Application Myo-Inositol Oxygenase Fluorescent Renal Proximal Tubular-Specific Mitochondrial AKT1 Transgenic Mice in Kidney Disease Research
- Bioengineering: Modeling, Diagnosis, Therapy
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
- 400 Bioengineering
- Lin, Hugo Y.-H., Kaohsiung Medical University Chung Ho Memorial Hospital, Kaohsiung, Taiwan
- Chen, I-Ya, Chang Gung University, Taoyuan, Taoyuan, Taiwan
- Wang, Tzu Ming, China Medical University Hospital, Taichung, Taiwan
- Chen, Yen-Hua, National Sun Yat-sen University, Kaohsiung, Taiwan
- Jiang, Si-Tse, National Applied Research Laboratories National Laboratory Animal Center, Tainan, Taiwan
Renal tubular AKT1 is activated and translocated into mitochondria upon renal injuries. To determine whether tubular mitochondrial AKT1 (mito-AKT) plays a protective role, we generated a novel transgenic mouse strain harboring inducible mitochondria-targeting constitutively active AKT1 (mcaAKT) driven by the renal proximal tubule-specific promoter.
The manufacturing process used a bacterial artificial chromosome clone containing mouse Miox as the transgene backbone and inserted the IRES-CreERT2-polyA and promoter CAGGS-loxP-GFP-polyA-loxP-MTS-AKT1(308T>D; 437S>D)-3XFLAG-polyA cassettes into the exon 5 and 3’ downstream of Miox, respectively, using Red/ET DNA recombination technology. The CreERT2 is tamoxifen-inducible Cre recombinase, the GFP is a green fluorescent protein, and the MTS is the mitochondria-targeting sequence. The modified transgenic construct was injected into mouse pronuclei and transferred into a surrogate mother to generate the transgenic mice.
The GFP was expressed ubiquitously and observed by epifluorescence in transgenic mice carrying the transgene. The expression of mcaAKT protein was only detected in the kidneys of tamoxifen (TAM)-injected KMioCAKT but not in corn oil-KMioCAKT mice or TAM-wildtype (WT) mice. The mcaAKT was exclusively expressed in kidneys but not in other organs in TAM-KMioCAKT. Moreover, we confirmed mcaAKT co-localized with mitochondria in the renal tubules with tissue staining. To measure the mitochondrial respiratory capacity, we used the Seahorse XF Analyzer and found significant differences in basal respiration, spare respiration, and ATP-dependent respiration (p<0.05) between TAM-KMioCAKT and corn oil-KmioCAKT.
These findings suggest that mitochondrial AKT1 is a renal tube bioenergy regulator. We will continue to explore whether it plays a protective role in renal tubules. These findings shed new light on the application in nephrology research. They may be used to screen for new drugs and develop new strategies for preventing and treating kidney diseases.
- Government Support – Non-U.S.