Abstract
Many surgical models are used to study kidney and other diseases in mice, yet the effects of the surgical procedure itself on the kidney and other tissues have not been elucidated. In the present study, we found that both sham surgery and unilateral nephrectomy (UNX), which is used as a model of renal compensatory hypertrophy, in mice resulted in increased mammalian target of rapamycin complex 1/2 (mTORC1/2) in the remaining kidney. mTORC1 is known to regulate lysosomal biogenesis and autophagy. Genes associated with lysosomal biogenesis and function were decreased in sham surgery and UNX kidneys. In both sham surgery and UNX, there was suppressed autophagic flux in the kidney as indicated by the lack of an increase in LC3-II or autophagosomes seen on immunoblot, IF and EM after bafilomycin A1 administration and a concomitant increase in p62, a marker of autophagic cargo. There was a massive increase in pro-inflammatory cytokines, which are known to activate ERK1/2, in the serum after sham surgery and UNX. There was a large increase in ERK1/2 in sham surgery and UNX kidneys, which was blocked by the MEK1/2 inhibitor, trametinib. Trametinib also resulted in a significant decrease in p62. In summary, there was an intense systemic inflammatory response, an ERK-mediated increase in p62 and suppressed autophagic flux in the kidney after sham surgery and UNX. It is important that researchers are aware that changes in systemic pro-inflammatory cytokines, ERK1/2 and autophagy can be caused by sham surgery as well as the kidney injury/disease itself.
Highlights
Compensatory renal hypertrophy is an important consequence in both glomeruli and tubules following partial or complete UNX performed for renal cancer or for living kidney donors
Unilateral nephrectomy and sham surgery-dependent mTOR activation are attenuated by rapamycin
Increased mTOR after sham surgery and unilateral nephrectomy is associated with a lysosomal defect mTOR is known to regulate transcription factor EB (TFEB), a master regulator of lysosomal biogenesis[3,9] Quantitative polymerase chain reaction analysis for Tfeb and known TFEBdownstream genes Atp6v0d2, a vATPase subunit localized to the lysosomal membrane involved in lysosomal acidification[10] and Lamp[211], a lysosomal-associated membrane protein, was performed
Summary
Compensatory renal hypertrophy is an important consequence in both glomeruli and tubules following partial or complete UNX performed for renal cancer or for living kidney donors. It is well known that pS6, a marker of mTORC1, increases as early as 30 min after UNX and that mTORC1 inhibition with rapamycin can blunt UNX-induced renal hypertrophy. The effect of UNX on lysosomal biogenesis and function, which are tightly tied to mTOR function, was determined. As mTOR and lysosomal function are crucial to autophagy, autophagic flux was determined in vivo in the kidney. According to the 2016 guidelines for the use and interpretation of assays for monitoring autophagy, if the basal increase in LC3-II is due to increased autophagosome production, it is expected that lysosomal inhibition will further increase LC3-II (i.e., increased autophagic flux)[4]. We measured the increase in LC3-II after lysosomal inhibition and used it to determine the effect of Official journal of the Cell Death Differentiation Association
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