Failure to sense energy depletion may be a novel therapeutic target in chronic kidney disease
2018; Elsevier BV; Volume: 95; Issue: 1 Linguagem: Inglês
10.1016/j.kint.2018.08.030
ISSN1523-1755
AutoresHiroaki Kikuchi, Emi Sasaki, Naohiro Nomura, Takayasu Mori, Yoji Andrew Minamishima, Yuki Yoshizaki, Naohiro Takahashi, Taisuke Furusho, Yohei Arai, Shintaro Mandai, Takahiro Yamashita, Fumiaki Ando, Yasuhiro Maejima, Kiyoshi Isobe, Tomokazu Okado, Tatemitsu Rai, Shinichi Uchida, Eisei Sohara,
Tópico(s)Bipolar Disorder and Treatment
ResumoThe kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD. The kidneys consume a large amount of energy to regulate volume status and blood pressure and to excrete uremic toxins. The identification of factors that cause energy mismatch in the setting of chronic kidney disease (CKD) and the development of interventions aimed at improving this mismatch are key research imperatives. Although the critical cellular energy sensor 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is known to be inactivated in CKD, the mechanism of AMPK dysregulation is unknown. In a mouse model of CKD, metabolome analysis confirmed a decrease in AMPK activation in the kidneys despite a high AMP: ATP ratio, suggesting that AMPK did not sense energy depletion. Similar AMPK inactivation was found in heart and skeletal muscle in CKD mice. Several uremic factors were shown to inactivate AMPK in vitro and in ex vivo preparations of kidney tissue. The specific AMPK activator A-769662, which bypasses the AMP sensing mechanism, ameliorated fibrosis and improved energy status in the kidneys of CKD mice, whereas an AMP analog did not. We further demonstrated that a low-protein diet activated AMPK independent of the AMP sensing mechanism, leading to improvement in energy metabolism and kidney fibrosis. These results suggest that a failure to sense AMP is the key mechanism underlying the vicious cycle of energy depletion and CKD progression and direct AMPK activation may be a novel therapeutic approach in CKD. Chronic kidney disease (CKD) is characterized by a progressive loss of kidney function and involves multiple pathological processes. In addition to the decline in glomerular filtration rate, CKD is associated with multiple organ damage, cardiovascular disease, and sarcopenia induction.1Hostetter T.H. Chronic kidney disease predicts cardiovascular disease.N Engl J Med. 2004; 351: 1344-1346Crossref PubMed Scopus (143) Google Scholar, 2Fouque D. Kalantar-Zadeh K. Kopple J. et al.A proposed nomenclature and diagnostic criteria for protein-energy wasting in acute and chronic kidney disease.Kidney Int. 2008; 73: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1306) Google Scholar Currently, there are no effective drugs available to prevent CKD progression; therefore, CKD treatment largely relies on dietary intervention, including protein restriction.3Knight E.L. Stampfer M.J. Hankinson S.E. et al.The impact of protein intake on renal function decline in women with normal renal function or mild renal insufficiency.Ann Intern Med. 2003; 138: 460-467Crossref PubMed Scopus (306) Google Scholar, 4Levey A.S. Greene T. Beck G.J. et al.for the Modification of Diet in Renal Disease Study GroupDietary protein restriction and the progression of chronic renal disease: what have all of the results of the MDRD study shown?.J Am Soc Nephrol. 1999; 10: 2426-2439Crossref PubMed Google Scholar The kidneys consume a large amount of energy to regulate body fluids and blood pressure and to excrete waste products and toxins.5Hallows K.R. Mount P.F. Pastor-Soler N.M. et al.Role of the energy sensor AMP-activated protein kinase in renal physiology and disease.Am J Physiol Renal Physiol. 2010; 298: F1067-F1077Crossref PubMed Scopus (126) Google Scholar Among various organs in the body, the kidney has the second highest energy consumption rate after the heart.6Kalim S. Rhee E.P. An overview of renal metabolomics.Kidney Int. 2017; 91: 61-69Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar Therefore, any mismatch between energy supply and demand is expected to cause kidney damage and lead to significant changes in various metabolites in the body. A recent study showed that impaired fatty acid oxidation (FAO) may cause tubulointerstitial fibrosis.7Kang H.M. Ahn S.H. Choi P. et al.Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development.Nat Med. 2015; 21: 37-46Crossref PubMed Scopus (715) Google Scholar Therefore, the identification of factors that cause energy mismatch and development of interventions aimed at improving the energy status in CKD kidney are key research imperatives. However, to date, the mechanisms leading to energy mismatch in CKD remain unclear. The energy-status sensor 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK) is an evolutionarily conserved serine-threonine kinase.8Oakhill J.S. Steel R. Chen Z.P. et al.AMPK is a direct adenylate charge-regulated protein kinase.Science. 2011; 332: 1433-1435Crossref PubMed Scopus (423) Google Scholar AMPK activity is exquisitely sensitive to cellular energy stress, which is reflected in the rising AMP level and an increase in the AMP–adenosine triphosphate (ATP) ratio, indicating that AMPK is a critical cellular energy sensor.8Oakhill J.S. Steel R. Chen Z.P. et al.AMPK is a direct adenylate charge-regulated protein kinase.Science. 2011; 332: 1433-1435Crossref PubMed Scopus (423) Google Scholar To maintain the body energy homeostasis, AMPK activates the pathways associated with ATP production.9Jeon S.M. Regulation and function of AMPK in physiology and diseases.Exp Mol Med. 2016; 48: e245Crossref PubMed Scopus (511) Google Scholar This has led to the emergence of a concept that AMPK activation may be a therapeutic target to slow down the progression of CKD.10Decleves A.E. Sharma K. Novel targets of antifibrotic and anti-inflammatory treatment in CKD.Nat Rev Nephrol. 2014; 10: 257-267Crossref PubMed Scopus (113) Google Scholar, 11Satriano J. Sharma K. Blantz R.C. et al.Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.Am J Physiol Renal Physiol. 2013; 305: F727-F733Crossref PubMed Scopus (53) Google Scholar Previous studies have shown that AMPK plays a role in early renal inflammation and the development of fibrosis in obesity-related kidney disease12Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar in a subtotal nephrectomy model of CKD11Satriano J. Sharma K. Blantz R.C. et al.Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.Am J Physiol Renal Physiol. 2013; 305: F727-F733Crossref PubMed Scopus (53) Google Scholar and in mice with tubule-specific liver kinase B1 (LKB1) deletion.13Han S.H. Malaga-Dieguez L. Chinga F. et al.Deletion of Lkb1 in renal tubular epithelial cells leads to CKD by altering metabolism.J Am Soc Nephrol. 2016; 27: 439-453Crossref PubMed Scopus (76) Google Scholar Several types of AMPK activators have been used in these studies. In a study of high-fat-induced obesity model, the stimulation of AMPK by 5-aminoimidazole 4-carbox-amide-1-beta-D-ribofuranoside (AICAR), which is an AMP mimic, reduced renal inflammation and albuminuria with the use of a high-fat diet.12Decleves A.E. Mathew A.V. Cunard R. et al.AMPK mediates the initiation of kidney disease induced by a high-fat diet.J Am Soc Nephrol. 2011; 22: 1846-1855Crossref PubMed Scopus (176) Google Scholar In a study using a rat model of subtotal nephrectomy, amelioration of the development of kidney fibrosis by the restoration of AMPK activity with metformin, which inhibits mitochondrial ATP synthesis, on the first surgery day of subtotal nephrectomy has been demonstrated.11Satriano J. Sharma K. Blantz R.C. et al.Induction of AMPK activity corrects early pathophysiological alterations in the subtotal nephrectomy model of chronic kidney disease.Am J Physiol Renal Physiol. 2013; 305: F727-F733Crossref PubMed Scopus (53) Google Scholar In a recent study, a specific AMPK agonist, A-769662, was shown to restore the fatty oxidation defect and reduce apoptosis in LKB1-deficient cultured cells.13Han S.H. Malaga-Dieguez L. Chinga F. et al.Deletion of Lkb1 in renal tubular epithelial cells leads to CKD by altering metabolism.J Am Soc Nephrol. 2016; 27: 439-453Crossref PubMed Scopus (76) Google Scholar However, to date, neither the mechanism of dysregulating AMPK activity in CKD nor the type of AMPK activator, which is efficient in the chronic stages of kidney injury, wherein the uremic condition was substantially established, has been elucidated. For the development of an ideal treatment targeting AMPK activation in the clinical setting, the proposed mechanism of AMPK inactivation in CKD needs to be clarified. For detecting early metabolic changes in the kidney caused by CKD, an animal model with persistently elevated levels of serum creatinine but with no severe renal cellular morphological changes was selected for this study. We used kidney samples from C57BL/6 mice that had undergone the sham-operation or 5/6 nephrectomy (5/6 Nx) at the age of 10 weeks (Figure 1a and b). At 8 weeks after sham-operation or 5/6 Nx, we performed the metabolome analysis using capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS). Although we observed subtle histological changes in the kidneys at 8 weeks after 5/6 Nx (Figure 1c), higher expressions of profibrotic markers, including Col1a1, Col3a1, and Fn, were observed in 5/6 Nx kidney samples by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), which indicated that profibrotic changes had certainly occurred at this phase (Figure 1d). Principal component analysis revealed remarkable differences of metabolome profiles in the 5/6 Nx kidney between sham-control mice and 5/6 Nx mice (Supplementary Figure S1A); these findings indicated that impaired renal function had substantially influenced the metabolite profile. Nucleoside triphosphates (i.e., ATP, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate) were all included in the list of top 30 decreased metabolites in 5/6 Nx mice kidney (Table 1), which confirmed the impaired energy status in 5/6 Nx mice kidneys.Table 1Top 30 list of decreased metabolites in 5/6 Nx mice kidney ordered by fold-changesKidneyFold changeP valueNADPH_divalent0.20.185Glutathione (GSH)0.30.3652,3-Diphosphoglyceric acid0.50.006Triethanolamine0.50.472Morpholine0.60.084S-Methylglutathione0.70.0045'-Deoxy-5'-methylthioadenosine0.70.031Phosphocreatine0.70.030Urocanic acid0.70.027Glucose 6-phosphate0.70.010Glutathione (GSSG)_divalent0.70.002UDP-glucuronic acid0.70.001CTPaNucleoside triphosphates (i.e., ATP, GTP, CTP, and UTP) were all included in the top 30 list of decreased metabolites in 5/6 Nx mice kidney. Statistical significance was evaluated using an unpaired t-test. n = 4 for each group. Reference = sham mice group.0.70.040UTPaNucleoside triphosphates (i.e., ATP, GTP, CTP, and UTP) were all included in the top 30 list of decreased metabolites in 5/6 Nx mice kidney. Statistical significance was evaluated using an unpaired t-test. n = 4 for each group. Reference = sham mice group.0.80.015GTPaNucleoside triphosphates (i.e., ATP, GTP, CTP, and UTP) were all included in the top 30 list of decreased metabolites in 5/6 Nx mice kidney. Statistical significance was evaluated using an unpaired t-test. n = 4 for each group. Reference = sham mice group.0.80.072ATPaNucleoside triphosphates (i.e., ATP, GTP, CTP, and UTP) were all included in the top 30 list of decreased metabolites in 5/6 Nx mice kidney. Statistical significance was evaluated using an unpaired t-test. n = 4 for each group. Reference = sham mice group.0.80.010Spermine0.80.089threo-β-Methylaspartic acid0.80.423Threonic acid0.90.132Phosphoenolpyruvic acid0.90.461Cystathionine0.90.034Pterin0.90.587Gln0.90.304Sedoheptulose 7-phosphate0.90.084Sorbitol 6-phosphate0.90.434Betaine aldehyde + H2O0.90.647Cyclohexylamine0.90.654Dyphylline0.90.654NAD+0.90.258γ-Glu-2-aminobutyric acid0.90.625ATP, adenosine triphosphate; CTP, Cytidine triphosphate; Gln, glutamine; GSH, reduced glutathione; GSSG, oxidized glutathione; GTP, guanosine triphosphate; NAD, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide; Nx, nephrectomy; UDP, uridine diphosphate; UTP, Uridine triphosphate.a Nucleoside triphosphates (i.e., ATP, GTP, CTP, and UTP) were all included in the top 30 list of decreased metabolites in 5/6 Nx mice kidney. Statistical significance was evaluated using an unpaired t-test. n = 4 for each group. Reference = sham mice group. Open table in a new tab ATP, adenosine triphosphate; CTP, Cytidine triphosphate; Gln, glutamine; GSH, reduced glutathione; GSSG, oxidized glutathione; GTP, guanosine triphosphate; NAD, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide; Nx, nephrectomy; UDP, uridine diphosphate; UTP, Uridine triphosphate. Actually, we observed that AMPK activity, which is reflected in the ratio of the phosphorylated form of AMPK at Thr 172 to the total AMPK (P-AMPK-AMPK), was significantly decreased in the kidney of 5/6 Nx mice compared with that in the sham-control (Figure 2a). Regarding the downstream target of AMPK, we evaluated the phosphorylated form of acetyl coenzyme A carboxylase (P-ACC) (the rate-limiting enzyme in fatty acid metabolism14Dasgupta B. Chhipa R.R. Evolving lessons on the complex role of AMPK in normal physiology and cancer.Trends Pharmacol Sci. 2016; 37: 192-206Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and phosphorylated Ulk1 at Ser 555 (P-Ulk1) (the first and key component involved in the initiation of autophagy cascade15Egan D.F. Shackelford D.B. Mihaylova M.M. et al.Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.Science. 2011; 331: 456-461Crossref PubMed Scopus (1815) Google Scholar). P-ACC and P-Ulk1 expression levels were decreased in 5/6 Nx mice kidney, which suggested that not only the failure of FAO but also the failure of the induction of autophagy contributed to energy failure in CKD (Figure 2a). Consistent with this observation, qRT-PCR analysis showed that expression levels of FAO-related genes, such as carnitine palmitoyl transferase (Cpt1, Cpt2) and acyl-coenzyme A oxidase (Acox) 1,2, and their key transcriptional regulator complex Pparα–Ppargc1A were markedly lower in 5/6 Nx kidney samples than in sham-control kidney (Figure 2b). As AMPK is well known to be inactivated by energy-repleted conditions, that is, a decrease in the AMP-ATP ratio,9Jeon S.M. Regulation and function of AMPK in physiology and diseases.Exp Mol Med. 2016; 48: e245Crossref PubMed Scopus (511) Google Scholar we assessed the AMP-ATP ratio in the kidney by CE-TOFMS. However, we found that the kidneys from 5/6 Nx mice had lower amounts of ATP and higher amounts of AMP compared with those from sham-control (Figure 2c), indicating AMPK sensing failure to AMP-ATP ratio. The phosphorylated LKB1 expression level at Ser 428 (P-LKB1), an upstream kinase that phosphorylates Thr 172 in AMPK,16Alessi D.R. Sakamoto K. Bayascas J.R. LKB1-dependent signaling pathways.Annu Rev Biochem. 2006; 75: 137-163Crossref PubMed Scopus (633) Google Scholar was not decreased in 5/6 Nx mice kidney (Figure 2a). Supporting this observation, 7-day treatment with AMP mimic, AICAR, did not increase AMPK phosphorylation at Thr 172 and did not change profibrotic and fatty acid oxidation-related markers in 5/6 Nx mice kidneys (Figure 2d, Supplementary Figure S2) despite increased AMPK phosphorylation in sham-control kidneys. To determine whether AMPK dysregulation in kidneys from 5/6 Nx mice is caused by systemic or local factors, we first compared P-AMPK-AMPK status between the sham right intact kidney and the left incised remnant (2/6 Nx) kidney of the sham-control mice. We did not observe a significant change in P-AMPK-AMPK between sham intact kidney and left 2/6 Nx kidney (Figure 3a), suggesting that the change is caused not by the local effects of surgery, but by systemic factors linked to the CKD status. To investigate whether these systemic factors affect distant organs, we compared AMPK activity in the skeletal muscle and heart between sham-control and 5/6 Nx mice. AMPK activity was significantly decreased both in muscle and heart tissues (Figure 3b and c). However, the AMP-ATP ratios of the muscles and heart did not significantly change in 5/6 Nx mice compared with those in sham-control mice (Figure 3d and e). These results strongly indicate the existence of systemic factors, which dysregulate AMPK activity by inducing energy status sensing failure in 5/6 Nx mice. As one of the of the main functions of kidney are to excrete the metabolic wastes and maintain acid–base homeostasis,17Toyohara T. Akiyama Y. Suzuki T. et al.Metabolomic profiling of uremic solutes in CKD patients.Hypertens Res. 2010; 33: 944-952Crossref PubMed Scopus (106) Google Scholar, 18Phisitkul S. Khanna A. Simoni J. et al.Amelioration of metabolic acidosis in patients with low GFR reduced kidney endothelin production and kidney injury, and better preserved GFR.Kidney Int. 2010; 77: 617-623Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 19de Brito-Ashurst I. Varagunam M. Raftery M.J. Yaqoob M.M. Bicarbonate supplementation slows progression of CKD and improves nutritional status.J Am Soc Nephrol. 2009; 20: 2075-2084Crossref PubMed Scopus (630) Google Scholar uremic metabolite accumulation and metabolic acidosis are expected to suppress AMPK activity. To identify uremic metabolites that systemically accumulate in 5/6 Nx mice, we performed CE-TOFMS analysis of muscle and heart metabolites from sham-control and 5/6 Nx mice and listed the top 30 candidates ordered according to fold-changes (Table 2). Principal component analysis revealed remarkable difference of metabolomic profiles in the 5/6 Nx skeletal muscle and heart between sham-control and the 5/6 Nx mice (Supplementary Figure S1B and C). In the top 30 candidates among the groups shown in Table 2, indoxyl 3-sulfate (IS), trimethylamine N-oxide (TMAO), creatinine, allantoin, 6-aminohexanoic acid, and asymmetric dimethylarginine (ADMA) were elevated in >2 organs (Table 2). In addition to these candidate uremic metabolites, we tested the change of pH because CKD patients are known to develop metabolic acidosis.18Phisitkul S. Khanna A. Simoni J. et al.Amelioration of metabolic acidosis in patients with low GFR reduced kidney endothelin production and kidney injury, and better preserved GFR.Kidney Int. 2010; 77: 617-623Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 19de Brito-Ashurst I. Varagunam M. Raftery M.J. Yaqoob M.M. Bicarbonate supplementation slows progression of CKD and improves nutritional status.J Am Soc Nephrol. 2009; 20: 2075-2084Crossref PubMed Scopus (630) Google Scholar We examined the effect of each of these factors on AMPK phosphorylation in HK-2 cells, which is a cultured cell line of human proximal tubular epithelial cells. Among these, ADMA and TMAO showed a strong correlation with the inhibition of AMPK activity, and low extracellular pH inhibited AMPK activity (Figure 4a). In addition, we examined the effect of these uremic factors on AMPK activity using ex vivo preparations of mice kidney slices. In our experiment, low extracellular pH and ADMA significantly suppressed AMPK activity (Supplementary Figure S3). Furthermore, consistent with the in vivo results (Figure 2d), acidic extracellular conditions, IS, and TMAO attenuated AMPK activation by AICAR in HK-2 cells, suggesting sensing failure of AMP-ATP ratio by AMPK under uremic conditions (Figure 4b). Because the spatial differentiation of metabolic profiles within the kidney is well-known,20Uchida S. Endou H. Substrate specificity to maintain cellular ATP along the mouse nephron.Am J Physiol. 1988; 255: F977-F983Crossref PubMed Google Scholar we examined whether this energy-sensing failure also exists in mouse podocyte cells, mpk collecting duct cells, and endothelial cells (human umbilical vein endothelial cells) (Supplementary Figure S4). The levels of sensing failure by uremic metabolites were relatively weaker in mouse podocyte cells compared with those in other cell types. Acidic condition and IS were strongly associated with sensing failure in mpk collecting duct cells, while acidic condition and TMAO were strongly associated with sensing failure in human umbilical vein endothelial cells. These results indicated that levels of sensing failure differed among individual cell types. To confirm the AMPK-inhibitory effects in other organs, we examined the effect of each of these factors on AMPK phosphorylation in murine skeletal myoblast cells (C2C12) and embryonic rat cardiomyocytes (H9C2). Similar to the results observed in HK-2 cells, low extracellular pH, ADMA, and TMAO inhibited AMPK activity. IS also inhibited AMPK activity in a dose-dependent manner in C2C12 and H9C2 cells, an effect that was not observed in HK-2 cells (Supplementary Figures S5A and S6A). In addition, similar to the results in the kidney, acidic extracellular conditions, IS, and TMAO attenuated AMPK activation by AICAR in C2C12 and H9C2 cells (Supplementary Figures S5B and S6B).Table 2Top 30 candidate metabolites accumulated in 5/6 Nx mice kidney, muscle, and heart ordered by fold changeKidneyFold changeP valueMuscleFold changeP valueHeartFold changeP value3-Indoxylsulfuric acidaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.9.20.029Trimethylamine N-oxideaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.3.40.001IMP2.40.258Cystine8.00.0582-Hydroxyvaleric acid1.93.9e−04Ribose 5-phosphate2.40.480Isovalerylalanine-2N-Acetylleucine-26.00.059Diphosphoglycerate1.90.137Homocitrulline2.4NATrimethylamine N-oxideaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.5.60.011Allantoin1.80.015Trimethylamine N-oxideaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.2.30.0101H-Imidazole-4-propionic acid5.10.076Uridine1.7NA6-Aminohexanoic acid2.20.077Phenaceturic acid4.60.039Urea1.64.5e−042-Hydroxyisobutyric acid2.14.9e−04Glucuronic acidGalacturonic acid4.40.073O-Acetylhomoserine2-Aminoadipic acid1.60.0092-Methylserine1.9NACysteine glutathione disulfide4.10.0163-HBA1.50.2053-Indoxylsulfuric acidaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.1.80.0244-Pyridoxic acid3.80.127N-Acetylglycine1.5NANADPH_divalent1.80.161Creatinine3.80.019Citric acid1.50.189Pipecolic acid1.70.007GalactosamineGlucosamine3.80.0891-Methylnicotinamide1.5NA1-Methylnicotinamide1.60.001Glutaric acid3.6NAOphthalmic acid1.40.0061-Methylhistidine3-Methylhistidine1.60.228Homovanillic acid3.60.008Methionine sulfoxide1.42.2e−04Creatinine1.50.0277-Methylguanine3.50.014Adenine1.40.0952-Hydroxyvaleric acid1.50.010Hippuric acid3.50.004GPCho1.40.223Urea1.50.0094-Aminohippuric acid3.40.037N-Acetylaspartic acid1.40.357Carnitine1.50.031Allantoin3.40.022Isethionic acid1.40.080Histamine1.50.1781-Methyl-4-imidazoleacetic acid3.30.0161-Methyladenosine1.30.152CMP1.50.078N-Acetylhistidine3.30.0252-Hydroxyglutaric acid1.30.3171-Methyladenosine1.50.0144-Acetamidobutanoic acid3.20.017Citrulline1.30.032CoA_divalent1.50.0014-Guanidinobutyric acid3.0NAN6-Methyllysine1.20.061Citrulline1.50.0376-Aminohexanoic acid3.00.044ADMAaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.1.20.085Pyridoxal1.40.117ADMAaIndoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs.2.90.008Triethanolamine1.20.489Gluconic acid1.40.013myo-Inositol 1-phosphatemyo-Inositol 3-phosphate2.90.0092-Oxoleucine2K3MVA1.20.5745-Hydroxylysine1.40.195IMP2.90.085PEP1.20.153Ascorbic acid1.40.498N8Oakhill J.S. Steel R. Chen Z.P. et al.AMPK is a direct adenylate charge-regulated protein kinase.Science. 2011; 332: 1433-1435Crossref PubMed Scopus (423) Google Scholar-Acetylspermidine2.91.1e−04Creatinine1.20.223Adenylosuccinic acid1.40.261ButyrylcarnitineIsobutyrylcarnitine2.80.285Trimethyllysine1.20.2272-Hydroxy-4-methylvaleric acid1.40.016Ergothioneine2.70.1384-Methylpyrazole1.2NAUMP1.40.141Adenosine2.70.060dCyt1.20.424N6Kalim S. Rhee E.P. An overview of renal metabolomics.Kidney Int. 2017; 91: 61-69Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar-Methyllysine1.30.232Gluconolactone2.70.010Pipecolic acid1.20.063Hydroxyproline1.30.067ADMA, asymmetric dimethylarginine; CMP, cytidine monophosphate; CoA, coenzyme A; dCyt, 2'-deoxycytidine; GPCho, glycerolphosphocholine; HBA, hydroxybutyrate; IMP, inosine monophosphate; NADPH, nicotinamide adenine dinucleotide phosphate; NA, not applicable; Nx, nephrectomy; PEP, phosphoenolpyruvic acid; UMP, uridine monophosphate.Statistical significance was evaluated using an unpaired t-test. n = 3 for 5/6 Nx mice heart group, and n = 4 for other groups. Reference =sham mice group.a Indoxyl 3-sulfate, trimethylamine N-oxide, and ADMA were elevated in >2 organs. Open table in a new tab ADMA, asymmetric dimethylarginine; CMP, cytidine monophosphate; CoA, coenzyme A; dCyt, 2'-deoxycytidine; GPCho, glycerolphosphocholine; HBA, hydroxybutyrate; IMP, inosine monophosphate; NADPH, nicotinamide adenine dinucleotide phosphate; NA, not applicable; Nx, nephrectomy; PEP, phosphoenolpyruvic acid; UMP, uridine monophosphate. Statistical significance was evaluated using an unpaired t-test. n = 3 for 5/6 Nx mice heart group, and n = 4 for other groups. Reference =sham mice group. As sodium bicarbonate supplementation is known to ameliorate metabolic acidosis,19de Brito-Ashurst I. Varagunam M. Raftery M.J. Yaqoob M.M. Bicarbonate supplementation slows progression of CKD and improves nutritional status.J Am Soc Nephrol. 2009; 20: 2075-2084Crossref PubMed Scopus (630) Google Scholar we treated sham-control and 5/6 Nx mice with sodium bicarbonate in vivo. As expected, AMPK activity was activated in sham-control kidney after the administration of a single i.p. injection of sodium bicarbonate solute. However, AMPK was not activated in 5/6 Nx mice kidney by sodium bicarbonate (Figure 4c), although pH was improved (Supplementary Figure S7), which suggests that AMPK inhibition in 5/6 Nx mice was not caused by a single factor but rather reflected an integrated effect of multiple systemic factors including uremic metabolites and acidic extracellular pH. Dietary protein restriction is known to retard disease progression in advanced CKD patients.3Knight E.L. Stampfer M.J. Hankinson S.E. et al.The impact of protein intake on renal function decline in women with normal renal function or mild renal insufficiency.Ann Intern Med. 2003; 138: 460-467Crossref PubMed Scopus (306) Google Scholar, 4Levey A.S. Greene T. Beck G.J. et al.for the Modification of Diet in Renal Disease Study GroupDietary protein restriction and the progression of chronic renal disease: what have all of the results of the MDRD study shown?.J Am Soc Nephrol. 1999; 10: 2426-2439Crossref PubMed Google Scholar Previous studies have also shown that protein restriction induces AMPK activity in liver and skeletal muscle.21Chotechuang N. Azzout-Marniche D. Bos C. et al.mTOR, AMPK, and GCN2 coordinate the adaptation of hepatic energy metabolic pathways in response to protein intake in the rat.Am J Physiol Endocrinol Metab. 2009; 297: E1313-E1323Crossref PubMed Scopus (84) Google Scholar, 22Mitsuishi M. Miyashita K. Muraki A. et al.Dietary protein decreases exercise endurance through rapamycin-sensitive suppression of muscle mitochondria.Am J Physiol Endocrinol Metab. 2013; 305: E776-E784Crossref PubMed Scopus (8) Google Scholar Here sham-control and 5/6 Nx mice at 6 weeks after 5/6 Nx were fed with a low-protein (LP) (7% protein), middle-protein (24% protein), or high-protein ([HP]; 45% protein) diet for 2 weeks and were then killed. Amount of food intake was almost identical among the groups. LP induced a marked increase in P-AMPK-AMPK, while HP caused a marked decrease in P-AMPK-AMPK in 5/6 Nx mice kidney; however, this phenomenon was not evident in sham-control (Figure 5a). Similar results were obtained from skeletal muscle and heart (Supplementary Figures S8 and S9). Neutrophil gelatinase-associated lipocalin, a marker of kidney damage, expression levels were elevated in inverse proportion to P-AMPK expression levels (Figure 5a). In parallel with significant decrease in P-AMPK expression in 5/6 Nx kidney from mice fed with a HP, P-LKB1 expression level was significantly suppressed. We observed significant reduction in P-ACC and P-Ulk1 expression l
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