Nox4 is a Target for Tuberin Deficiency Syndrome.

The mechanism by which TSC2 inactivation or deficiency contributes to the pathology of tuberous sclerosis complex (TSC) is not fully clear. We show that renal angiomyolipomas from TSC patients and kidney cortex from Tsc2+/- mice exhibit elevated levels of reactive oxygen species (ROS). Downregulation of tuberin (protein encoded by TSC2 gene) in renal proximal tubular epithelial cells significantly increased ROS concomitant with enhanced Nox4. Similarly, we found elevated levels of Nox4 in the renal cortex of Tsc2+/- mice and in the renal angiomyolipomas from TSC patients. Tuberin deficiency is associated with activation of mTORC1. Rapamycin, shRNAs targeting raptor, or inhibition of S6 kinase significantly inhibited the expression of Nox4, resulting in attenuation of production of ROS in tuberin-downregulated proximal tubular epithelial cells. In contrast, activation of mTORC1 increased Nox4 and ROS. These results indicate that Nox4 may be a potential target for tuberin-deficiency-derived diseases. Using a xenograft model from tuberin-null tubular cells in nude mice, both anti-sense Nox4 and GKT137831, a specific inhibitor of Nox1/4, significantly inhibited the tumor growth. Thus, our results demonstrate the presence of an antagonistic relationship between tuberin and Nox4 to drive oncogenesis in the tuberin deficiency syndrome and identify Nox4 as a target to develop a therapy for TSC.

Mouse Metanephric Mesenchymal Cell-Derived Angioblasts Undergo Vasculogenesis in Three-Dimensional Culture.

In vitro models for the investigation of renal vascular development are limited. We previously showed that isolated metanephric mesenchymal (MM) and ureteric bud (UB) cells grown in three-dimensional (3D) matrices formed organoids that consisted of primitive vascular structures surrounding a polarized epithelium. Here, we examined the potential of two principal effectors of vasculogenesis, vascular endothelial growth factor A (VEGF-A), and platelet-derived growth factor B chain (PDGF-BB), to stimulate MM cell differentiation. The results showed that MM cells possess angioblast characteristics by expressing phenotypic markers for endothelial and mesenchymal cells. UB cells synthesize VEGF-A and PDGF-BB proteins and RNA, whereas the MM cells express the respective cognate receptors, supporting their role in directional induction of vasculogenesis. VEGF-A stimulated proliferation of MM cells in monolayer and in 3D sponges but did not affect MM cell migration, organization, or vasculogenesis. However, PDGF-BB stimulated MM cell proliferation, migration, and vasculogenesis in monolayer and organization of the cells into primitive capillary-like assemblies in 3D sea sponge scaffolds in vitro. A role for PDGF-BB in vasculogenesis in the 3D MM/UB co-culture system was validated by direct interference with PDGF-BB or PDGF receptor-ß cell interactions to implicate PDGF-BB as a primary effector of MM cell vasculogenesis. Thus, MM cells resemble early renal angioblasts that may provide an ideal platform for the investigation of renal vasculogenesis in vitro.

Tryptophan Metabolism in Patients With Chronic Kidney Disease Secondary to Type 2 Diabetes: Relationship to Inflammatory Markers.

OBJECTIVE: Type 2 diabetes (T2D) is the primary case of chronic kidney disease (CKD). Inflammation is associated with metabolic dysregulation in patients with T2D and CKD. Tryptophan (TRP) metabolism may have relevance to the CKD outcomes and associated symptoms. We investigated the relationships of TRP metabolism with inflammatory markers in patients with T2D and CKD. METHODS: Data were collected from a well-characterized cohort of type 2 diabetic individuals with all stages of CKD, including patients on hemodialysis. Key TRP metabolites (kynurenine [KYN], kynurenic acid [KYNA], and quinolinic acid [QA]), proinflammatory cytokines (tumor necrosis factor-α [TNF-α] and interleukin-6 [IL-6]), and C-reactive protein were measured in plasma. The KYN/TRP ratio was utilized as a surrogate marker for indoleamine 2,3-dioxygenase 1 (IDO1) enzyme activity. RESULTS: There was a significant inverse association between circulating TRP level and stages of CKD (P< 0.0001). Downstream bioactive TRP metabolites KYN, KYNA, and QA were positively and robustly correlated with the severity of kidney disease (P < 0.0001). In multiple linear regression, neither TNF-α nor IL-6 was independently related to KYN/TRP ratio after adjusting for estimated glomerular filtration rate (eGFR). Only TNF-α was independently related to KYN after taking into account the effect of eGFR. CONCLUSIONS: Chronic kidney disease secondary to T2D may be associated with accumulation of toxic TRP metabolites due to both inflammation and impaired kidney function. Future longitudinal studies to determine whether the accumulation of KYN directly contributes to CKD progression and associated symptoms in patients with T2D are warranted.

Spleen contributes significantly to increased circulating levels of fibroblast growth factor 23 in response to lipopolysaccharide-induced inflammation.

Background: Circulating levels of fibroblast growth factor 23 (FGF23) increase progressively and correlate with systemic inflammation in chronic kidney disease (CKD). The aim of this study was to identify and characterize the causal relationship between FGF23 and inflammation in CKD. Methods: Circulating FGF23 and inflammatory cytokines were correlated in healthy subjects and patients with varying levels of CKD. In addition, FGF23 expression in blood and solid organs was measured in normal mice that were exposed acutely (one time) or chronically (2-week) to low-dose lipopolysaccharide (LPS); chronic exposure being either sustained (subcutaneous pellets), intermittent (daily injections) or combined sustained plus acute (subcutaneous pellets plus acute injection on the day of sacrifice). Blood was analyzed for both terminal (cFGF23) and intact (iFGF23) FGF23 levels. Solid tissues were investigated with immunohistochemistry, enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Results: FGF23 levels correlated significantly with neutrophil gelatinase-associated lipocalin ( r = 0.72, P < 0.001), C-reactive protein ( r = 0.38, P < 0.001), tumor necrosis factor-α ( r = 0.32, P = 0.001) and interleukin-6 ( r = 0.48, P < 0.001). Acute LPS administration increased tissue FGF23 mRNA and plasma levels of cFGF23 but not iFGF23. Neither chronic sustained nor chronic pulsatile LPS increased the tissue or circulating levels of FGF23. However, acute on chronic LPS raised tissue FGF23 mRNA and both circulating cFG23 and iFGF23. Interestingly, the spleen was the major source of FGF23. Conclusion: Acute on chronic exposure to LPS stimulates FGF23 production in a normal mouse model of inflammation. We provide the first evidence that the spleen, under these conditions, contributes substantially to elevated circulating FGF23 levels.

Layer-Specific Manganese-Enhanced MRI of the Diabetic Rat Retina in Light and Dark Adaptation at 11.7 Tesla.

PURPOSE: To employ high-resolution manganese-enhanced MRI (MEMRI) to study abnormal calcium activity in different cell layers in streptozotocin-induced diabetic rat retinas, and to determine whether MEMRI detects changes at earlier time points than previously reported. METHODS: Sprague-Dawley rats were studied 14 days (n = 8) and 30 days (n = 5) after streptozotocin (STZ) or vehicle (n = 7) injection. Manganese-enhanced MRI at 20 × 20 × 700 µm, in which contrast is based on manganese as a calcium analogue and an MRI contrast agent, was obtained in light and dark adaptation of the retina in the same animals in which one eye was covered and the fellow eye was not. The MEMRI activity encoding of the light and dark adaptation was achieved in awake conditions and imaged under anesthesia. RESULTS: Manganese-enhanced MRI showed three layers, corresponding to the inner retina, outer retina, and the choroid. In normal animals, the outer retina showed higher MEMRI activity in dark compared to light; the inner retina displayed lower activity in dark compared to light; and the choroid showed no difference in activity. Manganese-enhanced MRI activity changed as early as 14 days after hyperglycemia and decreased with duration of hyperglycemia in the outer retina in dark relative to light adaptation. The choroid also had altered MEMRI activity at 14 days, which returned to normal by 30 days. No differences in MEMRI activity were detected in the inner retina. CONCLUSIONS: Manganese-enhanced MRI detects progressive reduction in calcium activity with duration of hyperglycemia in the outer retina as early as 14 days after hyperglycemia, earlier than any other time point reported in the literature.

Reciprocal induction of simple organogenesis by mouse kidney progenitor cells in three-dimensional co-culture.

Kidney development is regulated by a coordinated reciprocal induction of metanephric mesenchymal (MM) and ureteric bud (UB) cells. Here, established MM and UB progenitor cell lines were recombined in three-dimensional Matrigel implants in SCID mice. Differentiation potential was examined for changes in phenotype, organization, and the presence of specialized proteins using immunofluorescence and bright-field and electron microscopy. Both cell types, when grown alone, did not develop into specialized structures. When combined, the cells organized into simple organoid structures of polarized epithelia with lumens surrounded by capillary-like structures. Tracker experiments indicated the UB cells formed the tubuloid structures, and the MM cells were the source of the capillary-like cells. The epithelial cells stained positive for pancytokeratin, the junctional complex protein ZO-1, collagen type IV, as well as UB and collecting duct markers, rearranged during transfection (RET), Dolichos biflorus lectin, EndoA cytokeratin, and aquaporin 2. The surrounding cells expressed α-smooth muscle actin, vimentin, platelet endothelial cell adhesion molecule 1 (PECAM), and aquaporin 1, a marker of vasculogenesis. The epithelium exhibited apical vacuoles, microvilli, junctional complexes, and linear basement membranes. Capillary-like structures showed endothelial features with occasional pericytes. UB cell epithelialization was augmented in the presence of MM cell-derived conditioned medium, glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), or fibronectin. MM cells grown in the presence of UB-derived conditioned medium failed to undergo differentiation. However, UB cell-derived conditioned medium induced MM cell migration. These studies indicate that tubulogenesis and vasculogenesis can be partially recapitulated by recombining individual MM and UB cell lineages, providing a new model system to study organogenesis ex vivo.

The tuberin/mTOR pathway promotes apoptosis of tubular epithelial cells in diabetes.

Apoptosis contributes to the development of diabetic nephropathy, but the mechanism by which high glucose (HG) induces apoptosis is not fully understood. Because the tuberin/mTOR pathway can modulate apoptosis, we studied the role of this pathway in apoptosis in type I diabetes and in cultured proximal tubular epithelial (PTE) cells exposed to HG. Compared with control rats, diabetic rats had more apoptotic cells in the kidney cortex. Induction of diabetes also increased phosphorylation of tuberin in association with mTOR activation (measured by p70S6K phosphorylation), inactivation of Bcl-2, increased cytosolic cytochrome c expression, activation of caspase 3, and cleavage of PARP; insulin treatment prevented these changes. In vitro, exposure of PTE cells to HG increased phosphorylation of tuberin and p70S6K, phosphorylation of Bcl-2, expression of cytosolic cytochrome c, and caspase 3 activity. High glucose induced translocation of the caspase substrate YY1 from the cytoplasm to the nucleus and enhanced cleavage of PARP. Pretreatment the cells with the mTOR inhibitor rapamycin reduced the number of apoptotic cells induced by HG and the downstream effects of mTOR activation noted above. Furthermore, gene silencing of tuberin with siRNA decreased cleavage of PARP. These data show that the tuberin/mTOR pathway promotes apoptosis of tubular epithelial cells in diabetes, mediated in part by cleavage of PARP by YY1.

Novel mechanism of reducing tumourigenesis: upregulation of the DNA repair enzyme OGG1 by rapamycin-mediated AMPK activation and mTOR inhibition.

Inhibition of mTOR by rapamycin is an important approach in cancer therapy. In early clinical trials, tuberous sclerosis complex (TSC)-related kidney tumours were found to regress following rapamycin treatment. Since loss of function of the DNA repair OGG1 enzyme has a major role in multistep carcinogenesis of the kidney and other organs, we investigated the effect of rapamycin on OGG1 regulation. Treatment of HK2 cells, mouse Tsc-deficient cells and human VHL-deficient cells (786-O) with rapamycin resulted in decrease in p70S6K phosphorylation at Thr(389), and increase in the expression of NF-YA and OGG1 proteins. In addition, rapamycin increased OGG1 promoter activity in cells transfected with OGG1 promoter construct. Furthermore, rapamycin increased the phosphorylation at Thr(172) of the energy sensor AMPK. Downregulation of AMPK phosphorylation by high glucose (HG) increases the phosphorylation of p70S6K and decreases the protein expression of NF-YA and OGG1. Pretreatment of the cells with rapamycin before exposure to HG reversed the effects of HG. However, downregulation of AMPK by dominant negative (DN)-AMPK in Tsc2(+/-) cells abolished AMPK and decreased OGG1 expression. In contrast, transfection of Tsc2(+/-) cells with DN-S6K abolished p70S6K phosphorylation and increased OGG1 expression, a response enhanced by rapamycin. Treatment of Tsc2(+/-) mice with rapamycin resulted in activation of AMPK, downregulation of phospho-p70S6K and enhanced OGG1 expression. Our data show that inhibition of mTOR can activate AMPK and lead to upregulation of DNA repair enzyme OGG1. These data comprise the first report to provide one mechanism whereby rapamycin might prevent or inhibit the formation and progression of certain cancers.

S6K1 is a multifaceted regulator of Mdm2 that connects nutrient status and DNA damage response.

p53 mediates DNA damage-induced cell-cycle arrest, apoptosis, or senescence, and it is controlled by Mdm2, which mainly ubiquitinates p53 in the nucleus and promotes p53 nuclear export and degradation. By searching for the kinases responsible for Mdm2 S163 phosphorylation under genotoxic stress, we identified S6K1 as a multifaceted regulator of Mdm2. DNA damage activates mTOR-S6K1 through p38alpha MAPK. The activated S6K1 forms a tighter complex with Mdm2, inhibits Mdm2-mediated p53 ubiquitination, and promotes p53 induction, in addition to phosphorylating Mdm2 on S163. Deactivation of mTOR-S6K1 signalling leads to Mdm2 nuclear translocation, which is facilitated by S163 phosphorylation, a reduction in p53 induction, and an alteration in p53-dependent cell death. These findings thus establish mTOR-S6K1 as a novel regulator of p53 in DNA damage response and likely in tumorigenesis. S6K1-Mdm2 interaction presents a route for cells to incorporate the metabolic/energy cues into DNA damage response and links the aging-controlling Mdm2-p53 and mTOR-S6K pathways.

Mechanism of oxidative DNA damage in diabetes: tuberin inactivation and downregulation of DNA repair enzyme 8-oxo-7,8-dihydro-2'-deoxyguanosine-DNA glycosylase.

OBJECTIVE: To investigate potential mechanisms of oxidative DNA damage in a rat model of type 1 diabetes and in murine proximal tubular epithelial cells and primary culture of rat proximal tubular epithelial cells. RESEARCH DESIGN AND METHODS: Phosphorylation of Akt and tuberin, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) levels, and 8-oxoG-DNA glycosylase (OGG1) expression were measured in kidney cortical tissue of control and type 1 diabetic animals and in proximal tubular cells incubated with normal or high glucose. RESULTS: In the renal cortex of diabetic rats, the increase in Akt phosphorylation is associated with enhanced phosphorylation of tuberin, decreased OGG1 protein expression, and 8-oxodG accumulation. Exposure of proximal tubular epithelial cells to high glucose causes a rapid increase in reactive oxygen species (ROS) generation that correlates with the increase in Akt and tuberin phosphorylation. High glucose also resulted in downregulation of OGG1 protein expression, paralleling its effect on Akt and tuberin. Inhibition of phosphatidylinositol 3-kinase/Akt significantly reduced high glucose-induced tuberin phosphorylation and restored OGG1 expression. Hydrogen peroxide stimulates Akt and tuberin phosphorylation and decreases OGG1 protein expression. The antioxidant N-acetylcysteine significantly inhibited ROS generation, Akt/protein kinase B, and tuberin phosphorylation and resulted in deceased 8-oxodG accumulation and upregulation of OGG1 protein expression. CONCLUSIONS: Hyperglycemia in type 1 diabetes and treatment of proximal tubular epithelial cells with high glucose leads to phosphorylation/inactivation of tuberin and downregulation of OGG1 via a redox-dependent activation of Akt in renal tubular epithelial cells. This signaling cascade provides a mechanism of oxidative stress-mediated DNA damage in diabetes.