of TRACP5b in cortical bone tissue was significantly higher in CKD-3 compared to CKD-1 group (p<0.05), and it was positively associated with PTH levels (R =0.597, p =0.004). There was no difference in ALP activity between the studied groups, and ALP was not associated with TRACP5b in this bone region. The expression of ATF4 and PTH1R genes was significantly increased in the CKD-3 group compared with the appropriate control (p<0.01 and p<0.05; respectively) and with the CKD-1 group (p<0.05 and p<0.01; respectively). The expression of ATF4 was inversely correlated with ALP and TRACP5b in trabecular bone (R = -0.534, and R = -0.528, both p<0.05), whereas it was positively related to TRACP5b in cortical bone region (R =0.418, p<0.05). The femoral length was significantly increased during 3-month of CKD development (p<0.000), and it was positively associated with PTH levels (R =0.571, p =0.007), cortical TRACP5b activity (R =0.609, p =0.002) and tended to be related with ATF4 gene expression (R =0.409, p =0.065). In contrast, the femoral length was inversely related to ALP activity in trabecular bone tissue (R= -0.656, p =0.0007). Conclusion The endogenous PTH, through PTH1R/ATF4 axis, inhibited trabecular bone remodeling. In contrary, PTH/PTH1R/ATF4 system intensified bone resorption in cortical bone region. This opposite effect of PTH on bone remodeling was associated with the intensification of growth process in the long bones of young rats with CKD.

P0096RENAL EXPRESSION AND CIRCULATING LEVELS OF CANONICAL WNT PATHWAY INHIBITORS IN THE EXPERIMENTAL MODEL OF EARLY CHRONIC KIDNEY DISEASE

Abstract Background and Aims Molecular mechanisms implicated in the initial stages of inorganic phosphate (Pi) imbalance in chronic kidney disease (CKD) remain poorly understood. The aim of the study was to evaluate whether canonical Wnt pathway inhibitors (iWnt) involved in early response to Pi retention in CKD. Method Mild CKD was induced by 3/4 nephrectomy (NE, n=10) in spontaneously hypertensive rats (SHR) fed rat chow diet containing 0.8 % phosphate. Controls were sham operated SHR (SO, n=9). Duration of experimental exposure (NE or SO) was 4 months. Creatinine (Cr), albumin/ creatinine ratio (Alb/Cr), inorganic phosphate (Pi), fractional (FEPi) and daily Pi excretion (uPi24), intact parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), Klotho protein, sclerostin (SOST) and Dickkopf-1 (DKK1), SOST and DKK1 mRNA expression in kidney, renal interstitial fibrosis (RF) were analyzed. Results Levels of Alb/Cr, serum Cr, RF and indices of Pi exchange in the experimental model corresponded to mild CKD with Pi retention (Table). No differences were observed in serum levels of Klotho, FGF23 and PTH in experimental group vs controls (Table). Serum SOST and DKK1 concentrations were significantly higher in NE group while the kidney expression of SOST and DKK1 mRNAs decreased compared to SO (Table). Conclusion Early stages of experimental CKD are characterized by increased serum concentrations of sclerostin and Dickkopf-1 in the absence of Klotho, FGF23 or PTH levels alterations. The increase of circulating iWnt levels seems not to be associated with their renal biosynthesis and likely have a bone origin.

Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease

BackgroundTubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury.MethodsHuman primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/− mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo.ResultsImmunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/− mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis.ConclusionData suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD.-4b_UPmq1WosguM_gnU3qmVideo Graphical abstractIn proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-β1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.

Vitamin D and fibroplastic processes in myocardium in spontaneously hypertensive rats with initial kidney dysfunction

Background. Even a moderate decrease in glomerular filtration rate leads to an increased risk of cardiovascular diseases (CVD), which is the leading cause of mortality in patients with chronic kidney disease (CKD). Left ventricular hypertrophy (LVH) underlies CVD development in renal dysfunction. The prevalence of LVH in patients with CKD stages 2–4 is 50–70 % and reaches 95 % at the beginning of dialysis, which significantly exceeds the number of cases in general population (15–21 %). Common hemodynamic factors associated with chronic kidney damage —hypertension (HTN), activation of the renin-angiotensin system, anemia, fluid and sodium retention, and others largely explain the high prevalence of LVH among patients with CKD. Nevertheless, the existence of additional non-hemodynamic mechanisms of myocardial remodeling (MR) is evident.Objective. To investigate the associations between the MR physiological/histological characteristics and laboratory parameters of calcium-phosphate metabolism in the initial stages of experimental CKD. Design and methods. Four groups of spontaneously hypertensive rats (SHR) were studied (n = 35): 3/4 nephrectomized rats (Nx) one month exposed after surgery (Nx(1), n = 9), 5/6 Nx two months after surgery (Nx(2), n = 8), sham operated rats one month after surgery (SO(1), n = 9) and two months after surgery (SO(2), n = 9). Myocardial mass index (MMI), systolic blood pressure (BP), proteinuria, creatinine (Cr) concentration, total calcium (Ca) and inorganic phosphate (Pi), 25-OH vitamin D (25OHD) and parathyroid hormone (PTH) in serum, myocardial morphology were studied in all experimental animals.Results. The models corresponded to the 1–3 stages CKD. There were no significant changes in serum total Ca (p = 0,066), Pi (p = 0,051) and PTH (p = 0,015) concentrations, the level of 25OHD was significantly lower in Nx(2) rats vs control (p = 0,015). MMI increased in all nephrectomized rats (p = 0,008). The cardiomyocytes (CM) thickness increased in Nx(1) and Nx(2) animals compared to the corresponding controls (p = 0,010, p = 0,002). A significant increase in interstitial (IF) and perivascular (PF) fibrosis occurred in Nx(2) rats with more damaging influence (p = 0,017, p = 0,004). CM thickness, IF and PF increased with the elevation of BP (r = 0,39, p = 0,038, r = 0,47, p = 0,026, r = 0,49, p = 0,031) and serum Cr (r = 0,68, p = 0,001, r = 0,61, p = 0,003, r = 0,69, p = 0,001), and the decrease in serum 25OHD concentration (r = –0,045, p = 0,047, r = –0,50, p = 0,020, r = –0,52, p = 0,012). Multiple linear regression analysis showed, that 25OHD is an independent predictor of myocardial fibrosis (IF: β = –0,38 ± 0,18, p = 0,047, PF: β = –0,34 ± 0,15, p = 0,032).Conclusions. The initial stages of CKD accompanied with HTN are associated with serum 25OHD concentration decrease CM hypertrophy and myocardial fibrosis. The CM growth is an earlier event in relation to the interstitial fibrosis. The obtained data suggest a possible role of vitamin D deficiency in the development of myocardial fibrotic lesions.

Metformin arrests the progression of established kidney disease in the subtotal nephrectomy model of chronic kidney disease.

Metformin, an AMP-activated protein kinase (AMPK) activator, has been shown in previous studies to reduce kidney fibrosis in different models of experimental chronic kidney disease (CKD). However, in all of these studies, the administration of metformin was initiated before the establishment of renal disease, which is a condition that does not typically occur in clinical settings. The aim of the present study was to investigate whether the administration of metformin could arrest the progression of established renal disease in a well-recognized model of CKD, the subtotal kidney nephrectomy (Nx) model. Adult male Munich-Wistar rats underwent either Nx or sham operations. After the surgery (30 days), Nx rats that had systolic blood pressures of >170 mmHg and albuminuria levels of >40 mg/24 h were randomized to a no-treatment condition or to a treatment condition with metformin (300 mg·kg-1·day-1) for a period of either 60 or 120 days. After 60 days of treatment, we did not observe any differences in kidney disease parameters between Nx metformin-treated and untreated rats. However, after 120 days, Nx rats that had been treated with metformin displayed significant reductions in albuminuria levels and in markers of renal fibrosis. These effects were independent of any other effects on blood pressure or glycemia. In addition, treatment with metformin was also able to activate kidney AMPK and therefore improve mitochondrial biogenesis. It was concluded that metformin can arrest the progression of established kidney disease in the Nx model, likely via the activation of AMPK.

Renal Deafferentation Improves Renal Function and Blood Pressure, but not Cardiac Parameters in an experimental model chronic kidney disease.

Chronic kidney disease is highly prevalent and its mortality is associated with cardiovascular disease. Experimental evidence shows that the renal nerves contribute to arterial hypertension and renal sympathetic hyperactivity worsening the disease; renal denervation has shown renoprotective and cardioprotective effects. However, renal sympathetic innervation plays a fundamental role in renal homeostasis and its removal is associated with impaired compensatory mechanisms during hemodynamic changes. Thus, the aim of this work was to investigate the effects of selective renal denervation of afferent fibers on renal and cardiovascular repercussions in chronic kidney disease (CKD) induced by 5/6 nephrectomy in Wistar rats. The use of animals was approved by the Federal University of São Paulo Animal Care and Use Committee and performed according to the National Institutes of Health guidelines for the conduct of experiments in animals. After five weeks of nephrectomy, denervation of renal afferent fibers with a periaxonal application of 33 mM capsaicin was performed. Cardiovascular and renal function parameters were evaluated three weeks after deafferentation.ResultsCKD induced increase in blood pressure (CTL 113±7.2 × CKD 185±14 mmHg), creatinine plasma levels (CTL 0.5±0.1 × CKD 1.2±0.5 mg/dl) and proteinuria (CTL 14±10 × 300±129 mg/24h). It was also observed an increase in cardiac hypertrophy, reduction in baroreflex sensitivity and in parasympathetic tone after autonomic double blockade. Afferent renal denervation reduced blood pressure (165±21 mmHg), improved creatinine plasma levels (0.8 ±0.2 mg/dl) and proteinuria (178±102 mg/24h). No alterations were observed after deafferentation over baroreflex sensitivity, cardiac hypertrophy and autonomic control to the heart. Together these results suggest that renal deafferentation improves blood pressure and renal function, without, however, improving the cardiac changes found in this CKD model. It is possible that the time expected after deafferentation or the stage of the disease in which it was performed was not sufficient to induce cardiac improvements. Further studies are necessary to elucidate this question.Support or Funding InformationCoordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; by the São Paulo Research Foundation (FAPESP 18/02671‐3) and by the Brazilian National Research Council (CNPq)

Oxidative stress and the antioxidant system in salivary glands of rats with experimental chronic kidney disease

ObjectiveThis study aims to analyze the presence of oxidative stress and activity of the antioxidant system in the parotid and submandibular salivary glands of rats with Chronic Kidney Disease (CKD). DesignSixteen male wistar rats were divided into two groups (n = 8, each): control rats and rats with CKD. CKD was induced by 5/6 nephrectomy. Blood urea nitrogen and serum creatinine clearance were quantified. Malondialdehyde, superoxide dismutase, glutathione peroxidase, glutathione reductase, catalase, total antioxidant status, ascorbic acid, α-tocopherol, superoxide anion, and hydrogen peroxide concentrations were assessed. ResultsIn CKD rats, blood urea nitrogen, serum creatinine, and proteinuria concentrations were increased, while creatinine clearance was reduced. In the submandibular gland, superoxide anion concentration was increased significantly (p < 0.05). Hydrogen peroxide and superoxide anion concentrations were reduced in the parotid gland. CKD rats presented increased malondialdehyde concentration, total antioxidant status, superoxide dismutase, and glutathione reductase activities only in the parotid gland (p < 0.05). ConclusionOxidative stress and changes in the antioxidant system were found in the parotid and submandibular salivary glands in an experimental model of CKD induced by 5/6 nephrectomy.

Gum arabic reduces inflammation, oxidative, and nitrosative stress in the gastrointestinal tract of mice with chronic kidney disease.

The aim of this study was to investigate some biochemical indices of inflammation and oxidative and nitrosative stresses in the gastrointestinal tract of mice with experimental chronic kidney disease (CKD) and treated with gum arabic (GA). Male CD1 mice (n = 28) were randomly distributed into four groups and treated for four consecutive weeks: group 1: Control: received the same diet without treatment until the end of the study; group 2: Adenine: switched to a powder diet containing adenine (0.2% w/w in feed); group 3: Gum acacia (GA): given normal feed and GA in drinking water at a concentration of 15% w/v; and group 4: Adenine + GA: given adenine in the feed as in the second group plus GA in the drinking water at concentration of 15% w/v. CKD was induced to mice by adenine feeding and concomitantly treated with the prebiotic dietary fiber gum acacia, GA (15% in drinking water). Duodenal mucosa from CKD mice had significantly higher concentrations of TNF-alfa, IL- 6, and TGF-beta-1 and lipid peroxidation. Moreover, low concentrations of IL-10, some antioxidants (catalase, glutathione reductase, total antioxidant capacity, and superoxide dismutase), and nuclear factor erythroid 2-related factor 2 were found in the duodenum. The levels of nitrosative stress (nitrite, nitrate, and total nitrate) were significantly increased by CKD, as well as the concentrations of ammonia and urea creatinine in the cecal content. Concomitant GA treatment significantly mitigated these harmful effects. Taken together, GA reduces inflammation and duodenal oxidative and nitrosative stress in the gastrointestinal tract of mice with CKD.

Chromogranin A pathway: from pathogenic molecule to renal disease.

Chromogranin A (CHGA) is an index granin protein critical for biogenesis and exocytotic release of catecholamine storage granules. It is elevated in plasma of patients with sympathetic over-activity and kidney dysfunction. Several CHGA polymorphisms are associated with hypertensive kidney disease. Previously, we unraveled the molecular mechanism by which CHGA expression is regulated in African Americans carrying a genetic variation associated with hypertensive chronic kidney disease (CKD). Experimental CKD mouse model were created by 5/6th nephrectomy (Npx) using wild-type and Chga-/- knockout mouse strains to delineate the role of CHGA in CKD. Wild-type-Npx mice expressing Chga developed exacerbated azotemia and fibrosis as compared with their knockout-Npx counterparts. Gene expression profiling revealed downregulation of mitochondrial respiratory complexes genes consistent with maladaptive mitochondria in wild-type-Npx mice, contrasted to knockout-Npx. In healthy individuals, an inverse relationship between circulating CHGA levels and glomerular function was observed. In vitro, mesangial cells treated with CHGA-triggered nitric oxide release by a signaling mechanism involving scavenger receptor SR-A. The CHGA-treated and untreated mesangial cells displayed differential expression of cytokine, chemokine, complement, acute phase inflammatory and apoptotic pathway genes. Thus, build-up of plasma CHGA because of kidney injury served as an insult to the mesangial cells resulting in expression of genes promoting inflammation, fibrosis, and progression of CKD. These findings improve understanding of the role of elevated CHGA in the progression of CKD and reveal novel pathways that could be exploited for therapeutic strategies in hypertensive kidney disease.