CKD Mice Research Articles

Etelcalcetide ameliorates bone loss in chronic kidney disease-mineral and bone disorder by activation of IRF7 and necroptosis pathways

Chronic kidney disease-mineral and bone disorder (CKD-MBD) is a multifaceted clinical syndrome characterized by mineral imbalances, abnormalities in bone metabolism, chronic inflammation and vascular calcification. Etelcalcetide, a second-generation intravenous calcimimetic agent, has been approved for treating high-turnover renal osteodystrophy, effectively targeting the pathophysiological mechanisms underlying this condition. We investigate the impacts of etelcalcetide on osteoclast (OC) differentiation and functionality in CKD-MBD via three critical mechanisms: inflammation initiated by interferon regulatory factor 7 (IRF7), receptor-interacting protein (RIP)-mediated necroptosis and apoptosis-induced cell death. The low-dose (CKD + L) or high-dose (CKD + H) of etelcalcetide groups significantly improved biochemical markers compared to the CKD control mice. Additionally, etelcalcetide-treated CKD mice significantly improved cortical and trabecular bone parameters. In an in vitro study, etelcalcetide was observed to bolster the IRF7-mediated IFNβ response in OC differentiation. Furthermore, it stimulated RIP-mediated necroptosis via RIP and MLKL activation, inhibiting bone resorption. Moreover, the drug increased levels of caspases 3 and 9, inducing cell death in OCs. These findings suggest that etelcalcetide regulates bone metabolism and reduces skeletal issues in CKD-MBD. Etelcalcetide likely enhances bone parameters in CKD-MBD mice by regulating IRF7 pathways and inhibiting OC differentiation. It also improves bone health and promotes RIP-mediated necroptosis and apoptosis pathways within OCs.

#911 A mice model of SHPT with bone abnormalities in CKD and potential functional role of sphingolipid in renal osteodystrophy

Abstract Background and Aims Renal osteodystrophy (ROD) is the skeletal disease of chronic kidney disease associated mineral and bone disorder (CKD-MBD). ROD is characterized by abnormal bone mineralization and impaired bone remodeling, leading to increased risk of fractures and mortality in patients with CKD. To date, the pathology of ROD remains poorly understood due to the lack of a suitable animal model. Hence, we successfully established a stable mouse model of ROD using an optimized adenine diet and explored the possible signaling based on RNA-seq expression analyses. Method Blood biochemical analysis, micro-computed tomography (micro-CT) and bone histomorphometry were performed to analyze the skeletal characteristics. RNA sequencing (RNA-seq) was used to investigate the mechanisms involved in ROD. The functions of differentially expressed genes (DEGs) were evaluated by Gene Ontology (GO) analyses, Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and Gene Set Enrichment Analyses (GSEA). DEGs were validated by quantitative real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results The mice with ROD induced by optimized adenine diet showed severe abnormalities in serum creatinine (Scr) and blood urea nitrogen (BUN) along with marked hyperparathyroidism and hyperphosphatemia. The bone mineral density (BMD) of femurs was significantly lower in the ROD mice than that in the control (CTL) mice. The trabecular and cortical bone parameters of femurs significantly showed bone loss and severe cortical porosity. ROD mice showed high bone turnover with increased osteoid, eroded, osteoblast, and osteoclast perimeters as well as increased bone formation rate and mineral apposition rate. Transcriptomic profiling identified 1286 genes were upregulated in the femurs of ROD mice compared to the CTL mice and 333 genes that were downregulated. GO, KEGG and GSEA pathway analyses indicated that these genes were highly enriched in functions related to lipid metabolism pathway, sphingolipid signaling pathway, among others. The differential expression of alkaline ceramidase 1(Acer1), ceramide synthase 4 (Cers4) sphingomyelin phosphodiesterase 3 (Smpd3), sphingosine-1-phosphate phosphatase 2 (Sgpp2) in skeletal tissue and serum of CKD patients were validated by qRT-PCR. Since sphingolipid pathways have been recently implicated in bone metabolism, these genes are potential target genes in the settings of ROD. Conclusion In conclusion, our findings demonstrated that the CKD mice with bone abnormalities induced by optimized adenine diet may serve as a useful model for skeletal analysis in ROD. Based on the RNA-seq, we identified that sphingolipid might play a crucial role in the pathogenesis of ROD and provided new therapeutic targets for future study.

Acute and Chronic Kidney Disease Worsen Outcomes in Experimental Sepsis.

Infection is a leading cause of morbidity in individuals with acute and chronic kidney disease (AKD, CKD). However, there is significant difficulty in modeling infection into an animal host with preexisting kidney disease. We report a novel method of peritoneal infection induced via cecal slurry inoculation deployed into mice with experimental aristolochic acid-induced AKD and CKD. AKD, CKD, and paired control mice were injected with sham, low, or higher doses of donor-recipient matched cecal slurry solution. Animal survival, sepsis severity, and change in glomerular filtration rate was tracked longitudinally throughout the study. Histology for kidney injury, flow cytometry, plasma cytokines, and evidence of indirect organ injury from sepsis were also assessed. Infected AKD mice experienced significantly heightened sepsis severity, with 100% mortality by 24 hours after high cecal slurry doses versus no mortality in control mice. Additionally, AKD mice receiving lower cecal slurry doses developed dramatically increased pro-inflammatory cytokines and persistent cytopenias. Infected CKD mice also had worse outcomes than paired CKD-controls, though less severe than in AKD mice. Interestingly, animals with AKD had worse outcomes than mice with CKD after any cecal slurry dose or time-point after inoculation, despite higher baseline kidney function and less uremic sequela. These data confirm that acute bacterial infection can be modeled in animals with established kidney disease; and suggest that the clinical state of kidney disease (AKD versus CKD) may influence host susceptibility to infection more than the degree of kidney failure alone.

Circulating miR-423-5p levels are associated with carotid atherosclerosis in patients with chronic kidney disease

Background and aimsCarotid atherosclerosis is associated with an elevated risk of stroke in patients with chronic kidney disease. However, the molecular basis for the incidence of carotid atherosclerosis in patients with CKD is poorly understood. Here, we investigated whether circulating miR-423-5p is a crucial link between CKD and carotid atherosclerosis. Methods and resultsWe recruited 375 participants for a cross-sectional study to examine the occurrence of carotid plaque and plaque thicknesses. Levels of miR-423-5p were determined by qPCR analysis. We found that non-dialysis CKD patients had higher circulating exosomal and plasma miR-423-5p levels, and dialysis-dependent patients had lower miR-423-5p levels than non-dialysis CKD patients. After excluding for the influence of dialysis patients, linear regression analysis indicated that levels of circulating miR-423-5p are negatively correlated with eGFR (P < 0.001). Higher plasma miR-423-5p levels were associated with the incidence and severity of carotid plaques. In parallel, we constructed a murine model of CKD with a 5/6 nephrectomy protocol and performed RNA sequencing studies of aortic tissues. Consistent with these findings in CKD patients, circulating exosomal miR-423-5p levels in CKD mice were elevated. Furthermore, our RNA-seq studies indicated that the putative target genes of miR-423-5p were related to oxidative stress functions for aorta of CKD mice. ConclusionLevels of miR-423-5p are associated with the presence and severity of carotid plaque in CKD. Data from our mouse model suggests that miR-423-5p likely influences gene expression programs related to oxidative stress in aorta of CKD mice.

Shen Qi Wan ameliorates nephritis in chronic kidney disease via AQP1 and DEFB1 regulation

Shen Qi Wan (SQW) has been proven to exert anti-inflammatory effects in the kidneys of CKD models accompanied by unclear therapeutic mechanisms. This study aims to evaluate the kidney-protective and anti-inflammatory effects of SQW and to elucidate its fundamental mechanisms for CKD treatment. Firstly, the main active components of SQW were identified by UPLC-Q-TOF/MS technique. Subsequently, we evaluated inflammatory factors, renal function and renal pathology changes following SQW treatment utilizing adenine-induced CKD mice and aquaporin 1 knockout (AQP1-/-) mice. Additionally, we conducted RNA-seq analysis and bioinformatics analysis to predict the SQW potential therapeutic targets and anti-nephritis pathways. Simultaneously, WGCNA analysis method and machine learning algorithms were used to perform a clinical prognostic analysis of potential biomarkers in CKD patients from the GEO database and validated through clinical samples. Lipopolysaccharide-induced HK-2 cells were further used to explore the mechanism. We found that renal collagen deposition was reduced, serum inflammatory cytokine levels decreased, and renal function was improved after SQW intervention. It can be inferred that β-defensin 1 (DEFB1) may be a pivotal target, as confirmed by serum and renal tissue samples from CKD patients. Furthermore, SQW assuages inflammatory responses by fostering AQP1-mediated DEFB1 expression was confirmed in in vitro and in vivo studies. Significantly, the renal-protective effect of SQW is to some extent attenuated after AQP1 gene knockout. SQW could reduce inflammatory responses by modulating AQP1 and DEFB1. These findings underscore the potential of SQW as a promising contender for novel prevention and treatment strategies within the ambit of CKD management.

Gut-Derived Uremic Toxins Correlate with Anxiety and Decreased Locomotor Activity in CKD Mice

Gut-Derived Uremic Toxins Correlate with Anxiety and Decreased Locomotor Activity in CKD Mice

Mitochondrial Aldehyde Dehydrogenase rs671 Single Nucleotide Polymorphism Increases the Susceptibility of Kidney Injury in Adenine-Induced CKD Mice

Mitochondrial Aldehyde Dehydrogenase rs671 Single Nucleotide Polymorphism Increases the Susceptibility of Kidney Injury in Adenine-Induced CKD Mice

#3454 IMPACT OF SYMBIOTICS ON UREMIC TOXINS PRODUCTION, RENAL FUNCTION AND PEW SYNDROME IN SHIME AND CKD MICE MODELS

Abstract Background and Aims Alterations in gut microbiota in CKD patients have been linked to CKD progression, cachexia, and mortality. Gut dysbiosis is associated by modification of gut-microbiota-derived metabolites, including a decrease of short-chain fatty acids (SCFAs), an increase in gut-derived uremic toxins. Therefore, modulation of gut microbiota seems to be an attractive therapeutic approach. In silico studies led to the selection of bacterial species able to increase SCFAs production, improve gut function without having the enzymatic machinery for producing uremic toxins. The propose of this study is to indentify the impact of two different symbiotics containing the selected bacteria: 1) on both uremic toxins and SCFAs production using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME) colonized with fresh feces from CKD or healthy controls (HC) and subsequently 2) on renal function, metabolic parameters, intestinal microbiota and gut-microbiota-derived metabolites in a mouse model of CKD (5/6 nephrectomy). Method 1) The SHIME® system manufactured by ProDigest (Ghent, Belgium) consists of systems comprising five double-wall biovessels simulating the stomach (ST), small intestine (SI), ascending colon (AC), transverse colon (TC), and descending colon (DC). The supernatant of the homogenized fecal slurry was inoculated into the AC (25 mL), TC (40 mL), and DC (30 mL), respectively. After fecal inoculation, SHIME system were fed in ST twice day with two different symbiotics (P1 or P2) for 10 days. After 7 days, we applied a diet amino acid (AA) challenge to all the groups. SCFAs and uremic toxins were quantified every day. 2) CKD mice received either P1 or P2 admix for 6 weeks. We measured food intake, body composition and renal function including renal fibrosis quantification. Plasma metabolomic and feces metagenomic analyses were performed. Results In vitro, the concentration of precursor of uremic toxins (indols and p-cresol) tended to be higher in DC colonized by feces from CKD patients and AA challenge increased uremic toxins production more significantly in CKD compared to HC. CKD microbiome showed a lower butyrate concentration at baseline and higher propionate and acetate production. Both P1 and P2 significantly limited uremic toxins production during AA challenge and increase SCFA production. In vivo, both symbiotics increased body weight (+14%) and food intake (+31%) compared to CKD mice on chow diet. Treatment did not impact lipid or glucose metabolism but fat accretion in epididymal was restored with P1. Both diets, but particularly P1, significantly reduced plasmatic urea, proteinuria, and kidney fibrosis. Symbiotics improved intestinal barrier with a greater expression of Occludin in the ileum. Metagenomic analyses of the gut microbiota indicated that the alfa-diversity was not different across groups while the beta-diversity was similar to the sham group if CKD mice were treated with symbiotics. Both symbiotics reduced significantly plasma levels of deleterious uremic toxins such as p-cresyl sulfate and indoxyl sulfate and symbiotics reduce fecal module of trypotophan degradation and other modules related to AA metabolism. Both diets increased fecal bacteria modules related to SCFAs production. Conclusion We have shown both in vitro and in vivo that a symbiotic association can reduce uremic toxins production and increase SCFA production by inducing a significant change in gut microbiota composition and function. These metabolic changes are associated with improved appetite, decreased uremic cachexia, and preserved renal function. This data needs be confirmed in a human clinical trial.

#6741 TRANSCRIPTION FACTOR ATF3 EXACERBATES RENAL FIBROSIS THROUGH RECRUITING HISTONE ACETYLASES

Abstract Background and Aims Chronic kidney disease (CKD) threatens public health around the world. Renal fibrosis is a final common pathology in renal diseases of different etiologies. However, no efficient, broadly applicable anti-fibrotic therapies exist. Histone acetylation is an epigenetic modification involved in the regulation of multiple disease-specific gene expressions, balanced by histone acetyltransferases and histone deacetylase (HDAC) enzymes. Transcription factors (TF) could recruit histone-modifying enzymes and alter gene expression. But now, much remains unknown about the role of histone acetylation and whether there exits the regulator of it in fibrosis of CKD. Here, we for the first time explored a novel aspect of ATF3 promoting kidney fibrosis via recruiting the histone acetylases to fibrosis-related genes in CKD. Method We constructed a CKD mouse model by using adenine and oxonate. Serum were taken for the measurement of BUN and creatinine. Kidneys were harvested and processed for histological, sequencing, qRT-PCR, and western blotting analysis. RNA-seq and ChIP-seq (antibody: H3K27ac) of mouse kidneys were conducted for profiling the differential gene expression and H3K27ac. GO and KEGG analysis was performed for annotation of the function. Motif analysis was applied for searching the regulator of H3K27ac. Biopsy samples of patients with common CKD were used for immunohistochemical analysis. ATF3-KO mice were also conducted for defining the function of ATF3 in CKD. To profile the ATF3 binding site on chromatin, ChIP-seq of ATF3 in mice kidneys of normal control and CKD were also performed. Besides, the data contained ATF3 interact protein in databases (ChIP-atlas and BioGrid) and the results of protein interaction prediction were integrated for finding the protein-protein interaction of ATF3. TGF-β- stimulated TCMK-1 cell served as the cell model for further mechanism validation. CO-IP in cells clarifies that ATF3 could recruit the histone acetyltransferases. ChIP-qPCR of the fibrosis gene was performed to further verify the mechanism of ATF3-regulated gene expression. Results Firstly, compared with the normal control, histone modification H3K27ac repertoire was changed in chronic kidney disease. 11859 H3K27ac peaks specific to CKD, and 14996 H3K27ac peaks specific to healthy mice kidneys were found. The binding intensity of these regions has altered dramatically in the kidney of CKD. Pearson correlation analysis and function annotation showed that CKD-specific changes of H3K27ac were associated with fibrosis gene expression. Motif analysis showed that ATF3 in the top 10 TFs that may interact with H3K27ac. Among those TFs, ATF3 elicited the highest expression. Besides, ATF3 could co-localize with H3K27ac along the mice genome globally. By immunohistochemical staining analysis, we observed the upregulation of ATF3 in the kidneys of patients with biopsy-proven CKD. We found that knocking out the ATF3 could significantly reduce kidney damage in CKD mice. In parallel with improved morphological injuries, the level of kidney fibrosis gene expression was also significantly reduced in ATF3–/– mice. Furthermore, the H3K27ac on the mice chromatin altered along with the knockout of ATF3, and several fibrosis-related gene expressions which elevated in CKD were decreased by deficiency of ATF3. On the region of gene cd2, H3K27ac disappeared with the knockout of ATF3 in CKD. We found that ATF3 could interact with histone acetyltransferases (CBP, KAT7, P300) to promote the H3K27ac. Further evidenced by ChIP-qPCR of fibrosis gene cd2, we found that ATF3 could recruit the histone acetyltransferases on the fibrosis gene to promote the H3K27ac to provoke the gene expression in the cell model. Conclusion We profiled the landscape of H3K27ac in CKD, explored the epigenetic regulator of H3K27ac, and revealed that ATF3 promotes renal fibrosis in CKD. Notably, we for the first time explored a novel mechanism of ATF3 about recruiting histone acetyltransferases to regulate the H3K27ac on fibrosis gene. Our data highlighted that ATF3 might represent a potential therapeutic target against fibrotic kidney diseases.

Renal function is a major predictor of circulating acyl-CoA-binding protein/diazepam-binding inhibitor.

Acyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has lately been described as an endocrine factor affecting food intake and lipid metabolism. ACBP is dysregulated in catabolic/malnutrition states like sepsis or systemic inflammation. However, regulation of ACBP has not been investigated in conditions with impaired kidney function, so far. Serum ACBP concentrations were investigated by enzyme-linked immunosorbent assay i) in a cohort of 60 individuals with kidney failure (KF) on chronic haemodialysis and compared to 60 individuals with a preserved kidney function; and ii) in a human model of acute kidney dysfunction (AKD). In addition, mACBP mRNA expression was assessed in two CKD mouse models and in two distinct groups of non-CKD mice. Further, mRNA expression of mACBP was measured in vitro in isolated, differentiated mouse adipocytes - brown and white - after exposure to the uremic agent indoxyl sulfate. Median [interquartile range] serum ACBP was almost 20-fold increased in KF (514.0 [339.3] µg/l) compared to subjects without KF (26.1 [39.1] µg/l) (p<0.001). eGFR was the most important, inverse predictor of circulating ACBP in multivariate analysis (standardized β=-0.839; p<0.001). Furthermore, AKD increased ACBP concentrations almost 3-fold (p<0.001). Increased ACBP levels were not caused by augmented mACBP mRNA expression in different tissues of CKD mice in vivo or in indoxyl sulfate-treated adipocytes in vitro. Circulating ACBP inversely associates with renal function, most likely through renal retention of the cytokine. Future studies need to investigate ACBP physiology in malnutrition-related disease states, such as CKD, and to adjust for markers of renal function.

Involvement of Dmp1 in the Precise Regulation of Hair Bundle Formation in the Developing Cochlea.

Dentin matrix protein 1 (Dmp1) is a highly phosphorylated, extracellular matrix protein that is extensively expressed in bone and teeth but also found in soft tissues, including brain and muscle. However, the functions of Dmp1 in the mice cochlea are unknown. Our study showed that Dmp1 was expressed in auditory hair cells (HCs), with the role of Dmp1 in those cells identified using Dmp1 cKD mice. Immunostaining and scanning electron microscopy of the cochlea at P1 revealed that Dmp1 deficiency in mice resulted in an abnormal stereociliary bundle morphology and the mispositioning of the kinocilium. The following experiments further demonstrated that the cell-intrinsic polarity of HCs was affected without apparent effect on the tissue planer polarity, based on the observation that the asymmetric distribution of Vangl2 was unchanged whereas the Gαi3 expression domain was enlarged and Par6b expression was slightly altered. Then, the possible molecular mechanisms of Dmp1 involvement in inner ear development were explored via RNA-seq analysis. The study suggested that the Fgf23-Klotho endocrine axis may play a novel role in the inner ear and Dmp1 may regulate the kinocilium-stereocilia interaction via Fgf23-Klotho signaling. Together, our results proved the critical role of Dmp1 in the precise regulation of hair bundle morphogenesis in the early development of HCs.

Serum and Urinary Soluble α-Klotho as Markers of Kidney and Vascular Impairment.

This study was designed to investigate the controversy on the potential role of sKlotho as an early biomarker in Chronic Kidney Disease-Mineral Bone Disorder (CKD-MBD), to assess whether sKlotho is a reliable marker of kidney α-Klotho, to deepen the effects of sKlotho on vascular smooth muscle cells (VSMCs) osteogenic differentiation and to evaluate the role of autophagy in this process. Experimental studies were conducted in CKD mice fed a normal phosphorus (CKD+NP) or high phosphorus (CKD+HP) diet for 14 weeks. The patients' study was performed in CKD stages 2-5 and in vitro studies which used VSMCs exposed to non-calcifying medium or calcifying medium with or without sKlotho. The CKD experimental model showed that the CKD+HP group reached the highest serum PTH, P and FGF23 levels, but the lowest serum and urinary sKlotho levels. In addition, a positive correlation between serum sKlotho and kidney α-Klotho was found. CKD mice showed aortic osteogenic differentiation, together with increased autophagy. The human CKD study showed that the decline in serum sKlotho is previous to the rise in FGF23. In addition, both serum sKlotho and FGF23 levels correlated with kidney function. Finally, in VSMCs, the addition of sKlotho prevented osteogenic differentiation and induced autophagy. It can be concluded that serum sKlotho was the earliest CKD-MBD biomarker, a reliable indicator of kidney α-Klotho and that might protect against osteogenic differentiation by increasing autophagy. Nevertheless, further studies are needed to investigate the mechanisms of this possible protective effect.

Chronic kidney disease promotes cerebral microhemorrhage formation

BackgroundChronic kidney disease (CKD) is increasingly recognized as a stroke risk factor, but its exact relationship with cerebrovascular disease is not well-understood. We investigated the development of cerebral small vessel disease using in vivo and in vitro models of CKD.MethodsCKD was produced in aged C57BL/6J mice using an adenine-induced tubulointerstitial nephritis model. We analyzed brain histology using Prussian blue staining to examine formation of cerebral microhemorrhage (CMH), the hemorrhagic component of small vessel disease and the neuropathological substrate of MRI-demonstrable cerebral microbleeds. In cell culture studies, we examined effects of serum from healthy or CKD patients and gut-derived uremic toxins on brain microvascular endothelial barrier.ResultsCKD was induced in aged C57BL/6J mice with significant increases in both serum creatinine and cystatin C levels (p < 0.0001) without elevation of systolic or diastolic blood pressure. CMH was significantly increased and positively correlated with serum creatinine level (Spearman r = 0.37, p < 0.01). Moreover, CKD significantly increased Iba-1-positive immunoreactivity by 51% (p < 0.001), induced a phenotypic switch from resting to activated microglia, and enhanced fibrinogen extravasation across the blood–brain barrier (BBB) by 34% (p < 0.05). On analysis stratified by sex, the increase in CMH number was more pronounced in male mice and this correlated with greater creatinine elevation in male compared with female mice. Microglial depletion with PLX3397 diet significantly decreased CMH formation in CKD mice without affecting serum creatinine levels. Incubation of CKD serum significantly reduced transendothelial electrical resistance (TEER) (p < 0.01) and increased sodium fluorescein permeability (p < 0.05) across the endothelial monolayer. Uremic toxins (i.e., indoxyl sulfate, p-cresyl sulfate, and trimethylamine-N-oxide) in combination with urea and lipopolysaccharide induced a marked drop in TEER compared with the control group (p < 0.0001).ConclusionsCKD promotes the development of CMH in aged mice independent of blood pressure but directly proportional to the degree of renal impairment. These effects of CKD are likely mediated in part by microglia and are associated with BBB impairment. The latter is likely related to gut-derived bacteria-dependent toxins classically associated with CKD. Overall, these findings demonstrate an important role of CKD in the development of cerebral small vessel disease.

Sulfasalazine Prevents Arteriovenous Fistula Failure and Inhibits Endothelial-Mesenchymal Transition

INTRODUCTION: Arteriovenous fistulae (AVF) are the preferred vascular access for hemodialysis; however, up to 60% fail within one year due to aggressive neointimal hyperplasia that is characterized by inflammation. Sulfasalazine is an anti-inflammatory drug that is used to treat inflammatory diseases. We hypothesized that sulfasalazine suppresses neointimal hyperplasia by inhibition of TGF-β signaling. METHODS: Aortocaval AVF were created in C57BL/6J mice, with control mice having sham procedures. CKD was created via 5/6 nephrectomy (Nx), with control mice having no (0/6) nephrectomy, three weeks prior to AVF operation. CKD mice with AVF were divided into two groups with or without sulfasalazine treatment. AVF diameter was determined using ultrasound at postoperative days 7, 14, and 21. AVF were harvested at days 7 or 21 and examined with histology, Immunofluorescence and Western blot. RESULTS: AVF+5/6Nx had significantly increased wall thickness (p < 0.005) and increased TGF-β1 (p = 0.0217) compared to AVF+0/6Nx (day 21; n = 6; t-test). Endothelial cells in AVF+5/6Nx had significantly increased immunoreactivity of the mesenchymal markers notch3 (p < 0.005), vimentin (p < 0.005) and FSP-1 (p = 0.0189; day 21; n = 6; t-test). The sulfasalazine treatment group had significantly decreased FSP-1 (p < 0.005), vimentin (p < 0.005); notch3 (p < 0.005; day 21; n = 6; t-test), consistent with reduced EndMT, as well as decreased wall thickness (p < 0.005;) and increased diameter (p < 0.05; t-test; day 21; n = 6-15). CONCLUSION: CKD promotes neointimal hyperplasia via EndMT during AVF maturation leading to AVF failure; Sulfasalazine could be a therapeutic drug to improve AVF maturation.

Critical Role of Osteopontin in Maintaining Urinary Phosphate Solubility in CKD.

Nephron loss dramatically increases tubular phosphate to concentrations that exceed supersaturation. Osteopontin (OPN) is a matricellular protein that enhances mineral solubility in solution; however, the role of OPN in maintaining urinary phosphate solubility in CKD remains undefined. Here, we examined (1) the expression patterns and timing of kidney/urine OPN changes in CKD mice, (2) if tubular injury is necessary for kidney OPN expression in CKD, (3) how OPN deletion alters kidney mineral deposition in CKD mice, (4) how neutralization of the mineral-binding (ASARM) motif of OPN alters kidney mineral deposition in phosphaturic mice, and (5) the in vitro effect of phosphate-based nanocrystals on tubular epithelial cell OPN expression. Tubular OPN expression was dramatically increased in all studied CKD murine models. Kidney OPN gene expression and urinary OPN/Cr ratios increased before changes in traditional biochemical markers of kidney function. Moreover, a reduction of nephron numbers alone (by unilateral nephrectomy) was sufficient to induce OPN expression in residual nephrons and induction of CKD in OPN-null mice fed excess phosphate resulted in severe nephrocalcinosis. Neutralization of the ASARM motif of OPN in phosphaturic mice resulted in severe nephrocalcinosis that mimicked OPN-null CKD mice. Lastly, in vitro experiments revealed calcium-phosphate nanocrystals to induce OPN expression by tubular epithelial cells directly. Kidney OPN expression increases in early CKD and serves a critical role in maintaining tubular mineral solubility when tubular phosphate concentrations are exceedingly high, as in late-stage CKD. Calcium-phosphate nanocrystals may be a proximal stimulus for tubular OPN production.

Drp1 activates ROS/HIF-1α/EZH2 and triggers mitochondrial fragmentation to deteriorate hypercalcemia-associated neuronal injury in mouse model of chronic kidney disease

BackgroundChronic kidney disease (CKD), characterized as renal dysfunction, is regarded as a major public health problem which carries a high risk of cardiovascular diseases. The purpose of this study is to evaluate the functional significance of Drp1 in hypercalcemia-associated neuronal damage following CKD and the associated mechanism.MethodsInitially, the CKD mouse models were established. Next, RT-qPCR and Western blot analysis were performed to measure expression of Fis1 and Drp1 in CKD. Chromatin immunoprecipitation (ChIP) assay and dual-luciferase reporter gene assay were utilized to explore the relationship among Drp1, HIF-1α, EZH2, and ROS with primary cortical neurons isolated from neonatal mice. Next, CKD mice were subjected to calcitonin treatment or manipulation with adenovirus expressing sh-Drp1, so as to explore the effects of Drp1 on hypercalcemia-induced neuronal injury in CKD. TUNEL assay and immunofluorescence staining were performed to detect apoptosis and NeuN-positive cells (neurons) in prefrontal cortical tissues of CKD mice.ResultsIt was found that hypercalcemia could induce neuronal injury in CKD mice. An increase of Fis1 and Drp1 expression in cerebral cortex of CKD mice correlated with mitochondrial fragmentation. Calcitonin suppressed Drp1/Fis1-mediated mitochondrial fragmentation to attenuate hypercalcemia-induced neuronal injury after CKD. Additionally, Drp1 could increase EZH2 expression through the binding of HIF-1α to EZH2 promoter via elevating ROS generation. Furthermore, Drp1 knockdown inhibited hypercalcemia-induced neuronal injury in CKD while overexpression of EZH2 could reverse this effect in vivo.ConclusionTaken together, the key findings of the current study demonstrate the promotive role of Drp1 in mitochondrial fragmentation which contributes to hypercalcemia-induced neuronal injury in CKD.

Chrysophanol, a main anthraquinone from Rheum palmatum L. (rhubarb), protects against renal fibrosis by suppressing NKD2/NF-κB pathway

PurposeChronic kidney disease (CKD), characterized as renal dysfunction and multi-system damage, has become a serious public health problem with high prevalence and mortality. Rheum palmatum L. (rhubarb) is one of the most widely used Chinese herb with renal protective activity. However, the active components and underlying mechanisms of rhubarb remain unknown. In this work, we tried to explore the pharmacological mechanism of chrysophanol, a main anthraquinone from rhubarb, against CKD by in vivo and in vitro models. Study designThe therapeutic effect of chrysophanol and its underlying mechanism were investigated using CKD mouse model induced by unilateral ureteral occlusion (UUO), and human kidney 2 (HK-2) cells stimulated by TGF-β1 in vivo. MethodsThe impact of chrysophanol on renal function, inflammation, fibrosis of CKD mice were evaluated. Then, the protein expressions of FN1, collagen ɑI, α-SMA, NF-κB and naked keratinocyte homolog 2 (NKD2) were investigated. In vitro studies, the inhibition on inflammation and fibrogenesis by chrysophanol was further validated in TGF-β1-stimulated HK2 cells, and the regulation of chrysophanol on NKD2/NF-κB pathway was analyzed. Moreover, NKD2 was overexpressed in HK-2 cells to confirm the role of NKD2/NF-κB pathway in chrysophanol-mediated efficacy. Finally, the binding mode of chrysophanol with NKD2 was studied using in silico molecular docking and microscale thermophoresis (MST) assay. ResultsChrysophanol could significantly improve the kidney dysfunction, alleviate renal pathology, and reverse the elevated levels of renal fibrosis markers such as FN1, collagen ɑI and α-SMA. Furthermore, chrysophanol effectively inhibited TNF-α, IL-6, and IL-1β production, and suppressed NF-κB activation and NKD2 expression. The findings of in vitro study were consistent with those of animal expriment. Using NKD2-overexpressing HK-2 cells, we also demonstrated that overexpression of NKD2 significantly compromised the anti-fibrotic effects of chrysophanol. In addition, molecular docking and MST analysis revealed that NKD2 was a direct target of chrysophanol. ConclusionTogether, our work demonstrated for the first time that chrysophanol could effectively ameliorate renal fibrosis by inhibiting NKD2/NF-κB pathway. Chrysophanol can potentially prevent CKD by suppressing renal NKD2 expression directly.

Indoleamine 2,3-dioxygenase-1, a Novel Therapeutic Target for Post-Vascular Injury Thrombosis in CKD.

CKD, characterized by retained uremic solutes, is a strong and independent risk factor for thrombosis after vascular procedures . Urem ic solutes such as indoxyl sulfate (IS) and kynurenine (Kyn) mediate prothrombotic effect through tissue factor (TF). IS and Kyn biogenesis depends on multiple enzymes, with therapeutic implications unexplored. We examined the role of indoleamine 2,3-dioxygenase-1 (IDO-1), a rate-limiting enzyme of kynurenine biogenesis, in CKD-associated thrombosis after vascular injury. IDO-1 expression in mice and human vessels was examined. IDO-1-/- mice, IDO-1 inhibitors, an adenine-induced CKD, and carotid artery injury models were used. Both global IDO-1-/- CKD mice and IDO-1 inhibitor in wild-type CKD mice showed reduced blood Kyn levels, TF expression in their arteries, and thrombogenicity compared with respective controls. Several advanced IDO-1 inhibitors downregulated TF expression in primary human aortic vascular smooth muscle cells specifically in response to uremic serum. Further mechanistic probing of arteries from an IS-specific mouse model, and CKD mice, showed upregulation of IDO-1 protein, which was due to inhibition of its polyubiquitination and degradation by IS in vascular smooth muscle cells. In two cohorts of patients with advanced CKD, blood IDO-1 activity was significantly higher in sera of study participants who subsequently developed thrombosis after endovascular interventions or vascular surgery. Leveraging genetic and pharmacologic manipulation in experimental models and data from human studies implicate IS as an inducer of IDO-1 and a perpetuator of the thrombotic milieu and supports IDO-1 as an antithrombotic target in CKD.

MO461FGF19 IMPROVES GLUCOSE METABOLISM IN CKD MICE

Abstract Background and Aims Chronic kidney disease (CKD) is associated with high cardiovascular mortality rate, especially because of altered glucose metabolism and insulin resistance. Fibroblast growth factor 19 (FGF19), an intestinal postprandial hormone, has been shown to improve metabolic disturbances and insulin resistance. In CKD, postprandial secretion of FGF19 seems blunted in hemodialyzed patients but the role and regulation of FGF19 in this population is unknown. The aim of our study is to ascertain if FGF19 can improve glucose homeostasis in a mouse model of CKD induced by 5/6 subtotal nephrectomy. Method Four weeks post-surgery, CKD mice or sham mice were treated during 18 days with subcutaneous injections of placebo or human recombinant FGF19 (0.01mg/kg of body weight). The effect on metabolic disturbances was estimated in vivo by glucose tolerance test (GTT, 1g/kg/body weight). Results Fasted hyperglycemia was observed in CKD groups compared to sham mice (151.3 +/- 10.6mg/l and 170.7 +/- 10.9mg/L, p &amp;lt; 0.001 in sham and CKD mice, respectively) and FGF19 tended to improve fasting glycemia in CKD mice (158.9 +/- 18mg/L, p&amp;lt;0.09). The GTT area under the curve (AUC) was significantly decreased in CKD-FGF19 group compared to CKD placebo (P &amp;lt; 0.0001) and control groups (P = 0.0063). Urea blood level was significantly higher in CKD groups (9.6 +/- 1.4 mmol/L and 25.5 +/- 5.6 mmol/L in sham and CKD, respectively) and was not affected by FGF19 treatment (24.8 +/- 5.8 mmol/L, p = 0.91). Conclusion Our results show that FGF19 could contribute to improve glucose intolerance observed in CKD and suggest that this hormone could be a novel therapeutic target for reducing mortality associated with CKD. The comprehension of the molecular and cellular events through which FGF19 exerted its benefits needs however further studies.

Novel predictive biomarkers for acute injury superimposed on chronic kidney disease

Introduction and objectivesChronic kidney disease (CKD) is a risk factor for the development of acute kidney injury (AKI). Recent studies have revealed numerous biomarkers eligible for AKI prediction. However, the expression and performance of AKI biomarkers in acute injury superimposed on preexisting CKD (AonC) remain elusive. The aim of this study was to evaluate whether biomarkers which robustly expressed in acute kidney injury could predict acute injury based on CKD. Materials and methodsMice were classified into cohorts: AKI, CKD, AonC and sham. The AonC model mice were subjected to renal bilateral ischemia/reperfusion (I/R) injury fourteen days after intraperitoneally administrated with 20mg/kg aristolochic acid. Severity of acute ischemic injury was stratified by clamping the dissected bilateral renal arteries with non-traumatic microvascular clips for 20 or 35min. The AKI mice were induced with renal bilateral I/R injury and CKD mice were crafted with 20mg/kg aristolochic acid administrated intraperitoneally. Histology, genetic and protein expression of biomarkers were measured in three cohorts. ResultsWe found that serum creatinine dramatically increased in severe (sAonC) but not in moderate (mAonC) injury mice. Upregulation of Kidney injury molecule-1 (KIM-1) mRNA, tissue inhibitor of metalloproteinase-2 (TIMP-2), Syndecan-1 (SDC-1) mRNA and insulin-like growth factor binding protein-7 (IGFBP7) protein indicated the onset of mAonC. An increase in neutrophil gelatinase-associated lipocalin (NGAL), rhomboid-like protein 2 (RHBDL2), Syndecan-1 (SDC-1) mRNA and protein, and a decrease in IGFBP7 protein were associated with sAonC. ConclusionsOur study revealed the variational expression of AKI biomarkers in AonC kidneys, and uncovered IGFBP7 protein can be used as a sensitive biomarker to predict and differentiate AonC severity. The performance of RHBDL2 and SDC-1 in predicting severe AonC was promising, providing new biomarkers for predicting AonC.