Time-Dependent Regulation of Cystogenesis and Disease Severity by AMPK in Polycystic Kidney Disease Cells and Mice
Authors: Hallows Kenneth , Li Hui , Pham Jessica , Sepehr Saman , Solaimanpour Kayvon , Chen Yu-Wei , Mancino Valeria 
Published: May 2024

Autosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic kidney disease, has limited treatment options and is caused by mutations in the PKD1 or PKD2 genes encoding the polycystin proteins PC1 and PC2. ADPKD presents with progressive development of renal cysts, kidney enlargement, and eventual end-stage kidney disease in ~50% of patients by the 6th decade of life. Although many cellular signaling pathways are dysregulated in ADPKD, cells with loss of PC function are hyper-proliferative with major metabolic derangements that may contribute to formation/expansion of kidney epithelial cysts, including increased glycolysis and impaired fatty acid oxidation. In addition, reduced cellular activity of the metabolic sensor AMP-activated protein kinase (AMPK) has been implicated as a key feature of ADPKD and other chronic kidney diseases. We and others have shown that AMPK activators like metformin may reduce disease severity in mouse and other animal ADPKD models, yet other studies have failed to show benefits of metformin in other animal models with treatment at different phases of the disease. Moreover, a recent study using a transgenic mouse model expressing a constitutive activating AMPK mutation led to the development of a PKD-like cystic kidney phenotype. Thus, the significance of AMPK activity in ADPKD is unclear with the possibility that AMPK could have distinct roles during different stages of the disease. To directly test the role of AMPK in ADPKD, we performed in vivo studies using the hypomorphic, slowly progressive Pkd1RC/RC mouse model for human ADPKD, which contains a human disease-causing missense mutation in the mouse Pkd1 gene. We compared various ADPKD disease features at 10 and 12 months of age in male and female Pkd1RC/RC mice with or without additional constitutive knockout (KO) of the AMPK-β1 subunit, which effectively reduces AMPK activity in kidney by >80%. Based on the prevailing view that AMPK activation should be beneficial in reducing disease severity, we hypothesized that the double transgenic AMPK-β1−/−;Pkd1RC/RC mice would manifest more severe disease features. Surprisingly, however, we found that AMPK KO in the Pkd1RC/RC mice had beneficial effects in ameliorating a variety of ADPKD disease features. Specifically, morphometric analyses revealed that AMPK KO lowered % cystic index in female mouse kidneys at 12 months of age relative to controls (14.2 ± 2.9% vs. 32.5 ± 4.0%; P<0.0001). Females had more severe disease features than males, as previously reported in the Pkd1RC/RC mouse model. Similarly, AMPK KO in females significantly lowered % total kidney weight-to-body weight relative to controls at both 10 months (1.82 ± 0.11% vs. 3.04 ± 0.20%; P<0.001) and 12 months (2.11 ± 0.12% vs. 4.03 ± 0.15%; P<0.0001) of age. Kidney function, as measured by plasma BUN levels and by live GFR measurements using the FITC-sinistrin technique, were also significantly improved at 12 months in the AMPK KO mice relative to controls. Various tissue biomarkers were measured by immunoblotting of kidney homogenates harvested at the time of euthanasia. In 12-month-old females, kidney PC1 and PC2 protein expression were each increased ~2-fold in the AMPK-β1−/−;Pkd1RC/RC mice relative to Pkd1RC/RC control mice ( P<0.0001) without any significant difference in Pkd1 or Pkd2 transcript levels, suggesting enhanced PC1/2 protein synthesis or stability in the AMPK KO mice. Furthermore, the kidney injury biomarkers KIM-1 and NGAL were decreased, the inflammatory marker IL-6 was decreased, cellular proliferation markers CDK1, PCNA, and pERK/tERK were decreased, the fibrosis marker COL1A1 was decreased, and the mitochondrial biogenesis marker PGC-1α was increased in the AMPK KO mouse kidneys relative to controls. To begin to explore mechanistically the potential time-dependent role of AMPK in cyst initiation vs. progression in vitro, we used a 3D Matrigel culture model of Pkd1-null mouse proximal tubule-derived (PN24) cells that were stably transduced using the pSMARTTM vector system for doxycycline (Dox)-inducible shRNA for knockdown of AMPK-β1 by ~50% (vs. non-silencing controls) and performed cyst growth assays over a 10-day period. We compared cyst growth/sizes of the cell population at 10 days in culture in the absence of Dox induction (no Dox), with early Dox induction at day 1, and with late Dox induction at day 7 in culture, the time at which of small cysts have just started to form. In PN24 non-silencing control cells, there were no substantial differences among the three Dox treatment conditions. However, in PN24 AMPK-β1 KD cells, early Dox induction significantly inhibited mean cyst size by >60% at 10 days relative to both no Dox induction and late Dox induction ( P<0.0001). In addition, late Dox induction increased cyst size by ~15% relative to no Dox ( P<0.01), suggesting that AMPK inhibition enhanced cellular proliferation and cyst growth once cysts have started to form. In conclusion, together with prior published studies, our findings here suggest that AMPK inhibition early in disease may be beneficial through inhibition of cystogenesis, whereas reduced AMPK activity in later stages of ADPKD could worsen disease manifestations, and thus the use of AMPK activators during advanced disease may be of therapeutic benefit.

Supported by UKRO and U.S. Department of Defense.

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.