No rim, a maior parte da carga de glicose filtrada é reabsorvida na porção inicial do túbulo proximal, no qual são co-expressos os transportadores: SGLT2 e GLUT2. No diabetes mellitus ocorre aumento no fluxo transepitelial de glicose, o que decorre de aumento na expressão desses transportadores, e pode ser revertido pelo tratamento com insulina. Os fatores transcricionais HNF1A, HNF4A e FOXA2 são descritos como potenciais reguladores do gene Slc2a2. A proteína AKT medeia efeitos da insulina, e é capaz de ativar fatores transcricionais. O objetivo deste estudo foi investigar em linhagem celular IRPTC, o efeito da alta concentração de glicose e da insulina sobre a expressão de Slc2a2/GLUT2 e Slc5a2/SGLT2, assim como a participação da AKT e dos fatores transcricionais. Observamos que a alta concentração de glicose aumentou a expressão do Slc2a2/GLUT2 e a atividade de ligação dos fatores transcricionais HNF4A e FOXA2 na região promotora do gene Slc2a2, por mecanismo mediado pela AKT. A insulina reverteu o efeito sobre o Slc2a2, porém não alterou o conteúdo de GLUT2. / Glucose filtrated load is reabsorbed in renal proximal tubule by the coordinate action of the glucose transporters SGLT2 and GLUT2. In diabetes, renal glucose reabsorption increases; that involves overexpression of the glucose transporters, and is reversed by insulin therapy. The transcription factors HNF1A, HNF4A and FOXA2 have been proposed as modulators of Slc2a2 gene expression. The AKT protein is an important mediator of insulin action, and has been able to activate transcription factors. The present study investigates in immortalized rat proximal tubule cells the effects of high glucose and insulin concentrations upon the Slc2a2/GLUT2 and Slc5a2/SGLT2 expression, as well as the participation of AKT, HNF1A, HNF4A and FOXA2. On the other hand, 25 mM glucose increased the expression of Slc2a2GLUT2, which was accompanied by increased HNF4A and FOXA2 binding in the Slc2a2 promoter, in an AKT-mediated way. Insulin reversed the Slc2a2 mRNA regulation, but did not alter GLUT2 content.

No rim, a maior parte da carga de glicose filtrada é reabsorvida na porção inicial do túbulo proximal, no qual são co-expressos os transportadores: SGLT2 e GLUT2. No diabetes mellitus ocorre aumento no fluxo transepitelial de glicose, o que decorre de aumento na expressão desses transportadores, e pode ser revertido pelo tratamento com insulina. Os fatores transcricionais HNF1A, HNF4A e FOXA2 são descritos como potenciais reguladores do gene Slc2a2. A proteína AKT medeia efeitos da insulina, e é capaz de ativar fatores transcricionais. O objetivo deste estudo foi investigar em linhagem celular IRPTC, o efeito da alta concentração de glicose e da insulina sobre a expressão de Slc2a2/GLUT2 e Slc5a2/SGLT2, assim como a participação da AKT e dos fatores transcricionais. Observamos que a alta concentração de glicose aumentou a expressão do Slc2a2/GLUT2 e a atividade de ligação dos fatores transcricionais HNF4A e FOXA2 na região promotora do gene Slc2a2, por mecanismo mediado pela AKT. A insulina reverteu o efeito sobre o Slc2a2, porém não alterou o conteúdo de GLUT2. / Glucose filtrated load is reabsorbed in renal proximal tubule by the coordinate action of the glucose transporters SGLT2 and GLUT2. In diabetes, renal glucose reabsorption increases; that involves overexpression of the glucose transporters, and is reversed by insulin therapy. The transcription factors HNF1A, HNF4A and FOXA2 have been proposed as modulators of Slc2a2 gene expression. The AKT protein is an important mediator of insulin action, and has been able to activate transcription factors. The present study investigates in immortalized rat proximal tubule cells the effects of high glucose and insulin concentrations upon the Slc2a2/GLUT2 and Slc5a2/SGLT2 expression, as well as the participation of AKT, HNF1A, HNF4A and FOXA2. On the other hand, 25 mM glucose increased the expression of Slc2a2GLUT2, which was accompanied by increased HNF4A and FOXA2 binding in the Slc2a2 promoter, in an AKT-mediated way. Insulin reversed the Slc2a2 mRNA regulation, but did not alter GLUT2 content.

Sodium-Glucose Linked Transporter 2 (SGLT2) inhibitors block renal glucose reabsorption and have shown marked cardiac protection in type 2 diabetics, and surprisingly, also in non-diabetics. However, the mechanism by which these drugs improve cardiovascular outcomes is unknown. Metabolic heart disease, which is characterized by cardiac hypertrophy and diastolic dysfunction, is associated with obesity and insulin resistance and leads to adverse cardiovascular outcomes including heart failure with a preserved ejection fraction. A high fat, high sucrose “Western” diet can induce metabolic syndrome, an aggregate of obesity-driven clinical phenotypes including insulin resistance, elevated triglycerides, hypertension, and abnormal cholesterol. Using a mouse model of metabolic syndrome and adult rat ventricular myocytes (ARVMs) in vitro, we aim to determine if the SGLT2 inhibitor Ertugliflozin (ERTU) can prevent metabolic syndrome-induced cardiac pathophysiology and whether ERTU can exert a direct action on cardiomyocytes, a cell type lacking SGLT2. SGLT2 inhibitors have been proposed to act directly on the Sodium-Hydrogen Exchanger 1 (NHE1) and thus could have direct action on cardiomyocytes that may mediate cardioprotective effects. Mice were fed either a control diet (CD) or a high fat high sucrose (HFHS) diet ± ERTU for 16 weeks. Echocardiography was performed and heart weights were obtained. ARVMs were used to assess ERTU’s effect on insulin sensitivity in vitro in a high-palmitate, insulin resistance model, and to test the efficacy of the known NHE1 inhibitor Cariporide (NHEi). A NHE1 activity ammonium chloride pulse assay was performed in HEK293 cells over-expressing either wild-type (WT) or a known NHEi-insensitive point mutant NHE1 ± NHEi or ERTU. In HFHS-fed mice, ERTU attenuated weight gain and restored blood glucose, insulin, hemoglobin A1c, and HOMA-IR to CD levels. HFHS-induced cardiac hypertrophy and diastolic dysfunction were prevented with ERTU. In vitro, high palmitate media decreased insulin stimulated AKT signaling compared to low palmitate media and was rescued by either ERTU or NHEi treatment. ERTU inhibited WT NHE1 activity in HEK293 cells by 67%, whereas activity of the NHEi-insensitive mutant NHE1 was unaffected by ERTU treatment. ERTU prevented the hallmarks of diet-induced metabolic heart disease (cardiac hypertrophy and diastolic dysfunction) in mice. These benefits exceed the expected consequences of glucose control alone. The actions of ERTU on ARVMs in vitro suggest the favorable effects on cardiac structure and function may be due, at least in part, to the direct action of the drug on cardiomyocytes. Furthermore, mutational overexpression studies show that ERTU can directly affect NHE1 in cardiac myocytes. Taken together, this thesis provides evidence that the direct cardioprotective effects of ERTU could be via inhibition of NHE1, a critical modulator of intracellular pH and sodium in the cardiomyocyte, with known implications in the pathophysiology of diabetes and heart failure. / 2022-06-04T00:00:00Z

Indiana University-Purdue University Indianapolis (IUPUI) / Recent clinical outcome studies demonstrate that Sodium glucose cotransporter 2 inhibitors (SGLT2i) significantly reduce major adverse cardiovascular events and heart failure outcomes in subjects with type 2 diabetes mellitus. At present, several hypotheses have been proposed to explain the observed cardiovascular benefit of SGLT2i, however, the mechanisms responsible remain to be elucidated. This investigation tested the hypothesis that SGLT2i improves cardiac function and efficiency during acute, regional ischemia/reperfusion injury via preferential shifts in myocardial substrate selection and/or inhibition of cardiac sodium/hydrogen exchanger-1 (NHE-1). Our initial investigation evaluated the effects of 24 hour pretreatment of the SGLT2i canagliflozin on cardiac contractile function, substrate utilization, and efficiency before and during regional myocardial ischemia/reperfusion injury in healthy swine. At the onset of ischemia, canagliflozin increased left ventricular end diastolic and systolic volumes which returned to baseline with reperfusion. This increased end diastolic volume was directly associated with increased stroke volume and stroke work relative to controls during ischemia. Canagliflozin also increased cardiac work efficiency during ischemia relative to control swine. No differences in myocardial substrate uptake of glucose, lactate, fatty acids or ketones were detected between groups. In separate experiments using a longer 60 min coronary occlusion, canagliflozin significantly diminished myocardial infarct size. Subsequent studies investigated the effect of an acute administration (15-30 min pre-treatment) of canagliflozin and the NHE-1i cariporide on cardiac contractile function efficiency in response to myocardial ischemia/reperfusion injury. Similar to our initial studies, canagliflozin increased diastolic filling, stroke work and improved cardiac work efficiency relative to untreated control hearts during the ischemic period. In contrast, cariporide did not alter ventricular filling volume, cardiac output or work efficiency at any time point. Additional examination of AP-1 cells transfected with wild-type NHE-1 showed dose-dependent inhibition of NHE-1 activity by cariporide, while canagliflozin had minimal effect on overall activity. This investigation demonstrates that SGLT2i improves cardiac function and efficiency during acute, regional ischemia in healthy swine. However, the present data fail to support the hypothesis that these SGLT2i-mediated improvements involve either preferential alterations in myocardial substrate utilization or the inhibition of NHE-1 activity.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13558号 / 論医博第2287号 / 新制||医||1067(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 岩田 想, 教授 中川 一路, 教授 妹尾 浩 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Akut hjärtsvikt definieras som en debut eller försämring av hjärtsvikt. Hjärtsvikt är ett allvarligt tillstånd där hjärtat inte upprätthåller adekvat hjärtminutvolym för att möta kroppens metaboliska behov vilket leder till trötthet och försämrad livskvalitet. Symtom som relaterar till systemisk vätskeansamling är vanliga vid akut hjärtsvikt. Vätskeansamling och hypoperfusion ökar morbiditet och mortalitet. Patienter med akut hjärtsvikt behöver således snabba och effektiva behandlingar, men också långtidsbehandling efter utskrivning. Avsvällande/vattendrivande behandling är hörnstenen i den terapeutiska behandlingen och loopdiuretika anses som det mest effektiva och snabbverkande läkemedlet. I dagsläget är empagliflozin ett rekommenderat läkemedel för behandling av kronisk hjärtsvikt med nedsatt ejektionsfraktion, då empagliflozins diuretiska egenskap kan stärka effekten av loopdiuretika.   Syftet med detta examensarbete var att beskriva effekter hos empagliflozin vid behandling av akut hjärtsvikt som en litteraturstudie. En sökning av randomiserade kliniska prövningar gjordes i PubMed där nio artiklar valdes ut för att ingå i detta examenarbete. Resultatet av de granskade artiklarna visade att empagliflozin som tilläggsbehandling till loopdiuretika ökade urinutsöndring hos patienterna. Resultatet visade också en statistiskt signifikant minskning av NT-proBNP i studierna vars behandlingstid var 5, 7, 15 och 30 dagar jämfört med placebo. Vidare minskade empagliflozin också plasmavolymen och mängden urinsyran i plasman. Två av studierna rapporterade en statistiskt signifikant ökning i andel röda blodkroppar i behandlingsgruppen. Insättning av empagliflozin, hos kliniskt stabila sjukhusvårdade patienterna i studien EMPULSE, gav en statistiskt signifikant klinisk nytta definierat som ett hierarkiskt kompositmått av död oavsett orsak, antal hjärtsviktsepisoder, tid till första hjärtsviktsepisod och klinisk meningsfull förbättring i KCCQ-TSS med vinstratio 1,36. Vidare gav empagliflozin en positiv effekt på viktminskning, ökad diuretisk respons, ödemlindring och ökad livskvalitet under 90 dagars behandling. Hos patienter som fick empagliflozin sänktes eGFR något till en början men återställdes senare. Det fanns dock inte någon statistiskt signifikant skillnad i eGFR mellan grupperna. En av studierna visade på en signifikant minskning av biomarkörer hos patienter som fick empagliflozin vilket gav en njurfunktionsskyddande effekt mot akut njurskada.  Slutsatsen var att empagliflozin gav gynnsamma effekter såsom till exempel avsvällande/vätskedrivande effekt och klinisk fördel hos patienter med såväl akut de novo som med dekompenserad kronisk hjärtsvikt vilket betydde att läkemedlet var både säkert och effektivt. I framtiden behöver effekterna dock styrkas genom ytterligare studier med större antal deltagare och under längre behandlingstid efter sjukhusutskrivning.

Yes / Recent clinical trials of the hypoglycaemic sodium-glucose co-transporter-2 (SGLT2) inhibitors, which inhibit renal glucose reabsorption, have reported beneficial cardiovascular outcomes. Whether SGLT2 inhibitors directly affect cardiovascular tissues, however, remains unclear. We have previously reported that the SGLT2 inhibitor canagliflozin activates AMP-activated protein kinase (AMPK) in immortalised cell lines and murine hepatocytes. As AMPK has anti-inflammatory actions in vascular cells, we examined whether SGLT2 inhibitors attenuated inflammatory signalling in cultured human endothelial cells. Incubation with clinically-relevant concentrations of canagliflozin, but not empagliflozin or dapagliflozin activated AMPK and inhibited IL-1β-stimulated adhesion of pro-monocytic U937 cells and secretion of IL-6 and monocyte chemoattractant protein-1 (MCP-1). Inhibition of MCP-1 secretion was attenuated by expression of dominant-negative AMPK and was mimicked by the direct AMPK activator, A769662. Stimulation of cells with either canagliflozin or A769662 had no effect on IL-1β-stimulated cell surface levels of adhesion molecules or nuclear factor-κB signalling. Despite these identical effects of canagliflozin and A769662, IL-1β-stimulated IL-6/MCP-1 mRNA was inhibited by canagliflozin, but not A769662, whereas IL-1β-stimulated c-jun N-terminal kinase phosphorylation was inhibited by A769662, but not canagliflozin. These data indicate that clinically-relevant canagliflozin concentrations directly inhibit endothelial pro-inflammatory chemokine/cytokine secretion by AMPK-dependent and -independent mechanisms without affecting early IL-1β signalling. / Project Grant (PG/13/82/30483 to IPS and TMP) and PhD studentships (FS/16/55/32731 and FS/14/61/31284 to DB and AS) from the British Heart Foundation and an equipment grant (BDA11/0004309 to IPS and TMP) from Diabetes UK. OJK was supported by a Scholarship from the Iraqi Ministry of Higher Education and Scientific Research. TAA was supported by a Libyan Ministry of Education PhD Studentship.

La néphropathie diabétique (DN) est l’une des premières causes de maladie rénale en phase terminale (ESKD). L’ESKD est un important facteur de risque d'insuffisance cardiaque et d'accidents vasculaires cérébraux. La dysfonction du système rénine-angiotensine intrarénal (iRAS) est considérée comme étant l'une des principales causes du développement de la DN. Tous les composants du iRAS sont identifiés dans les cellules épithéliales des tubules rénaux proximaux (RPTCs), y compris l'angiotensinogène (Agt), le seul précurseur de toutes les angiotensines. Notre laboratoire a rapporté précédemment que la surexpression spécifique de l’Agt dans les RPTCs provoque l’hypertension, la protéinurie, la fibrose rénale, l’apoptose et des lésions rénales. Nrf2 (Nuclear factor erythroid 2-related factor 2) est un facteur de transcription qui est exprimé de façon abondante dans les RPTCs et a été considéré comme étant un régulateur central de l'équilibre redox dans les réponses cytoprotectrices cellulaires. Le rôle de l’activation du Nrf2 dans la DN, toutefois, est controversé. L’objectif général de cette thèse est de comprendre le rôle physiologique du Nrf2 dans la DN et d’étudier le(s) mécanisme(s) moléculaire(s) de l’action de Nrf2. Premièrement, nous avons démontré que la délétion génétique de Nrf2 ou l’inhibition pharmacologique de Nrf2 avec de la trigonelline chez les souris Akita diabétiques de type 1 régule à la hausse la voie Ace2/MasR et supprime l’expression de Agt/ACE dans les RPTCs, ce qui a pour effet d'atténuer l’hypertension systémique et les lésions rénales. Conformément, dans les cellules immoratalisées de tubule proximal de rat (IRPTC) en culture, la transfection de ARNsi ou le traitement à la trigonelline empêche la régulation positive de Agt/ACE induite par le HG, avec une baisse subséquente de l’expression des gènes Ace2/MasR. Ces données identifient un nouveau mécanisme dans lequel l’activation de Nrf2 stimule l’expression et l’activation des gènes du iRAS, menant au développement de l’hypertension et de la néphropathie dans le diabète. Deuxièmement, nous avons généré des souris Nrf2 transgéniques qui surexprime spécifiquement Nrf2 dans les RPTCs (souris Nrf2RPTC Tg), sous le contôle du promoteur KAP (kidney specific androgen-regulated protein). Nous avons ensuite croisé les souris Nrf2RPTC Tg avec les 6 souris Akita Nrf2-/- pour générer des souris Akita Nrf2-/- /Nrf2RPTC Tg. Nous avons trouvé que la surexpression de Nrf2 dans les RPTCs des souris Akita Nrf2-/- augmentait significativement l’expression du gène SGLT2, entraînant une élévation du glucose sanguin, du taux de filtration glomérulaire, du rapport albumine/créatinine urinaire et de la fibrose tubulo-interstitielle. Dans les cellules tubulaires proximales humaines immortalisées (HK2), le traitement à l’oltipraz ou la transfection de l’ADNc du NRF2 stimule l’expression de l’ARNm du SGLT2 et l’activité de son promoteur. De plus, des tests de retard sur gel et d’immunoprécipitation de chromatine ont montrés que NRF2 se lie au NRF2-RE du promoteur du SGLT2. En outre, une expression plus élevée de NRF2 et SGLT2 est observée dans les RPTCs de reins de patients diabétiques que dans les reins de patients non diabétiques. Ces données ont établi un nouveau mécanisme de la régulation du NRF2 sur l’expression et l’activation du gène SGLT2, menant à une exacerbation du glucose sanguin, de l’hyperfiltration et des lésions rénales dans le diabète. En somme, cette thèse a démontré que le stress oxidatif (hyperglycémie) induisait l’activation du Nrf2 qui stimulait le iRAS et l’expression de SGLT2, contribuant ainsi à la progression de la DN. Ces études suggèrent que le Nrf2 pourrait être une cible thérapeutique potentielle dans le traitement de la DN et pourront fournir de valabless données pré-cliniques pour les essais cliniques en cours avec le bardoxolone méthyle (un activateur de Nrf2). / Diabetic nephropathy (DN) is one of the leading causes of end-stage kidney disease (ESKD). ESKD is a major risk factor for heart failure and stroke. Dysfunction of intrarenal renin angiotensin system (iRAS) is considered as one of the main reasons that caused the DN. All components of the iRAS are identified in the renal proximal tubule cells (RPTCs), including angiotensinogen (Agt), the sole precursor of all angiotensins. Our lab has previously reported that specific overexpression of Agt in RPTCs induces hypertension, proteinuria, kidney fibrosis, apoptosis and kidney injury. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that abundantly expresses in RPTCs and has been considered as a master regulator of redox balance in cellular cytoprotective responses. The role of Nrf2 activation in DN, however, is not clear. The overall aim of this study is to understand the physiological role of Nrf2 in DN and investigate the molecular mechanism(s) of Nrf2 action. First, we have demonstrated that genetic deletion of Nrf2 or pharmacological blockade of Nrf2 with trigonelline in type 1 diabetic Akita mice effectively upregulates Ace2/MasR and suppresses Agt/ACE expression in isolated RPTCs, resulting in attenuation of systemic hypertension and kidney injury. Consistently, in cultured IRPTCs, Nrf2 siRNA transfection or trigonelline treatment prevents high glucose-induced upregulation of Agt/ACE with downregulation of Ace2/MasR gene expression. These data identified a novel mechanism in which Nrf2 activation stimulates iRAS gene expression and activation, leading to the development of hypertension and nephropathy in diabetes. Second, we have generated Nrf2 transgenic mice under the kidney specific androgen regulated protein (KAP) promoter which specifically overexpress Nrf2 in RPTCs (Nrf2RPTC Tg mice). We further crossbred the Nrf2RPTC Tg mice with Akita Nrf2-/- mice to generate Akita Nrf2-/- /Nrf2RPTC Tg mice. We have found that overexpression of Nrf2 in RPTCs of Akita Nrf2-/- mice significantly unregulated sodium-glucose transporter-2 (SGLT2) expression, resulting in elevation of blood glucose, glomerular filtration rate, albumin-creatinine ratio and tubulointerstitial fibrosis. In 8 immortalized human proximal tubular cells (HK2), oltipraz treatment or NRF2 cDNA transfection stimulated SGLT2 mRNA expression and its promoter activity. Furthermore, NRF2 bound to NRF2- RE of SGLT2 promoter were identified by gel mobility shift assay and chromatin immunoprecipitation assay. Moreover, human diabetic kidneys exhibited higher expression of NRF2 and SGLT2 in RPTCs than non-diabetic kidneys. These data established a novel mechanism of NRF2’s regulation on SGLT2, leading to exacerbation of blood glucose, hyperfiltration and kidney injury in diabetes. In summary, this study documented that activation of Nrf2 in hyperglycemia contributed to the progression of DN via regulation of iRAS and SGLT2, suggesting that Nrf2 might be a potential therapeutic target in the treatment of DN.

Background: Diabetic kidney disease is the leading cause of end-stage renal disease. Administration of ACE inhibitors or/and SGLT2 inhibitors show renoprotective effects in diabetic and other kidney diseases. The underlying renoprotective mechanisms of SGLT2 inhibition, especially in combination with ACE inhibition, are incompletely understood. We used longitudinal intravital microscopy to directly elucidate glomerular hemodynamics on a single nephron level in response to the ACE inhibitor enalapril or/and the SGLT2 inhibitor empagliflozin. Methods: Five weeks after the induction of diabetes by streptozotocin, male C57BL/6 mice were treated with enalapril, empagliflozin, enalapril/empagliflozin or placebo for 3 days. To identify hemodynamic regulation mechanisms, longitudinal intravital multiphoton microscopy was employed to measure single nephron glomerular filtration rate (snGFR) and afferent/efferent arteriole width. Results: Diabetic mice presented a significant hyperfiltration. Compared to placebo treatment, snGFR was reduced in response to enalapril, empagliflozin, or enalapril/empagliflozin administration under diabetic conditions. While enalapril treatment caused significant dilation of the efferent arteriole (12.55 ± 1.46 µm vs. control 11.92 ± 1.04 µm, p < 0.05), empagliflozin led to a decreased afferent arteriole diameter (11.19 ± 2.55 µm vs. control 12.35 ± 1.32 µm, p < 0.05) in diabetic mice. Unexpectedly under diabetic conditions, the combined treatment with enalapril/empagliflozin had no effects on both afferent and efferent arteriole diameter change. Conclusion: SGLT2 inhibition, besides ACE inhibition, is an essential hemodynamic regulator of glomerular filtration during diabetes mellitus. Nevertheless, additional mechanisms—independent from hemodynamic regulation—are involved in the nephroprotective effects especially of the combination therapy and should be further explored in future studies.