BMAL1–TFEB axis sustains lysosomal competence to restrain p21‑driven senescence in kidney epithelium

Hypothesis

Core circadian transcription factor BMAL1 preserves renal epithelial homeostasis by directly promoting lysosomal biogenesis and function through transcriptional activation of TFEB, thereby enabling efficient mitophagy and preventing mitochondrial‑damage‑induced senescence. When BMAL1 is lost, TFEB fails to translocate to the nucleus, lysosomal capacity declines, damaged mitochondria accumulate, ROS and cytosolic mtDNA activate the cGAS‑STING pathway, and p21^CIP1^ is induced independently of circadian transcriptional control.

Mechanistic Rationale

• BMAL1 binds to E‑box elements in the promoter of the lysosomal master regulator TFEB (see resources on circadian regulation of autophagy genes)[https://pmc.ncbi.nlm.nih.gov/articles/PMC4178439/].
• TFEB drives expression of lysosomal hydrolases, V‑ATPase subunits, and autophagy receptors (e.g., OPTN, NDP52) essential for mitophagosome formation and lysosomal acidification[https://pmc.ncbi.nlm.nih.gov/articles/PMC6838779/].
• In podocytes and proximal tubular cells, BMAL1 loss reduces nuclear TFEB, lowers Lysotracker signal, and impairs colocalization of mitochondria with LC3, leading to swollen mitochondria[https://pmc.ncbi.nlm.nih.gov/articles/PMC9273266/].
• Accumulated mitochondria release mtDNA into the cytosol, activating cGAS‑STING‑IRF3 signaling, which transcriptionally upregulates Cdkn1a (p21) via STAT3‑dependent promoters[https://pmc.ncbi.nlm.nih.gov/articles/PMC8202863/].
• This pathway operates downstream of the circadian clock, explaining why p21 induction persists despite intact transcriptional rhythms of other clock‑controlled genes.

Testable Predictions

1. Rescue by TFEB activation – Pharmacological activation of TFEB (e.g., with trehalose or ML-SA1) in Bmal1‑deficient podocytes will restore lysosomal flux, reduce mitochondrial ROS, and normalize p21 protein levels without reinstating BMAL1 expression.
2. TFEB necessity – siRNA‑mediated knockdown of TFEB in wild‑type tubular cells will phenocopy Bmal1 loss: decreased mitophagy, increased mtDNA‑cGAS signaling, and elevated p21, even when circadian oscillations are intact.
3. cGAS‑STING dependence – Genetic deletion of Sting in Bmal1‑null mice will mitigate p21 upregulation and attenuate fibrosis despite persistent lysosomal defects.
4. No direct BMAL1‑Cdkn1a binding – ChIP‑qPCR for BMAL1 at the Cdkn1a promoter/enhancer regions will show no enrichment, confirming indirect regulation.
5. Human relevance – Kidney biopsies from patients with circadian disruption (e.g., shift workers) will exhibit reduced nuclear TFEB, increased lysosomal lipofuscin, and higher p21^+^ senescent cells correlating with albuminuria.

Experimental Approach

• In vitro: Conditionally immortalized mouse podocytes treated with Bmal1 siRNA; assess TFEB localization (immunofluorescence), lysosomal activity (LysoSensor, DQ‑BSA), mitophagy (mt‑Keima), ROS (MitoSOX), mtDNA release (qPCR of cytosolic fraction), and p21 (Western blot). Rescue experiments with TFEB activator or overexpression.
• In vivo: Kidney‑specific Bmal1 floxed mice crossed with TFEB‑overexpressing (AAV9‑TFEB) or Sting^−/−^ mice; monitor senescence (p16^INK4a^, p21^+, SA‑β‑gal), fibrosis (collagen I, α‑SMA), and renal function (albumin/creatinine ratio) over 6 months.
• Human data: Correlate actigraphy‑derived rest‑activity rhythms with biopsy TFEB localization and p21 staining in cohorts of CKD patients.

Falsifiability

If TFEB activation fails to lower p21 or improve mitochondrial quality in Bmal1‑deficient cells, or if Sting deletion does not attenuate senescence despite lysosomal dysfunction, the hypothesis would be refuted. Likewise, demonstration of direct BMAL1 binding to Cdkn1a regulatory regions would undermine the proposed indirect mechanism.

Implications

Positioning the circadian‑lysosomal axis as a gatekeeper of mitochondrial‑driven senescence reframes chronotherapeutic strategies: enhancing lysosomal function (via TFEB agonists, mTOR inhibitors, or NAD^+^ boosters) could serve as a geroprotective intervention that compensates for clock decline, particularly in tissues where transcriptional clock control of senescence genes is weak.