PI3K/AKT/mTOR pathway plays a major pathogenetic role in glycogen accumulation and tumor development in renal distal tubules of rats and men.

Activation of the PI3K/AKT/mTOR pathway is a crucial molecular event in human clear cell renal cell carcinoma (ccRCC), and is also upregulated in diabetic nephropathy. In diabetic rats metabolic changes affect the renal distal tubular epithelium and lead to glycogen-storing Armanni-Ebstein lesions (AEL), precursor lesions of RCC in the diabetes induced nephrocarcinogenesis model. These lesions resemble human sporadic clear cell tubules (CCT) and tumor cells of human ccRCC.Human sporadic CCT were examined in a collection of 324 nephrectomy specimen, in terms of morphologic, metabolic and molecular alterations, and compared to preneoplastic CCT and RCC developed in the rat following streptozotocin-induced diabetes or N-Nitrosomorpholine administration. Diabetic and non-diabetic rats were subjected to the dual PI3K/mTOR inhibitor, NVP/BEZ235.Human sporadic CCT could be detected in 17.3% of kidney specimens. Human and rat renal CCT display a strong induction of the PI3K/AKT/mTOR pathway and related metabolic alterations. Proteins involved in glycolysis and de novo lipogenesis were upregulated. In in vivo experiments, dual inhibition of PI3K and mTOR resulted in a reduction of proliferation of rat diabetes related CCT and increased autophagic activity.The present data indicate that human sporadic CCT exhibit a pattern of morphologic and metabolic alterations similar to preneoplastic lesions in the rat model. Activation of the PI3K/AKT/mTOR pathway in glycogenotic tubuli is a remarkable molecular event and suggests a preneoplastic character of these lesions also in humans.


Processing of human and rat renal tissue
Samples of renal tumor and non-tumor tissue were rapidly processed after being explanted and fixed in buffered 4.5% formaldehyde, dehydrated, and embedded in paraffin. Serial sections of 2-3 μm thickness were stained with hematoxylin & eosin (H&E) and the periodic acid Schiff's reaction (PAS) for assessing the glycogen content. For immunohistochemistry and western blot analysis, slices of about 1.5 cm × 1.5 cm × 0.5 cm were immediately frozen in -120°C cold isopentane and stored at -80°C. Some specimens of 2 mm 3 were cut with a razor blade, fixed in 2.5% glutaraldehyde, embedded in Glycidether 100 (formerly called Epon 812), cut with diamond knifes with a Leica ultratome to 500 and 750 nm thick semi-thin slides and stained according to Richardson [22]. Ultrathin sections of 70-90 nm were stained with uranyl acetate and lead citrate and examined with a Libra 120 electron microscope from Carl Zeiss (Jena, Germany). Glycogenotic tubules were identified as lesions of enlarged distal tubule epithelial cells with pale cytoplasm in H&E staining due to extensive glycogen storage, which stains positive for PAS staining [1,5,6]. The corresponding lesions in the enzyme-and immunostained-sections were detected by comparison with H&E-stained sections.

Western blot analysis
Six ccRCC and corresponding non-tumor tissue were homogenized in lysis buffer containing the Complete Protease Inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN) and sonicated. Protein concentrations were measured with the Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) using bovine serum as standard. For immunoblotting, aliquots of 40 μg were denatured by boiling in Tris-glycine SDS Sample buffer (Life Technologies, Grand Island, NY), separated by SDS-PAGE and transferred to nitrocellulose membranes by electroblotting. Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 for 1 h and probed with specific antibodies against AKT, p-AKT, Hexokinase 2, Pyruvate Kinase M2 (PKM2), Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH), Lactate dehydrogenase (LADH A/C), fatty acid synthase (FASN), phosphorylated ATP citrate lyase (p-ACLY), acyl-CoA dehydrogenase for acyl chains of medium length (ACADM), squalene synthetase (SQS) (Supporting table 1b) followed by incubation with a secondary horseradish peroxidase-conjugated antibody.

Immunohistochemistry of rat CCT and tumors and human CCT (frozen sections)
Frozen tissue was cut in 10 μm thick slices and stained with H&E and PAS. Serial cryostat sections were also incubated with a battery of primary antibodies against proteins of the PI3K/AKT/mTOR signaling pathway and enzymes of glycolysis, de novo lipogenesis, and cholesterol synthesis (Supporting Table 2). Endogeneous peroxidase was quenched with 1% hydrogen peroxide, and positive reactivity of primary antibodies was identified using the Ultravision LP detection system HRP polymer and DAB as the chromogen substrate (Thermoscientific, Waltham, MA, USA). Immunohistochemical signal intensity in CCT and tumors was estimated semiquantitatevely comparing to corresponding surrounding unaltered renal tissue. Negative controls were stained without a primary antibody.

Tissue microarrays (TMAs) of human ccRCC
A total of 102 cases of ccRCC were collected at the Institut für Pathologie, Universitätsspital Basel (Basel, Switzerland) and used for the construction of renal TMAs. Tumor grade was defined according to the Fuhrman grading system: 4 cases were Fuhrman grade I; 28 cases Fuhrman grade II; 37 cases Fuhrman grade III and 32 cases Fuhrman grade IV. The study was approved by the Institutional Review Board of the Universities of Greifswald and Basel. Renal TMA was constructed using formalin-fixed, paraffinembedded tissue. Paraffin blocks containing representative tumor areas were identified on corresponding H&E stained sections. The source block was cored, and a 1-mm core was transferred to the recipient "master block" using the Beecher Tissue Microarrayer (Beecher Instruments, Silver Spring, MD). 1-2 representative cores of tumor were arrayed per specimen.

Immunohistochemistry of TMAs
Immunohistochemical staining was performed for nine selected proteins belonging to the PI3K/AKT/mTOR pathway. Dilutions and manufacturers' information are listed in Supporting Table 3. Immunohistochemical staining was scored as positive if more than 10% of the tumor cells showed at least a focal weak cytoplasmatic and/or membranous staining. Staining patterns were compared to non-neoplastic renal cortical parenchyma.

Proliferation and apoptosis assays
RCC cell lines were seeded at 2 × 10 3 cells/well in a 96-well plate, allowed to attach and adjust for the next 12 hours (corresponding to 0 hour time point in the graphs) and grown for additional 48 hours. Proliferation was assessed at these two time points with the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology) by measuring the absorbance at 450 nm following the manufacturer's protocol. To measure apoptosis, cell lines were plated at 2 × 10 3 cells/well in a 96-well plate, incubated for 12 hours, subjected to 24 h serum deprivation (corresponding to 0 hour time point in the graphs), and continued to grow for additional 48 hours. Apoptosis was assessed using the Cell Death Detection ELISA-Plus Kit (Roche Molecular Biochemicals) by measuring the absorbance at 405 nm following manufacturer's instructions. Experiments were conducted at least three times in triplicate. Data are reported as percent viable or nonviable, respectively, tumor cells in each condition as compared with untreated cells or cells treated with DMSO alone.

Western blot analysis
Cells were pretreated as described above for ccRCC. After transfer of isolated proteins membranes were probed with specific antibodies against AKT, p-AKT, phosphorylated eukaryotic translation initiation factor 4E binding protein 1 (p-4E-BP1), phosphorylated ribosomal protein S6 (p-RPS6) and fatty acid synthase (FASN) (Supporting table 1a) followed by incubation with a secondary horseradish peroxidase-conjugated antibody.