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Cited by 112Chronic Kidney Disease and DiabetesAcute Kidney Injury ResearchNitric Oxide and Endothelin Effects

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Canagliflozin ameliorates renal oxidative stress and inflammation by stimulating AMPK-Akt-eNOS pathway in the isoprenaline-induced oxidative stress model

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Hasan, Raquibul, Lasker, Shoumen, Hasan, Ahasanul, Zerin, Farzana, Zamila, Mushfera, Parvez, Faisal, Rahman, Md. Mizanur, Khan, Ferdous, Subhan, Nusrat, Alam, Md. Ashraful·2020·Scientific Reports
DOI 10.1038/s41598-020-71599-2PMC PMC7474058
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Abstract

Diabetes is a leading cause of chronic kidney disease, and the high prevalence of sympathetic nervous system (SNS) hyperactivity in diabetic patients makes them further susceptible to SNS-mediated oxidative stress and accelerated kidney damage. Here, we investigated if canagliflozin can reverse isoprenaline (ISO)-induced renal oxidative damage in rats, a model that mimics SNS overstimulation-induced organ injuries in humans. We found that ISO administration elevates renal oxidative stress markers including malondialdehyde (MDA), advanced protein oxidation product (APOP), myeloperoxidase (MPO) and nitric oxide (NO), while depleting levels of endogenous antioxidants such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH). Strikingly, canagliflozin treatment of ISO-treated rats not only prevents elevation of oxidative stress markers but also rescues levels of depleted antioxidants. Our results also show that canagliflozin stimulates antioxidant/anti-inflammatory signaling pathways involving AMP-activated protein kinase (AMPK), Akt and eNOS, and inhibits iNOS and NADPH oxidase isoform 4 (NOX4), all of which are associated with oxidative stress and inflammation. Further, canagliflozin prevents ISO-induced apoptosis of kidney cells by inhibiting Bax protein upregulation and caspase-3 activation. Histological examination of kidney sections reveal that canagliflozin attenuates ISO-mediated increases in inflammatory cell infiltration, collagen deposition and fibrosis. Finally, consistent with these findings, canagliflozin treatment improves kidney function in ISO-treated rats, suggesting that the antioxidant effects may be clinically translatable.

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Figure 1 Canagliflozin (Canaglif) treatment reduces ISO-induced elevation of oxidative stress markers in kidney and plasma. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 1 Canagliflozin (Canaglif) treatment reduces ISO-induced elevation of oxidative stress markers in kidney and plasma. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central
Figure 2 Canagliflozin (Canaglif) treatment rescues ISO-induced reduction of endogenous antioxidants. Statistical significance was determined using one-way ANOVA with Newman–Keuls post hoc test. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 2 Canagliflozin (Canaglif) treatment rescues ISO-induced reduction of endogenous antioxidants. Statistical significance was determined using one-way ANOVA with Newman–Keuls post hoc test. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central
Figure 3 Canagliflozin (Canaglif) stimulates AMPK, Akt and eNOS phosphorylation. (A) Representative Western blot images of phosphorylated (p) proteins normalized to their total protein expression for AMPK, Akt and eNOS. Full-length blot images are presented in supplementary figure 1. (B–D) Mean data comparing relative changes of phosphorylation normalized to total protein expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4–5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 3 Canagliflozin (Canaglif) stimulates AMPK, Akt and eNOS phosphorylation. (A) Representative Western blot images of phosphorylated (p) proteins normalized to their total protein expression for AMPK, Akt and eNOS. Full-length blot images are presented in supplementary figure 1. (B–D) Mean data comparing relative changes of phosphorylation normalized to total protein expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4–5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central
Figure 4 Canagliflozin stimulates AMPK, Akt and eNOS phosphorylation in human renal proximal tubule epithelial cells. (A,B) Representative Western blot images of phosphorylated (p) proteins normalized to their total protein expression for AMPK, Akt and eNOS. Full-length images are presented in supplementary figure 2. (C–E) Mean data comparing relative changes of AMPK, Akt and eNOS phosphorylation normalized to total protein expression. DM and A-443 indicate dorsomorphin and A-443654, respectively. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO, $ indicates p < 0.05 vs ISO + Canaglif.
Figure 4 Canagliflozin stimulates AMPK, Akt and eNOS phosphorylation in human renal proximal tubule epithelial cells. (A,B) Representative Western blot images of phosphorylated (p) proteins normalized to their total protein expression for AMPK, Akt and eNOS. Full-length images are presented in supplementary figure 2. (C–E) Mean data comparing relative changes of AMPK, Akt and eNOS phosphorylation normalized to total protein expression. DM and A-443 indicate dorsomorphin and A-443654, respectively. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO, $ indicates p < 0.05 vs ISO + Canaglif.CC BY · PubMed Central
Figure 5 Canagliflozin (Canaglif) reduces ISO-mediated upregulation of NOX4 and iNOS. (A) Representative Western blot image showing a reduction of Nox4 protein expression by canagliflozin. Full-length Western blot images are presented in supplementary figure 3. (B) Mean data for changes of Nox4 expression normalized to α-actin. n = 4 for each group. (C) Representative Western blot image showing a reduction of ISO-mediated iNOS upregulation by canagliflozin treatment. (D) Mean data for changes of iNOS expression normalized to α-actin or GAPDH expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 5 Canagliflozin (Canaglif) reduces ISO-mediated upregulation of NOX4 and iNOS. (A) Representative Western blot image showing a reduction of Nox4 protein expression by canagliflozin. Full-length Western blot images are presented in supplementary figure 3. (B) Mean data for changes of Nox4 expression normalized to α-actin. n = 4 for each group. (C) Representative Western blot image showing a reduction of ISO-mediated iNOS upregulation by canagliflozin treatment. (D) Mean data for changes of iNOS expression normalized to α-actin or GAPDH expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central
Figure 6 Canagliflozin (Canaglif) treatment attenuates apoptosis of kidney cells by ISO. (A) Representative Western blot image illustrating a reduction of Bax protein expression by canagliflozin. Full-length blot images are presented in supplementary figure 4. (B) Mean data for changes of Bax protein levels relative to anti-apoptotic protein Bcl-2. (C) Example Western blot image showing that canagliflozin treatment lowered cleaved caspase-3 expression. (D) Mean data comparing relative changes of cleaved caspase-3 protein expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 6 Canagliflozin (Canaglif) treatment attenuates apoptosis of kidney cells by ISO. (A) Representative Western blot image illustrating a reduction of Bax protein expression by canagliflozin. Full-length blot images are presented in supplementary figure 4. (B) Mean data for changes of Bax protein levels relative to anti-apoptotic protein Bcl-2. (C) Example Western blot image showing that canagliflozin treatment lowered cleaved caspase-3 expression. (D) Mean data comparing relative changes of cleaved caspase-3 protein expression. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 4 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central
Figure 7 Canagliflozin (Canaglif) treatment reduces inflammation and fibrosis and improves kidney function. (A–D) Representative images of H&E staining showing presence or absence of inflammatory cell infiltration in kidney tissue sections under different conditions. (E–H) Representative images of picrosirius red staining showing presence and/or extent of fibrosis in kidney tissue sections. (I) Mean data illustrating % of area fibrosis under different conditions, n = 12 for each group. (J,K) Mean data showing that canagliflozin abolished elevation of renal function markers uric acid and creatinine in the plasma. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.
Figure 7 Canagliflozin (Canaglif) treatment reduces inflammation and fibrosis and improves kidney function. (A–D) Representative images of H&E staining showing presence or absence of inflammatory cell infiltration in kidney tissue sections under different conditions. (E–H) Representative images of picrosirius red staining showing presence and/or extent of fibrosis in kidney tissue sections. (I) Mean data illustrating % of area fibrosis under different conditions, n = 12 for each group. (J,K) Mean data showing that canagliflozin abolished elevation of renal function markers uric acid and creatinine in the plasma. One-way ANOVA with Newman–Keuls post hoc test was used for multiple group comparisons. Data are presented as mean ± SEM. n = 5 for each group. * indicates p < 0.05 vs control, # indicates p < 0.05 vs ISO.CC BY · PubMed Central

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Authors
Hasan, R., Lasker, S., Hasan, A., Zerin, F., Zamila, M., Parvez, F., Rahman, M. M., Khan, F., Subhan, N., & Alam, M. A.
Affiliations
  • Mercer University Health Sciences Center (US)
  • North South University (BD)
Corresponding author
  • Hasan, Raquibul
  • Alam, Md. Ashraful
ORCID
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2020-09-04
Volume
10
Issue
1
Pages
14659-14659
ISSN
2045-2322 (ISSN-L)
DOI
10.1038/s41598-020-71599-2
PMC
PMC7474058
OpenAlex
W3083174088
Cited by
111
References
42
Citations by year
2026: 11 · 2025: 15 · 2024: 20 · 2023: 24 · 2022: 28 · 2021: 13
FWCI
10.30
Type
journal article
Language
en
Open access
Licence
Locations
  • Scientific Reports (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Greater South Information System
Funders
  • Mercer University College of Pharmacy
  • North South University
Topics
  • Health Sciences › Medicine › Nephrology › Chronic Kidney Disease and Diabetes
  • Health Sciences › Medicine › Nephrology › Acute Kidney Injury Research
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
Keywords
canagliflozin, renal oxidative stress, renal inflammation, sympathetic hyperactivity, diabetic nephropathy, nitric oxide, NOX4, inducible nitric oxide synthase, apoptosis, Bax, caspase-3, renal fibrosis, antioxidant enzymes, SGLT2 inhibitors
MeSH terms
Canagliflozin, Animals, Anti-Inflammatory Agents, Antioxidants, Cells, Cultured, Disease Models, Animal, Epithelial Cells, Humans, Inflammation, Isoproterenol, Kidney Tubules, Male, Oxidative Stress, Rats, Long-Evans, Proto-Oncogene Proteins c-akt, Rats, Renal Insufficiency, Nitric Oxide Synthase Type III, AMP-Activated Protein Kinases

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