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Effects of canagliflozin on renal and urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db diabetic mice

Thanekar, Unmesha Hemant
http://orcid.org/0000-0002-6118-825X
http://rave.ohiolink.edu/etdc/view?acc_num=wright1567169063911723

Abstract Details

2019, Master of Science (MS), Wright State University, Pharmacology and Toxicology.
Diabetes is one of the major causes of chronic kidney disease (CKD) which could lead to end-stage renal disease. Angiotensin-II (Ang-II), a major bioactive peptide of renin angiotensin system (RAS) plays a pivotal role in the pathogenesis and progression of diabetic nephropathy. The reno-degenerative effects of Ang-II can be partially counteracted by Ang (1-7). Therefore, Ang (1-7) forming enzymes, angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP), are hypothesized to have reno-protective effects. In our previous studies, we have demonstrated increased renal shedding of ACE2 and NEP in the urine of type-2 diabetic mice which was attenuated by normalizing hyperglycemia and glycosuria with peroxisome-proliferator activated receptor- γ (PPAR-γ) agonist, rosiglitazone and pioglitazone. Sodium glucose co-transporter-2 (SGLT-2) inhibitors, control hyperglycemia by inhibiting renal glucose reabsorption and inducing glycosuria. The aim of this study is to determine the effect of the SGLT-2 inhibitor, canagliflozin on the metabolic and renal parameters in db/db mice and investigate whether it will attenuate urinary albuminuria and ACE2 shedding in this mice. In addition, we investigated whether glycosuria induced by canagliflozin in non-diabetic normal mice will impact urinary shedding of ACE2 and NEP and cause renal injury. We also investigated the effect of canagliflozin on sirtuin-1 (SIRT1) and some of the markers of renal injury. Six-week old db/db mice and their lean control littermates were fed either normal chow or chow contain canagliflozin (20 mg/kg/day) for 15 weeks. Measurement of urinary albumin, glucose and RAS enzymes expression and activity were performed using 24 hours urine. The db/db mice demonstrated hyperglycemia and early-onset of albuminuria compared to lean control mice (p <0.001). At 5 weeks of age, db/db mice demonstrated significant increase in the urinary expression and activity of ACE2 and NEP, prior to onset of albuminuria. Treatment with canagliflozin for 15 weeks significantly decreased blood glucose in db/db compared to untreated mice (206.58 ± 38.35 vs. 461.82 ± 40.63 mg/dL, p<0.0001), with no effect in lean mice. The blood glucose lowering effect was seen as early as 2 weeks of canagliflozin treatment. Our previous studies demonstrated that treatment with rosiglitazone and pioglitazone, normalizes hyperglycemia and glycosuria and attenuated urinary ACE2 shedding in the db/db mice. However, management of hyperglycemia with canagliflozin was not associated with attenuation of urinary albumin and ACE2. One of the novel findings of this study is the increased urinary NEP expression and activity in the urine of both lean and db/db mice. Since canagliflozin treatment significantly increased glycosuria in both lean and db/db mice, there could be a possible association between NEP and glycosuria. Neutrophil gelatinase-associated lipocalin (NGAL) is one of the well-studied urinary biomarker for CKD. At 5 weeks of age, there was a significant increase in the urinary NGAL expression in the db/db mice compared to the age-matched control mice. Treatment with canagliflozin for 12 weeks attenuated urinary NGAL expression in the db/db mice. There was a significant increase in renal expression and activity of ACE2 in the db/db mice compared to non-diabetic lean control mice. Further, db/db mice demonstrated a significant decrease in the renal expression and activity of NEP compared to lean mice. Treatment with canagliflozin for 15 weeks had no effect on renal and plasma ACE2 and NEP activity. The source of the urinary ACE2 and NEP is unlikely to be plasma since the renal ACE2 and NEP activity almost 100 folds higher compared to plasma ACE2 (0.02 vs. 5 nmol/hr/μg protein) and NEP (0.4 vs.12 nmol/hr/μg protein) activity. This finding supports the notion that kidney is the major source of urinary ACE2 and NEP in the urine. Recently, arginase-II has been viewed as a mediator of diabetic nephropathy and proposed to be a potential biomarker of CKD. We demonstrated upregulation of renal arginase-II protein expression in db/db mice. Further, we showed that treatment of db/db mice with canagliflozin significantly downregulated renal expression of arginase-II (p<0.05). Sirtuins-1(SIRT1), a life-spanning peptide induced on calorie restriction is recently proposed to play a reno-protective role in diabetic nephropathy. In the present study, we demonstrated significant downregulation of renal SIRT1 in db/db mice. Further, we showed that treatment with canagliflozin markedly augmented renal SIRT1 expression. Thus it is tempting to speculate that the reno-protective effect of canagliflozin could be at least partially mediated via upregulation of renal SIRT1. In conclusion, at the dose of 20 mg/kg/day canagliflozin effectively lowered hyperglycemia level in db/db mice. The glycosuria induced by canagliflozin had no effect on urinary albumin and ACE2, but it increased urinary NEP expression and activity. Further, we showed that the reno-protective effects of canagliflozin could be mediated by upregulation of renal SIRT1 expression and downregulation of renal injury biomarkers, arginase-II and NGAL. Canagliflozin treatment did not attenuated albuminuria and urinary ACE2 shedding and activity, thus could not provide complete reno-protection. This finding provide new insights for possible mechanism through which canagliflozin exhibits reno-protection.
Khalid M. Elased, Pharm.D., Ph.D. (Advisor)
Mauricio Di Fulvio, Ph.D. (Committee Member)
Keiichiro Susuki, M.D., Ph.D. (Committee Member)
118 p.
Pharmacology
Urinary Biomarker; Canagliflozin; Neprilysin; Angiotensin Converting Enzyme 2; Renin Angiotensin System

Recommended Citations

Citations

  • Thanekar, U. H. (2019). Effects of canagliflozin on renal and urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db diabetic mice [Master's thesis, Wright State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=wright1567169063911723

    APA Style (7th edition)

  • Thanekar, Unmesha. Effects of canagliflozin on renal and urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db diabetic mice. 2019. Wright State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=wright1567169063911723.

    MLA Style (8th edition)

  • Thanekar, Unmesha. "Effects of canagliflozin on renal and urinary angiotensin converting enzyme 2 (ACE2) and neprilysin (NEP) in db/db diabetic mice." Master's thesis, Wright State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=wright1567169063911723

    Chicago Manual of Style (17th edition)

wright1567169063911723
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This open access ETD is published by Wright State University and OhioLINK.