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Abstract: FR-PO1000

Metabolomics and Mechanistic Approaches to Identify Therapeutic Targets for Progressive Kidney Disease

Session Information

  • CKD: Pathobiology - I
    November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
    Abstract Time: 10:00 AM - 12:00 PM

Category: CKD (Non-Dialysis)

  • 2203 CKD (Non-Dialysis): Mechanisms

Authors

  • Saliba, Afaf, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
  • Ye, Hongping, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States
  • Sharma, Kumar, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States

Group or Team Name

  • Center for Precision Medicine, The Laboratory of Dr. Kumar Sharma
Background

The role of MDM2 in chronic kidney disease has not yet been clarified. Thus, we aim to (1) establish the link between MDM2 loss and skewed metabolic pathways in the DKD kidney and (2) screen precise therapeutic pathways that improve mitochondrial function.

Methods

(1) To elucidate a metabolic mechanism through which MDM2 absence reduces mitochondrial biogenesis and promotes kidney dysfunction, we developed transgenic 3-4 months mice with doxycycline-inducible deletion of MDM2 in renal tubule cells (MDM2cKO). We performed ZipChip CE MS metabolomics analysis on MDM2cKO kidneys and plasma samples.
(2) To determine if AMPK activation could benefit tubular epithelial cells, we transfected HK2 cells with siRNA-MDM2 (HK2; MDM2KD) vs. siRNA-scramble, treated the cells with AICAR (AMPK activator) and performed Annexin V apoptosis assay.

Results

(1) While the control group (n=19) survived at 100% during the doxycycline treatment, the MDM2cKO mice group (n=8) had a 65% survival percentage at day 6 and 0% survival beyond day 8. In addition to substantial increase in the Blood Urea Nitrogen (BUN) (300-fold increase) in the cKO compared to control mice, we observed a significant decrease in PGC-1a (master regulator of mitochondrial biogenesis) and MCCC2 kidney mRNA levels. MCCC2 is an essential enzyme for Leucine (Leu) metabolism. ZipChip MS analysis revealed a significant decrease in Alanine (Ala) levels in the MDM2cKO plasma. Both Leu and Ala are essential to support mitochondrial biogenesis.
(2) Annexin V assay on MDM2KD HK2 cells showed a significant decrease in apoptotic cells with AMPK stimulation (HK2; MDM2KD: 66.67% ± 7.572; HK2; MDM2KD + AICAR 1mmol/L: 30.67%± 9.452; p=0.0068).

Conclusion

MDM2cKO mice are models for metabolic progressive kidney disease associated with decreased mitochondrial biogenesis and altered Leu and Ala metabolism. AMPK stimulation in vitro significantly improved cell survival upon loss of MDM2 in HK2 cells. We are currently exploring mechanisms that connect MDM2 to Leu and Ala and testing AMPK activation role in improving kidney function, metabolic outcome, and survival of MDMcKO mice. The outcomes of this study will lead to improved treatment approaches for metabolic-related kidney diseases such as DKD.

Funding

  • Other NIH Support