Surgical procedures suppress autophagic flux in the kidney.

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.

The effect of trehalose on autophagy-related proteins and cyst growth in a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder characterized by kidney cyst growth often resulting in end-stage renal disease. There is growing attention on understanding the role of impaired autophagy in ADPKD. Trehalose (TRE) has been shown to increase both protein stability and aggregate clearance and induce autophagy in neurodegenerative diseases. TRE treatment in wild type mice compared to vehicle resulted in increased expression in the kidney of Atg12-5 complex and increased Rab9a, autophagy-related proteins that play a role in the formation of autophagosomes. Thus, the aim of the study was to determine the effect of TRE on cyst growth and autophagy-related proteins, in the hypomorphic Pkd1RC/RC mouse model of ADPKD. Pkd1RC/RC mice were treated 2% TRE in water from days 50 to 120 of age. TRE did not slow cyst growth or improve kidney function or affect proliferation and apoptosis in Pkd1RC/RC kidneys. In Pkd1RC/RC vs. wild type kidneys, expression of the Atg12-5 complex was inhibited by TRE resulting in increased free Atg12 and TRE was unable to rescue the deficiency of the Atg12-5 complex. Rab9a was decreased in Pkd1RC/RC vs. wild type kidneys and unaffected by TRE. The TRE-induced increase in p62, a marker of autophagic cargo, that was seen in normal kidneys was blocked in Pkd1RC/RC kidneys. In summary, the autophagy phenotype in Pkd1RC/RC kidneys was characterized by decreases in crucial autophagy-related proteins (Atg12-5 complex, Atg5, Atg16L1), decreased Rab9a and increased mTORC1 (pS6S240/244, pmTORS2448) proteins. TRE increased Atg12-5 complex, Rab9a and p62 in normal kidneys, but was unable to rescue the deficiency in autophagy proteins or suppress mTORC1 in Pkd1RC/RC kidneys and did not protect against cyst growth.

Increased mTOR and suppressed autophagic flux in the heart of a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease.

Cardiac hypertrophy is common in autosomal dominant polycystic kidney disease (ADPKD) patients. We found increased heart weight in Pkd1RC/RC and Pkd2WS25/+ mouse models of ADPKD. As there is a link between increased heart weight and mammalian target of rapamycin (mTOR), the aim of the study was to determine mTOR complex 1 and 2 signaling proteins in the heart in the Pkd1RC/RC mouse model of PKD. In 70 day old Pkd1RC/RC hearts, on immunoblot analysis, there was a large increase in p-AMPKThr172, a known autophagy inducer, and an increase in p-AktSer473 and p-AktThr308, but no increase in other mTORC1/2 proteins (p-S6Ser240/244, p-mTORSer2448). In 150 day old Pkd1RC/RC hearts, there was an increase in mTORC1 (p-S6Ser240/244) and mTOR-related proteins (p-AktThr308, p-GSK3ßSer9, p-AMPKThr172). As the mTOR pathway is the master regulator of autophagy, autophagy proteins were measured. There was an increase in p-Beclin-1 (BECN1), an autophagy regulator and activating molecule in Beclin-1-regulated autophagy (AMBRA1), a regulator of Beclin that play a role in autophagosome formation, an early stage of autophagy. There was a defect in the later stage of autophagy, the fusion of the autophagosome with the lysosome, known as autophagic flux, as evidenced by the lack of an increase in LC3-II, a marker of autophagosomes, with the lysosomal inhibitor bafilomycin, in both 70 day old and 150 day old hearts. To determine the role of autophagy in causing increased heart weight, Pkd1RC/RC were treated with 2-deoxyglucose (2-DG) or Tat-Beclin1 peptide, agents known to induce autophagy. 2-DG treatment from 150 to 350 days of age, a time period when increased heart weight developed, did not reduce the increased heart weight. Unexpectedly, Tat-Beclin 1 peptide treatment from 70 to 120 days of age resulted in increased heart weight. In summary, there is suppressed autophagic flux in the heart at an early age in Pkd1RC/RC mice. Increased mTOR signaling in older mice is associated suppressed autophagic flux. There was a large increase in p-AMPKThr172, a known autophagy inducer, in both young and old mice. 2-DG treatment did not impact increased heart weight and Tat-Beclin1 peptide increased heart weight.

The effect of MEK1/2 inhibitors on cisplatin-induced acute kidney injury (AKI) and cancer growth in mice.

In a clinically-relevant model of 4 week, low-dose cisplatin-induced AKI, mice were injected subcutaneously with non small cell lung cancer (NSCLC) cells that harbor an activating Kirsten rat sarcoma viral oncogene homolog (KRAS)G12V mutation. Phospho extracellular signal-regulated kinase1/2 (pERK1/2) expression in kidney and tumors was decreased by the MEK1/2 inhibitors, U0126 and trametinib, that potently inhibit pERK1/2. U0126 resulted in a significant improvement in kidney function, acute tubular necrosis (ATN) and tubular cell apoptosis in mice with AKI. Genes that were significantly decreased by U0126 were heat shock protein 1, cyclin-dependent kinase 4 (CDK4) and stratifin (14-3-3σ). U0126 resulted in a significant decrease in tumor weight and volume and significantly increased the chemotherapeutic effect of cisplatin. Trametinib, a MEK1/2 inhibitor that is FDA-approved for the treatment of cancer, did not result in functional protection against AKI or worse AKI, but dramatically decreased tumor growth more than cisplatin. Smaller tumors in cisplatin or MEK1/2 inhibitor-treated mice were not related to changes in microtubule-associated proteins 1A/1B light chain 3B (LC3-II), p62, cleaved caspase-3, granzyme B, or programmed death-ligand 1 (PD-L1). In summary, despite ERK inhibition by both U0126 and trametinib, only U0126 protected against AKI suggesting that the protection against AKI by U0126 was due to an off-target effect independent of ERK inhibition. The effect of U0126 to decrease AKI may be mediated by inhibition of heat shock protein 1, CDK4 or stratifin (14-3-3σ). Trametinib was more effective than cisplatin in decreasing tumor growth, but unlike cisplatin, trametinib did not cause AKI.

The consequences of increased 4E-BP1 in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disease, characterized by cyst formation and growth. Hyperproliferation is a major contributor to cyst growth. At the nexus of regulating proliferation, is 4E-BP1. We demonstrate that ADPKD mouse and rat models, ADPKD patient renal biopsies and PKD1-/- cells exhibited hyperphosphorylated 4E-BP1, a biomarker of increased translation and proliferation. We hypothesized that expression of constitutively active 4E-BP1 constructs (4E-BP1F113A and 4E-BP1R13AF113A) would decrease proliferation and reduce cyst expansion. Utilizing the Pkd1RC/RC mouse, we determined the effect of 4E-BP1F113A on PKD. Unexpectedly, 4E-BP1F113A resulted in increased cyst burden and suppressed apoptosis markers, increased anti-apoptotic Bcl-2 protein and increased mitochondrial proteins. Exogenous 4E-BP1 enhanced proliferation, decreased apoptosis, increased anti-apoptotic Bcl-2 protein, impaired NADPH oxidoreductase activity, increased mitochondrial proteins and increased superoxide production in PKD patient-derived renal epithelial cells. Reduced 4E-BP1 expression suppressed proliferation, restored apoptosis and improved cellular metabolism. These findings provide insight into how cyst-lining cells respond to 4E-BP1.