Chronic Kidney Disease Induction Research Articles

Combining raloxifene and mechanical loading improves bone composition and mechanical properties in a murine model of chronic kidney disease (CKD)

IntroductionPatients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. MethodsSixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 μƐ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. ResultsRegardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. DiscussionAn effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties.

Effects of exogenous advanced glycation end products on oxidative stress and renal injury in healthy mice

AbstractThis study evaluated the biological effects of exogenous advanced glycation end products (AGEs) on the induction of chronic kidney disease and the dose‐effect relationship. Male C57BL/6 mice were placed on four diets, including saline and three other diets differing only in AGEs content (low‐AGEs [LA], medium‐AGEs [MA], and high‐AGEs [HA] ratio, 1:3:5) for 4 weeks. With the increasing intake of AGEs, mice developed a significant increase in blood glucose and lipid levels, the fluorescence intensity of AGEs, Nε‐(carboxymethyl)‐lysine, Nε‐(carboxyethyl)‐lysine, and malondialdehyde levels, whereas their superoxide dismutase activity and glutathione levels were decreased significantly. HA had the highest urinary protein levels and the lowest creatinine clearance compared to the other groups. These suggested that AGEs are an essential contributor to increasing oxidative stress levels and intake of high‐level AGEs induces more severe kidney function impairment. Meanwhile, the AGEs intake damaged the kidney structure in a dose‐dependent manner, as evidenced by granular degeneration of kidney tubular epithelial cells and inflammatory cell infiltration. These findings shed light on the detrimental impacts of AGEs on human kidneys, which also will help reveal a dose‐effect relationship of AGEs.

#5485 CHRONIC KIDNEY DISEASE NOR STROMAL CELL-DERIVED FACTOR 1Α PEPTIDE (SDF-1A) BIO-FUNCTIONALIZATION ALTER RODENT VASCULAR IN SITU TISSUE FORMATION

Abstract Background and Aims The systemic pro-inflammatory environment of chronic kidney disease (CKD) could influence neo-tissue formation of in situ tissue engineered vascular access grafts. Here, we studied in-graft tissue formation and inflammation in a rat CKD model. Moreover, we explored graft bio-functionalization with stromal cell-derived factor 1α peptides (SDF-1α), a progenitor cell chemotactic which may improve cell engraftment. Method Pristine or SDF-1α bio-functionalized vascular grafts (1,2 mm ID, 2 cm length, 250–300 μm wall thickness) were created from biodegradable, polycarbonate-bisurea (PC-BU) electrospun meshes. Abdominal aorta interposition grafts were implanted in female Sprague Dawley rats (n = 53) that underwent sham surgery or CKD induction by 5/6th nephrectomy. Explanations and analyses were performed after two (n = 25) or twelve (n = 9) weeks. Results At two weeks, survival and graft patency were 100%. Explant cellularity and collagen content were not significantly different between sham and CKD rats and were not influenced by SDF-1α. Endothelial cell coverage (RECA+) and smooth muscle cell presence (αSMA+) were visually similar irrespective of disease or bio-functionalization. Elastin+ (p = 0,11), pan-(CD68+, p = 0,08) and anti-inflammatory macrophage (CD163+, p = 0,52) surface area, as well as (anti-)inflammatory gene expression were not significantly different between groups. The twelve-week CKD-group was taken out of experiment prematurely due to rapid disease progression. In addition, preliminary death due to vascular rupture occurred in four animals (n = 2 pristine and n = 2 SDF-1α) in the twelve-week sham animals. While patent, all remaining sham explants at twelve weeks (n = 9) showed severe vascular dilatation and calcifications (Von Kossa+). No differences in inflammatory and neo-tissue markers were found between twelve-week sham pristine and SDF-1α grafts or between explants over time. Conclusion CKD did not significantly alter inflammation or tissue formation of in situ tissue engineered vascular grafts. Additionally, SDF-1α bio-functionalization did not significantly alter cell engraftment or tissue formation. Mechanical stability of the graft appears to be the primary driver of neo-tissue formation. Future emphasis on in vitro to in vivo translation should be on the fine balance between tissue formation on one hand and fiber resorption on the other hand.

438 Platelets, Inflammation and Thrombosis in Chronic Kidney Disease

OBJECTIVES/GOALS: Platelets reside at the nexus of thrombosis and inflammation which make them an ideal target of investigation to understand mechanisms underlying chronic kidney disease (CKD)-related inflammatory and thrombotic dysregulation. Our objective is to determine whether a pro-inflammatory state in CKD is exacerbated by platelets. METHODS/STUDY POPULATION: Aim 1 will investigate effects of engineered reduction in the interaction of platelets with leukocytes [by disruption of one of the platelet surface receptor (GPIb-IX)] in the development of CKD in murine models. Aim 2 will investigate effects of platelet inhibitors on the development of CKD in murine models. RESULTS/ANTICIPATED RESULTS: We anticipate that the proposed studies in Aim 1 will demonstrate reduction in the interaction of platelets with leukocytes results in exacerbation of kidney injury upon CKD induction with cisplatin. We also anticipate that inhibition of platelets in Aim 2 with P2Y12 receptor inhibitors results in reduction in kidney injury upon CKD induction with cisplatin. DISCUSSION/SIGNIFICANCE: Upon successful completion of the proposed studies, we shall be able to better describe the role of platelets as modulators of inflammation in CKD. This will be a significant stride towards understanding the pathophysiology of a pro-inflammatory state in CKD and how platelets exacerbate inflammation and thrombosis in this population.

Mitochondrial dysfunction in kidney cortex and medulla of subtotally nephrectomized rats.

Five-sixths nephrectomy is a widely used experimental model of chronic kidney disease (CKD) that is associated with severe mitochondrial dysfunction of the remnant tissue. In this study, we assessed the effect of CKD on mitochondrial respiration separately in the rat kidney cortex and medulla 10 weeks after induction of CKD by subtotal 5/6 nephrectomy (SNX). Mitochondrial oxygen consumption was evaluated on mechanically permeabilized samples of kidney cortex and medulla using high-resolution respirometry and expressed per mg of tissue wet weight or IU citrate synthase (CS) activity. Mitochondrial respiration in the renal cortex of SNX rats was significantly reduced in all measured respiratory states if expressed per unit wet weight and remained lower if recalculated per IU citrate synthase activity, i.e. per mitochondrial mass. In contrast, the profound decrease in the activity of CS in SNX medulla resulted in significantly elevated respiratory states expressing the OXPHOS capacity when Complexes I and II or II only are provided with electrons, LEAK respiration after oligomycin injection, and Complex IV-linked oxygen consumption per unit CS activity suggesting compensatory hypermetabolic state in remaining functional mitochondria that is not sufficient to fully compensate for respiratory deficit expressed per tissue mass. The results document that CKD induced by 5/6 nephrectomy in the rat is likely to cause not only mitochondrial respiratory dysfunction (in the kidney cortex), but also adaptive changes in the medulla that tend to at least partially compensate for mitochondria loss.

Oxidized-LDL Deteriorated the Renal Residual Function and Parenchyma in CKD Rat through Upregulating Epithelial Mesenchymal Transition and Extracellular Matrix-Mediated Tubulointerstitial Fibrosis-Pharmacomodulation of Rosuvastatin.

This study tested the hypothesis that intrarenal arterial transfusion of oxidized low-density lipoprotein (ox-LDL) jeopardized the residual renal function and kidney architecture in rat chronic kidney disease ((CKD), i.e., induced by 5/6 nephrectomy) that was reversed by rosuvastatin. Cell culture was categorized into A1 (NRK-52E cells), A2 (NRK-52E + TGF-β), A3 (NRK-52E + TGF-β + ox-LDL) and A4 (NRK-52E + TGF-β + ox-LD). The result of in vitro study showed that cell viability (at 24, 48 and 72 h), NRK-52E ox-LDL-uptake, protein expressions of epithelial-mesenchymal-transition (EMT) markers (i.e., p-Smad2/snail/α-SMA/FSP1) and cell migratory and wound healing capacities were significantly progressively increased from A1 to A4 (all p < 0.001). SD rats were categorized into group 1 (sham-operated control), group 2 (CKD), group 3 (CKD + ox-LDL/0.2 mg/rat at day 14 after CKD induction) and group 4 (CKD + ox-LDL-treated as group 3+ rosuvastatin/10 mg/kg/day by days 20 to 42 after CKD induction) and kidneys were harvested at day 42. The circulatory levels of BUN and creatinine, ratio of urine-protein to urine-creatinine and the protein expressions of the above-mentioned EMT, apoptotic (cleaved-caspase3/cleaved-PARP/mitochondrial-Bax) and oxidative-stress (NOX-1/NOX-2/oxidized-protein) markers were lowest in group 1, highest in group 3 and significantly higher in group 4 than in group 2 (all p < 0.0001). Histopathological findings demonstrated that the kidney injury score, fibrotic area and kidney injury molecule-1 (KIM-1) displayed an identical pattern, whereas the cellular expression of podocyte components (ZO-1/synaptopodin) exhibited an opposite pattern of EMT markers (all p < 0.0001). In conclusion, ox-LDL damaged the residual renal function and kidney ultrastructure in CKD mainly through augmenting oxidative stress, EMT and fibrosis that was remarkably reversed by rosuvastatin.

Abstract 15473: A New Metabolic Phenotype in a Mouse Model of Chronic Kidney Disease and Atherosclerosis

Introduction: Patients with chronic kidney disease (CKD) experience significant cardiovascular morbidity and mortality. Vascular calcification is a hallmark of CKD and a strong predictor of atherosclerotic cardiovascular disease. Hypothesis: We hypothesize that CKD-induced vascular calcification potentiates atherosclerosis. Methods: First, wished to conduct a pilot study to confirm that a co-treatment of the low-density protein receptor mutant mice (Ldlr whc ) with a 0.2% adenine in a western diet will produce accelerated atherosclerosis, consistent with a well-known phenotype in the 5/6 nephrectomy model on an ApoE or Ldlr knockout background. Then we developed a two-step non-surgical mouse model in which the induction of calcification by a 0.2% adenine treatment was followed by western diet-induced hyperlipidemia. Results: Contrary to expected, the adenine and western diet (W+A) co-treatment resulted in a striking reduction of plasma triglycerides (TG), aortic root atherosclerosis, and liver lipids despite a fully penetrant CKD phenotype with tubulointerstitial damage, increased blood urea nitrogen, hyperphosphatemia, polyuria, vascular calcification, and osteoporosis. The two-step induction of CKD and atherosclerosis demonstrated partially normalized but reduced plasma triglycerides and liver lipid content, polyurea, and polydipsia in CKD mice, and there was no difference in atherosclerotic burden or plaque calcification between the groups. Interestingly we observed an increase in food and calorie intake and 24-hour glucose excretion in the urine in the pre-treated group compared with mice on a western diet without pre-treatment, while plasma glucose levels were not different between the groups. Conclusion: The paradoxical lack of dyslipidemia in mice treated with adenine provides an insight into the potential effects of tubulointerstitial kidney disease and nephrogenic diabetes incipits on lipid metabolism and atherosclerosis. 1

Towards a better understanding of arterial calcification disease progression in CKD: investigation of early pathological alterations.

Cardiovascular disease remains the leading cause of death in chronic kidney disease (CKD) patients, especially in those undergoing dialysis and kidney transplant surgery. CKD patients are at high risk of developing arterial media calcifications (AMC) and arterial stiffness. We hypothesized that investigation of disease progression at an early stage could provide novel insights in understanding AMC etiology. An adenine diet was administered to male Wistar rats to induce AMC. Rats were sacrificed after 2, 4 and 8 weeks. AMC was measured by assessment of aortic calcium and visualized using histology. Arterial stiffness was measured in vivo by ultrasound and ex vivo by applying cyclic stretch of physiological magnitude on isolated arterial segments, allowing us to generate the corresponding pressure-diameter loops. Further, ex vivo arterial reactivity was assessed in organ baths at 2 and 4 weeks to investigate early alterations in biomechanics/cellular functionality. CKD rats showed a time-dependent increase in aortic calcium which was confirmed on histology. Accordingly, ex vivo arterial stiffness progressively worsened. Pressure-diameter loops showed a gradual loss of arterial compliance in CKD rats. Additionally, viscoelastic properties of isolated arterial segments were altered in CKD rats. Furthermore, after 2 and 4 weeks of adenine treatment, a progressive loss in basal, nitric oxide (NO) levels was observed, which was linked to an increased vessel tonus and translates into an increasing viscous modulus. Our observations indicate that AMC-related vascular alterations develop early after CKD induction prior to media calcifications being present. Preventive action, related to restoration of NO bioavailability, might combat AMC development.

Aerobic Exercise Attenuates Kidney Injury, Improves Physical Performance, and Increases Antioxidant Defenses in Lungs of Adenine-Induced Chronic Kidney Disease Mice.

The association between chronic kidney disease (CKD) and pulmonary pathophysiological changes is well stablished. Nevertheless, the effects of aerobic exercise (AE) on lungs of CKD need further clarification. Thus, Swiss mice were divided in control, AE, CKD, and CKD + AE groups. CKD was induced by 0.2% adenine intake during 8weeks (4weeks of CKD induction and 4weeks of AE). AE consisted in running on treadmill, at moderate intensity, 30min/day, 5days/week, during 4weeks. Twenty-four hours after the last training day, functional capacity test was performed, and 48h after the test, mice were euthanized. CKD mice showed a significant increase in urine output, serum urea, and creatinine concentrations, and decreased body weight and urine density, besides oxidative damage (p = 0.044), edema area (p < 0.001), leukocyte infiltration (p = 0.040), and collagen area in lung tissue (p = 0.004). AE resulted in an increase of distance traveled (p = 0.049) and maximum speed (p = 0.046), increased activity of catalase (p = 0.031) and glutathione peroxidase (p = 0.048) in lungs, increased levels of nitric oxide (NOx) in serum (p = 0.001) and bronchoalveolar lavage fluid (p = 0.047), and decreased kidney histological injury (p = 0.018) of CKD mice. However, AE also increased oxidative damage (p = 0.003) and did not change collagen content or perivascular edema in lungs (p > 0.05) of CKD mice. Therefore, AE attenuated kidney injury and improved antioxidants defenses in lungs. Despite no significantchanges in pulmonary damage, AE significantly improvedphysical performance in CKD mice.

MO554: Roxadustat Improves Renal Osteodystrophy by Dual Regulating Bone Remodeling

Abstract BACKGROUND AND AIMS Renal osteodystrophy (ROD) is a complication of chronic kidney disease (CKD) resulting in increased susceptibility to fractures. Therefore, developing effective drugs to prevent and treat ROD is vital. Accumulating evidence suggested stabilization of hypoxia-inducible factor-1α (HIF-1α) could promote bone formation. Roxadustat is a novel HIF stabilizer to treat renal anemia in CKD patients. This study aims to investigate the effects of roxadustat on bone remodeling using a CKD rat model. METHOD A total of 24 8-week-old male Sprague–Dawley rats were divided into three groups (n = 8/group): the normal group (Normal), CKD group and CKD + Roxadustat group. For induction of CKD, the diet containing 0.5% (w/w) adenine was given to the rats for the first 4 weeks. Roxadustat (10 mg/kg, by gavage) was given every other day from the third week of CKD induction for 4 weeks. Then blood was collected to test hemoglobin (Hb) concentrations, serum levels of creatinine (Cr), calcium (Ca), phosphorus (P), intact parathyroid hormone (iPTH) and bone turnover markers including bone alkaline phosphatase (BAP), osteocalcin (OCN) and tartrate-resistant acid phosphatase 5b (TRACP-5b). Left femurs were subjected to micro-computed tomography (micro-CT) to evaluate bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp). Left tibiae were collected to calculate osteoclast surface/bone surface (Oc.S/BS), osteoclast number/bone surface (N.Oc/BS), osteoblast surface/bone surface (Ob.S/BS) and osteoblast number/bone surface (N.Ob/BS). One-way analysis of variance followed by Tukey HSD post hoc test was used for comparisons. P &amp;lt; 0.05 was statistically significant. RESULTS CKD rats exhibited remarkably higher serum Cr, P, iPTH, TRACP-5b levels and obviously lower Hb and serum Ca levels compared with the normal group. Whereas roxadustat treatment significantly decreased iPTH, TRACP-5b and increased Hb, BAP and OCN levels in CKD rats. CKD induced remarkably decreased BMD, BV/TV, Tb.N, Tb.Th and dramatically increased Tb.Sp compared with the normal group. However, roxadustat treatment improved these changes in CKD rats. CKD rats had increased Oc.S/BS, N.Oc/BS, Ob.S/BS and N.Ob/BS compared with the normal group. Roxadustat decreased Oc.S/BS, N.Oc/BS but further increased Ob.S/BS and N.Ob/BS in CKD rats. CONCLUSION Roxadustat alleviated bone loss and bone microstructure deterioration of CKD rats by promoting bone formation and inhibiting bone resorption.

MO587: Chronic Kidney Disease Induces Endotoxin-Related Activation of The Innate Immune System

Abstract BACKGROUND AND AIMS Chronic inflammation is common in patients with chronic kidney disease (CKD) and is associated with poor outcomes. Although the etiology is only partly understood, the gut–kidney axis is considered an important contributor [1]. Lipopolysaccharide (LPS), also known as endotoxin, is a well-characterized pyrogen found in the exterior cell membrane of most of the gram-negative bacteria. It plays an important role in promoting intestinal inflammatory responses. When absorbed through the intestinal epithelium, LPS induces inflammation by activating macrophages and monocytes. Due to short half-life and semi-quantitative assay characteristics of the Limulus Amebocyte Lysate (LAL) assay, direct quantification of LPS is not suitable to quantify activation of the innate immune system. Lipoprotein-binding protein (LBP), a more stable biomarker, is an acute phase protein produced mostly by the hepatocytes and intestinal epithelial cells and is an essential component of an effective innate immune response to LPS. Circulating LBP significantly enhances the sensitivity of CD14 + cells (mostly monocytes and macrophages) to stimulation by LPS. LBP has been shown to facilitate binding of LPS to CD14 receptor. Levels of LBP peak in serum shortly after endotoxemia and remain increased up to 72 h later. An increased plasma LBP indicates gut epithelial barrier dysfunction [2]. Whether LBP is altered by CKD is not known. METHOD We analyzed the effects of CKD on LBP plasma concentrations. We used samples from the Leuven mild-to-moderate CKD cohort (NCT00441623). To study causality, we used animal models of experimentally induced CKD. To exclude model-related bias, two different rat models of CKD, i.e. 5/6th nephrectomy and adenine supplementation, were used. LBP was measured using commercially available ELISA kits (Hycultbiotech, The Netherlands). RESULTS In a cohort of 460 patients with CKD, we found a significant increase in LBP levels across the different stages of CKD (ANOVA, P: 0.001). When analyzed as a continuous variable, LBP is significantly inversely correlated with eGFR (P &amp;lt; 0.001; Spearman, r: −0.221), and positively correlated with CRP (P &amp;lt; 0.001; Spearman, r: 0.592). During a median follow-up of 56 (IQR, 53–59) months, 70 patients died, with more deaths observed among patients with LBP in higher tertiles [tertiles 1–3: 11, 22, and 37 events, respectively (Fig. 1)]. In univariate Cox proportional hazard analysis, plasma LBP was significantly associated with mortality [hazard ratio (HR) of 1.032; 95% confidence interval (95% CI): 1.014– 1.046; P &amp;lt; 0.001). This association remained significant in multivariate models with adjustment for age, sex and BMI. In two different rat models, induction of CKD resulted in a significant increase in LBP (P &amp;lt; 0.001). In these animals, we observed a significant inverse association between eGFR (and measured creatinine clearance) and LBP concentrations (P &amp;lt; 0.001; Spearman, r: −0.460). CONCLUSION Patients with CKD have higher levels of LBP, with higher levels of plasma LBP present in patients with more advanced CKD. Plasma LBP is an independent predictor of mortality. Experimentally induced CKD equally results in significantly increased LBP. Our data suggest that CKD leads to increased passage of LPS across the intestinal barrier. These findings strengthen the hypothesis of the gut–kidney axis as a source of chronic inflammation.

MicroRNAs are differentially expressed in the serum and renal tissues of cats with experimentally induced chronic kidney disease: a preliminary study.

To identify differentially expressed microRNA in the serum and renal tissues of cats with experimentally induced chronic kidney disease (CKD). Banked renal tissues and serum from 4 cats. Cats previously underwent 90-minute unilateral ischemia with delayed contralateral nephrectomy 3 months after ischemia. Tissues were collected from the contralateral kidney at the time of nephrectomy and from the ischemic kidney 6 months after nephrectomy (study end). Serum was collected prior to ischemia (baseline serum) and at study end (end point serum). Total RNA was isolated from tissues and serum, and microRNA sequencing was performed with differential expression analysis between the contralateral and ischemic kidney and baseline and end point serum. 20 microRNAs were differentially expressed between ischemic and contralateral kidneys, and 52 microRNAs were differentially expressed between end point and baseline serum. Five microRNAs were mutually differentially expressed between ischemic and contralateral kidneys and baseline and end point serum, with 4 (mir-21, mir-146, mir-199, and mir-235) having increased expression in both the ischemic kidney and end point serum and 1 (mir-382) having increased expression in the ischemic kidney and decreased expression in end point serum. Predicted target search for these microRNA revealed multiple genes previously shown to be involved in the pathogenesis of feline CKD, including hypoxia-inducible factor-1α, transforming growth factor-β, hepatocyte growth factor, fibronectin, and vascular endothelial growth factor A. MicroRNAs were differentially expressed after CKD induction in this preliminary study. Regulation of renal fibrosis in feline CKD may occur through microRNA regulation of mRNAs of pro- and anti-fibrotic genes.

Treatment of Chronic Kidney Disease with Extracellular Vesicles from Mesenchymal Stem Cells and CD133+ Expanded Cells: A Comparative Preclinical Analysis.

Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133+ cells (eCD133+) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133+ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133+ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.

Abstract 87: Chronic Kidney Disease Induces Cerebral Microhemorrhages In Aged Mice

Introduction: Chronic kidney disease (CKD) is increasingly recognized as a risk factor of cerebral microvascular disease, but its association with common neuropathologic changes is not well understood. We investigated the relationship between CKD and development of cerebral microhemorrhages (CMH) in a mouse model of aging. We also examined the effect of CKD on endothelial cell function in an in vitro blood-brain barrier (BBB) model. Methods: CKD was produced in aged C57BL/6J mice using an adenine-induced tubulointerstitial nephritis model. We performed standard histology using Prussian blue staining to examine CMH formation. Correlations between CMH burden and serum creatinine levels were assessed. In cell culture studies, human brain microvascular endothelial cells (ihBMECs) derived from induced pluripotent stem cell line IMR90-4 were treated with serum from healthy or CKD patient donors for up to 3 days. Transendothelial electrical resistance (TEER) and tracer (sodium fluorescein) permeability across the ihBMEC monolayer were measured to assess the integrity of the in vitro BBB. Results: CKD induction in aged C57BL/6J mice caused a significant increase in both serum creatinine level (0.09±0.01 mg/dL to 0.43±0.03 mg/dL, p&lt;0.001) and CMH number (p&lt;0.05), without alteration of systolic or diastolic blood pressure. Number of CMH was positively correlated with serum creatinine level (r=0.41, p&lt;0.001). Incubation of CKD serum with ihBMECs significantly reduced TEER by 26% (p&lt;0.05) and increased sodium fluorescein permeability by 100% (p&lt;0.01) across the ihBMEC monolayer. Conclusions: Adenine-induced CKD promotes the development of CMH in aged C57BL/6J mice independent of blood pressure, and extent of CMH development is directly proportional to degree of renal insufficiency. Moreover, serum-derived factors in CKD disrupt BBB integrity in vitro. These findings suggest CKD provokes microvascular injury at the capillary level, leading to CMH formation in this model.

Influences of renal insufficiency and ischemia on mitochondrial bioenergetics and limb dysfunction in a novel murine iliac arteriovenous fistula model

ObjectiveHand disability after hemodialysis access surgery has been common yet has remained poorly understood. Arteriovenous fistula (AVF) hemodynamic perturbations have not reliably correlated with the observed measures of hand function. Chronic kidney disease (CKD) is known to precipitate myopathy; however, the interactive influences of renal insufficiency and ischemia on limb outcomes have remained unknown. We hypothesized that CKD would contribute to access-related hand dysfunction via altered mitochondrial bioenergetics. Using a novel murine AVF model, we sought to characterize the skeletal muscle outcomes in mice with and without renal insufficiency. MethodsMale, 8-week-old C57BL/6J mice were fed either an adenine-supplemented diet to induce renal insufficiency (CKD) or a casein-based control chow (CON). After 2 weeks of dietary intervention, the mice were randomly assigned to undergo iliac AVF surgery (n = 12/group) or a sham operation (n = 5/group). Measurements of aortoiliac hemodynamics, hindlimb perfusion, and hindlimb motor function were collected for 2 weeks. The mice were sacrificed on postoperative day 14 to assess skeletal muscle histopathologic features and mitochondrial function. To assess the late outcome trends, 20 additional mice had undergone CKD induction and sham (n = 5) or AVF (n = 15) surgery and followed up for 6 weeks postoperatively before sacrifice. ResultsThe adenine-fed mice had had a significantly reduced glomerular filtration rate and elevated blood urea nitrogen, confirming the presence of CKD. The sham mice had a 100% survival rate and AVF cohorts an 82.1% survival rate with an 84.4% AVF patency rate. The aorta and inferior vena cava velocity measurements and the vessel diameter had increased after AVF creation (P < .0001 vs sham). The AVF groups had had a 78.4% deficit in paw perfusion compared with the contralateral limb after surgery (P < .0001 vs sham). Mitochondrial function was influenced by the presence of CKD. The respiratory capacity of the CKD-sham mice (8443 ± 1509 pmol/s/mg at maximal energy demand) was impaired compared with that of the CON-sham mice (12,870 ± 1203 pmol/s/mg; P = .0001). However, this difference was muted after AVF creation (CKD-AVF, 4478 ± 3685 pmol/s/mg; CON-AVF, 5407 ± 3582 pmol/s/mg; P = .198). The AVF cohorts had had impairments in grip strength (vs sham; P < .0001) and gait (vs sham; P = .012). However, the presence of CKD did not significantly alter the measurements of gross muscle function. The paw perfusion deficits had persisted 6 weeks postoperatively for the AVF mice (P < .0001 vs sham); however, the myopathy had resolved (grip strength, P = .092 vs sham; mitochondrial respiration, P = .108 vs sham). ConclusionsCKD and AVF-induced distal limb ischemia both impaired skeletal muscle mitochondrial function. Renal insufficiency was associated with a baseline myopathy that was exacerbated by the acute ischemic injury resulting from AVF creation. However, ischemia was the primary driver of the observed phenotype of gross motor impairment. This model reliably reproduced the local and systemic influences that contribute to access-related hand dysfunction and provides a platform for further mechanistic and therapeutic investigation.

Effect of curcumin on acute chronic kidney disease due to ischemia-reperfusion syndrome.

To evaluate the effect of curcumin on renal function, hemodynamics, and renal oxidative profile of rats with chronic kidney disease (CKD) subjected to renal ischemia-reperfusion injury (IRI). Wistar rats, 250-300 g, distributed in four groups: Sham (n = 5), CKD simulation; CKD (n = 5), 5/6 renal ablation for CKD induction; CKD + IRI (n = 5), CKD and renal pedicle clamping for 30 minutes; and CKD + IRI+curcumin (n = 5), CKD + IRI, curcumin administration 30 mg/kg/day, orally, for 10 days. Renal function (inulin clearance, urine flow, plasma creatinine), hemodynamics (blood pressure), and oxidative profile (peroxides, TBARS, and urine nitrate, non-protein soluble thiols in renal tissue) were evaluated. The CKD + IRI + curcumin group showed increased inulin clearance and reduced plasma creatinine, decreased RVR and increased RBF, decreased oxidative metabolites in urine and increased thiols in renal tissue when compared with the CKD + IRI group. The treatment with curcumin preserved renal function and hemodynamics of animals with acute CKD, improving oxidative profile, with reduction of oxidants and preservation of antioxidant reserve.

A systematic review and meta-analysis of murine models of uremic cardiomyopathy

Chronic kidney disease (CKD) triggers the risk of developing uremic cardiomyopathy as characterized by cardiac hypertrophy, fibrosis and functional impairment. Traditionally, animal studies are used to reveal the underlying pathological mechanism, although variable CKD models, mouse strains and readouts may reveal diverse results. Here, we systematically reviewed 88 studies and performed meta-analyses of 52 to support finding suitable animal models for future experimental studies on pathological kidney-heart crosstalk during uremic cardiomyopathy. We compared different mouse strains and the direct effect of CKD on cardiac hypertrophy, fibrosis and cardiac function in "single hit" strategies as well as cardiac effects of kidney injury combined with additional cardiovascular risk factors in "multifactorial hit" strategies. In C57BL/6 mice, CKD was associated with a mild increase in cardiac hypertrophy and fibrosis and marginal systolic dysfunction. Studies revealed high variability in results, especially regarding hypertrophy and systolic function. Cardiac hypertrophy in CKD was more consistently observed in 129/Sv mice, which express two instead of one renin gene and more consistently develop increased blood pressure upon CKD induction. Overall, "multifactorial hit" models more consistently induced cardiac hypertrophy and fibrosis compared to "single hit" kidney injury models. Thus, genetic factors and additional cardiovascular risk factors can "prime" for susceptibility to organ damage, with increased blood pressure, cardiac hypertrophy and early cardiac fibrosis more consistently observed in 129/Sv compared to C57BL/6 strains.

The Effect of Chronic Kidney Disease Induction Upon Affective Factors on Muscle Apoptosis in Rats and the Role of Aerobic Training in Its Modulation

Background and Objectives Chronic Kidney Disease (CKD) is characterized by progressive and irreversible loss of renal function. One of the complications of this disease is muscle wasting and atrophy. The aim of this study was to determine the effect of exercise training on CKD-induced muscle apoptosis in rats. Subjects and Methods In the present developmental study, 30 rats were selected and divided into 3 groups: disease training, disease control and healthy controls. Induction of CKD was through primary and final 1.3 resection of one kidney and complete resection of the other kidney. The training program consisted of eight weeks of continuous running training with an intensity of 45-55% VO2max. After removal of EDL muscle, protein level and Bcl-2 and Bax gene expression were measured by immunohistochemistry and real-time PCR, respectively. Results Induction of CKD caused a significant increase in protein level and gene expression of Bax and Bax/Bcl-2 and a significant decrease in Bcl-2 compared to the healthy control group (P&lt;0.001). Also significant decrease was funded in the expression of genes and proteins Bax and Bax/Bcl-2 (P&lt;0.001) and a significant increase was funded in protein Bcl-2 (P&lt;0.001), without significant changes in the gene expression of Bcl-2 (P=0.935) in disease training compared to the disease control. Conclusion The results of the present study support the anti-apoptotic effect of exercise on muscle tissue of CKD patients; therefore, it is recommended that clinical research be conducted in this regard for exercise recommendations for these patients.

Chronic Kidney Disease-Induced Arterial Media Calcification in Rats Prevented by Tissue Non-Specific Alkaline Phosphatase Substrate Supplementation Rather Than Inhibition of the Enzyme.

Patients with chronic kidney disease (CKD) suffer from arterial media calcification and a disturbed bone metabolism. Tissue-nonspecific alkaline phosphatase (TNAP) hydrolyzes the calcification inhibitor pyrophosphate (PPi) into inorganic phosphate (Pi) and thereby stimulates arterial media calcification as well as physiological bone mineralization. This study investigates whether the TNAP inhibitor SBI-425, PPi or the combination of both inhibit arterial media calcification in an 0.75% adenine rat model of CKD. Treatments started with the induction of CKD, including (i) rats with normal renal function (control diet) treated with vehicle and CKD rats treated with either (ii) vehicle, (iii) 10 mg/kg/day SBI-425, (iv) 120 µmol/kg/day PPi and (v) 120 µmol/kg/day PPi and 10 mg/kg/day SBI-425. All CKD groups developed a stable chronic renal failure reflected by hyperphosphatemia, hypocalcemia and high serum creatinine levels. CKD induced arterial media calcification and bone metabolic defects. All treatments, except for SBI-425 alone, blocked CKD-related arterial media calcification. More important, SBI-425 alone and in combination with PPi increased osteoid area pointing to a less efficient bone mineralization. Clearly, potential side effects on bone mineralization will need to be assessed in any clinical trial aimed at modifying the Pi/PPi ratio in CKD patients who already suffer from a compromised bone status.

METABOLIC EFFECT OF A MINERALOCORTICOID RECEPTOR ANTAGONIST IN A NEW MODEL OF DIABETIC NEPHROPATHY

Objective: Diabetic nephropathy (DN) is the most prevalent form of chronic kidney disease (CKD) and is associated with cardiovascular diseases. Several studies reported beneficial effects of mineralocorticoid receptor (MR) antagonists in DN, indicating the importance of aldosterone/MR axis in this pathology We investigated the metabolic alterations associated to a new model of DN and explored the effect of the MR antagonist canrenoate and conditional MR knock-down Design and method: One month after CKD induction by 5/6 nephrectomy, mice were split in 4 groups: SHAM, CKD, CKD with 60% fat diet (CKD-HFD) and CKD-HFD treated with 30 mg/kg/day canrenoate (CKD-HFD-CAN). Another series was split in 2 groups CKD-HFD Ctrl and CKD-HFD-AdipoMRKO (in which the MR gene is deleted in adipocytes only). Glycated hemoglobin (HbA1c) was measured and glucose tolerance test (ipGTT) and insulin tolerance test (ipITT) were performed between 5 and 7 weeks after the starting of the diet/treatment. The whole body fat content was determined by MRI (Brucker). The comparisons were performed by one-way ANOVA and two-way ANOVA for the insulin and glucose tolerance tests. Results were considered significant with p &lt; 0.05. Results: The body weight, the fat content, the epididymal visceral adipose tissue (EVAT) size and HbAc1 were significantly higher in the CKD-HFD group vs SHAM or CKD, an effect prevented by the treatment with canrenoate. The glucose tolerance and the response to insulin were impaired in CKD-HFD vs SHAM or CKD, an effect ameliorated by canrenoate (Table). The impact on body weight, fat content and EVAT size was similar in CKD-HFD-adipoMRKO mice when compared to CKD-HFD Ctrl mice, however the improvement of the glucose tolerance was milder (Table). Conclusions: MR antagonism improves metabolic alterations associated to DN, partially due to its effect on adipose tissue, which could be translated in a better prognosis of the disease