Adenine Diet Research Articles

#4331 UPREGULATION OF ADIPOSE TISSUE FATTY ACID-BINDING PROTEIN 4 IN PATIENTS WITH CHRONIC KIDNEY DISEASE: IMPLICATIONS FOR DYSFUNCTIONAL VASCULAR CELLS

Abstract Background and Aims FABP4, a fatty acid-binding protein, is predominantly expressed in adipose tissue (AT) and has been shown to be elevated in patients with chronic kidney disease (CKD). Although elevated levels of FABP4 have been linked to cardiovascular disease in end-stage renal disease (ESRD) patients, the cause of this increase is unclear. The aim of this study was to evaluate the expression of AT FABP4 under uremic conditions and its impact on the function of macrophages and vascular smooth muscle cells (VSMC). Method We measured the levels of FABP4 in the blood and urine of CKD patients. The expression of AT FABP4 under uremic conditions was analyzed using omental AT obtained from healthy kidney donors and patients with ESRD who received peritoneal catheter insertion. The effect of FABP4 on the function of macrophages and VSMC was also assessed. Results The levels of FABP4 in the blood and urine of CKD patients were inversely correlated with their estimated glomerular filtration rate (eGFR). An increase in FABP4 in the blood was detected before its increase in the urine. FABP4 expression was found to be higher in mature adipocytes in visceral AT compared to the stromal vascular fraction (SVF). Adipocytes treated with p-cresol and adipocytes isolated from CKD patients showed higher levels of FABP4 expression compared to healthy individuals. Single-cell RNA sequencing of SVF showed increased FABP4 expression in progenitor cells and macrophages from CKD patients. In THP-1 cells, FABP4 induced more foam cells and increased levels of inflammatory mediators in the presence of palmitic acid. VSMC treated with p-cresol or VSMC isolated from CKD mice induced by an adenine diet showed a pro-calcific phenotype, as indicated by increased calcium content and bone-related gene expression, which was further enhanced by FABP4. Conclusion The elevated levels of FABP4 in CKD patients are not solely a result of decreased renal clearance but also due to increased production in AT. These higher levels of circulating FABP4 may be associated with dysfunction in vascular cells.

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Fate mapping reveals compartment-specific clonal expansion of mononuclear phagocytes during kidney disease

Beneficiary Effects of Colchicine on Inflammation and Fibrosis in a Mouse Model of Kidney Injury

Introduction: Low-grade inflammation is seen in many chronic illnesses, including chronic kidney disease (CKD). We have recently reported on beneficiary effects of anti-inflammatory treatment in the interleukin (IL-) 1 pathway on anemia as well as CKD extent in a mouse model. Colchicine has been shown to have beneficiary effects in several inflammatory conditions through various mechanisms, including inhibition of tubulin polymerization as well as caspase-1-mediated IL-1 activation. Methods: Kidney injury (KI) was induced by administering an adenine diet to 8-week-old C57BL/6J mice treated with colchicine (Col) (30 µg/kg) or saline injections for 3 weeks, generating 4 groups: C, Ccol, KI, and KIcol. Results: KI animals had an increase in inflammation indices in the blood (neutrophils), liver, and kidneys (uromodulin, IL-6, pSTAT3). Increased kidney tubulin polymerization and caspase-1 in KI, as well as kidney Mid88 and IRAK4 (downstream of IL-1), were inhibited in KIcol. Kidney macrophage and polymorphonuclear infiltration (positive for F4/80 and MPO, respectively), the percentage of fibrotic area, and TGFβ mRNA levels were lower in KIcol versus KI. Conclusions: Colchicine inhibited tubulin polymerization and caspase-1 activation and attenuated kidney inflammation and fibrosis in a mouse model of adenine-induced KI. Given its reported safety profile for long-term anti-inflammatory therapy without increasing infection tendency, it may serve as novel therapeutic approach in CKD.

Dentoalveolar Alterations in an Adenine-Induced Chronic Kidney Disease Mouse Model.

Chronic kidney disease (CKD) is characterized by kidney damage and loss of renal function. CKD mineral and bone disorder (CKD-MBD) describes the dysregulation of mineral homeostasis, including hyperphosphatemia and elevated parathyroid hormone (PTH) secretion, skeletal abnormalities, and vascular calcification. CKD-MBD impacts the oral cavity, with effects including salivary gland dysfunction, enamel hypoplasia and damage, increased dentin formation, decreased pulp volume, pulp calcifications, and altered jaw bones, contributing to clinical manifestations of periodontal disease and tooth loss. Underlying mechanisms are not fully understood, and CKD mouse models commonly require invasive procedures with high rates of infection and mortality. We aimed to characterize the dentoalveolar effects of an adenine diet (AD)-induced CKD (AD-CKD) mouse model. Eight-week-old C57BL/6J mice were provided either a normal phosphorus diet control (CTR) or adenine and high-phosphorus diet CKD to induce kidney failure. Mice were euthanized at 15 weeks old, and mandibles were collected for micro-computed tomography and histology. CKD mice exhibited kidney failure, hyperphosphatemia, and hyperparathyroidism in association with porous cortical bone in femurs. CKD mice showed a 30% decrease in molar enamel volume compared to CTR mice. Enamel wear was associated with reduced ductal components, ectopic calcifications, and altered osteopontin (OPN) deposition in submandibular salivary glands of CKD mice. Molar cusps in CKD mice were flattened, exposing dentin. Molar dentin/cementum volume increased 7% in CKD mice and pulp volume decreased. Histology revealed excessive reactionary dentin and altered pulp-dentin extracellular matrix proteins, including increased OPN. Mandibular bone volume fraction decreased 12% and bone mineral density decreased 9% in CKD versus CTR mice. Alveolar bone in CKD mice exhibited increased tissue-nonspecific alkaline phosphatase localization, OPN deposition, and greater osteoclast numbers. AD-CKD recapitulated key aspects reported in CKD patients and revealed new insights into CKD-associated oral defects. This model has potential for studying mechanisms of dentoalveolar defects or therapeutic interventions. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Astragaloside IV attenuates indoxyl sulfate-induced injury of renal tubular epithelial cells by inhibiting the aryl hydrocarbon receptor pathway.

Astragalus membranaceus Fisch. ex Bunge has long been used to treat chronic kidney disease (CKD) in China. However, the mechanism of action requires further study. Indoxyl sulfate accumulation is the key cause of CKD progression. The aryl hydrocarbon receptor (AhR) plays an essential role in the renal tubular injury induced by indoxyl sulfate (IS). We explored the effects of Astragaloside IV (AS-IV), a minor component of the flowering perennial Astragalus membranaceus Fisch. ex Bunge, on AhR activity during IS-induced injury of renal tubular epithelial cells. C57BL/6 mice fed a 0.2% adenine diet (adenine+IS) and intraperitoneally injected with IS were used to study the protective effects of AS-IV, and specifically the effect on the AhR. In addition, apoptosis (annexin/PI), oxidative stress and the AhR pathway were investigated in IS-stimulated HK-2cells treated with AS-IV. The binding of AS-IV to the AhR was assessed in a molecular docking analysis. AhR knockdown using AhR siRNA allowed determination of the effects of AS-IV in IS-stimulated HK-2cells. AS-IV inhibited tubulointerstitial injury in adenine+IS mice. While AS-IV did not reduce serum IS levels, it did inhibit AhR expression in the kidney. In IS-stimulated HK-2cells, AS-IV also dramatically reduced apoptosis, decreased oxidative stress responses and inhibited the expression of the AhR pathway. The molecular docking analysis showed surface binding of AS-IV to the AhR. Following AhR knockdown in HK-2cells, IS-induced apoptosis was reduced and could not be further reduced by AS-IV. By targeting the AhR, AS-IV may alleviate IS-induced renal tubular injury, thus offering a novel therapeutic approach to the treatment of chronic renal failure.

Impact of N-acetylcysteine supplementation on skeletal muscle myopathy in a mouse model of access-related hand dysfunction

Introduction: Hemodialysis is a life-line therapy for end-stage kidney disease patients that requires long-term arteriovenous access. Arteriovenous fistula (AVF) is the most durable and commonly employed access strategy. However, access-related hand dysfunction (ARHD) including ischemia, weakness, and neuromotor discoordination is prevalent (~50%) following AVF placement. Unfortunately, there are no pre-emptive non-surgical therapeutic studies available. We hypothesized that N-acetylcysteine (NAC) supplementation would attenuate hindlimb disability in a mouse model of ARHD. Methods: Adult male and female C57BL/6J mice (N=9/group) were fed adenine diet to induce kidney disease. Thereafter, animals were randomly allocated to receive NAC (150 mg/kg in 0.2 ml of 0.9% saline) or placebo (0.2 ml of saline) via oral gavage beginning 3 days prior to the AVF surgery and until euthanasia. Limb perfusion to the ischemic hindlimb and hindlimb grip strength were assessed post-operatively (POD) in vivo. Muscle contractile and mitochondrial function, and histological assessments of muscle and the neuromuscular junction were also performed. Results: Laser Doppler perfusion to the ventral paw was decreased immediately after AVF creation on the left common iliac artery (~25% of the contralateral limb) and recovered (~40-60%) at POD13 without a treatment effect. NAC treatment improved muscle mass in the tibialis anterior (TA) (+13~21%, P<0.05) and soleus (+17~21%, P<0.05) muscles in both male and female mice compared with placebo-treated animals. NAC female, not male, mice had significant improvement in grip strength (~20% greater at POD4 and POD12, P<0.05) and maximal mitochondrial respiration (~30% higher at POD14, P<0.05) compared with control animals. Muscle contractile function and myofiber area and capillarization in TA and soleus muscles were similar between NAC and placebo groups (P>0.05). Interestingly, the area of the post-synaptic acetylcholine receptor clusters was significantly greater in the animals that received NAC treatment (+10~22%, P<0.05) in both male and female mice. Conclusion: Daily treatment with a thiol reducing antioxidant, NAC, has beneficial effects on the recovery of muscle mass and neuromuscular junction morphology in both male and female mice with additional improvement in grip strength and mitochondrial function in female mice suggesting a potential therapeutic strategy targeting antioxidant defense to ameliorate ARHD. Funding Support: National Institutes of Health grant R01HL148597 and American Heart Association grant POST903198. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Urate-Lowering Therapy Inhibits Thoracic Aortic Aneurysm and Dissection Formation in Mice.

Thoracic aortic aneurysm and dissection (TAAD) is a highly lethal vascular disease without effective drug therapy. Whether elevated serum concentrations of uric acid are involved in TAAD development remains unclear. Serum uric acid levels were detected in different TAAD mouse models and patients. The urate-lowering drug allopurinol was administered in the drinking water of TAAD mice. Adenine diet-induced mice were established to investigate the role of hyperuricemia in TAAD formation and RNA-sequencing of thoracic aortas from these mice was performed. We found serum uric acid levels were elevated in various mouse TAAD models, including mice fed a β-aminopropionitrile diet, Marfan mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+), and ApoE-/- mice infused with Ang II (angiotensin II), as well as in patients with TAAD. Administration of urate-lowering drug allopurinol in the drinking water significantly alleviated TAAD formation in β-aminopropionitrile-treated mice, Fbn1C1041G/+ mice, and Ang II-infused ApoE-/- mice. Moreover, an adenine diet was used to induce hyperuricemia in mice. Intriguingly, a 4-week adenine diet feeding directly induced TAAD formation characterized by increased maximal thoracic aortic diameters and severe elastin degradation, which were ameliorated by allopurinol. Unbiased RNA-sequencing in mouse thoracic aortas suggested that FcγR (Fc gamma receptor) was upregulated upon adenine diet, but reciprocally repressed by allopurinol. Mechanistically, hyperuricemia activated FcγR-mediated ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation to induce macrophage inflammation and TAAD development, which was abrogated by allopurinol or FcγR deficiency. This study uncovered an important and previously unrecognized role of hyperuricemia in mediating the pathogenesis of TAAD, and uric acid-lowering drug may represent a promising therapeutic approach for TAAD.

Effects of Mouse Kidney Parvovirus on Pharmacokinetics of Chemotherapeutics and the Adenine Model of Chronic Kidney Disease.

Mouse kidney parvovirus (MKPV) causes inclusion body nephropathy in severely immunocompromised mice and renal interstitial inflammation in immunocompetent mice. Here we sought to determine the effects of MKPV on pre-clinical murine models that depend on renal function. To assess the effects of MKPV infection on the pharmacokinetics of 2 renally excreted chemotherapeutic agents, methotrexate and lenalidomide, we measured drug concentrations in the blood and urine of MKPV-infected or uninfected immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) and immunocompetent C57BL/6NCrl (B6) female mice. No differences in plasma pharmacokinetics were observed for lenalidomide. However, the AUC of methotrexate was 1.5-fold higher in uninfected NSG mice compared with infected NSG mice, 1.9-fold higher in infected B6 mice compared with uninfected B6 mice, and 4.3-fold higher in uninfected NSG mice compared with uninfected B6 mice. MKPV infection did not significantly affect the renal clearance of either drug. To assess effects of MKPV infection on the adenine diet model of chronic kidney disease, MKPV-infected and uninfected B6 female mice were fed a 0.2% adenine diet, and clinical and histopathologic features of disease were assessed over 8 wk. MKPV infection did not significantly alter urine chemistry results, hemogram findings, or serum concentrations of BUN, creatinine, or symmetric dimethylarginine. However, infection did influence histologic outcomes. As compared with uninfected mice, MKPV-infected mice had more interstitial lymphoplasmacytic infiltrates after 4 and 8 wk of diet consumption and less interstitial fibrosis at week 8. Macrophage infiltrates and renal tubular injury were similar between in infected and uninfected mice. These findings indicate that MKPV infection had minimal effects on the renal excretion of 2 chemotherapeutics and on serum biomarkers of renal function. However, infection significantly influenced two histologic features of the adenine diet model of chronic renal disease. MKPV-free mice are critically important in studies evaluating renal histology as an experimental outcome.

Skeletal muscle single fiber force production declines early in juvenile male mice with chronic kidney disease.

Children with chronic kidney disease (CKD) frequently exhibit delayed physical development and reduced physical performance, presumably due to skeletal muscle dysfunction. However, the cellular and molecular basis of skeletal muscle impairment in juvenile CKD remains poorly understood. Cellular (single fiber) and molecular (myosin-actin interactions and myofilament properties) function was examined ex vivo in slow (soleus) and fast (extensor digitorum longus) contracting muscles of juvenile male (6 weeks old) CKD and control mice. CKD was induced by 0.2% adenine diet for 3 weeks starting at 3 weeks of age. Specific tension (maximal isometric force divided by cross-sectional area) was reduced in larger myosin heavy chain (MHC) I and IIA fibers and in all IIB fibers in juvenile male mice with CKD due to fewer strongly bound myosin-actin cross-bridges. Fiber cross-sectional area in juvenile CKD mice was unchanged in MHC I and IIB fibers and increased in MHC IIA fibers, compared to controls. CKD slowed cross-bridge kinetics (slower rate of myosin force production and longer myosin attachment time, ton ) in MHC IIA fibers, and accelerated kinetics (shorter ton ) in MHC IIB fibers, which may indicate fiber type dependent shifts in contractile velocity in juvenile CKD. Overall, our findings show that single fiber myopathy is an early event during juvenile CKD, manifesting prior to the development of cellular atrophy as reduced force generation due to fewer strongly bound myosin heads. These results warrant clinical translation and call for early interventions to preserve physical function in children with CKD.

Echinatin maintains glutathione homeostasis in vascular smooth muscle cells to protect against matrix remodeling and arterial stiffening

Decreased vascular compliance of the large arteries as indicated by increased pulse wave velocity is shown to be associated with atherosclerosis and the related cardiovascular events. The positive correlation between arterial stiffening and disease progression derives a hypothesis that softening the arterial wall may protect against atherosclerosis, despite that the mechanisms controlling the cellular pathological changes in disease progression remain unknown. Here, we established a mechanical-property-based screening to look for compounds alleviating the arterial wall stiffness through their actions on the interaction between vascular smooth muscle cells (VSMCs) and the wall extracellular matrix (ECM). We found that echinatin, a chalcone preferentially accumulated in roots and rhizomes of licorice (Glycyrrhiza inflata), reduced the stiffness of ECM surrounding cultured VSMCs. We examined the potential beneficial effects of echinatin on mitigating arterial stiffening and atherosclerosis, and explored the mechanistic basis by which the compound exert the effects. Administration of echinatin in mice fed on an adenine diet and in hyperlipidemia mice subjected to 5/6 nephrectomy mitigated arterial stiffening and atherosclerosis. Mechanistic insights were gained from the RNA-sequencing results showing that echinatin upregulated the expression of glutamate cysteine ligases (GCLs), both the catalytic (GCLC) and modulatory (GCLM) subunits. Further study indicated that upregulation of GCLC/GCLM in VSMCs by echinatin maintains the homeostasis of glutathione (GSH) metabolism; adequate availability of GSH is critical for counteracting arterial stiffening. As a consequence of regulating the GSH synthesis, echinatin inhibits ferroptosis and matrix remodeling that being considered two contributors of arterial stiffening and atherosclerosis. These data demonstrate a pivotal role of GSH dysregulation in damaging the proper VSMC-ECM interaction and uncover a beneficial activity of echinatin in preventing vascular diseases.

Magnesium Improves Cardiac Function in Experimental Uremia by Altering Cardiac Elastin Protein Content.

Cardiovascular complications are accompanied by life-threatening complications and represent the major cause of death in patients with chronic kidney disease (CKD). Magnesium is important for the physiology of cardiac function, and its deficiency is common in CKD. In the present study, we investigated the impact of oral magnesium carbonate supplementation on cardiac function in an experimental model of CKD induced in Wistar rats by an adenine diet. Echocardiographic analyses revealed restoration of impaired left ventricular cardiac function in animals with CKD. Cardiac histology and real-time PCR confirmed a high amount of elastin protein and increased collagen III expression in CKD rats supplemented with dietary magnesium as compared with CKD controls. Both structural proteins are crucial in maintaining cardiac health and physiology. Aortic calcium content increased in CKD as compared with tissue from control animals. Magnesium supplementation numerically lowered the increases in aortic calcium content as it remained statistically unchanged, compared with controls. In summary, the present study provides evidence for an improvement in cardiovascular function and aortic wall integrity in a rat model of CKD by magnesium, as evidenced by echocardiography and histology.

Pharmacological interventions improve myosin-actin cross-bridge function in mice with advanced chronic kidney disease.

Skeletal muscle fiber force production in adult mice with late-stage chronic kidney disease (CKD) is significantly impaired, although by different myofilament and myosin-actin cross-bridge disease responses in the various fiber types. To reverse some of the CKD-induced contractile deficits, we examined the effects of the ATP analog, 2-deoxyadenosine triphosphate (dATP), to enhance cross-bridge kinetics and the troponin sensitizer, Tirasemtiv (2 µM), to increase cross-bridge formation. Single fiber force production, myosin-actin interactions and myofilament properties were measured at 25°C in fibers from soleus and extensor digitorum longus muscles of 16-week-old CKD mice (N=5) and controls (N=5). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. dATP in myosin heavy chain (MHC) I and IIA fibers with CKD increased cross-bridge kinetics, returning them to control values or faster, leading to more strongly bound cross-bridges for maximal Ca2+-activation (pCa 4.5, pH 7.0, 5 mM Phosphate) and fatiguing (pCa 4.5, pH 6.2, 30 mM Phosphate) conditions. Specific tensions in CKD were also improved 12-21% under maximal and fatiguing conditions, except in maximally Ca2+-activated MHC I fibers, but fell short of control values. Notably, other studies have found that improvements of 13-25% in knee extensor strength in CKD patients improved their physical function, suggesting changes of this magnitude are clinically meaningful. At submaximal Ca2+-activation (pCa 6.0), Tirasemtiv in MHC IIB fibers with CKD increased myofilament stiffness 520%, number of strongly bound cross-bridges 705%, and specific tension 370%, returning specific tension back to control values. This study provides evidence that cellular and molecular contractile function in CKD may be enhanced through use of dATP or Tirasemtiv, which should be tested to potentially improve physical function and overall quality of life in late-stage CKD patients.

Skeletal muscle fiber force declines with chronic kidney disease in juvenile and adult mice.

Juvenile and adult chronic kidney disease (CKD) patients frequently have compromised physical performance, which worsens with disease severity. Examining the underlying mechanisms behind this contractile dysfunction can be difficult to impossible in humans, especially in juveniles, but can be accomplished using animal models. We characterized CKD-induced changes in cellular (single fiber force) and molecular (myosin-actin interactions, myofilament properties) function in soleus and extensor digitorum longus muscles of 6-week-old (juvenile, control = 5, CKD = 6) and 16-week-old (adults, control = 10, CKD = 12) mice. CKD was induced by 0.2% adenine diet for 3 weeks (early-stage CKD) for juveniles and 8 weeks (late-stage CKD) for adults. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced in all myosin heavy chain (MHC) I, IIA, and IIB fibers in juvenile (23-30%) and adult (36-51%) mice with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges. Additionally, myofilament stiffness was reduced in MHC IIA fibers from juveniles and adults as well as MHC IIB fibers from adults, potentially reducing force transmission. For adults, myosin-actin interactions were slower in MHC I and IIA fibers. In juveniles, myosin-actin interactions were slower only in MHC IIA fibers but faster in MHC IIB fibers. Fiber cross-sectional area with CKD was increased or unchanged in all fiber types, except for a decrease in MHC IIB fibers from adults. Overall, juvenile early-stage and adult late-stage CKD showed similar molecular mechanisms leading to reduced force production and cross-bridge kinetics, although alterations were generally larger in adult late-stage CKD. Thus, physical function in juvenile early-stage and adult late-stage CKD is being impaired on the molecular level due to intrinsic contractile deficits independent of skeletal muscle atrophy.

Iron supplementation improves skeletal muscle contractile properties in mice with chronic kidney disease.

Adult chronic kidney disease (CKD) patients commonly display decreased physical function, leading to increased mortality, which is exacerbated with iron deficiency and anemia. Interventions to mitigate CKD myopathy are scarce, potentially because the underlying contractile deficits have not been fully investigated. We examined single fiber force production, myosin-actin interactions and myofilament properties in soleus and extensor digitorum longus muscles of 16-week-old CKD mice without (CKD, N=12) and with iron supplementation (CKDFE, N=6, weekly injections of 0.2 mg/kg iron dextran) and controls (N=10). Late-stage CKD was induced by 0.2% adenine diet for 8 weeks. CKD with iron supplementation (CKDFE) improved anemia status compared to CKD, but not back to control levels. Maximally activated (pCa 4.5, 25°C) specific tension (maximal isometric force divided by cross-sectional area) was reduced 36-51% with CKD due, in a large part, to a reduction in strongly-bound myosin-actin cross-bridges in all myosin heavy chain (MHC) I, IIA, and IIB fibers. Iron supplementation improved specific tension and number of strongly-bound cross-bridges in MHC IIA and IIB fibers. Myosin-actin cross-bridge kinetics were slower with CKD in MHC I and IIA fibers, with iron supplementation restoring kinetics closer to control values in MHC IIA fibers. Higher hemoglobin levels were correlated to improvements in specific tension and strongly-bound cross-bridges in MHC IIA and IIB fibers, and with faster cross-bridge kinetics in MHC IIA fibers. However, CKD induced decreases in myofilament lattice stiffness for MHC IIA and IIB fibers were not improved with iron supplementation. Our findings show that iron supplementation improved various aspects of the cellular and molecular CKD-induced myopathy in fast-contracting fiber types. Thus, correction of iron imbalance and/or anemia may be an effective countermeasure to reduce contractile deficits and improve quality of life in late-stage CKD patients.

Effects of long-term tubular HIF-2α overexpression on progressive renal fibrosis in a chronic kidney disease model

Renal fibrosis is the final manifestation of chronic kidney disease (CKD) regardless of etiology. Hypoxia-inducible factor-2 alpha (HIF-2α) is an important regulator of chronic hypoxia, and the late-stage renal tubular HIF-2α activation exerts protective effects against renal fibrosis. However, its specific role in progressive renal fibrosis remains unclear. Here, we investigated the effects of the long-term tubular activation of HIF-2α on renal function and fibrosis, using in vivo and in vitro models of renal fibrosis. Progressive renal fibrosis was induced in renal tubular epithelial cells (TECs) of tetracycline-controlled HIF-2α transgenic (Tg) mice and wild-type (WT) controls through a 6-week adenine diet. Tg mice were maintained on doxycycline (DOX) for the diet period to induce Tg HIF-2α expression. Primary TECs isolated from Tg mice were treated with DOX (5 μg/ml), transforming growth factor-β1 (TGF-β1) (10 ng/ml), and a combination of both for 24, 48, and 72 hr. Blood was collected to analyze creatinine (Cr) and blood urea nitrogen (BUN) levels. Pathological changes in the kidney tissues were observed using hematoxylin and eosin, Masson's trichrome, and Sirius Red staining. Meanwhile, the expression of fibronectin, E-cadherin and α-smooth muscle actin (α-SMA) and the phosphorylation of p38 mitogenactivated protein kinase (MAPK) was observed using western blotting. Our data showed that serum Cr and BUN levels were significantly lower in Tg mice than in WT mice following the adenine diet. Moreover, the protein levels of fibronectin and E-cadherin and the phosphorylation of p38 MAPK were markedly reduced in the kidneys of adenine-fed Tg mice. These results were accompanied by attenuated fibrosis in Tg mice following adenine administration. Consistent with these findings, HIF-2α overexpression significantly decreased the expression of fibronectin in TECs, whereas an increase in α-SMA protein levels was observed after TGF-β1 stimulation for 72 hr. Taken together, these results indicate that long-term HIF-2α activation in CKD may inhibit the progression of renal fibrosis and improve renal function, suggesting that long-term renal HIF-2α activation may be used as a novel therapeutic strategy for the treatment of CKD. [BMB Reports 2023; 56(3): 196-201].

Iodomethylcholine Inhibits Trimethylamine-N-Oxide Production and Averts Maternal Chronic Kidney Disease-Programmed Offspring Hypertension.

Chronic kidney disease (CKD) affects 10% of the global population, including pregnant women. Adverse maternal conditions determine the developmental programming of many diseases later in life. We previously demonstrated that adult rat offspring born to dams with CKD developed hypertension and renal hypertrophy. Trimethylamine-N-oxide (TMAO), a uremic toxin derived from the gut microbiota, has been linked to hypertension. This study assesses the effects of TMAO inhibition by iodomethylcholine (IMC) treatment on offspring hypertension programmed by maternal CKD. Female rats were fed either a control or a 0.5% adenine diet before conception, with or without IMC treatment during pregnancy and lactation. Maternal IMC treatment averted maternal CKD-primed offspring hypertension and renal hypertrophy in 12-week-old offspring. Offspring hypertension is associated with increases in the plasma TMAO concentration and oxidative stress and shifts in gut microbiota. The beneficial effects of IMC are related to a reduction in TMAO; increases in genera Acetatifactor, Bifidobacterium, and Eubacterium; and decreases in genera Phocacecola and Bacteroides. Our findings afford insights into the targeting of the gut microbiota to deplete TMAO production, with therapeutic potential for the prevention of offspring hypertension programmed by maternal CKD, although these results still need further clinical translation.

Deletion of the P2Y2 receptor aggravates internal elastic lamina calcification in chronic kidney disease mice through upregulation of alkaline phosphatase and lipocalin-2.

Calcification of the medial layer, inducing arterial stiffness, contributes significantly to cardiovascular mortality in patients with chronic kidney disease (CKD). Extracellular nucleotides block the mineralization of arteries by binding to purinergic receptors including the P2Y2 receptor. This study investigates whether deletion of the P2Y2 receptor influences the development of arterial media calcification in CKD mice. Animals were divided into: (i) wild type mice with normal renal function (control diet) (n= 8), (ii) P2Y2 R-/- mice with normal renal function (n= 8), (iii) wild type mice with CKD (n= 27), and (iv) P2Y2 R-/- mice with CKD (n= 22). To induce CKD, animals received an alternating (0.2-0.3%) adenine diet for 7 weeks. All CKD groups developed a similar degree of chronic renal failure as reflected by high serum creatinine and phosphorus levels. Also, the presence of CKD induced calcification in the heart and medial layer of the aortic wall. However, deletion of the P2Y2 receptor makes CKD mice more susceptible to the development of calcification in the heart and aorta (aortic calcium scores (median ± IQR), CKD-wild type: 0.34 ± 4.3mg calcium/g wet tissue and CKD-P2Y2 R-/- : 4.0± 13.2mg calcium/g wet tissue). As indicated by serum and aortic mRNA markers, this P2Y2 R-/- mediated increase in CKD-related arterial media calcification was associated with an elevation of calcification stimulators, including alkaline phosphatase and inflammatory molecules interleukin-6 and lipocalin 2. The P2Y2 receptor should be considered as an interesting therapeutic target for tackling CKD-related arterial media calcification.

Cognitive impairment is associated with increased central arterial stiffness in an animal model of chronic kidney disease.

AbstractBackgroundThe prevalence of cognitive impairment (CI) and dementia in end‐stage chronic kidney disease (CKD) has been estimated at 30‐60%. Cardiovascular diseases (CVD) including increased central arterial stiffness (CAS) accompany CKD development and contribute to CI. Elevated pulse wave velocity (PWV) is associated with higher mortality in CVD and CKD independently of blood pressure (BP). Marinobufagenin (MBG) is a pro‐fibrotic factor, which increases in CKD and contributes to CAS. The aim of this study was to determine whether CKD potentiates CI development via CAS and cardiovascular remodeling in a rat model of CKD.MethodFour months old male Sprague‐Dawley rats were fed with 0.25% adenine (n=19; CKD group) diet to induce CKD or regular diet (n=16; CTRL group) for 8 weeks. Body weight (BW), BP, heart rate (HR), aortic PWV (aPWV), and behavioral tests were conducted at the end of the study. The level of anxiety was tested in an open field test (OFT). Morris water maze (MWM) was used to test spatial memory. Blood was collected for measurements of plasma sodium, potassium, blood urea nitrogen (BUN), hematocrit and MBG. The data were analyzed using two‐tailed unpaired t‐test and linear regression.ResultAnimals in the CKD group had higher levels of plasma creatinine and BUN, which indicate the development of CKD, higher plasma sodium, potassium, and MBG, and higher aPWV, lower hemoglobin, hematocrit, BW, HR, enlarged hearts, aortae, and kidneys, vs. CTRL group (Table 1). BP was similar in both groups. .Rats in CKD group spent less time in the center of open field compared to their CTRL counterparts, indicating increased anxiety‐like behavior (Figure 1 A, B). Although there was no difference in performance in MWM between the groups, there was a positive correlation between aortic weight and ability to find a hidden platform in the CKD group (Figure 1C).ConclusionWe demonstrated that CKD development in young Sprague‐Dawley rats was accompanied by increased CAS, estimated as aPWV, which developed in blood pressure‐independent manner. Higher PWV was associated with a higher level of anxiety, and aortic tissue remodeling was associated with spatial memory in CKD model.Supported by NIH/NIA IRP

DISC-0974, an Anti-Hemojuvelin (HJV) Monoclonal Antibody, Reduced Hepcidin and Improved Anemia in a Rat Model of Chronic Kidney Disease

Introduction DISC-0974 is a humanized monoclonal antibody against hemojuvelin (HJV) and is currently in Phase I clinical studies. HJV is a pathway-specific coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. Loss of function mutations of the HFE2 gene encoding HJV in humans cause profound reductions in hepcidin synthesis and severe iron overload. Consequently, it is hypothesized that pharmacological reduction of HJV activity will lead to reduction of hepcidin synthesis and increased iron availability. This hypothesis has been confirmed in studies of DISC-0974 in rodents and non-human primates and in healthy human volunteers. We have conducted studies to evaluate the effect of DISC-0974 in an animal model of CKD anemia. Anemia is a common complication in patients with CKD and has been associated with multiple adverse outcomes in this population. In CKD hepcidin is increased because of both reduced renal clearance and increased synthesis. This increase is believed to be a central contributor to the development of anemia by reducing the availability of iron from systemic iron stores and by reducing dietary iron absorption. Supporting this hypothesis, treatment with hepcidin lowering drugs has been demonstrated to increase serum iron and to increase hemoglobin (Sheetz et al, Br J Clin Pharmacol. 2019;85:935-948). We hypothesize that by downregulating hepcidin, DISC-0974 will have beneficial effects in treating anemia in the CKD patients because it will make iron available for improved erythropoiesis and increased hemoglobin synthesis as well as reducing erythropoietin resistance. Material and Methods The effect of DISC-0974 on improving anemia was evaluated in an adenine-induced rat model of CKD. In this study, renal impairment was induced by giving Sprague Dawley rats a diet containing 0.75% adenine for 3 weeks to induce kidney injury and then switched to a control diet for the remainder of the study (i.e., 3 more weeks). DISC-0974 (20 mg/kg) or vehicle (n-5/group) was administered intravenously once per week starting on Day 7 and continuing to Day 35. An additional group of rats (n=5/group) was fed with the control diet for the entire study and dosed intravenously with vehicle. Rats were euthanized on Day 42. Blood was collected on Days 0, 7, 21, 35, and 42 for evaluation of hematology parameters, as well as serum iron and hepcidin. Livers were also collected at study end for evaluation of hepcidin gene (HAMP) mRNA expression level. Results Consistent with a previous report (Sun et al, Nephrol Dial Transplant. 2013;28:1733-1743), the 0.75% adenine diet induced kidney dysfunction, as evidenced by marked increase in both serum creatinine and blood urea nitrogen. Rats on adenine diet developed reduced serum iron, reduced red cell production, and lowered hemoglobin with a hypochromic microcytic profile reflecting reduced iron availability. CKD rats appeared to tolerate DISC-0974 treatment well, as DISC-0974 @ 20mg/kg did not affect body weight. Consistent with the mechanism of action of blocking the formation of the BMP/BMPR/HJV complex, DISC-0974 reduced HAMP gene expression in the liver as measured at the end the study, which led to reduced serum hepcidin and increased serum iron concentration. Cellular hemoglobinization measured as reticulocyte Hemoglobin was increased. The maximum improvement in hemoglobin by DISC-0974 compared to the vehicle group was 17g/L in the rat CKD model. Conclusions We conclude that treatment with DISC-0974 at a dose that reduces hepcidin gene expression is able to increase iron availability and substantially prevents the reduction in hemoglobin that is seen in animals that develop adenine-induced renal impairment. This study supports the development of DISC-0974 for the treatment of patients with CKD anemia. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Perinatal Garlic Oil Supplementation Averts Rat Offspring Hypertension Programmed by Maternal Chronic Kidney Disease

Garlic (Allium sativum) is a functional food, having hydrogen sulfide (H2S)-releasing capacity, which exhibits considerable effects on hypertension and gut microbiota. H2S is strongly associated with hypertension and chronic kidney disease (CKD). Maternal CKD leads to hypertension in adult rat progeny, which was linked to disruption of the gut microbiota. This study validated the benefits of perinatal garlic oil supplementation against offspring hypertension induced by maternal CKD via modulation of H2S signaling, nitric oxide (NO), and the gut microbiota. Before pregnancy, female rats received a 0.5% adenine diet for 3 weeks to develop an animal model to mimic human CKD. Garlic oil (100 mg/kg/day) or vehicle was administered to pregnant rats by oral gavage during gestation and lactation. Perinatal garlic oil supplementation protected against maternal CKD-induced hypertension in offspring at 12 weeks of age. The beneficial effects of garlic oil are associated with enhanced H2S signaling, increased NO bioavailability, and shifts in gut microbiota. Perinatal garlic oil supplementation reduces abundance of genera Variovorax, Nocardia, Sphingomonas, and Rhodococcus. Our findings provide insight into the role of early H2S-targeted intervention as a preventive strategy in hypertension for further translational research.