Sodium–glucose cotransporter 2 inhibition normalizes glucose metabolism and suppresses oxidative stress in the kidneys of diabetic mice
2018; Elsevier BV; Volume: 94; Issue: 5 Linguagem: Inglês
10.1016/j.kint.2018.04.025
ISSN1523-1755
AutoresShinji Tanaka, Yuki Sugiura, Hisako Saito, Mai Sugahara, Yoshiki Higashijima, Junna Yamaguchi, Reiko Inagi, Makoto Suematsu, Masaomi Nangaku, Tetsuhiro Tanaka,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoIt is unclear whether long-term sodium–glucose cotransporter 2 (SGLT2) inhibition such as that during the treatment of diabetes has deleterious effects on the kidney. Therefore, we first sought to determine whether abnormal glucose metabolism occurs in the kidneys of 22-week-old BTBR ob/ob diabetic mice. Second, the cumulative effect of chronic SGLT2 inhibition by ipragliflozin and 30% calorie restriction, either of which lowered blood glucose to a similar extent, on renal glucose metabolism was evaluated. Mass spectrometry–based metabolomics demonstrated that these diabetic mice exhibited an abnormal elevation in the renal pools of tricarboxylic acid cycle metabolites. This was almost completely nullified by SGLT2 inhibition and calorie restriction. Moreover, imaging mass spectrometry indicated an increased level of the tricarboxylic acid cycle intermediate, citrate, in the cortex of the diabetic mice. SGLT2 inhibition as well as calorie restriction almost completely eliminated citrate accumulation in the cortex. Furthermore, imaging mass spectrometry revealed that the accumulation of oxidized glutathione in the cortex of the kidneys, prominent in the glomeruli, was also canceled by SGLT2 inhibition and calorie restriction. Effects of these beneficial interventions were consistent with improvements in glomerular damage, such as albuminuria, glomerular hyperfiltration, and mesangial expansion. Tubulointerstitial macrophage infiltration and fibrosis were ameliorated only by calorie restriction, which may have been due to autophagy activation, which was observed only with calorie restriction. Thus, chronic SGLT2 inhibition is efficient in normalizing the levels of accumulated tricarboxylic acid cycle intermediates and increased oxidative stress in the kidneys of diabetic mice. It is unclear whether long-term sodium–glucose cotransporter 2 (SGLT2) inhibition such as that during the treatment of diabetes has deleterious effects on the kidney. Therefore, we first sought to determine whether abnormal glucose metabolism occurs in the kidneys of 22-week-old BTBR ob/ob diabetic mice. Second, the cumulative effect of chronic SGLT2 inhibition by ipragliflozin and 30% calorie restriction, either of which lowered blood glucose to a similar extent, on renal glucose metabolism was evaluated. Mass spectrometry–based metabolomics demonstrated that these diabetic mice exhibited an abnormal elevation in the renal pools of tricarboxylic acid cycle metabolites. This was almost completely nullified by SGLT2 inhibition and calorie restriction. Moreover, imaging mass spectrometry indicated an increased level of the tricarboxylic acid cycle intermediate, citrate, in the cortex of the diabetic mice. SGLT2 inhibition as well as calorie restriction almost completely eliminated citrate accumulation in the cortex. Furthermore, imaging mass spectrometry revealed that the accumulation of oxidized glutathione in the cortex of the kidneys, prominent in the glomeruli, was also canceled by SGLT2 inhibition and calorie restriction. Effects of these beneficial interventions were consistent with improvements in glomerular damage, such as albuminuria, glomerular hyperfiltration, and mesangial expansion. Tubulointerstitial macrophage infiltration and fibrosis were ameliorated only by calorie restriction, which may have been due to autophagy activation, which was observed only with calorie restriction. Thus, chronic SGLT2 inhibition is efficient in normalizing the levels of accumulated tricarboxylic acid cycle intermediates and increased oxidative stress in the kidneys of diabetic mice. Diabetic kidney disease is a leading cause of end-stage kidney disease in many countries, although its pathophysiology remains unclear. Sodium–glucose cotransporter 2 (SGLT2) inhibitors have emerged as a new class of antidiabetic drugs. Filtered glucose in the glomerulus is reabsorbed together with sodium, mostly in the S1/S2 segments of the proximal tubules via SGLT2 expressed on the brush border membrane. The reabsorbed glucose then exits via glucose transporter 2 expressed on the basolateral membrane and reenters the circulation. SGLT2 inhibitors block the reabsorption of filtered glucose, increase glucose excretion into urine, and lower blood glucose.1Gilbert R.E. Sodium-glucose linked transporter-2 inhibitors: potential for renoprotection beyond blood glucose lowering?.Kidney Int. 2014; 86: 693-700Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Several concerns have been raised that long-term SGLT2 inhibition may have deleterious effects because it exposes the S3 segment and distal parts of the tubules to increased urine glucose concentrations. Indeed, SGLT2 inhibition increases glucose reabsorption by SGLT1 in the S3 segment,2Rieg T. Masuda T. Gerasimova M. et al.Increase in SGLT1-mediated transport explains renal glucose reabsorption during genetic and pharmacological SGLT2 inhibition in euglycemia.Am J Physiol Renal Physiol. 2014; 306: F188-F193Crossref PubMed Scopus (187) Google Scholar and acute SGLT inhibition decreases medullary oxygen tension,3O'Neill J. Fasching A. Pihl L. et al.Acute SGLT inhibition normalizes O2 tension in the renal cortex but causes hypoxia in the renal medulla in anaesthetized control and diabetic rats.Am J Physiol Renal Physiol. 2015; 309: F227-F234Crossref PubMed Scopus (150) Google Scholar possibly via the increased reabsorption of sodium in the distal part of the tubules. However, how the cellular glucose metabolism in the kidney is altered by long-term SGLT2 inhibition and whether the alteration has deleterious effects remain unknown. So far, most animal studies have failed to distinguish between the direct effect of SGLT2 inhibition and the protective effect caused by lowered blood glucose levels. Only a few studies have investigated the differences between animals with similar blood glucose levels that were treated with SGLT2 inhibitors or insulin, but these studies yielded inconsistent results.4Kojima N. Williams J.M. Takahashi T. et al.Effects of a new SGLT2 inhibitor, luseogliflozin, on diabetic nephropathy in T2DN rats.J Pharmacol Exp Ther. 2013; 345: 464-472Crossref PubMed Scopus (128) Google Scholar, 5Gangadharan Komala M. Gross S. Mudaliar H. et al.Inhibition of kidney proximal tubular glucose reabsorption does not prevent against diabetic nephropathy in type 1 diabetic eNOS knockout mice.PLoS One. 2014; 9: e108994Crossref PubMed Scopus (53) Google Scholar In this study, we investigated the long-term effects of ipragliflozin treatment in a mouse model of type 2 diabetes, with a focus on glucose metabolism in the kidney, using mass spectrometry (MS)-based metabolomics as well as metabolite imaging by imaging mass spectrometry (IMS). A calorie restriction model was utilized to achieve blood glucose levels similar to those obtained with ipragliflozin treatment because SGLT2 inhibitors are possibly a “calorie restriction mimetic,”6Kalra S. Gupta Y. Patil S. Sodium-glucose cotransporter-2 inhibition and the insulin: glucagon ratio: unexplored dimensions.Indian J Endocrinol Metab. 2015; 19: 426-429Crossref PubMed Scopus (24) Google Scholar, 7Kalra S. Jacob J.J. Gupta Y. Newer antidiabetic drugs and calorie restriction mimicry.Indian J Endocrinol Metab. 2016; 20: 142-146Crossref PubMed Scopus (20) Google Scholar given that these drugs enhance glucose (calorie) excretion into urine. Because our pilot study using BTBR ob/ob mice suggested that 30% calorie restriction resulted in a blood glucose–lowering effect similar to that of ipragliflozin treatment, we studied the effects of ipragliflozin in comparison with 30% calorie restriction and vehicle treatment in BTBR ob/ob mice as well as in BTBR wild-type mice as control subjects. These interventions were applied for 18 weeks (from 4 to 22 weeks of age) until killing. We first attempted to visualize the distribution of chronically administered ipragliflozin in the kidneys of 22-week-old BTBR ob/ob mice by employing Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS)-based imaging. The ion signal for the drug at m/z 403.102 corresponding to the [M-H]- ion of C21H21FO5S was observed only in the kidneys of ipragliflozin-treated mice (Figure 1a). The signal was prominent in the superficial aspect of the cortex and was not evident in the glomeruli, which is in accordance with the fact that SGLT2 is selectively expressed in the S1 and S2 segments of the proximal tubules (Figure 1b–g). Then, we characterized the phenotypes of each mouse group. Food intake in the ipragliflozin group was approximately 25% greater than that in the vehicle group during the observation period (Figure 2a), which is in accordance with previous studies, although the mechanism is unknown.8Nagata T. Fukuzawa T. Takeda M. et al.Tofogliflozin, a novel sodium-glucose co-transporter 2 inhibitor, improves renal and pancreatic function in db/db mice.Br J Pharmacol. 2013; 170: 519-531Crossref PubMed Scopus (75) Google Scholar, 9Terami N. Ogawa D. Tachibana H. et al.Long-term treatment with the sodium glucose cotransporter 2 inhibitor, dapagliflozin, ameliorates glucose homeostasis and diabetic nephropathy in db/db mice.PLoS One. 2014; 9: e100777Crossref PubMed Scopus (231) Google Scholar, 10Ferrannini G. Hach T. Crowe S. et al.Energy balance after sodium-glucose cotransporter 2 inhibition.Diabetes Care. 2015; 38: 1730-1735Crossref PubMed Scopus (228) Google Scholar The body weight increased in the following order: wild type < calorie restriction < vehicle < ipragliflozin (Figure 2b). Despite the higher body weight, the blood glucose level in the ipragliflozin group was significantly lower than that in the vehicle group and comparable with that in the calorie restriction group at 10, 16, and 22 weeks of age (Figure 2c). BTBR ob/ob mice excreted a significant amount of glucose in the urine at as early as 4 weeks of age, whereas wild-type mice did not exhibit such glucosuria (Figure 2d). Calorie restriction significantly reduced urinary glucose levels at all time points examined compared with the vehicle group. By contrast, the urinary glucose levels in the ipragliflozin group, which were significantly higher than those in the calorie restriction group at all time points, were higher than those in the vehicle group at 10 weeks and similar to those in the vehicle group at 16 and 22 weeks, perhaps reflecting that the effects of reduction in blood glucose (filtered glucose) levels were eliminated by the inhibition of glucose reabsorption in the ipragliflozin group. The blood and urine glucose data of the ipragliflozin and calorie restriction groups emphasize that SGLT2 inhibition markedly alters renal glucose handling and exposes tubular cells to significantly elevated urinary glucose levels. Other parameters and expression levels of glucose transporters in the kidney at 22 weeks of age are summarized in Supplementary Figure S1 and Supplementary Table S1. To determine the metabolic fates of excessive glucose in the diabetic kidney, we performed MS-based metabolome analysis using whole kidney tissues, with a focus on glucose metabolic pathways. We then assessed the effects of ipragliflozin treatment and calorie restriction on the detected metabolites. We found decreased tissue glucose content in diabetic kidneys, whereas fructose was detected only in the kidneys of ob/ob mice (Figure 3a), indicating that the glucose taken into cells was immediately metabolized into downstream pathways, including the polyol pathway. Neither ipragliflozin nor calorie restriction affected the amount of glucose or fructose in the kidney. The pie charts in Figure 3b summarize the fractional contributions of each pathway to the total glucose metabolic intermediates detected. The tricarboxylic acid (TCA) cycle (blue) occupied the largest fraction in the kidneys of the ob/ob vehicle group, whereas glycolysis (green) was dominant in the other 3 groups. By quantifying the metabolites of each pathway (Figure 3c), we noted the remarkably increased pool size of the TCA cycle in ob/ob mice, indicating that excessive glucose in the diabetic kidney was predominantly metabolized through the TCA cycle. Interestingly, this elevation was almost completely normalized by both ipragliflozin treatment and calorie restriction.Figure 3Metabolic shift to the tricarboxylic acid (TCA) cycle in the diabetic kidney (metabolomics analysis). All samples are of the whole kidneys (a–d) or urine (e) from 22-week-old mice in each group. (a) Amounts of glucose and fructose in the kidneys. . (b) Pie graphs showing the fraction of each metabolite in the entire glucose metabolites. The inner and outer pies indicate the fractions of metabolites that belong to the specified metabolic pathways and those of each metabolite species. The circle area reflects the absolute amount of metabolites (per ng kidney). Experiments were repeated at least 3 times with consistent results. Representative pie charts are shown. (c) The total amount of glucose metabolites (nmol/ng kidney) in each pathway (total, glycolysis, TCA cycle, and pentose phosphate pathway [PPP]). (d) Representative citrate imaging mass spectrometry data (m/z 191.0) and metabolomics data of the TCA cycle. The amount of TCA cycle metabolites is shown as nmol/ng kidney. Bar = 1 mm. (e) The amount of metabolites of the TCA cycle in the urine. The value was corrected according to urine creatinine concentration. Data are shown as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. n = 3 to 5 per group (a–d); n = 5 to 8 per group (e). CR, calorie restriction; ipra, ipragliflozin; ND, •••; WT, wild type. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Metabolic shift to the tricarboxylic acid (TCA) cycle in the diabetic kidney (metabolomics analysis). All samples are of the whole kidneys (a–d) or urine (e) from 22-week-old mice in each group. (a) Amounts of glucose and fructose in the kidneys. . (b) Pie graphs showing the fraction of each metabolite in the entire glucose metabolites. The inner and outer pies indicate the fractions of metabolites that belong to the specified metabolic pathways and those of each metabolite species. The circle area reflects the absolute amount of metabolites (per ng kidney). Experiments were repeated at least 3 times with consistent results. Representative pie charts are shown. (c) The total amount of glucose metabolites (nmol/ng kidney) in each pathway (total, glycolysis, TCA cycle, and pentose phosphate pathway [PPP]). (d) Representative citrate imaging mass spectrometry data (m/z 191.0) and metabolomics data of the TCA cycle. The amount of TCA cycle metabolites is shown as nmol/ng kidney. Bar = 1 mm. (e) The amount of metabolites of the TCA cycle in the urine. The value was corrected according to urine creatinine concentration. Data are shown as mean ± SEM. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001. n = 3 to 5 per group (a–d); n = 5 to 8 per group (e). CR, calorie restriction; ipra, ipragliflozin; ND, •••; WT, wild type. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) One limitation of our method is that the pool size of the “glycolysis pathway” indeed reflects both glycolysis and gluconeogenesis. Thus, gluconeogenesis-related gene expression (Pepck and G6pc) was assessed in the kidney. These enzymes tended to be decreased under diabetic conditions and increased by ipragliflozin treatment and calorie restriction (Supplementary Figure S2). These changes in gluconeogenesis provide supporting evidence that the glycolysis pathway itself (relative to TCA cycle) was down-regulated in diabetic kidneys and up-regulated by ipragliflozin treatment and calorie restriction. Furthermore, IMS provided spatial information to identify renal region(s) responsible for the up-regulation of the TCA cycle. Here we focused on citrate, which showed remarkable accumulation in the diabetic kidney, as well as its reduction in response to ipragliflozin treatment and calorie restriction (Figure 3d). Citrate accumulation in the cortex was prominent only in the ob/ob vehicle group, whereas that in the inner medulla was observed in all groups. Importantly, the citrate distribution of the ipragliflozin group was similar to that of the calorie restriction group and did not show any peculiar pattern in each tubular segment. Concomitantly, organic acids of the TCA cycle, including citrate, in the urine were increased by diabetes (Figure 3e), which is in accordance with previous results obtained for Akita mice11You Y.H. Quach T. Saito R. et al.Metabolomics reveals a key role for fumarate in mediating the effects of NADPH oxidase 4 in diabetic kidney disease.J Am Soc Nephrol. 2016; 27: 466-481Crossref PubMed Scopus (127) Google Scholar and db/db mice.12Li M. Wang X. Aa J. et al.GC/TOFMS analysis of metabolites in serum and urine reveals metabolic perturbation of TCA cycle in db/db mice involved in diabetic nephropathy.Am J Physiol Renal Physiol. 2013; 304: F1317-F1324Crossref PubMed Scopus (72) Google Scholar Ipragliflozin treatment and calorie restriction decreased the tissue content of many TCA cycle intermediates in the kidneys (Figure 3d), although they did not change the urine levels of TCA cycle intermediates (Figure 3e). Enhanced oxidative stress is an important pathophysiological index for the progression of diabetic kidney disease. The elevation of TCA cycle metabolites (Figure 3) might result in increased oxidative stress via oxidative phosphorylation (OxPhos) acceleration. We therefore measured reduced glutathione (GSH) and oxidized glutathione (GSSG) levels in whole kidney tissues. Compared with the wild-type mice, significantly diminished GSH levels as well as elevated GSSG levels and GSSG-GSH ratios were detected in the ob/ob mice (Figure 4a), demonstrating enhanced oxidative stress in the diabetic kidney. Both ipragliflozin treatment and calorie restriction attenuated these alterations, although this result was not statistically significant.Figure 4Amelioration of oxidative stress in the cortex by ipragliflozin (ipra) treatment and calorie restriction (CR). (a) Amounts of reduced glutathione (GSH) and oxidized glutathione (GSSG) and the ratio of GSSG-GSH in the whole kidneys (n = 3 to 5 per group). (b) Representative GSSG imaging mass spectrometry data (m/z 611.1). Note that there are many “hot spots” (numbered arrowheads) in which the GSSG level is higher than that in other parts, especially in the ob/ob vehicle group. A merged image of GSSG imaging mass spectrometry and periodic acid–Schiff staining in the kidney of the ob/ob vehicle group is also shown. Bar = 1 mm. Expanded images of the “hot spots” colocalized in the glomeruli are shown at the bottom. . (c) Representative GSH imaging mass spectrometry data (m/z 306.1). A merged image of GSH imaging mass spectrometry and hematoxylin and eosin staining in the kidney of the ob/ob ipragliflozin group is also shown. Bar = 1 mm. (d) Amounts of malondialdehyde in the kidney cortex (n = 5 to 6 per group). Data are shown as mean ± SEM. ∗P < 0.05. WT, wild type. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because IMS allows us to analyze metabolic molecules within microtissue regions, we employed this technology to identify the tissue regions responsible for alterations of GSSG and GSH. GSSG accumulated in the cortex of ob/ob mice, and this accumulation was ameliorated by ipragliflozin treatment and calorie restriction, whereas GSSG accumulation in the inner medulla was not different among the groups (Figure 4b). These distribution patterns are in accordance with those of citrate, which may support a connection between the TCA cycle and oxidative stress, possibly via OxPhos. Interestingly, many “hot spots” of GSSG accumulation were observed in the cortex of the ob/ob vehicle group. By merging the IMS images with periodic acid–Schiff–stained images obtained from the same section, we found that many of the GSSG “hot spots” were colocalized in the glomeruli (Figure 4b). Here ipragliflozin treatment and calorie restriction successfully reduced the intensity of GSSG signals as well as the number of GSSG-accumulated glomeruli. Additionally, GSH was mainly distributed in the glomeruli within the kidney cortex (Figure 4c). The GSH signal in the glomeruli was not evident in the ob/ob vehicle group; however, ipragliflozin treatment and calorie restriction restored the signal. Malondialdehyde, another marker of oxidative stress, was significantly increased in the cortex of ob/ob mice, which was almost completely eliminated by ipragliflozin treatment and calorie restriction (Figure 4d). Taken together, the cortex of diabetic kidneys was exposed to accelerated oxidative stress, and the glomerulus was among the most sensitive components. These results indicate that ipragliflozin treatment and calorie restriction may exhibit renal protection by ameliorating oxidative stress. Markedly decreased oxidative stress in the glomeruli by SGLT2 inhibition and calorie restriction prompted us to evaluate the change in glomerular damage. BTBR ob/ob mice exhibited remarkably increased urinary albumin levels from 10 weeks of age (more than 10 times that in wild-type mice13Brosius F.C. Alpers C.E. Bottinger E.P. et al.Mouse models of diabetic nephropathy.J Am Soc Nephrol. 2009; 20: 2503-2512Crossref PubMed Scopus (433) Google Scholar) (Figure 5a). Both ipragliflozin treatment and calorie restriction significantly reduced urinary albumin levels at 10, 16, and 22 weeks of age, with a greater reduction observed in the calorie restriction group. At 22 weeks of age, ob/ob mice exhibited notable increases in the glomerular filtration rate compared with wild-type mice, suggesting the occurrence of glomerular hyperfiltration (Figure 5b). Ipragliflozin treatment and calorie restriction decreased glomerular filtration rate equally. In addition, mesangial expansion was also equally improved by ipragliflozin treatment and calorie restriction (Figure 5c), although the glomerular tuft area, which was increased in ob/ob mice, was reduced only by calorie restriction (Figure 5d). These findings suggest that both interventions ameliorated glomerular damage, which is in accordance with suppressed oxidative stress in the glomeruli. Finally, we examined whether the normalized glucose metabolism and decreased oxidative stress in the cortex by ipragliflozin treatment resulted in the amelioration of tubulointerstitial lesion. Tubulointerstitial inflammation, which is characterized by macrophage infiltration, is a key step in the progression of diabetic kidney disease because it is closely linked to fibrosis.14Wada J. Makino H. Innate immunity in diabetes and diabetic nephropathy.Nat Rev Nephrol. 2016; 12: 13-26Crossref PubMed Scopus (243) Google Scholar, 15Lin M. Yiu W.H. Li R.X. et al.The TLR4 antagonist CRX-526 protects against advanced diabetic nephropathy.Kidney Int. 2013; 83: 887-900Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 16Lin M. Yiu W.H. Wu H.J. et al.Toll-like receptor 4 promotes tubular inflammation in diabetic nephropathy.J Am Soc Nephrol. 2012; 23: 86-102Crossref PubMed Scopus (274) Google Scholar The number of infiltrating macrophages in the cortex was significantly increased in diabetes and decreased only by calorie restriction (Figure 6a). Only a few macrophages were found within the glomeruli. In accordance with this result, the Ccl2 mRNA expression level in the cortex was decreased by calorie restriction but not by ipragliflozin treatment (Figure 6b). Picrosirius red staining revealed modest but significant tubulointerstitial fibrosis in BTBR ob/ob mice (Figure 6c). This fibrosis was improved with calorie restriction but not with ipragliflozin treatment, which was corroborated by the mRNA levels of fibrosis-related genes (Col1a1 and Acta2) showing similar changes (Figure 6d). Kidney weight was significantly higher in ob/ob mice than in wild-type mice, and calorie restriction but not ipragliflozin treatment significantly decreased the kidney weight (Supplementary Figure S3). These findings suggest that normalized glucose metabolism and decreased oxidative stress in the cortex by ipragliflozin treatment are independent of tubulointerstitial inflammation. Given that autophagy activation by calorie restriction was associated with an improvement in kidney hypertrophy, macrophage infiltration, and fibrosis among diabetic and aged animals,17Kitada M. Takeda A. Nagai T. et al.Dietary restriction ameliorates diabetic nephropathy through anti-inflammatory effects and regulation of the autophagy via restoration of Sirt1 in diabetic Wistar fatty (fa/fa) rats: a model of type 2 diabetes.Exp Diabetes Res. 2011; 2011: 908185Crossref PubMed Scopus (190) Google Scholar, 18Kume S. Uzu T. Horiike K. et al.Calorie restriction enhances cell adaptation to hypoxia through Sirt1-dependent mitochondrial autophagy in mouse aged kidney.J Clin Invest. 2010; 120: 1043-1055Crossref PubMed Scopus (489) Google Scholar we investigated whether autophagy was activated in the kidneys. Immunoblotting using cortex samples demonstrated that p62 accumulation was reduced (Figure 7a), and the LC3 IILC3 I ratio was increased (Figure 7b) only with calorie restriction. The expression of Sirt1, a positive regulator of autophagy,19Lee I.H. Cao L. Mostoslavsky R. et al.A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy.Proc Natl Acad Sci U S A. 2008; 105: 3374-3379Crossref PubMed Scopus (1113) Google Scholar also increased only with calorie restriction (Figure 7c). These results indicate that calorie restriction (but not ipragliflozin treatment) activates autophagy at least in part via restored Sirt1 expression, which may explain the difference in the amelioration of tubulointerstitial lesions between ipragliflozin treatment and calorie restriction. To the best of our knowledge, this is the first study to investigate the long-term effect of SGLT2 inhibition on renal glucose metabolism in the diabetic kidney by employing state-of-the-art MS techniques, including IMS. The obtained data highlights that excessive glucose caused the accumulation of TCA cycle intermediates, and both SGLT2 inhibition and calorie restriction ameliorated this metabolic alteration. Notably, IMS data showed that citrate, a major TCA cycle intermediate, accumulated in the cortex of ob/ob mice, which was ameliorated by both SGLT2 inhibition and calorie restriction. Furthermore, enhanced oxidative stress in the cortex of diabetic kidneys, especially in the glomeruli, was also nullified by these 2 interventions. These results may indicate that in diabetic kidneys, accelerated OxPhos caused by excessive glucose exposure results in high levels of oxidative stress. Reduced oxidative stress in the glomeruli by SGLT2 inhibition and calorie restriction was accompanied by the amelioration of albuminuria, hyperfiltration, and mesangial expansion. Although decreased blood glucose levels by SGLT2 inhibition are likely to contribute to these preferable changes in the glomeruli, the possibility that SGLT2 inhibition in the S1 and S2 segments can directly affect the glomeruli merits further investigation.20Hasegawa K. Wakino S. Simic P. et al.Renal tubular Sirt1 attenuates diabetic albuminuria by epigenetically suppressing claudin-1 overexpression in podocytes.Nat Med. 2013; 19: 1496-1504Crossref PubMed Scopus (312) Google Scholar Tubulointerstitial lesions, including macrophage infiltration, was ameliorated only by calorie restriction but not by ipragliflozin treatment. This suggests that autophagy activation, observed only in the mice undergoing calorie restriction, may be necessary to ameliorate tubulointerstitial lesions (Figure 8). One of the primary findings of this study is that excessive glucose was predominantly metabolized in the TCA cycle in the diabetic kidney, and SGLT2 inhibition is effective in normalizing this metabolic shift to the same extent as calorie restriction. As is well known, SGLT2 inhibition causes unusual glucose handling in the kidney, which has raised concerns regarding its long-term deleterious effect on the kidney, especially the S3 segment and distal parts of the tubules.3O'Neill J. Fasching A. Pihl L. et al.Acute SGLT inhibition normalizes O2 tension in the renal cortex but causes hypoxia in the renal medulla in anaesthetized control and diabetic rats.Am J Physiol Renal Physiol. 2015; 309: F227-F234Crossref PubMed Scopus (150) Google Scholar, 21Layton A.T. Vallon V. Edwards A. Modeling oxygen consumption in the proximal tubule: effects of NHE and SGLT2 inhibition.Am J Physiol Renal Physiol. 2015; 308: F1343-F1357Crossref PubMed Scopus (92) Google Scholar However, here we demonstrated that SGLT2 inhibition does not have such a deleterious effect at least in terms of glucose metabolism in the kidney. Moreover, our results also imply that renal glucose metabolism is primarily affected by blood glucose levels and not by urine glucose levels, given the significant difference in urine glucose levels between
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