RESUMO
The redox regulator NRF2 is hyperactivated in a large percentage of non-small cell lung cancer (NSCLC) cases, which is associated with chemotherapy and radiation resistance. To identify redox vulnerabilities for KEAP1/NRF2 mutant NSCLC, we conducted a CRISPR-Cas9-based negative selection screen for antioxidant enzyme genes whose loss sensitized cells to sub-lethal concentrations of the superoxide (O2â¢-) -generating drug ß-Lapachone. While our screen identified expected hits in the pentose phosphate pathway, the thioredoxin-dependent antioxidant system, and glutathione reductase, we also identified the mitochondrial superoxide dismutase 2 (SOD2) as one of the top hits. Surprisingly, ß-Lapachone did not generate mitochondrial O2â¢- but rather SOD2 loss enhanced the efficacy of ß-Lapachone due to loss of iron-sulfur protein function, loss of mitochondrial ATP maintenance and deficient NADPH production. Importantly, inhibition of mitochondrial electron transport activity sensitized cells to ß-Lapachone, demonstrating that these effects may be translated to increase ROS sensitivity therapeutically.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antioxidantes/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , OxirreduçãoRESUMO
An increase of glomerular filtration rate (GFR) is a common observation in early diabetes and is considered a key risk factor for subsequent kidney injury. However, the mechanisms underlying diabetic hyperfiltration have not been fully clarified. Here, we tested the hypothesis that macula densa neuronal nitric oxide synthase (NOS1) is upregulated via sodium glucose cotransporter type 1 (SGLT1) in diabetes, which then inhibits tubuloglomerular feedback (TGF) promoting glomerular hyperfiltration. Therefore, we examined changes in cortical NOS1 expression and phosphorylation, nitric oxide production in the macula densa, TGF response, and GFR during the early stage of insulin-deficient (Akita) diabetes in wild-type and macula densa-specific NOS1 knockout mice. A set of sophisticated techniques including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of kidney tubules in vivo, and clearance kinetics of plasma fluorescent-sinistrin were employed. Complementary studies tested the role of SGLT1 in SGLT1 knockout mice and explored NOS1 expression and phosphorylation in kidney biopsies of cadaveric donors. Diabetic mice had upregulated macula densa NOS1, inhibited TGF and elevated GFR. Macula densa-selective NOS1 knockout attenuated the diabetes-induced TGF inhibition and GFR elevation. Additionally, deletion of SGLT1 prevented the upregulation of macula densa NOS1 and attenuated inhibition of TGF in diabetic mice. Furthermore, the expression and phosphorylation levels of NOS1 were increased in cadaveric kidneys of diabetics and positively correlated with blood glucose as well as estimated GFR in the donors. Thus, our findings demonstrate that the macula densa SGLT1-NOS1-TGF pathway plays a crucial role in the control of GFR in diabetes.
Assuntos
Diabetes Mellitus Experimental , Transportador 1 de Glucose-Sódio/metabolismo , Animais , Diabetes Mellitus Experimental/metabolismo , Retroalimentação , Taxa de Filtração Glomerular/fisiologia , Glomérulos Renais/metabolismo , Túbulos Renais/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo I/metabolismoRESUMO
[Figure: see text].
