Autophagic Clearance of Mitochondria in the Kidney Copes with Metabolic Acidosis

Abstract

Metabolic acidosis, a common complication of CKD, causes mitochondrial stress by undefined mechanisms. Selective autophagy of impaired mitochondria, called mitophagy, contributes toward maintaining cellular homeostasis in various settings. We hypothesized that mitophagy is involved in proximal tubular cell adaptations to chronic metabolic acidosis. In transgenic mice expressing green fluorescent proteintagged microtubule-associated protein 1 light chain 3 (GFP-LC3), NH 4 Cl loading increased the number of GFP puncta exclusively in the proximal tubule. In vitro, culture in acidic medium produced similar results in proximal tubular cell lines stably expressing GFP-LC3 and facilitated the degradation of SQSTM1/p62 in wild-type cells, indicating enhanced autophagic flux. Upon acid loading, proximal tubule-specific autophagydeficient (Atg5-deficient) mice displayed significantly reduced ammonium production and severe metabolic acidosis compared with wild-type mice. In vitro and in vivo, acid loading caused Atg5-deficient proximal tubular cells to exhibit reduced mitochondrial respiratory chain activity, reduced mitochondrial membrane potential, and fragmented morphology with marked swelling in mitochondria. GFP-LC3-tagged autophagosomes colocalized with ubiquitinated mitochondria in proximal tubular cells cultured in acidic medium, suggesting that metabolic acidosis induces mitophagy. Furthermore, restoration of Atg5-intact nuclei in Atg5-deficient proximal tubular cells increased mitochondrial membrane potential and ammoniagenesis. In conclusion, metabolic acidosis induces autophagy in proximal tubular cells, which is indispensable for maintaining proper mitochondrial functions including ammoniagenesis, and thus for adapted urinary acid excretion. Our results provide a rationale for the beneficial effect of alkali supplementation in CKD, a condition in which autophagy may be reduced, and suggest a new therapeutic option for acidosis by modulating autophagy.

Time-dependent dysregulation of autophagy: Implications in aging and mitochondrial homeostasis in the kidney proximal tubule

“…[17][18][19][20] We observed slight hypertrophy with an accumulation of cytosolic amorphous substrates in the proximal tubular cells of the kidney of atg5 F/F -KAP mice after the age of 6 mo. Kidney function is comparable in atg5 F/F -KAP mice and littermate controls up to 9 mo of age.…”

NH4Cl Treatment Prevents Tissue Calcification in Klotho Deficiency

“…[34][35][36][37] Moreover, clinical studies in humans demonstrate that acidosis accelerates and alkali treatment retards the progression of CKD. 34,39,[74][75][76] Along those lines, decreased bicarbonate concentrations are associated with decline of eGFR in community-living older persons. 77 Our observations may shed light on vascular calcification in further clinical conditions.…”

High-Fat Diet–Induced Lysosomal Dysfunction and Impaired Autophagic Flux Contribute to Lipotoxicity in the Kidney

“…23 Briefly, we used fresh cryosections (5 mm thick) of kidney tissues. For COX staining, the sections were incubated in the solution composed of 2 mg/ml of catalase (Sigma-Aldrich), 1 mg/ml of cytochrome c (Sigma-Aldrich), and 0.5 mg/ml of diaminobenzidine (Wako) in 0.1 M of phosphate buffer (pH 7.4) for 1 hour at 37°C.…”

“…23 To prove mitophagy, PTCs stably expressing GFP-MAP1LC3 were stained with 25 nM of MitoTracker Red FM for 15 minutes at 37°C.…”

“…[20][21][22][23][24] More recently, we have established methods for monitoring autophagic activity in vivo. 25 Given that autophagic activity can be downregulated by lipid overload, cellular functions may be jeopardized in pathologic settings in which properly enhanced autophagy should exert a compensatory or protective role.…”