Dopamine 1 receptor activation protects mouse diabetic podocytes injury via regulating the PKA/NOX-5/p38 MAPK axis

Shao, Xiaoting; Zhang, Xinying; Hu, Jing; Gao, Tielei; Chen, Junting; Xu, Chi; Wei, Can

doi:10.1016/j.yexcr.2020.111849

Cited by 17 publications

(12 citation statements)

References 36 publications

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“…Sometimes the most known genes, such as ACTB and GAPDH, are used for samples and conditions that do not support their use. Even in the most recent years, researchers still normalize their qPCR data of in vitro studies based on the most frequently used genes, as ACTB for podocytes and GAPDH for mesangial cells (opposite to the finding we had in our analysis), without literature support for this choice (28)(29)(30)(31)(32)(33). Unfortunately, some studies do not clearly provide which reference gene was used to normalize the data, or even if there was a data normalization.…”

Section: Discussionmentioning

confidence: 61%

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Hosni

Anauate

Boim

2021

Preprint

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BackgroundReal-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high quality qPCR, providing strong support for results, as well as performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used so far to enhance validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g. Losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cells and podocytes studies of an in vitro DKD model of disease and its treatment.MethodsFive software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n=5; 5mM glucose), mannitol (n=5; 30mM, as osmotic control), HG (n=5; 30mM glucose), and HG + losartan (n=5; 30mM glucose and 10-4 mM of losartan). Real-time PCR was performed according to MIQE guidelines.ResultsWe identified that the use of 2 genes is the best combination for qPCR normalization for both mesangial cell and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results.ConclusionThis analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR technique, while for podocytes, HPRT and GAPDH should be chosen.

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Section: Discussionmentioning

confidence: 61%

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Hosni

Anauate

Boim

2021

Preprint

View full text Add to dashboard Cite

show abstract

“…Sometimes the most known genes, such as ACTB and GAPDH , are used for samples and conditions that do not support their use. Even in the most recent years, researchers still normalize their qPCR data of in vitro studies based on the most frequently used genes, such as ACTB for podocytes and GAPDH for mesangial cells (opposite to the finding we had in our analysis), without literature support for this choice [ 34 – 39 ]. Unfortunately, some studies do not clearly provide which reference gene was used to normalize the data, or even if there was data normalization.…”

Section: Discussionmentioning

confidence: 87%

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

2021

View full text Add to dashboard Cite

Background Real-time PCR remains currently the gold standard method for gene expression studies. Identification of the best reference gene is a key point in performing high-quality qPCR, providing strong support for results, and performing as a source of bias when inappropriately chosen. Mesangial cells and podocytes, as essential cell lines to study diabetic kidney disease (DKD) physiopathology, demand accurate analysis of the reference genes used thus far to enhance the validity of gene expression studies, especially regarding high glucose (HG) and DKD treatments, with angiotensin II receptor blockers (e.g., losartan) being the most commonly used. This study aimed to evaluate the suitability and define the most stable reference gene for mesangial cell and podocyte studies of an in vitro DKD model of disease and its treatment. Methods Five software packages (RefFinder, NormFinder, GeNorm, Bestkeeper, and DataAssist) and the comparative ΔCt method were selected to analyze six different candidate genes: HPRT, ACTB, PGAM-1, GAPDH, PPIA, and B2M. RNA was extracted, and cDNA was synthesized from immortalized mouse mesangial cells and podocytes cultured in 4 groups: control (n = 5; 5 mM glucose), mannitol (n = 5; 30 mM, as osmotic control), HG (n = 5; 30 mM glucose), and HG + losartan (n = 5; 30 mM glucose and 10−4 mM losartan). Real-time PCR was performed according to MIQE guidelines. Results We identified that the use of 2 genes was the best combination for qPCR normalization for both mesangial cells and podocytes. For mesangial cells, the combination of HPRT and ACTB presented higher stability values. For podocytes, HPRT and GAPDH showed the best results. Conclusion This analysis provides support for the use of HPRT and ACTB as reference genes in mouse mesangial cell studies of gene expression via real-time PCR, while for podocytes, HPRT and GAPDH should be chosen.

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“…45 Interestingly, MAPK and AMPK signaling pathway plays critical roles in proliferation, apoptosis and autophagy of podocytes. [46][47][48][49][50] Diana et al, reported that EGF promotes HK-2 cells proliferation by MAPK (ERK1/2) signaling activation 51 . Our founding indicated that the differentially DNA methylation genes were enriched in the MAPK and AMPK signaling pathway, suggesting that EGF-mediated protective effect may be regulated via MAPK or AMPK signaling pathway in high glucose environment.…”

Section: Discussionmentioning

confidence: 99%

Epidermal Growth Factor Protects Against High Glucose-Induced Podocyte Injury Possibly via Modulation of Autophagy and PI3K/AKT/mTOR Signaling Pathway Through DNA Methylation

Sun¹,

Deng

et al. 2021

DMSO

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Aim: Diabetic nephropathy (DN) is a serious health problem worldwide. Epidermal growth factor (EGF) has suggested as a potential biomarker for the progression of chronic kidney disease. In this study, we examined the effects of EGF on the high glucose (HG)-induced podocyte injury and explored the underlying molecular mechanisms. Methods: The cell proliferation, toxicity, and cell apoptosis of podocytes were determined by CCK-8 assay, lactate dehydrogenase release assay, and flow cytometry, respectively, and protein levels in the podocytes were determined by Western blot assay. Mechanistically, DNA methylation analysis, bioinformatic analysis, methylation-specific PCR and quantitative real-time PCR were used to analyze functional pathways in differentially methylated genes and the expression of the key methylated genes in the podocytes after different interventions.Results: EGF treatment significantly increased the protein expression level of LC3 and decreased the protein level of P62 in HG-stimulated podocytes, which was attenuated by autophagy inhibitor, 3-methyladenine. EGF increased the cell proliferation and the protein expression levels of nephrin and synaptopodin, but reduced cell toxicity and cell apoptosis and protein expression level of cleaved caspase-3, which was partially antagonized by 3-methyladenine. DNA methylation expression profiles revealed the differential hypermethylation sites and hypomethylation sites among podocytes treated with normal glucose, HG and HG+EGF. GO enrichment analysis showed that DNA methylation was significantly enriched in negative regulation of phosphorylation, cellcell junction and GTPase binding. KEGG pathway analysis showed that these genes were mainly enriched in PI3K-Akt, Hippo and autophagy pathways. Further validation studies revealed that six hub genes (ITGB1, GRB2, FN1, ITGB3, FZD10 and FGFR1) may be associated with the protective effects of EGF on the HG-induced podocyte injury. Conclusion:In summary, our results demonstrated that EGF exerted protective effects on HG-induced podocytes injury via enhancing cell proliferation and inhibiting cell apoptosis. Further mechanistic studies implied that EGF-mediated protective effects in HG-stimulated podocytes may be associated with modulation of autophagy and PI3K/AKT/mTOR signaling pathway.

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Dopamine 1 receptor activation protects mouse diabetic podocytes injury via regulating the PKA/NOX-5/p38 MAPK axis

Cited by 17 publications

References 36 publications

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Reference genes for mesangial cell and podocyte qPCR gene expression studies under high-glucose and renin-angiotensin-system blocker conditions

Epidermal Growth Factor Protects Against High Glucose-Induced Podocyte Injury Possibly via Modulation of Autophagy and PI3K/AKT/mTOR Signaling Pathway Through DNA Methylation

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