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PHLPP1/Nrf2–Mdm2 axis induces renal apoptosis via influencing nucleo-cytoplasmic shuttling of FoxO1 during diabetic nephropathy

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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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Correspondence to Poonam Kakkar.

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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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