Kidney Week

Abstract: FR-OR029

ATR Deletion Drives TOR-Autophagy Spatial Coupling Compartment (TASCC) Formation and Kidney Fibrosis

Session Information

• AKI: Mechanisms - Inflammation and AKI-CKD Transition
  November 08, 2019 | Location: 202, Walter E. Washington Convention Center
  Abstract Time: 06:06 PM - 06:18 PM

Category: Acute Kidney Injury

• 103 AKI: Mechanisms

Authors

• Brooks, Craig R., Vanderbilt University Medical Center/Division of Nephrology & Hpertension, Nashville, Tennessee, United States

Craig R. Brooks, PhD



Acute kidney injury (AKI) occurs in ~13% of hospitalized patients and is associated with a fourfold increase in mortality. AKI also predisposes patients to chronic kidney disease (CKD), which affects ~10% of the world's population. Renal proximal tubule epithelial cells (PTCs) are vital to the function of the kidney and are also integral to the pathology of the injured kidney, as the most sensitive kidney cell type to ischemia and nephrotoxic insults. The overall goal of my research is to delineate the molecular mechanisms of PTC cell injury/death, survival and subsequent regeneration to identify therapeutic targets to treat AKI. To this end, I investigated mechanisms of PTC cell death. I was the first to demonstrate pathological mitochondrial fragmentation in PTCs during kidney injury, leading to cell death, (Brooks et al. JCI 2009) and demonstrate the therapeutic potential of inhibiting mitochondrial fragmentation using small molecule inhibitors. I also demonstrated a direct interaction between Bcl-2 family proteins and mitochondrial morphology proteins (Brooks et al., PNAS, 2007; Brooks et al., AJP Cell 2011). Next, I focused on the role of PTCs in regulating inflammatory processes during AKI. I found tubular cell phagocytosis suppressed inflammation (Yang, Brooks et al., J. Clinical Inv., 2015) as well as induced autophagy, which in turn, inhibited proximal tubular cell activation of T cells through MHC presentation (Brooks et al., EMBO, 2015). More recently, we identified the role of an atypical cyclin, cyclin G1, in the transition of AKI to CKD and CKD progression (Canaud et al., STM 2019). Current research interests include: Further delineating the mechanism and therapeutic potential of autophagy activation in the regulation of immune processes in AKI; Determining if the activation of novel cell cycle regulators following AKI contributes to fibrosis and chronic kidney disease through induction of mTOR-LC3 associated pro-secretory phenotype; and examining if mitochondrial dynamics proteins, such as mitofusins, modulate autophagic flux in AKI.

• Kishi, Seiji, Kawasaki Medical School, Kurashiki, Japan

Seiji Kishi,

• Taguchi, Kensei, Department of Nephrology and Hypertension, Nashville, Tennessee, United States

Kensei Taguchi,

• Bonventre, Joseph V., Brigham and Women's Hospital, Boston, Massachusetts, United States

Joseph V. Bonventre,

Background

Acute kidney injury (AKI) occurs in ~20% of hospitalized patients. While the kidney can functionally recover from AKI, AKI predisposes patients to subsequent chronic kidney disease (CKD), which effects 13% of the global population. Our lab, and others, have shown the AKI-CKD transition involves maladaptive repair leading to G2/M arrest regulated by DNA damage response (DDR) genes. Recently, we have identified target of rapamycin–autophagy spatial coupling compartments (TASCCs) as components of the secretory response in G2/M arrested cells. To determine how the DDR modulates AKI-CKD transition, we tested if the loss of ataxia telangiectasia and Rad3-related (ATR) regulates TASCC formation and fibrosis.

Methods

1: ATR flox/flox mice were breed to SLC34a1-Cre-ERT2 mice to generate proximal tubular cell (PTC) specific ATR deletion (ATR^RPTC-/-) upon tamoxifen injection. ATR floxed mice lacking the Cre acted as control (ATR^Ctrl). ATR^Ctrl and ATR^RPTC-/- received unilateral ureteral obstruction (UUO). Kidneys were taken at day 7 and analyzed for fibrosis, G2/M arrest markers, injury markers and TASCC formation by immunostaining. TASCCs were identified by super-resolution microscopy. 2: Fucci2a mice were breed to γGT-Cre mice to generate PTC specific Fucci2a expression. The PTCs were then isolated and treated with aristolochic acid (AA), with/without the ATR inhibitor VE-821. Cells were analyzed for increased connective tissue growth factor (CTGF) by western blot.

Results

Inhibition of ATR in PTCs resulted in increased numbers of G2/M arrested cells following AA treatment, as measured by the Fucci cell cycle reporter, and greater production of CTGF, in vitro. In vivo, ATR^RPTC-/- mice had more sever and rapidly progressing fibrosis compared to ATR^Ctrl mice. Deletion of ATR from PTCs resulted in increased tubular cell injury, caspase 3 staining, and G2/M arrest in response to UUO. G2/M arrest was associated with formation of TASCCs in PTCs. ATR^RPTC-/- mice had greater numbers of TASCC/cell compared to controls.

Conclusion

ATR deletion sensitized PTCs to injury and G2/M arrest, which drove TASCC formation and production of CTGF. These data indicate the DDR modulates the AKI-CKD transition through a pathway involving G2/M arrest, TASCC formation and profibrotic factor secretion, as well as identifying novel targets for therapeutic intervention.

Funding

• NIDDK Support