Sitai Liang

Novel protective mechanism of reducing renal cell damage in diabetes: Activation AMPK by AICAR increased NRF2/OGG1 proteins and reduced oxidative DNA damage

ABSTRACT Exposure of renal cells to high glucose (HG) during diabetes has been recently proposed to be involved in renal injury. In the present study, we investigated a potential mechanism by which AICAR treatment regulates the DNA repair enzyme, 8-oxoG-DNA glycosylase (OGG1) in renal proximal tubular mouse cells exposed to HG and in kidney of db/db mice. Cells treated with HG for 2 days show inhibition in OGG1 promoter activity as well as OGG1 and Nrf2 protein expression. In addition, activation of AMPK by AICAR resulted in an increase raptor phosphorylation at Ser792 and leads to increase the promoter activity of OGG1 through upregulation of Nrf2. Downregulation of AMPK by DN-AMPK and raptor and Nrf2 by siRNA resulted in significant decease in promoter activity and protein expression of OGG1. On the other hand, downregulation of Akt by DN-Akt and rictor by siRNA resulted in significant increase in promoter activity and protein expression of Nrf2 and OGG1. Moreover, gel shift analysis shows reduction of Nrf2 binding to OGG1 promoter in cells treated with HG while cells treated with AICAR reversed the effect of HG. Furthermore, db/db mice treated with AICAR show significant increased in AMPK and raptor phosphroylation as well as OGG1 and Nrf2 protein expression that associated with significant decrease in oxidative DNA damage (8-oxodG) compared to non-treated mice. In summary, our data provide a novel protective mechanism by which AICAR prevents renal cell damage in diabetes and the consequence complications of hyperglycemia with a specific focus on nephropathy.

Is mTOR inhibitor good enough for treatment all tumors in TSC patients

Tuberous sclerosis complex (TSC) is an autosomal dominant and multi-system genetic disorder in humans. TSC affects around 25,000 to 40,000 individuals in the United States and about 1 to 2 million individuals worldwide, with an estimated prevalence of one in 6,000 newborns. TSC occurs in all races and ethnic groups, and in both genders. TSC is caused by defects or mutations in two genes, TSC1 and TSC2. Loss of TSC1/TSC2 leads to dysregulation of mTOR, resulting in aberrant cell differentiation and development, and abnormal enlargement of cells. TSC is characterized by the development of benign and/or malignant tumors in several organs including renal/liver angiomyolipomas, facial angiofibroma, lymphangiomyomatosis, cardiac rhabdomyomas, retinal astrocytic, renal cell carcinoma, and brain subependymal giant cell astrocytomas (SEGA). In addition, TSC disease causes disabling neurologic disorders, including epilepsy, mental retardation and autism. Particularly problematic are the development of renal angiomyolipomas, which tend to be larger, bilateral, multifocal and present at a younger age compared with sporadic forms. In addition, SEGA block the flow of fluid within the brain, causing a buildup of fluid and pressure that leads to blurred vision and seizures. In the current review, we describe the pathology of TSC disease in key organs and summarize the use of mTOR inhibitors to treat tumors in TSC patients.

Novel mechanism of regulation of fibrosis in kidney tumor with tuberous sclerosis

BackgroundDeficiency in tuberin results in activation the mTOR pathway and leads to accumulation of cell matrix proteins. The mechanisms by which tuberin regulates fibrosis in kidney angiomyolipomas (AMLs) of tuberous sclerosis patients are not fully known.MethodIn the present study, we investigated the potential role of tuberin/mTOR pathway in the regulation of cell fibrosis in AML cells and kidney tumor tissue from tuberous sclerosis complex (TSC) patients.ResultsAML cells treated with rapamycin shows a significant decrease in mRNA and protein expression as well as in promoter transcriptional activity of alpha-smooth muscle actin (α-SMA) compared to untreated cells. In addition, cells treated with rapamycin significantly decreased the protein expression of the transcription factor YY1. Rapamycin treatment also results in the redistribution of YY1 from the nucleus to cytoplasm in AML cells. Moreover, cells treated with rapamycin resulted in a significant reduce of binding of YY1 to the αSMA promoter element in nuclear extracts of AML cells. Kidney angiomyolipoma tissues from TSC patients showed lower levels of tuberin and higher levels of phospho-p70S6K that resulted in higher levels of mRNA and protein of αSMA expression compared to control kidney tissues. In addition, most of the α-SMA staining was identified in the smooth muscle cells of AML tissues. YY1 was also significantly increased in tumor tissue of AMLs compared to control kidney tissue suggesting that YY1 plays a major role in the regulation of αSMA.ConclusionsThese data comprise the first report to provide one mechanism whereby rapamycin might inhibit the cell fibrosis in kidney tumor of TSC patients.

Novel mechanism of transcriptional regulation of cell matrix protein through CREB

The transcription mechanism(s) of renal cell matrix accumulation in diabetes does not explored. Phosphorylation of the transcription factor cAMP-responsive element binding protein (CREB) significantly increased in cells treated with high glucose (HG) compared to cell grown in normal glucose (NG). Cells pretreated with rapamycin before exposure to HG showed significant decrease phosphorylation of CREB, increase in AMPK activity and decrease protein/mRNA and promoter activity of fibronectin. In addition, cells transfected with siRNA against CREB showed significant increase in AMPK activity, decrease in protein/mRNA and promoter activity of fibronectin. Cells treated with HG showed nuclear localization of p-CREB while pretreated cells with rapamycin reversed HG effect. Moreover, gel shift analysis shows increase binding of CREB to fibronectin promoter in cells treated with HG while cells pretreated with rapamycin reversed the effect of HG. Furthermore, db/db mice treated with rapamycin showed significant increase in AMPK activity, decrease in expression of p-CREB and protein/mRNA of fibronectin. Strong staining of fibronectin and p-CREB was detected in kidney cortex of db/db mice while treated mice with rapamycin reversed hyperglycemia effect. In summary, our data provide a novel mechanism of transcriptional regulation of fibronectin through CREB that may be used as therapeutic approach to prevent diabetes complications.

Preclinical evidence of the enhanced effectiveness of combined rapamycin and AICAR in reducing kidney cancer

Loss of Von Hippel‐Lindau in renal carcinoma cells results in upregulation of the activity of hypoxia‐inducible factor (HIF‐α), a major transcription factor involved in kidney cancer. Rapamycin as mammalian target of rapamycin inhibitor and 5‐aminoimidazole‐4‐carboxamide‐riboside (AICAR) as AMPK activator are used separately to treat cancer patients. In the current study, the possible additive effect of drug combinations in reducing kidney tumorigenesis was investigated. Treatment with drug combinations significantly decreased cell proliferation, increased cell apoptosis, and abolished Akt phosphorylation and HIF‐2α expression in renal cell carcinoma cells, including primary cells isolated from kidney cancer patients. Significant decreases in cell migration and invasion were detected using drug combinations. Drug combinations effectively abolished binding of HIF‐2α to the Akt promoter and effected formation of the DNA‐protein complex in nuclear extracts from 786‐O cells, as demonstrated using electromobility shift assay and examination of Akt promoter activity. Importantly, we tested the effect of each drug and the combined drugs on kidney tumor size in the nude mouse model. Our data show that treatment with rapamycin, AICAR, and rapamycin+AICAR decreased tumor size by 38%, 36%, and 80%, respectively, suggesting that drug combinations have an additive effect in reducing tumor size compared with use of each drug alone. Drug combinations effectively decreased cell proliferation, increased apoptotic cells, and significantly decreased p‐Akt, HIF‐2α, and vascular endothelial growth factor expression in tumor kidney tissues from mice. These results show for the first time that drug combinations are more effective than single drugs in reducing kidney tumor progression. This study provides important evidence that may lead to the initiation of pre‐clinical trials in patients with kidney cancer.

Galectin-1 is a new fibrosis protein in type 1 and type 2 diabetes

Chronic exposure of tubular renal cells to high glucose contributes to tubulointerstitial changes in diabetic nephropathy. In the present study, we identified a new fibrosis gene called galectin‐1 (Gal‐1), which is highly expressed in tubular cells of kidneys of type 1 and type 2 diabetic mouse models. Gal‐1 protein and mRNA expression showed significant increase in kidney cortex of heterozygous Akita+/− and db/db mice compared with wild‐type mice. Mouse proximal tubular cells exposed to high glucose showed significant increase in phosphorylation of Akt and Gal‐1. We cloned Gal‐1 promoter and identified the transcription factor AP4 as binding to the Gal‐1 promoter to up‐regulate its function. Transfection of cells with plasmid carrying mutations in the binding sites of AP4 to Gal‐1 promoter resulted in decreased protein function of Gal‐1. In addition, inhibition of Gal‐1 by OTX‐008 showed significant decrease in p‐Akt/AP4 and protein‐promoter activity of Gal‐1 and fibronectin. Moreover, down‐regulation of AP4 by small interfering RNA resulted in a significant decrease in protein expression and promoter activity of Gal‐1. We found that kidney of Gal‐1−/− mice express very low levels of fibronectin protein. In summary, Gal‐1 is highly expressed in kidneys of type 1 and 2 diabetic mice, and AP4 is a major transcription factor that activates Gal‐1 under hyperglycemia. Inhibition of Gal‐1 by OTX‐008 blocks activation of Akt and prevents accumulation of Gal‐1, suggesting a novel role of Gal‐1 inhibitor as a possible therapeutic target to treat renal fibrosis in diabetes.—Al‐Obaidi, N., Mohan, S., Liang, S., Zhao, Z., Nayak, B. K., Li, B., Sriramarao, P., Habib, S. L. Galectin‐1 is a new fibrosis protein in type 1 and type 2 diabetes. FASEB J. 33, 373–387 (2019). www.fasebj.org

A new drug combination significantly reduces kidney tumor progression in kidney mouse model

Tuberous sclerosis complex (TSC) disease is associated with tumors in many organs, particularly angiomyolipoma (AML) in the kidneys. Loss or inactivation of TSC1/2 results in high levels of HIF-α activity and VEGF expression. mTOR inhibitor (rapamycin) and the AMPK activator 5-aminoimidazole-4-carboxamide (AICA)-riboside (AICAR) are currently used separately to treat cancer patients. Here, we investigated the effect of a novel combination of rapamycin and AICAR on tumor progression. Our data show that treatment of AML human cells with drug combinations resulted in 5-7-fold increase in cell apoptosis compared to each drug alone. In addition, drug combinations resulted in 4-5-fold decrease in cell proliferation compared to each drug alone. We found that drug combinations abolished Akt and HIF activity in AML cells. The drug combinations resulted in decrease in cell invasion and cell immigration by 70% and 84%, respectively in AML cells. The combined drugs also significantly decreased the VEGF expression compare to each drug alone in AML cells. Drug combinations effectively abolished binding of HIF-2α to the putative Akt site in the nuclear extracts isolated from AML cells. Treatment TSC mice with drug combinations resulted in 75% decrease in tumor number and 88% decrease in tumor volume compared to control TSC mice. This is first evidence that drug combinations are effective in reducing size and number of kidney tumors without any toxic effect on kidney. These data will provide evidence for initiating a new clinical trial for treatment of TSC patients.