Dysfunction of the circadian clock in the kidney tubule leads to enhanced kidney gluconeogenesis and exacerbated hyperglycemia in diabetes

The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes. The circadian clock is a ubiquitous molecular time-keeping mechanism which synchronizes cellular, tissue, and systemic biological functions with 24-hour environmental cycles. Local circadian clocks drive cell type- and tissue-specific rhythms and their dysregulation has been implicated in pathogenesis and/or progression of a broad spectrum of diseases. However, the pathophysiological role of intrinsic circadian clocks in the kidney of diabetics remains unknown. To address this question, we induced type I diabetes with streptozotocin in mice devoid of the circadian transcriptional regulator BMAL1 in podocytes (cKOp mice) or in the kidney tubule (cKOt mice). There was no association between dysfunction of the circadian clock and the development of diabetic nephropathy in cKOp and cKOt mice with diabetes. However, cKOt mice with diabetes exhibited exacerbated hyperglycemia, increased fractional excretion of glucose in the urine, enhanced polyuria, and a more pronounced kidney hypertrophy compared to streptozotocin-treated control mice. mRNA and protein expression analyses revealed substantial enhancement of the gluconeogenic pathway in kidneys of cKOt mice with diabetes as compared to diabetic control mice. Transcriptomic analysis along with functional analysis of cKOt mice with diabetes identified changes in multiple mechanisms directly or indirectly affecting the gluconeogenic pathway. Thus, we demonstrate that dysfunction of the intrinsic kidney tubule circadian clock can aggravate diabetic hyperglycemia via enhancement of gluconeogenesis in the kidney proximal tubule and further highlight the importance of circadian behavior in patients with diabetes. Translational StatementIn diabetes, the kidney contributes to the development of diabetic hyperglycemia by increasing glucose reabsorption from primary urine and by upregulating gluconeogenesis in the proximal tubule. However, these 2 processes are also controlled by the circadian clock, a mechanism that synchronizes a variety of specific kidney functions with daily light–dark cycles. Herein, we demonstrate that dysfunction of the intrinsic circadian clock in the renal tubule can aggravate diabetic hyperglycemia via enhancement of renal gluconeogenesis. These results highlight the importance of circadian effects in diabetic patients, with potential implications for glucose management. In diabetes, the kidney contributes to the development of diabetic hyperglycemia by increasing glucose reabsorption from primary urine and by upregulating gluconeogenesis in the proximal tubule. However, these 2 processes are also controlled by the circadian clock, a mechanism that synchronizes a variety of specific kidney functions with daily light–dark cycles. Herein, we demonstrate that dysfunction of the intrinsic circadian clock in the renal tubule can aggravate diabetic hyperglycemia via enhancement of renal gluconeogenesis. These results highlight the importance of circadian effects in diabetic patients, with potential implications for glucose management. Diabetes is a systemic disease in which the kidney plays a particular role. In diabetes, the kidney contributes to the development of diabetic hyperglycemia by increasing glucose reabsorption from the primary urine1Vallon V. Glucose transporters in the kidney in health and disease.Pflugers Arch. 2020; 472: 1345-1370Google Scholar and by enhancing glucose production via gluconeogenesis.2Gerich J.E. Role of the kidney in normal glucose homeostasis and in the hyperglycaemia of diabetes mellitus: therapeutic implications.Diabet Med. 2010; 27: 136-142Google Scholar,3Mithieux G. Gautier-Stein A. Rajas F. et al.Contribution of intestine and kidney to glucose fluxes in different nutritional states in rat.Comp Biochem Physiol B Biochem Mol Biol. 2006; 143: 195-200Google Scholar However, long-term elevation of blood glucose levels may, in turn, cause diabetic nephropathy (DN), one of the most serious complications of diabetes, characterized by glomerular, tubular, and vascular damage in the kidney. Although metabolic stress is the primary factor involved in pathogenesis and progression of DN, hyperglycemia alone does not lead to kidney insufficiency in most diabetic patients.4Gheith O. Farouk N. Nampoory N. et al.Diabetic kidney disease: world wide difference of prevalence and risk factors.J Nephropharmacol. 2016; 5: 49-56Google Scholar This suggests that combination with an intercurrent illness or presence of environmental, genetic, or epigenetic "second hits" may be required for initiation and/or accelerated progression of DN. Recent research has suggested that the circadian clock mechanism is at the intersection of several (patho)physiological processes in the kidney.5Firsov D. Bonny O. Circadian rhythms and the kidney.Nat Rev Nephrol. 2018; 14: 626-635Google Scholar Conditional perturbation of the circadian clock in different renal cell types in animal models results in the disruption of circadian rhythms in glomerular filtration rate (GFR),6Ansermet C. Centeno G. Nikolaeva S. et al.The intrinsic circadian clock in podocytes controls glomerular filtration rate.Sci Rep. 2019; 9: 16089Google Scholar partial loss of blood pressure control,7Stow L.R. Richards J. Cheng K.Y. et al.The circadian protein period 1 contributes to blood pressure control and coordinately regulates renal sodium transport genes.Hypertension. 2012; 59: 1151-1156Google Scholar,8Zuber A.M. Centeno G. Pradervand S. et al.Molecular clock is involved in predictive circadian adjustment of renal function.Proc Natl Acad Sci U S A. 2009; 106: 16523-16528Google Scholar substantial alterations in renal metabolic pathways,9Nikolaeva S. Ansermet C. Centeno G. et al.Nephron-specific deletion of circadian clock gene Bmal1 alters the plasma and renal metabolome and impairs drug disposition.J Am Soc Nephrol. 2016; 27: 2997-3004Google Scholar,10Tokonami N. Mordasini D. Pradervand S. et al.Local renal circadian clocks control fluid-electrolyte homeostasis and BP.J Am Soc Nephrol. 2014; 25: 1430-1439Google Scholar and accelerated progression of chronic kidney disease.11Motohashi H. Tahara Y. Whittaker D.S. et al.The circadian clock is disrupted in mice with adenine-induced tubulointerstitial nephropathy.Kidney Int. 2020; 97: 728-740Google Scholar In humans, shift work-related circadian misalignment between the biological clock and feeding and activity rhythms is associated with decreased GFR,12Charles L.E. Gu J.K. Fekedulegn D. et al.Association between shiftwork and glomerular filtration rate in police officers.J Occup Environ Med. 2013; 55: 1323-1328Google Scholar increased urinary albumin excretion,13Boogaard P.J. Caubo M.E. Increased albumin excretion in industrial workers due to shift work rather than to prolonged exposure to low concentrations of chlorinated hydrocarbons.Occup Environ Med. 1994; 51: 638-641Google Scholar nocturia and increased kidney urine production,14Kim S.J. Kim J.W. Cho Y.S. et al.Influence of circadian disruption associated with artificial light at night on micturition patterns in shift workers.Int Neurourol J. 2019; 23: 258-264Google Scholar and increased risk of chronic kidney disease.15Uhm J.Y. Kim H.R. Kang G.H. et al.The association between shift work and chronic kidney disease in manual labor workers using data from the Korea National Health and Nutrition Examination Survey (KNHANES 2011-2014).Ann Occup Environ Med. 2018; 30: 69Google Scholar Interestingly, the circadian clock in the kidney controls, or is interconnected with, many cellular pathways that are involved in the pathogenesis of diabetes and/or DN. For instance, renal tubule-specific knockout of the transcriptional activator BMAL1 (also named ARNTL), a central element of the circadian clock machinery, results in a marked increase in expression levels of mRNAs encoding proteins involved in renal glutamine gluconeogenesis, including glutamine transporter SNAT3 (SLC38A3), glutaminase (GLS), and glutamate dehydrogenase 1 (GLUD1).9Nikolaeva S. Ansermet C. Centeno G. et al.Nephron-specific deletion of circadian clock gene Bmal1 alters the plasma and renal metabolome and impairs drug disposition.J Am Soc Nephrol. 2016; 27: 2997-3004Google Scholar More important, these expression changes occur in normoglycemic knockout animals. Solocinski et al. have demonstrated that the circadian clock protein PER1 is involved in the transcriptional regulation of glucose transporter Sglt1 (Slc5a1) in proximal tubule cells,16Solocinski K. Richards J. All S. et al.Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells.Am J Physiol Renal Physiol. 2015; 309: F933-F942Google Scholar and Ansermet et al. have shown that the circadian clock in podocytes controls expression of the Arhgap24 gene associated with predisposition to DN in both type 1 and type 2 diabetes.6Ansermet C. Centeno G. Nikolaeva S. et al.The intrinsic circadian clock in podocytes controls glomerular filtration rate.Sci Rep. 2019; 9: 16089Google Scholar Both high glucose levels and tubular deficiency of BMAL1 have been shown to strongly induce expression of cyclin-dependent kinase inhibitor p21CIP1 (Cdkn1a), a critical factor in triggering cellular senescence in the diabetic kidney.9Nikolaeva S. Ansermet C. Centeno G. et al.Nephron-specific deletion of circadian clock gene Bmal1 alters the plasma and renal metabolome and impairs drug disposition.J Am Soc Nephrol. 2016; 27: 2997-3004Google Scholar,17Wolf G. Cell cycle regulation in diabetic nephropathy.Kidney Int Suppl. 2000; 77: S59-S66Google Scholar The nicotinamide adenine dinucleotide–dependent deacetylase sirtuin 1 (SIRT1), one of the key regulators of podocyte damage in DN,18Papadimitriou A. Silva K.C. Peixoto E.B. et al.Theobromine increases NAD(+)/Sirt-1 activity and protects the kidney under diabetic conditions.Am J Physiol Renal Physiol. 2015; 308: F209-F225Google Scholar has been identified as a master regulator of the energy feedback loop within the core clock network.19Perelis M. Ramsey K.M. Bass J. The molecular clock as a metabolic rheostat.Diabetes Obes Metab. 2015; 17: 99-105Google Scholar Another cellular mechanism that links DN with the circadian clock is the mammalian target of rapamycin, a kinase that coordinates cellular metabolism with circadian time-keeping.20Ramanathan C. Kathale N.D. Liu D. et al.mTOR signaling regulates central and peripheral circadian clock function.PLoS Genet. 2018; 14e1007369Google Scholar Altered mammalian target of rapamycin signaling has also been recognized as an important pathogenic factor in diabetes-induced damage of renal tubular cells,21Sakaguchi M. Isono M. Isshiki K. et al.Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice.Biochem Biophys Res Commun. 2006; 340: 296-301Google Scholar podocytes,22Godel M. Hartleben B. Herbach N. et al.Role of mTOR in podocyte function and diabetic nephropathy in humans and mice.J Clin Invest. 2011; 121: 2197-2209Google Scholar and glomerular endothelial23Lenoir O. Jasiek M. Henique C. et al.Endothelial cell and podocyte autophagy synergistically protect from diabetes-induced glomerulosclerosis.Autophagy. 2015; 11: 1130-1145Google Scholar and mesangial24Lu Q. Zhou Y. Hao M. et al.The mTOR promotes oxidative stress-induced apoptosis of mesangial cells in diabetic nephropathy.Mol Cell Endocrinol. 2018; 473: 31-43Google Scholar cells. Collectively, these observations suggest that perturbations of the circadian clock in the kidney may influence the development of diabetic hyperglycemia by stimulating renal tubular gluconeogenesis and/or glucose reabsorption, or by acting as a "second hit" contributing to the pathogenesis of DN. To address these hypotheses, we generated and characterized mice with streptozotocin (STZ)-induced type I diabetes and a specific deletion of the circadian clock coordinator Bmal1 in glomerular podocytes or in the renal tubule. Animals were maintained ad libitum on the standard laboratory chow diet (KLIBA NAFAG diet 3800). All experiments were performed on male mice. Inactivation of the Bmal1 (Arntl) gene was induced by 2-week treatment with doxycycline (DOX; 2 mg/ml in drinking water) of 8-week-old Bmal1lox/lox/Nphs2-rtTA/LC1 mice (cKOp mice) or of 8-week-old Bmal1lox/lox/Pax8-rtTA/LC1 mice (cKOt mice). Their littermate controls (Bmal1lox/lox mice) received the same DOX treatment. Both models have been previously described and validated.6Ansermet C. Centeno G. Nikolaeva S. et al.The intrinsic circadian clock in podocytes controls glomerular filtration rate.Sci Rep. 2019; 9: 16089Google Scholar,9Nikolaeva S. Ansermet C. Centeno G. et al.Nephron-specific deletion of circadian clock gene Bmal1 alters the plasma and renal metabolome and impairs drug disposition.J Am Soc Nephrol. 2016; 27: 2997-3004Google Scholar One week after the end of DOX treatment, type I diabetes was induced by i.p. injections of STZ (50 mg/kg body weight [BW]; daily for 5 days). Vehicle-treated mice received vehicle injections of phosphate-buffered saline. All experiments were performed 8 weeks after the last STZ or vehicle injection. In all experiments, tissue and blood collection was performed from mice sacrificed at ZT9 (ZT indicates Zeitgeber time units; ZT0 is the time of light on, and ZT12 is the time of light off). Mice were housed in individual metabolic cages (Tecniplast). Urine collection was performed over 24 hours after a 4-day adaptation period. Urinary and plasma Na+, K+, Ca2+, Mg2+, phosphate, creatinine, glucose, urate, and urea concentrations and osmolality were measured by the Laboratoire de prestations de Chimie Clinique of the Centre Hospitalier Universitaire Vaudois. Urinary pH was assessed with a pH meter (Metrohm). Blood pH and blood gases were measured on mixed arterial-venous blood using an epoc blood analyzer (Siemens Healthcare). Urinary ammonium was measured using the Berthelot method. Urinary titratable acid was measured using the method of Chan.25Chan J.C. The rapid determination of urinary titratable acid and ammonium and evaluation of freezing as a method of preservation.Clin Biochem. 1972; 5: 94-98Google Scholar Plasma aldosterone levels were measured by radioimmunoassay (DPC). Plasma insulin was determined using a kit from Mercodia. GFR was measured on anesthetized animals with inulin–fluorescein isothiocyanate, as previously described.26Eisner C. Faulhaber-Walter R. Wang Y. et al.Major contribution of tubular secretion to creatinine clearance in mice.Kidney Int. 2010; 77: 519-526Google Scholar After 15 hours of fasting, vehicle- or STZ-treated control and cKOt mice were i.p. injected with glucose (1 g/kg of BW). Glycemia was measured with glycemia reader in a drop of tail blood (Contour Next One; Bayer) before (time = 0) and 15, 30, 60, 90, 120, and 180 minutes after glucose injection. After 4 hours of food restriction, vehicle- or STZ-treated control and cKOt mice were i.p. injected with 0.5 U of insulin (human recombinant insulin; Sigma) per kg of BW. Glycemia was measured before insulin injection (time = 0) and 15, 30, 45, 60, 90, 120, 150, and 180 minutes after insulin injection. If glycemia decreased below 2 mM, mice were rescued by i.p. injection of 30 mg of glucose and were excluded from the analysis. RNA sequencing was performed as described in Ansermet et al.6Ansermet C. Centeno G. Nikolaeva S. et al.The intrinsic circadian clock in podocytes controls glomerular filtration rate.Sci Rep. 2019; 9: 16089Google Scholar Mus musculus GRCm38 .92 gene annotation was used. Gene counts were scaled using trimmed mean of M values (TMM) normalization and log transformed into counts per million (CPM) using the "cpm" function from the limma R package.27Ritchie M.E. Phipson B. Wu D. et al.limma powers differential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015; 43: e47Google Scholar Differential expression between knockout and control animals was computed for the untreated (phosphate-buffered saline) and the treated (STZ) conditions and the interaction between the 2. For each of the 3 comparisons, genes with a false discovery rate (FDR) < 5% were selected for a gene ontology "Biological Process" enrichment analysis with "clusterProfiler."28Yu G. Wang L.G. Han Y. et al.clusterProfiler: an R package for comparing biological themes among gene clusters.Omics. 2012; 16: 284-287Google Scholar Terms with a Q-value < 0.01 were considered as significant and further processed with the function "simplify" with default parameters to remove redundancy. All data are expressed as mean ± SEM. Statistical tests are described in figure legends and in Supplementary Table S10. P < 0.05 was considered significant. Statistical analysis was performed using GraphPad Prism software (version 8.2.1). Capillary Western blots were performed at the University of Lausanne Protein Analysis Facility (https://www.unil.ch/paf/home/menuinst/technologies/western-in-capillaries.html). Their quantitation was performed in a blinded manner by a technician from this facility. Podocyte-specific inactivation of Bmal1 (Arntl) was induced by 2-week treatment with DOX (2 mg/ml in drinking water) of 8-week-old Bmal1lox/lox/Nphs2-rtTA/LC1 mice (hereinafter referred to as cKOp mice).6Ansermet C. Centeno G. Nikolaeva S. et al.The intrinsic circadian clock in podocytes controls glomerular filtration rate.Sci Rep. 2019; 9: 16089Google Scholar Their littermate controls (Bmal1lox/lox mice; hereinafter referred to as control mice) received the same DOX treatment. As shown in Figure 1a, STZ treatment (see Methods) led to hyperglycemia, which was not different between control and cKOp mice. BW was lower in STZ-treated animals, but this effect was similar in mice of both genotypes (Figure 1b). The GFR, measured using inulin–fluorescein isothiocyanate clearance, was not different between STZ-treated control and cKOp mice (Figure 1c). Diabetic animals showed glucosuria (Figure 1d), polyuria (Figure 1e), low-molecular-weight proteinuria (Figure 1f), and slight albuminuria (Figure 1f). However, there was no difference in these parameters between STZ-treated control and cKOp mice. As shown in Figure 2a, BW was not different in diabetic control mice and mice devoid of BMAL1 in the renal tubule (DOX-treated Bmal1lox/lox/Pax8-rtTA/LC1 mice9Nikolaeva S. Ansermet C. Centeno G. et al.Nephron-specific deletion of circadian clock gene Bmal1 alters the plasma and renal metabolome and impairs drug disposition.J Am Soc Nephrol. 2016; 27: 2997-3004Google Scholar; hereinafter referred to as cKOt mice). Blood plasma analysis revealed that STZ-induced hyperglycemia is exacerbated in cKOt mice in both fed and fasted conditions (Figure 2b and c, respectively). None of the other measured plasma parameters, including osmolality and sodium, potassium, phosphate, calcium, magnesium, creatinine, urate, and aldosterone concentrations, was different between STZ-treated control and cKOt mice, except for increased plasma urea levels in cKOt mice (Supplementary Table S1). Plasma insulin levels were not different between STZ-treated control and cKOt fasted mice (Figure 2d). Glucose and insulin tolerance tests assessed as the slope of the declining glucose concentration did not reveal significant differences between mice of either genotype in both treatment conditions (Figure 2e and f, respectively). Diabetes-induced kidney hypertrophy was more pronounced in diabetic cKOt mice than in diabetic controls (Figure 2g). GFR was not different between STZ-treated mice of both genotypes (Figure 2h). The STZ-induced increase in water intake and urine volume was more pronounced in cKOt mice (Figure 3a and b, respectively), whereas urine osmolality was not different between diabetic mice of both genotypes (Figure 3c). Fractional excretion of glucose was higher and fractional excretion of sodium was lower in STZ-treated cKOt mice compared with STZ-treated controls (Figure 3d and e, respectively), whereas fractional excretion values of phosphate (Figure 3f), magnesium (Figure 3g), and calcium (Figure 3h) were not different between diabetic mice of both genotypes. Both control and cKOt mice treated with STZ had similar low-molecular-weight proteinuria but no albuminuria (Supplementary Figure S1). No major histologic differences were found between kidneys of vehicle- or STZ-treated control and cKOt mice (Supplementary Figure S2).Figure 3Water intake and urine characteristics of vehicle- or streptozotocin (STZ)-treated control and cKOt mice. (a) The 24-hour water intake. n = 8 for vehicle-treated control or cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (b) The 24-hour urine volume. n = 8 for vehicle-treated control or cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (c) Urine osmolality. n = 8 for vehicle-treated control mice, n = 7 for vehicle-treated cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (d) Fractional excretion (Fe) of glucose. n = 8 for vehicle-treated control or cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (e) Fe of sodium. n = 8 for vehicle-treated control mice, n = 7 for vehicle-treated cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (f) Fe of phosphate. n = 8 for vehicle-treated control mice, n = 7 for vehicle-treated cKOt mice, n = 8 for STZ-treated control mice, and n = 9 for STZ-treated cKOt mice. (g) Fe of magnesium. n = 8 for vehicle-treated control mice, n = 7 for vehicle-treated cKOt mice, and n = 9 for STZ-treated control or cKOt mice. (h) Fe of calcium. n = 8 for vehicle-treated control mice, n = 7 for vehicle-treated cKOt mice, and n = 9 for STZ-treated control or cKOt mice. Means ± SEM are given. Two-way analysis of variance with the Sidak multiple comparisons test was used. ∗P < 0.05, †P < 0.01, ‡P < 0.001. BW, body weight.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To identify molecular pathways associated with exacerbated hyperglycemia in STZ-treated cKOt mice, we performed RNA-sequencing analysis of kidney transcriptomes followed by whole-transcriptome pathway enrichment analysis and specific analyses of RNAs encoding proteins involved in renal gluconeogenesis and renal glucose reabsorption. Comparison of transcriptomes revealed 1833 transcripts differentially expressed between vehicle- and STZ-treated controls (Supplementary Table S2; FDR < 5%), 1784 transcripts differentially expressed between vehicle- and STZ-treated cKOt mice (Supplementary Table S3; FDR < 5%), 3107 transcripts differentially expressed between vehicle-treated control and cKOt mice (Supplementary Table S4; FDR < 5%), and 2667 transcripts differentially expressed between STZ-treated control and cKOt mice (Supplementary Table S5; FDR < 5%). Gene ontology analysis of differentially expressed transcripts revealed enrichment of pathways related to lipid, amino acid, and carboxylic acid metabolism, and organic anion transport between control and cKO mice in both vehicle and STZ treatment groups (Figure 4a and b, respectively, and Supplementary Tables S6 and S7, respectively). A total of 284 transcripts exhibited genotype-by-treatment interaction effects (Supplementary Table S8; FDR < 5%), including Glud1 and G6pc transcripts encoding enzymes involved in renal gluconeogenesis (see below). Gene ontology analysis of transcripts with significant interaction showed enrichment of only a limited number of pathways, mainly related to lipid metabolism and organic anion transport (Figure 4c and Supplementary Table S9). Among the genes encoding transporters involved in glucose reabsorption in the proximal tubule (Sglt1, Sglt2, Glut1, and Glut2), only Glut1 (Slc2a1) displayed higher expression in kidneys of diabetic cKOt mice compared with diabetic controls (Supplementary Table S5). The 2 main gluconeogenic precursors in the kidney are lactate and glutamine.29Gerich J.E. Meyer C. Woerle H.J. et al.Renal gluconeogenesis: its importance in human glucose homeostasis.Diabetes Care. 2001; 24: 382-391Google Scholar As shown in Figure 4d and e and Supplementary Table S5, expression levels of transcripts encoding proteins involved in renal glutamine gluconeogenesis (Snat3, Gls, and Glud1) were increased in kidneys of STZ-treated cKOt mice compared with STZ-treated controls. Conversely, expression of the mRNA encoding glutamate-ammonia ligase (GLUL), which reverses the GLS-driven initial step of glutaminolysis, was decreased. Similarly, expression levels of mRNAs encoding 2 enzymes in the common part of the gluconeogenic pathway (namely, phosphoenolpyruvate carboxykinase [Pck1] and glucose-6-phosphatase [G6pc]), were higher in STZ-treated cKOt mice. Higher expression of GLUD1 and PCK1 in kidneys of diabetic cKOt mice was confirmed at the protein level by capillary Western blot (Figure 4f and Supplementary Figure S3). In the liver of diabetic mice, the expression level of GLUD1 was not different between control and cKOt mice, and expression of PCK1 was lower in cKOt mice (Supplementary Figure S4). Of note, expression levels of Glud1, Snat3, fructose-1,6-biphospatase 2 (Fbp2), and Glut1 were higher and expression levels of Glul, Fbp1, and Glut2 were lower in kidneys of vehicle-treated cKOt mice compared with vehicle-treated controls. The gluconeogenic pathway in the kidney can be influenced by the circadian clock either directly, through transcriptional, translational, or posttranslational control of proteins involved in renal gluconeogenesis, or indirectly, by affecting other renal mechanisms that are intrinsically connected to glucose production in the proximal tubule. Transcriptional factors, coactivators, and corepressors that govern transcription of gluconeogenic enzymes have been partially characterized in liver, kidney, and other tissues. Figure 5a and b summarize changes in expression levels of transcripts encoding known transcriptional regulators of gluconeogenesis in kidneys of control and cKOt mice (see also Supplementary Tables S4 and S5). Among the analyzed transcripts, those encoding peroxisome proliferator-activated receptor δ (PPARδ) and cryptochromes 1 and 2 showed a substantial increase, whereas nuclear receptor NR1D1 (also known as REV-ERBα) was substantially decreased in kidneys of cKOt mice in both vehicle- and STZ-treated animals compared with controls with the same treatment. Expression of peroxisome proliferator-activated receptor γ coactivator 1-α (Pgc1α, Ppargc1a) was increased in STZ-treated cKOt mice compared with STZ-treated controls, and expression of glucocorticoid receptor (Gr, Nr3c1) was decreased in vehicle-treated cKOt mice compared with vehicle-treated controls. Expression of forkhead O box protein (Foxo1), hepatocyte nuclear factor 4 α (Hnf4a), cyclic adenosine monophosphate responsive element binding protein (Creb1), Pparα, Sirt1, CREB-binding protein (Cbp, Crebbp), CREB-regulated transcriptional coactivator 2 (Crtc2), and histone deacetylase 3 (Hdac3) was unaffected by treatment or genotype. Because acidosis is the major indirect stimulus for renal gluconeogenesis, we measured blood and urinary pH, blood gases, urinary excretion of ammonia (NH3/NH4+), and titratable acidity. As shown in Figure 6, blood pH was higher in vehicle-treated cKOt mice compared with vehicle-treated controls but was not different between diabetic mice of both genotypes. Plasma bicarbonate was lower in diabetic controls compared with vehicle-treated controls; however, no difference was found in plasma bicarbonate between diabetic control and cKOt mice. Plasma base excess and urine pH were not affected by treatment or genotype. However, the STZ-treated cKOt mice excreted increased amounts of ammonia and titratable acidity compared with STZ-treated controls. Analysis of transcripts encoding proteins involved in renal acid–base handling revealed decreased expression of sodium-hydrogen exchanger NHE3 (Slc9a3; see Figure 4d and e) and of anion exchanger AE1 (Slc4a1) and increased expression of carbonic anhydrase II (Car2) and b1 subunit of H+-ATPase (Atp6v1b1). However, no difference was observed in