Abstract
Impaired PI3K/Akt signaling (insulin resistance) and poor glycemic control (hyperglycemia) are the major risk factors involved in the progression of diabetic nephropathy (DN). This study was designed to identify factors influencing cell survival during DN. We found that high glucose exposure in renal proximal tubular cells (NRK52E) upregulated PHLPP1, an Akt phosphatase (Ser473), causing suppression in Akt and IGF1β phosphorylation leading to inhibition in insulin signaling pathway. Results demonstrate that sustained activation of PHLPP1 promoted nuclear retention of FoxO1 by preventing its ubiquitination via Mdm2, an Akt/ Nrf2-dependent E3 ligase. Thus, enhanced FoxO1 nuclear stability caused aberration in renal gluconeogenesis and activated apoptotic cascade. Conversely, gene silencing of PHLPP1-enhanced Nrf2 expression and attenuated FoxO1 regulated apoptosis compared to hyperglycemic cells. Mechanistic aspects of PHLPP1–Nrf2/FoxO1 signaling were further validated in STZ-nicotinamide-induced type 2 diabetic Wistar rats. Importantly, we observed via immunoblotting and dual immunocytochemical studies that treatment of Morin (2′,3,4′,5,7-Pentahydroxyflavone) during diabetes significantly augmented FoxO1 nuclear exclusion, resulting in its ubiquitination via Akt–Nrf2/Mdm2 pathway. Furthermore, lowering of PHLPP1 expression by Morin also prevented FoxO1/Mst1-mediated apoptotic signaling in vitro and in vivo. Morin treatment under the experimental conditions, effectively decreased blood glucose levels, ameliorated insulin resistance, alleviated oxidative stress and attenuated renal apoptosis in diabetic rats comparable to metformin thereby exhibiting tremendous potential against renal complications of diabetes. These novel results further acclaim that inhibition of PHLPP1/FoxO1–Mdm2 axis is critical in the pathogenesis of diabetic nephropathy.
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Abbreviations
- ARE:
-
Antioxidant response element
- AKT:
-
Akt murine thymoma viral oncogene
- BAX:
-
Bcl-2 associated X protein
- BCL2:
-
B-cell lymphoma 2
- BIM:
-
Bcl-2-like protein 11
- BUN:
-
Blood urea nitrogen
- Bwt.:
-
Body weight
- DN:
-
Diabetic nephropathy
- ELISA:
-
Enzyme-linked immunosorbent assay
- FoxO1:
-
Forkhead-box O1 protein
- G6P:
-
Glucose-6-phosphate
- G6K:
-
Glucose 6 kinase
- HG:
-
High glucose
- HO-1:
-
Hemeoxygenase 1
- IR:
-
Insulin resistance
- MAPK:
-
Mitogen activated protein kinase
- MDM2:
-
Mouse double minute 2 homolog
- MST1:
-
Mammalian sterile 20-like kinase 1
- NA:
-
Nicotinamide
- NEH:
-
Nrf2–ECH homology
- NQO1:
-
NAD(P)H dehydrogenase (quinone 1)
- PEPCK:
-
Phosphoenolpyruvate carboxykinase
- PIP3:
-
Phosphatidylinositol 3,4,5-triphosphate
- PKB:
-
Protein kinase B
- PHLPP1:
-
Pleckstrin homology domain and leucine rich repeat protein phosphatase 1
- PKC:
-
Protein kinase C
- PI3K:
-
Phosphoinositide 3-kinase
- PUMA:
-
P53 upregulated modulator of apoptosis
- STZ:
-
Streptozotocin
- T2D:
-
Type 2 diabetes
- Ub:
-
Ubiquitin
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Acknowledgements
The authors thank CSIR-IITR review committee for evaluation of the manuscript and providing the manuscript communication no.3592.
Funding
This study was supported by grants from CSIR project (HCP-0019). Senior Research fellowships to AM, MFK, VKP from CSIR and UGC is gratefully acknowledged.
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The study was conceptualized and designed by AM and PK. Experimentation, analysis and data interpretation was done by AM, VKP and MFK. Manuscript drafting and writing work was a joint effort of AM, VKP and PK. All authors have discussed and contributed to the final manuscript.
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All experiments on rats were conducted in accordance with the international guidelines and protocols approved by the Institutional Animal Ethics Committee (IAEC) of CSIR-IITR with approval no. IITR/IAEC/01/16–82/16.
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Mathur, A., Pandey, V.K., Khan, M.F. et al. PHLPP1/Nrf2–Mdm2 axis induces renal apoptosis via influencing nucleo-cytoplasmic shuttling of FoxO1 during diabetic nephropathy. Mol Cell Biochem 476, 3681–3699 (2021). https://doi.org/10.1007/s11010-021-04177-3
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DOI: https://doi.org/10.1007/s11010-021-04177-3