Mouse Model Of Chronic Kidney Disease Research Articles

Hyperphosphatemia Contributes to Skeletal Muscle Atrophy in Chronic Kidney Disease

Alterations in mineral metabolism involve changes in serum levels of phosphate as well as its key regulators, fibroblast growth factor 23 (FGF23), vitamin D, parathyroid hormone and klotho. Chronic Kidney Disease (CKD) is a public health epidemic that affects an estimated 37 million Americans, and is associated with elevated serum levels of phosphate (hyperphosphatemia) and FGF23 as well as various pathologies, such vascular calcification, cardiac hypertrophy, and skeletal muscle atrophy. We previously found that elevated FGF23 directly targets cardiac myocytes and contributes to cardiac hypertrophy. Furthermore, elevated phosphate has direct effects on smooth muscle cells and induces vascular calcification. In order to determine whether FGF23 and/or phosphate might also contribute to CKD-associated skeletal muscle atrophy, we tested if skeletal muscle cells can respond to elevations of FGF23 or phosphate. Since the skeletal muscle phenotype in CKD is not well characterized, we analyzed skeletal muscle on a functional, histological, and molecular level in two established mouse models of CKD. We studied the C2C12 skeletal muscle cell line during the differentiation process from myoblasts to myotubes as well as fully differentiated myotubes. Myoblast differentiation was induced with horse serum; cells were co-treated with 25 ng/ml FGF23 or 5 mM phosphate for 5 days, followed by the expression analysis of differentiation markers by qPCR. In a separate study, differentiated C2C12 myotubes were treated with FGF23 or phosphate for 24 hours, followed by the qPCR analysis of atrophy genes (atrogenes), including MT1, Murf1, and Fbox32. Furthermore, we studied mice with global deletion of collagen 4a3 (Col4a3-/-) and wildtype littermates at 10 weeks of age, as well as C57Bl/6 mice receiving an adenine-rich (0.2%) diet or regular chow for 12 weeks. In all mice we studied grip strength, hindlimb circumference by MRI, cross-sectional area of individual muscle fibers that were immuno-labeled with anti-laminin by fluorescence microscopy, and expression levels of atrogenes by qPCR. Phosphate but not FGF23 affected the differentiation process of C2C12 myoblasts and differentiated C2C12 myotubes. Phosphate treatments significantly reduced expression levels of MyoD and MyoG and increased expression of MT1. In both mouse models, grip strength and areas of hindlimb and myotubes were significantly reduced, and expression levels of MT1 and Murf1 were significantly elevated when compared to their controls. FGF23 does not target skeletal muscle cells suggesting that it cannot directly contribute to skeletal muscle atrophy. Elevated phosphate impairs myoblast differentiation and induces atrophy in vitro. We have characterized two CKD mouse models with associated skeletal muscle atrophy, where we will now determine the contribution of hyperphosphatemia to the injury process. Pharmacological approaches targeting phosphate uptake or excretion, or phosphate's direct actions on tissues might alleviate various CKD-associated pathologies.

Tracking Acoustic and Electrophysiological Changes Associated with CKD-induced Cardiac and Valvular Remodeling in a Mouse Model

Objective: Patients with chronic kidney disease (CKD) are at a higher risk of developing heart and valvular diseases than the general population. Non-invasive screening methods are needed to identify cardiovascular abnormalities in this population. In this study, we use a diet-induced CKD mouse to study AV and cardiac remodeling. We hypothesize that structural differences in AV and cardiac remodeling occur in CKD mice, and can be identified through phonocardiogram (PCG) and electrocardiogram (ECG) signals. Methods: Eight-week old C57BL/6J mice were assigned to the following groups: 1) control group, fed a normal chow diet for 12 weeks, and 2) CKD group, fed a chow containing 0.2% adenine, with normal (0.6%) phosphorus for 6 weeks to induce CKD, and 3) CKD+high phosphate (HP) group, fed CKD inducing diet for 6 weeks, and then fed for 6 weeks with a 0.2% adenine/1.8% phosphorus diet to induce cardiovascular calcification. PCG and ECG signals were recorded weekly and echocardiogram parameters were recorded at the experimental end-point. Results: The HP diet induced aortic valve calcification in the CKD+HP group. CKD and CKD+HP group had increased end-diastolic volume, which is indicative of left ventricular hypertrophy. Changes in ECG parameters indicate that CKD groups have delayed cardiac repolarization time, an early ECG marker associated with arrhythmias. PCG signals indicate the presence of a systolic and diastolic murmur in the CKD+HP group, a characteristic of both valve disease and cardiac dysfunction. Conclusions: We demonstrate that the diet-induced CKD mouse model can be used to study valve and cardiac remodeling, and ECG and PCG signals can noninvasively track valve and cardiac changes. Future studies will determine the adequacy of using these signals to predict cardiovascular diseases.

Therapeutic Effect of Endothelin-Converting Enzyme Inhibitor on Chronic Kidney Disease through the Inhibition of Endoplasmic Reticulum Stress and the NLRP3 Inflammasome.

Chronic inflammation and oxidative stress significantly contribute to the development and progression of chronic kidney disease (CKD). The NOD-like receptor family pyrin containing domain-3 (NLRP3) inflammasome plays a key role in the inflammatory response. The renal endothelin (ET) system is activated in all cases of CKD. Furthermore, ET-1 promotes renal cellular injury, inflammation, fibrosis and proteinuria. Endothelin-converting enzymes (ECEs) facilitate the final processing step of ET synthesis. However, the roles of ECEs in CKD are not clear. In this study, we investigated the effects of ETs and ECEs on kidney cells. We found that ET-1 and ET-2 expression was significantly upregulated in the renal tissues of CKD patients. ET-1 and ET-2 showed no cytotoxicity on human kidney tubular epithelial cells. However, ET-1 and ET-2 caused endoplasmic reticulum (ER) stress and NLRP3 inflammasome activation in tubular epithelial cells. The ECE inhibitor phosphoramidon induced autophagy. Furthermore, phosphoramidon inhibited ER stress and the NLRP3 inflammasome in tubular epithelial cells. In an adenine diet-induced CKD mouse model, phosphoramidon attenuated the progression of CKD by regulating autophagy, the NLRP3 inflammasome and ER stress. In summary, these findings showed a new strategy to delay CKD progression by inhibiting ECEs through autophagy activation and restraining ER stress and the NLRP3 inflammasome.

The Non-coding MicroRNA-223 is a Promising Biomarker of Chronic Kidney Disease

Renal diseases are consecutive to a deregulation of gene expression regulated by non-coding RNAs. These non-coding RNAs were discovered at the turn of the 21st century when it was established that post-transcriptional regulation was performed through small non-coding RNAs, known as microRNAs (miRNAs). Up to 3,000 miRNAs are expressed by human cells. They are small, single-stranded nucleic acids, which trigger translational repression of mRNA by base-pairing with the 3′ untranslated region of their mRNA targets. In addition to miRNA regulation, it was also demonstrated that 60,000 long non-coding RNAs are expressed in the human cell and that they are able to regulate gene expression at all levels. The roles of these various RNA families are just beginning to be understood in the field of nephrology. In the past decade, the authors and various others have published that several miRNAs are deregulated during the onset of chronic kidney disease (CKD) and are associated with cardiovascular damage. This review focuses on miRNA-223 (miR-223) as its expression is increased in vivo in the large vessels of a mouse model of CKD, whereas it is diminished in the serum of both mice and human patients with CKD. In patients, miR-223 expression was correlated with all-cause mortality, as well as cardiovascular and renal events. Molecular clues were given by a multi-omics approach, indicating that miR-223 modulates gene regulation at all levels including mRNA expression, protein amounts, and metabolic molecule accumulation. miR-223 is thus a potential target to prevent or treat complications of CKD pathogenesis.

Metformin Attenuates Renal Fibrosis in a Mouse Model of Adenine-Induced Renal Injury Through Inhibiting TGF-β1 Signaling Pathways.

It is well-known that all progressive chronic kidney disease (CKD) is pathologically characterized by tubulointerstitial fibrosis process. Multiple studies have shown the critical role of inflammation and fibrosis in the development of CKD. Hence strategies that target inflammatory and fibrotic signaling pathways may provide promising opportunities to protect against renal fibrosis. Metformin has been used as the first-line glucose-lowering agent to treat patients with type 2 diabetes mellitus (T2DM) for over 50 years. Accumulating evidence suggests the potential for additional therapeutic applications of metformin, including mitigation of renal fibrosis. In this study, the anti-fibrotic effects of metformin independent of its glucose-lowering mechanism were examined in an adenine -induced mouse model of CKD. Expressions of inflammatory markers MCP-1, F4/80 and ICAM, fibrotic markers type IV collagen and fibronectin, and the cytokine TGF-β1 were increased in adenine-induced CKD when compared to control groups and significantly attenuated by metformin treatment. Moreover, treatment with metformin inhibited the phosphorylation of Smad3, ERK1/2, and P38 and was associated with activation of the AMP-activated protein kinase (AMPK) in the kidneys of adenine-treated mice. These results indicate that metformin attenuates adenine-induced renal fibrosis through inhibition of TGF-β1 signaling pathways and activation of AMPK, independent of its glucose-lowering action.

Nicotinamide Attenuates the Progression of Renal Failure in a Mouse Model of Adenine-Induced Chronic Kidney Disease.

Nicotinamide adenine dinucleotide (NAD+) supplies energy for deoxidation and anti-inflammatory reactions fostering the production of adenosine triphosphate (ATP). The kidney is an essential regulator of body fluids through the excretion of numerous metabolites. Chronic kidney disease (CKD) leads to the accumulation of uremic toxins, which induces chronic inflammation. In this study, the role of NAD+ in kidney disease was investigated through the supplementation of nicotinamide (Nam), a precursor of NAD+, to an adenine-induced CKD mouse model. Nam supplementation reduced kidney inflammation and fibrosis and, therefore, prevented the progression of kidney disease. Notably, Nam supplementation also attenuated the accumulation of glycolysis and Krebs cycle metabolites that occurs in renal failure. These effects were due to increased NAD+ supply, which accelerated NAD+-consuming metabolic pathways. Our study suggests that Nam administration may be a novel therapeutic approach for CKD prevention.

Exosomes isolated from melatonin-stimulated mesenchymal stem cells improve kidney function by regulating inflammation and fibrosis in a chronic kidney disease mouse model

Chronic kidney disease (CKD) is defined as structural and functional abnormalities of the kidney due to inflammation and fibrosis. We investigated the therapeutic effects of exosomes secreted by melatonin-stimulated mesenchymal stem cells (Exocue) on the functional recovery of the kidney in a CKD mouse model. Exocue upregulated gene expression of micro RNAs (miRNAs) associated with anti-inflammatory and anti-fibrotic effects. Exocue-treated groups exhibited low tumor necrosis factor-α and transforming growth factor-β levels in serum and fibrosis inhibition in kidney tissues mediated through regulation of cell apoptosis and proliferation of fibrosis-related cells. Exocue treatment decreased the gene expression of CKD progression-related miRNAs. Moreover, the CKD severity was alleviated in the Exocue group via upregulation of aquaporin 2 and 5 levels and reduction of blood urea nitrogen and creatinine, resulting in functional recovery of the kidney. In conclusion, Exocue could be a novel therapeutic agent for treating CKD by regulating inflammation and fibrosis.

Glycolysis and Lactate Dehydrogenase A in Iron-Mediated Suppression of Osteoblast Function

Background/Objective: 
 An estimated 37 million Americans have Chronic Kidney Disease (CKD), a condition in characterized by gradual decline in kidney function1. Patients with CKD are often afflicted with skeletal fractures, increasing morbidity and mortality. CKD has also shown a strong correlation with iron-deficiency anemia. The underlying mechanisms of how iron-deficiency anemia of CKD affects bone loss are not well understood. Based on RNA-sequencing results, we hypothesize that lactate dehydrogenase A (LDHA) may play a role in iron-deficiency mediated suppression of osteoblast differentiation and function. 
 
 Methods: 
 Mouse Progenitor Cells (MPC2) were incubated in osteogenic media along with deferoxamine (DFO) to induce differentiation in chronic iron deficiency; samples were collected after 7 and 14 days. Quantitative real-time PCR and western blot were used to validate LDHA mRNA and protein levels in DFO treated MPCs versus control cells. RNA levels of osteoblast genes and LDHA were also assessed in a pre-clinical mouse model of CKD. 
 
 Results: 
 In vitro, MPCs cultured in DFO media showed a significant increase of LDHA mRNA at 7 days (p=0.015) and returned to near control levels by day 14. Western blots showed a slight increase of total LDHA protein in DFO treated MPCs at 7 days and a large increase of protein at the 14-day mark (p=0.051). In vivo, CKD bone marrow showed a reduction in osteoblast gene expression (osteocalcin and type 1 collagen; p<0.05). LDHA mRNA expression was increased in CKD mice bone marrow when compared to wild-type mice (p=0.051), suggesting an inverse relationship. 
 
 Conclusion and Potential Impact: 
 Inappropriate activation of glycolysis and LDHA appears to play a role in iron-deficiency mediated suppression of osteoblast function in relation to CKD, both in vitro and in vivo. Further exploration of this relationship could be critical to the development of improved treatment options to maintain bone homeostasis during CKD.

The HIF-PHI BAY 85-3934 (Molidustat) Improves Anemia and Is Associated With Reduced Levels of Circulating FGF23 in a CKD Mouse Model.

Fibroblast growth factor-23 (FGF23) is a critical factor in chronic kidney disease (CKD), with elevated levels causing alterations in mineral metabolism and increased odds for mortality. Patients with CKD develop anemia as the kidneys progressively lose the ability to produce erythropoietin (EPO). Anemia is a potent driver of FGF23 secretion; therefore, a hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) currently in clinical trials to elevate endogenous EPO to resolve anemia was tested for effects on iron utilization and FGF23-related parameters in a CKD mouse model. Mice were fed either a casein control diet or an adenine-containing diet to induce CKD. The CKD mice had markedly elevated iFGF23 and blood urea nitrogen (BUN), hyperphosphatemia, and anemia. Cohorts of mice were then treated with a patient-equivalent dose of BAY 85-3934 (BAY; Molidustat), which elevated EPO and completely resolved aberrant complete blood counts (CBCs) in the CKD mice. iFGF23 was elevated in vehicle-treated CKD mice (120-fold), whereas circulating iFGF23 was significantly attenuated (>60%) in the BAY-treated CKD mice. The BAY-treated mice with CKD also had reduced BUN, but there was no effect on renal vitamin D metabolic enzyme expression. Consistent with increased EPO, bone marrow Erfe, Transferrin receptor (Tfrc), and EpoR mRNAs were increased in BAY-treated CKD mice, and in vitro hypoxic marrow cultures increased FGF23 with direct EPO treatment. Liver Bmp-6 and hepcidin expression were downregulated in all BAY-treated groups. Femur trabecular parameters and cortical porosity were not worsened with BAY administration. In vitro, differentiated osteocyte-like cells exposed to an iron chelator to simulate iron depletion/hypoxia increased FGF23; repletion with holo-transferrin completely suppressed FGF23 and normalized Tfrc1. Collectively, these results support that resolving anemia using a HIF-PHI during CKD was associated with lower BUN and reduced FGF23, potentially through direct restoration of iron utilization, thus providing modifiable outcomes beyond improving anemia for this patient population. © 2021 American Society for Bone and Mineral Research (ASBMR).

Astragalus mongholicus Bunge and Panax Notoginseng Formula (A&P) Combined With Bifidobacterium Contribute a Renoprotective Effect in Chronic Kidney Disease Through Inhibiting Macrophage Inflammatory Response in Kidney and Intestine.

There is increasing evidence that Chronic Kidney Disease (CKD) can cause intestinal dysfunction, which in turn aggravates the progression of kidney disease. Studies have shown that the immune response of macrophage plays an important role in promoting inflammation in kidney and intestine of CKD. Astragalus mongholicus Bunge and Panax notoginseng formula (A&P) is a widely used traditional medicine for the treatment of CKD in China, however, the underlying mechanism is largely unclear. In this study, we aimed to explore the role of A&P and Bifidobacterium combination treatment in regulation of inflammatory response of macrophage in kidney and intestine of CKD mouse, as well as the potential molecular mechanism. We established a CKD mouse model with 5/6 nephrectomy and a macrophage inflammatory cellular model with LPS and urotoxin in vivo and in vitro. The results showed that A&P combined with Bifidobacterium significantly reduced the expression and secretion of IL-1β, IL-6, TNFα, and MCP-1 in kidney and blood, as well as in inflammatory macrophage. Interestingly, A&P combined with Bifidobacterium strongly improved the intestinal flora and protected the intestinal barrier. Notably, the maintainer of macrophage polarization, Mincle, was activated in kidney and intestine of CKD mouse as well as in urotoxin stimulated macrophage, that was effectively inhibited by the treatment of A&P and Bifidobacterium combination. Overexpression of Mincle by genetic modification can abolish the inhibitory effects of A&P combined with Bifidobacterium on inflammation in urotoxin stimulated RAW264.7 cells. In summary, these findings demonstrated that A&P combined with Bifidobacterium can protect kidney against CKD by down-regulating macrophage inflammatory response in kidney and intestine via suppressing Mincle signaling, which provides a new insight in the treatment of CKD with traditional medicine.

Kv1.3 Channel Inhibition Limits Uremia-Induced Calcification in Mouse and Human Vascular Smooth Muscle.

Chronic kidney disease (CKD) significantly increases cardiovascular risk. In advanced CKD stages, accumulation of toxic circulating metabolites and mineral metabolism alterations triggers vascular calcification, characterized by vascular smooth muscle cell (VSMC) transdifferentiation and loss of the contractile phenotype. Phenotypic modulation of VSMC occurs with significant changes in gene expression. Even though ion channels are an integral component of VSMC function, the effects of uremia on ion channel remodeling has not been explored. We used an in vitro model of uremia-induced calcification of human aorta smooth muscle cells (HASMCs) to study the expression of 92 ion channel subunit genes. Uremic serum-induced extensive remodeling of ion channel expression consistent with loss of excitability but different from the one previously associated with transition from contractile to proliferative phenotypes. Among the ion channels tested, we found increased abundance and activity of voltage-dependent K+ channel Kv1.3. Enhanced Kv1.3 expression was also detected in aorta from a mouse model of CKD. Pharmacological inhibition or genetic ablation of Kv1.3 decreased the amount of calcium phosphate deposition induced by uremia, supporting an important role for this channel on uremia-induced VSMC calcification.

Withaferin A protects against endoplasmic reticulum stress-associated apoptosis, inflammation, and fibrosis in the kidney of a mouse model of unilateral ureteral obstruction

BackgroundWithaferin A is a functional ingredient of a traditional medicinal plant, Withania somnifera, which has been broadly used in India for protecting against chronic diseases. This bioactive steroidal lactone possesses multiple functions such as anti-oxidation, anti-inflammation, and immunomodulation. Chronic kidney disease (CKD) is one of the major health problems worldwide with the high complication, morbidity, and mortality rates. The detailed effects and underlying mechanisms of withaferin A on CKD progression still remain to be clarified. PurposeWe aimed to investigate whether withaferin A treatment ameliorates the development of renal fibrosis and its related mechanisms in a CKD mouse model. MethodsA mouse model of unilateral ureteral obstruction (UUO) was used to mimic the progression of CKD. Male adult C57BL/6J mice were orally administered with 3 mg/kg/day withaferin A for 14 consecutive days after UUO surgery. Candesartan (5 mg/kg/day) was used as a positive control. ResultsBoth Withaferin A and candesartan treatments significantly ameliorated the histopathological changes and collagen deposition in the UUO kidneys. Withaferin A could significantly reverse the increases in the protein levels of pro-fibrotic factors (fibronectin, transforming growth factor-β, and α-smooth muscle actin), inflammatory signaling molecules (phosphorylated nuclear factor-κB-p65, interleukin-1β, and cyclooxygenase-2), and cleaved caspase-3, apoptosis, and infiltration of neutrophils in the UUO kidneys. The protein levels of endoplasmic reticulum (ER) stress-associated molecules (GRP78, GRP94, ATF4, CHOP, phosphorylated eIF2α, and cleaved caspase 12) were increased in the kidneys of UUO mice, which could be significantly reversed by withaferin A treatment. ConclusionWithaferin A protects against the CKD progression that is, at least in part, associated with the moderation of ER stress-related apoptosis, inflammation, and fibrosis in the kidneys of CKD. Withaferin A may serve as a potential therapeutic agent for the development of CKD.

Diet posttranslationally modifies the mouse gut microbial proteome to modulate renal function.

Associations between chronic kidney disease (CKD) and the gut microbiota have been postulated, yet questions remain about the underlying mechanisms. In humans, dietary protein increases gut bacterial production of hydrogen sulfide (H2S), indole, and indoxyl sulfate. The latter are uremic toxins, and H2S has diverse physiological functions, some of which are mediated by posttranslational modification. In a mouse model of CKD, we found that a high sulfur amino acid-containing diet resulted in posttranslationally modified microbial tryptophanase activity. This reduced uremic toxin-producing activity and ameliorated progression to CKD in the mice. Thus, diet can tune microbiota function to support healthy host physiology through posttranslational modification without altering microbial community composition.

Magnetic resonance imaging accurately tracks kidney pathology and heterogeneity in the transition from acute kidney injury to chronic kidney disease

Acute kidney injury (AKI) increases the risk for chronic kidney disease (CKD). However, there are few tools to detect microstructural changes after AKI. Here, cationic ferritin-enhanced magnetic resonance imaging (CFE-MRI) was applied to examine the heterogeneity of kidney pathology in the transition from AKI to CKD. Adult male mice received folic acid followed by cationic ferritin and were euthanized at four days (AKI), four weeks (CKD-4) or 12 weeks (CKD-12). Kidneys were examined by histologic methods and CFE-MRI. In the CKD-4 and CKD-12 groups, glomerular number was reduced and atubular cortical lesions were observed. Apparent glomerular volume was larger in the AKI, CKD-4 and CKD-12 groups compared to controls. Glomerular hypertrophy occurred with ageing. Interglomerular distance and glomerular density were combined with other MRI metrics to distinguish the AKI and CKD groups from controls. Despite significant heterogeneity, the noninvasive (MRI-based) metrics were as accurate as invasive (histological) metrics at distinguishing AKI and CKD from controls. To assess the toxicity of cationic ferritin in a CKD model, CKD-4 mice received cationic ferritin and were examined one week later. The CKD-4 groups with and without cationic ferritin were similar, except the iron content of the kidney, liver, and spleen was greater in the CKD-4 plus cationic ferritin group. Thus, our study demonstrates the accuracy and safety of CFE-MRI to detect whole kidney pathology allowing for the development of novel biomarkers of kidney disease and providing a foundation for future invivo longitudinal studies in mouse models of AKI and CKD to track nephron fate.

Pro-neurotensin depends on renal function and is related to all-cause mortality in chronic kidney disease.

Patients with chronic kidney disease (CKD) have a high risk of premature cardiovascular diseases (CVD) and show increased mortality. Pro-neurotensin (Pro-NT) was associated with metabolic diseases and predicted incident CVD and mortality. However, Pro-NT regulation in CKD and its potential role linking CKD and mortality have not been investigated, so far. In a central lab, circulating Pro-NT was quantified in three independent cohorts comprising 4715 participants (cohort 1: patients with CKD; cohort 2: general population study; and cohort 3: non-diabetic population study). Urinary Pro-NT was assessed in part of the patients from cohort 1. In a 4th independent cohort, serum Pro-NT was further related to mortality in patients with advanced CKD. Tissue-specific Nts expression was further investigated in two mouse models of diabetic CKD and compared to non-diabetic control mice. Pro-NT significantly increased with deteriorating renal function (P < 0.001). In meta-analysis of cohorts 1-3, Pro-NT was significantly and independently associated with estimated glomerular filtration rate (P ≤ 0.002). Patients in the middle/high Pro-NT tertiles at baseline had a higher all-cause mortality compared to the low Pro-NT tertile (Hazard ratio: 2.11, P = 0.046). Mice with severe diabetic CKD did not show increased Nts mRNA expression in different tissues compared to control animals. Circulating Pro-NT is associated with impaired renal function in independent cohorts comprising 4715 subjects and is related to all-cause mortality in patients with end-stage kidney disease. Our human and rodent data are in accordance with the hypotheses that Pro-NT is eliminated by the kidneys and could potentially contribute to increased mortality observed in patients with CKD.

Abstract P231: Macrophage Neutrophil Gelatinase-associated Lipocalin Has A Critical Role In Aldosterone-induced Renal Fibrosis Via The Ccl5-il4 Pathway.

Introduction: Neutrophil Gelatinase-Associated Lipocalin (NGAL) (or lipocalin 2) is a novel mineralocorticoid biotarget in the cardiovascular system. NGAL is also described as an acute renal lesion biomarker and NGAL serum concentration is associated with the severity of renal damages patients with a chronic kidney disease (CKD). Lipocalin2 (Lcn2) gene invalidation in a CKD mouse model protects from proteinuria and renal lesions. Objective: We hypothesized that the NGAL from macrophages promotes expression of chemoattractant molecules involved in renal lesions induced by mineralocorticoid excess. Methods: The role of Lcn2 was analyzed using full Lcn2 knock out mice (NGAL KO) challenged with uni-Nephrectomy, Aldosterone 200μg/kg/day, Salt 1% (NAS model) during 6 weeks. Assessment of CCL5/IL4 in kidney fibrosis were studied using maraviroc administration (50mg/kg/d in chow diet) or by injections of anti-IL4 antibody (600μg/week). Results: NAS induced a significant increase in the expression (relative values, mean±SEM, compared to 1 in the control samples, p&amp;lt0.05) of extracellular matrix proteins such as collagen I (2.35±0.33), α-SMA (2.04±0.44) and fibronectin (3.38±0.42) in the kidney of WT mice associated with interstitial kidney fibrosis (6.49±0.70). These modifications were fully prevented by NGAL deletion. Expression of macrophages markers F4/80 , CD80 and CD86 was increased (5.11±0.46, 4.84±0.19 and 5.22±0.45 respectively) in WT NAS mice and partly prevented in NGAL KO mice. Macrophages isolated from NGAL KO or WT mice were co-treated with aldosterone (10 -8 M) and NaCl (40mM). In WT macrophages, expression of Lcn2 (2.81±0.30) and the CCL5 chemokine (2.48±0.32) was increased. The increase of CCL5 was prevented in NGAL KO macrophages. Such as total deletion of NGAL, CCL5 receptor blockade improved renal fibrosis and high level of Th2 CD4 + cell markers induced by NAS. Neutralization of IL4 Th2 cytokine in NAS mice injected with anti-IL4 antibody blunted kidney fibrosis and overexpression of profibrotic proteins such as collagen I, α-SMA and fibronectin. Conclusion: NGAL produced by macrophages plays a critical role in renal interstitial fibrosis through CCL5/IL4 pathways in mice exposed to mineralocorticoid excess.

Carbonyl iron and iron dextran therapies cause adverse effects on bone health in juveniles with chronic kidney disease

Anemia is a frequent complication of chronic kidney disease (CKD), related in part to the disruption of iron metabolism. Iron therapy is very common in children with CKD and excess iron has been shown to induce bone loss in non-CKD settings, but the impact of iron on bone health in CKD remains poorly understood. Here, we evaluated the effect of oral and parenteral iron therapy on bone transcriptome, bone histology and morphometry in two mouse models of juvenile CKD (adenine-induced and 5/6-nephrectomy). Both modalities of iron therapy effectively improved anemia in the mice with CKD, and lowered bone Fgf23 expression. At the same time, iron therapy suppressed genes implicated in bone formation and resulted in the loss of cortical and trabecular bone in the mice with CKD. Bone resorption was activated in untreated CKD, but iron therapy had no additional effect on this. Furthermore, we assessed the relationship between biomarkers of bone turnover and iron status in a cohort of children with CKD. Children treated with iron had lower levels of circulating biomarkers of bone formation (bone-specific alkaline phosphatase and the amino-terminal propeptide of type 1 procollagen), as well as fewer circulating osteoblast precursors, compared to children not treated with iron. These differences were independent of age, sex, and glomerular filtration rate. Thus, iron therapy adversely affected bone health in juvenile mice with CKD and was associated with low levels of bone formation biomarkers in children with CKD.

P0689LINACLOTIDE, THE GUANYLATE CYCLASE C AGONIST LIMITS CARDIAC FIBROSIS FORMATION IN MICE WITH CHRONIC KIDNEY DISEASE AFTER ISCHEMIA-REPERFUSION INJURY

Abstract Background and Aims Cardiovascular disease (CVD) is a major cause of mortality in patients with chronic kidney disease (CKD). Renal ischemia-reperfusion injury (IRI) is a clinically significant condition that progress to CKD after acute kidney injury (AKI). We have reported that the suppression of Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine, by linaclotide could prevent renal fibrosis and improve renal function in the chronic phase after renal IRI. TMAO has been linked directly with progression of CVD too. We investigated whether the reduction of TMAO by linaclotide limits cardiac fibrosis in mouse model of CKD after IRI. Method Linaclotide (100μg/kg) was administered for 2weeks before IRI and continued for 2weeks after IRI. After 2weeks since IRI, removed hearts sections were performed Azan stain to evaluate the level of fibrosis and western blotting to evaluate the expression level of fibronectin. Results The administration of linaclotide significantly improved cardiac fibrosis area (Fig1). Additionally, western blotting showed linaclotide suppress the expression level of fibronectin (Fig2). Conclusion The reduction of TMAO by linaclotide could prevent cardiac fibrosis in the chronic phase after renal IRI. Linaclotide may be a powerful tool for prevention of the cardiorenal syndrome after renal IRI.

Female AhR Knockout Mice Develop a Minor Renal Insufficiency in an Adenine-Diet Model of Chronic Kidney Disease.

Cardiovascular complications observed in chronic kidney disease (CKD) are associated with aryl hydrocarbon receptor (AhR) activation by tryptophan-derived uremic toxins—mainly indoxyl sulfate (IS). AhR is a ligand-activated transcription factor originally characterized as a receptor of xenobiotics involved in detoxification. The aim of this study was to determine the role of AhR in a CKD mouse model based on an adenine diet. Wild-type (WT) and AhR−/− mice were fed by alternating an adenine-enriched diet and a regular diet for 6 weeks. Our results showed an increased mortality rate of AhR−/− males. AhR−/− females survived and developed a less severe renal insufficiency that WT mice, reflected by urea, creatinine, and IS measurement in serum. The protective effect was related to a decrease of pro-inflammatory and pro-fibrotic gene expression, an attenuation of tubular injury, and a decrease of 2,8-dihydroxyadenine crystal deposition in the kidneys of AhR−/− mice. These mice expressed low levels of xanthine dehydrogenase, which oxidizes adenine into 2,8-dihydroxyadenine, and low levels of the IS metabolism enzymes. In conclusion, the CKD model of adenine diet is not suitable for AhR knockout mice when studying the role of this transcription factor in cardiovascular complications, as observed in human CKD.

Assessment of Thrombotic and Bleeding Tendency in Two Mouse Models of Chronic Kidney Disease: Adenine-Diet and 5/6th Nephrectomy.

The coexistence of bleeding and thrombosis in patients with chronic kidney disease (CKD) is frequent and poorly understood. Mouse models are essential to understand complications of CKD and to develop new therapeutic approaches improving the health of patients. We evaluated the hemostasis in two models of renal insufficiency: adenine-diet and 5/6th nephrectomy (5/6Nx). Compared with 5/6Nx mice, mice fed with 0.25% adenine had more severe renal insufficiency and so higher levels of prothrombotic uremic toxins like indoxyl sulfate. More severe renal inflammation and fibrosis were observed in the adenine group, as demonstrated by histological and reverse transcription quantitative polymerase chain reaction experiments. Liver fibrinogen γ chain expression and level of plasma fibrinogen were increased only in adenine mice. In both CKD mouse models, tissue factor (TF) expression was increased in kidney and aorta extracts. Immunochemistry analysis of kidney sections showed that TF is localized in the vascular walls. Thrombin–antithrombin complexes were significantly increased in plasma from both adenine and 5/6Nx mice. Tail bleeding time increased significantly only in adenine mice, whereas platelet count was not significant altered. Finally, results obtained by intravital microscopy after laser-induced endothelial injury showed impaired platelet function in adenine mice and an increase in fibrin generation in 5/6Nx mice. To summarize, adenine diet causes a more severe renal insufficiency compared with 5/6Nx. The TF upregulation and the hypercoagulable state were observed in both CKD models. Bleeding tendency was observed only in the adenine model of CKD that recapitulates the whole spectrum of hemostasis abnormalities observed in advanced human CKD.