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

Abstract

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.

Keywords: AKT/mTOR; clear cell tubules; glycogen; nephrocarcinogenesis.

Figure 1. Clear cell tubules (CCT, A–C) of streptozotocin-induced diabetic rats and human glycogenotic alterations of distal tubules (D–F)

A. Periodic acid-Schiff (PAS) reaction: distribution of PAS-positive rat AEL near the transition zone of renal cortex (upper part) and medulla (bottom). B. The cytoplasm of AEL cells is enlarged and appears clearly in hematoxylin and eosin (H & E) staining C. Magnification of PAS stain demonstrates the corresponding dense glycogen storage in distal AEL tubules. D, E. Human glycogenotic tubules (arrowheads) in a nephrectomy specimen. The similarity to rat CCT is easily recognizable in magnified H&E staining F. Glycogen is fast eluted in not perfused renal tissue, thus PAS staining is often patchy but still illustrates increased glycogen storage in enlarged tubules. Length of the lower edge A 1.25 mm, B, C, E, F 0.15 mm, D 0.5 mm.

Figure 2. Electron microscopic features of diabetes related clear cell tubules (CCT)

The overview micrograph A. illustrates a transverse section of an altered CCT with enlarged epithelium due to densely packed α-particles of glycogen in the cytoplasm B. in comparison to unaltered distal tubule epithelium C. with small amounts of glycogen and preserved loose distributed cell organelles. In CCT, masses of glycogen displace mitochondria (arrows) to the outer cell borders and the endoplasmatic reticulum (arrowheads) has nearly vanished and is only detectable beneath the nucleus. Length of the lower edge A 50 μm, B 7.5 μm, C 3 μm.

Figure 3. Autophagic activity in rat clear cell tubulus lesions

Immunohistochemical demonstration of microtubule associated light chain 3 A (LC3A) indicates autophagy activity in glycogenotic tubules of diabetic rats, without A. or after treatment with NVP/BEZ235 for 4 weeks B. Low autophagy activity was detected in control kidney samples of non diabetic rats following NVP/BEZ235 administration for 4 weeks C. Electron microscopic examination reveals segregation vacuoles in the cytoplasm of the distal tubular epithelium with membrane bound vacuoles (D. arrows), containing glycogen particles, thus representing autophagic vacuoles. In untreated diabetic rats E. they are small and often singular (segmentated square). After treatment with NVP/BEZ235 for 4 weeks F., G. vacuoles are multiple, large, filled with abundant amounts of glycogen and are membrane bound (arrow). Length of the lower edge: A-C 0.15 mm; D 1.5 μm, E 20 μm, F 17 μm, G 7 μm.

Figure 4. Representative immunohistochemistry in serial cryostat sections series of clear cell tubules (CCT) from streptozotocin-induced diabetic rats (A) and preneoplastic tubular lesions and renal carcinomas in rats after administration of N-Nitrosomorpholine (NNM) (B)

A. CCT of diabetic rats after 4 weeks (upper panel of each row) with clear cell feature in H&E staining, which is caused by glycogen storage, as shown by strong positivity to the PAS reaction. Overexpression of the insulin receptor (IR) leads to upregulation of activated/phosphorylated AKT and mTOR proteins. Glucose accumulation is due to upregulation of glucose transporters, i.e. GLUT4, a process dependent on insulin. Utilization of glucose in the glycolytic pathway (PFKL) is facilitated by activated/phosphorylated AKT and chREBP. Glycogenolysis is inhibited, as GSK-3β is phosphorylated/inactivated. Additionally, SREBP-1 mediates transcription of enzymes of de novo lipogenesis (MVK, FASN). Simultaneously, β-oxidation of fatty acids is activated, as acyl-CoA dehydrogenase for acyl chains of medium length (ACADM) is upregulated. CCT of rats treated with NVP/BEZ235 for 1 week (lower panel of each row) are small and narrowed and reveal lower expression of all members of the PI3K/AKT/mTOR pathway examined. B. Molecular and metabolic alterations in NNM-induced glycogenotic preneoplastic tubules (upper part of each picture) and solid renal cell carcinomas (lower part) are similar to those detected in diabetes-induced nephrocarcinogenesis (24 months old rat, NNM was administered as a single dose). Length of the lower edge: 4A H&E, PAS 0.25 mm; immunohistochemistry 0.125 mm; 4B 0.3 mm.

Figure 5. Morphological aspects of human clear cell tubules (CCT) and advanced lesions

Glycogenotic CCT (A. arrowhead) at the related glomerulus with its vas afferens and the transition (B. arrows) of distal tubule epithelial cells to glycogen storing CCT and multiple CCT lesions in the renal cortex (C. arrowheads) could be detected in the nephrectomy specimens. The glycogenotic tubules also form cell clusters near the related glomerulus D. or almost 1 mm large nodules. Nodular lesions with a basophilic (E. larger magnification of the square box) or acidophilic (F. larger magnification of the square box) cytoplasm, enlargement of nuclei and prominent nucleoli represent advanced lesions. Length of the lower edge: A, B 0.3 mm, C 1.67, D, E 1 mm, F, G 0.6 mm.

Figure 6. Representative immunohistochemical findings in serial cryostat sections series of human glycogenotic tubules

At low magnification and PAS reaction, human glycogenotic distal tubules can be easily detected in the transition zone of renal cortex and medulla. Their cytoplasm is swollen and appears “empty”, containing glycogen, as it can be demonstrated with purple staining in PAS-reaction. The insulin receptor (IR) and members of the PI3K/AKT/mTOR signaling pathway (phosphorylated AKT and mTOR, RPS6, and 4EBP1) are induced when compared with adjacent renal tissue (control H&E and representative negative immunohistochemistry staining). Activation of the IR increases glucose uptake via translocation to the cell membrane of GLUT1 and 4. Also, glycolysis (PKM2, PFKL), de novo lipogenesis (phosphorylated/inactivated AMPK, FASN, ACLY, SCD1 and chREBP) and β-oxidation of lipids (ACADM) are strongly induced in glycogenotic tubules. There is no expression of any of the investigated proteins in proximal or unaltered distal tubules of the same kidney (control), as shown by representative immunohistochemistry for PKM2. Length of the lower edge: PAS overview 0.4 mm; remaining stainings 0.14 mm.

Figure 7. Schematic representation of the molecular mechanisms responsible for the glycogenotic and lipogenic phenotype and promotion of cell growth in rat preneoplastic clear cell tubulus (CCT) lesions, human clear cell renal cell carcinoma and presumably human CCT

Deregulation of the PI3K/AKT/mTOR cascades induces de-novo-lipogenesis (chREBP, SREBP-1, p-ACLY, ACAC, FASN, USP-2A, SCD1), and glycolysis (IGLK, PKM2, PFKL). The glucose required for the aforementioned processes is obtained by the upregulation of GLUT1 and 4 glucose transporters. In addition, the AKT/mTOR cascade inactivates GSK-3β, AMPKα, 4E-BP1, and activates RPS6. Autophagy is also inhibited. Aberrant activation of lipogenesis and glycolysis by the PI3K/AKT/mTOR pathway leads to the upregulation of oncogenic signals, with consequent tubular epithelium proliferation and growth. Symbols: arrows: activation, blunted arrows: inhibition.