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Supplementary Figure S1. Tumor volumes in mice normalized to day 0; Supplementary Table S1. Macronutrient compositions of diets used in mouse study.

Supplemental Figure Legends

Supplementary legends

Supplemental figure 1. Lack of toxicity of KPT-9274 at 24 hours; Supplemental figure 2. Cell cycle analysis.

Further representative sections of normal right kidney (RK) and tumor tissues were subjected to immunohistochemical analysis for NRF2

Many cancers appear to activate intrinsic antioxidant systems as a means to counteract oxidative stress. Some cancers, such as clear cell renal cell carcinoma (ccRCC), require exogenous glutamine for growth and exhibit reprogrammed glutamine metabolism, at least in part due to the glutathione pathway, an efficient cellular buffering system that counteracts reactive oxygen species and other oxidants. We show here that ccRCC xenograft tumors under the renal capsule exhibit enhanced oxidative stress compared with adjacent normal tissue and the contralateral kidney. Upon glutaminase inhibition with CB-839 or BPTES, the RCC cell lines SN12PM-6-1 (SN12) and 786-O exhibited decreased survival and pronounced apoptosis associated with a decreased GSH/GSSG ratio, augmented nuclear factor erythroid-related factor 2, and increased 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of DNA damage. SN12 tumor xenografts showed decreased growth when treated with CB-839. Furthermore, PET imaging confirmed that ccRCC tumors exhibited increased tumoral uptake of 18F-(2S,4R)4-fluoroglutamine compared with the kidney in the orthotopic mouse model. This technique can be utilized to follow changes in ccRCC metabolism in vivo. Further development of these paradigms will lead to new treatment options with glutaminase inhibitors and the utility of PET to identify and manage patients with ccRCC who are likely to respond to glutaminase inhibitors in the clinic. Cancer Res; 77(23); 6746–58. ©2017 AACR.

Figure legends for S1-S5; S1. Further representative sections of normal right kidney; S2. Glucose (Glu), glutamine (Gln), glutathione (GSH), and cell viability in RCC cells; S3. Apoptosis flow data; S4. Bioluminescence imaging, measurements, and animal weights; S5. Coronal PET MIP images overlaid with CT images.

The advent of multikinase inhibitors targeting the vascular endothelial growth factor (VEGF) receptor has revolutionized the treatment of highly angiogenic malignancies such as renal cell carcinoma. Interestingly, several such inhibitors are commercially available, and they each possess diverse specific beneficial and adverse effect profiles. In examining the structure of sorafenib, it was hypothesized that this compound would possess inhibitory effects on the soluble epoxide hydrolase, an enzyme with pleiotropic effects on inflammation and vascular disease. We now show that sorafenib but not another VEGF receptor targeted inhibitor sunitinib is a potent inhibitor of the human soluble epoxide hydrolase in vitro (KI = 17 ± 4 nmol/L). Furthermore, sorafenib causes the expected in vivo shift in oxylipid profile resulting from soluble epoxide hydrolase inhibition, evidence of a reduction in the acute inflammatory response. Lipopolysaccharide-induced hypotension was reversed with sorafenib but not sunitinib treatment, suggesting that soluble epoxide hydrolase inhibition accounts for at least part of the anti-inflammatory effect of sorafenib. The pharmacokinetic studies presented here in light of the known potency of sorafenib as a soluble epoxide hydrolase inhibitor indicate that the soluble epoxide hydrolase will be largely inhibited at therapeutic doses of sorafenib. Thus, it is likely that soluble epoxide hydrolase inhibition contributes to the beneficial effects from the inhibition of the VEGF receptor and other kinases during treatment with sorafenib. [Mol Cancer Ther 2009;8(8):2193–203]

Supplemental Figures Legend

Absolute values and statistics for quantitative acylcarnitine data. Absolute values and statistics for quantitative GSH and GSSG data.

PDF file - 24K, averaged tumor + kidney weights at sacrifice for each animal

1. Cytosolic lipid storage is decreased in RCC tissue with increasing tumor grade 2. Glutamine depletion attenuates RCC cell viability, decreases the anti-oxidant system, and results in evidence of increased oxidative stress

XLS file - 27K, All identified metabolites in tissue

PDF file - 74K, The PPAR-alpha agonist Wy-14,643 decreases tryptophan and increases nicotinamide levels in vitro 2. The IDO inhibitor 1-MT increases tryptophan levels in vitro

XLS file - 56K, Variable Importance in Projection (VIP) data for Tissue, Serum and Urine

PDF file - 74K, Summary of all Supplemental data and Detailed methods: Non-targeted metabolomic analysis part of Supplemental Data

Objective Nephrectomy, the standard of care for localized renal cell carcinoma (RCC), may lead to kidney function loss. Our goal was to identify prognostic biomarkers of postoperative renal function using metabolomics. Methods Metabolomics data from benign kidney parenchyma were collected prospectively from 138 patients with RCC who underwent nephrectomy at a single institution. The primary endpoint was the difference between the postoperative and preoperative estimated glomerular filtration (eGFR) rate divided by the elapsed time (eGFR slope). eGFR slope was calculated ∼2 years post-nephrectomy (GFR1), and at last follow-up (GFR2). A multivariate regularized regression model identified clinical characteristics and abundance of metabolites in baseline benign kidney parenchyma that were significantly associated with eGFR slope. Findings were validated by associating gene expression data with eGFR slope in an independent cohort (n = 58). Results Data were compiled on 78 patients (median age 62.6 years, 65.4% males). The mean follow-up was 25 ± 3.4 months for GFR1 and 69.5 ± 23.5 months for GFR2 and 17 (22%) and 32 (41%) patients showed eGFR recovery, respectively. Nephrectomy type, blood lipids, gender and 23 metabolites from benign parenchyma were significantly associated with eGFR slope. Some metabolites associated with eGFR slope overlapped with previously reported chronic kidney disease-related processes. Subgroup analysis identified unique ‘metabolite signatures’ by older age, nephrectomy type and preoperative eGFR. Conclusions Nephrectomy type, gender, blood lipids and benign parenchyma metabolites at nephrectomy were associated with long-term kidney function. On further study, these metabolites may be useful as potential biomarkers and to identify novel therapeutic targets for malignancy-associated renal disease.

BACKGROUND: Kidney cancer (or renal cell carcinoma, RCC) is the sixth most common malignancy in the United States and is increasing in incidence. Despite new therapies, including targeted therapies and immunotherapies, most RCCs are resistant to treatment. Thus, several laboratories have been evaluating new approaches to therapy, both with single agents as well as combinations. Although we have previously shown efficacy of the dual PAK4/nicotinamide phosphoribosyltransferase (NAMPT) inhibitor KPT-9274, and the immune checkpoint inhibitors (CPI) have shown utility in the clinic, there has been no evaluation of this combination either clinically or in an immunocompetent animal model of kidney cancer. METHODS: In this study, we use the renal cell adenocarcinoma (RENCA) model of spontaneous murine kidney cancer. Male BALB/cJ mice were injected subcutaneously with RENCA cells and, after tumors were palpable, they were treated with KPT-9274 and/or anti–programmed cell death 1 (PDCD1; PD1) antibody for 21 days. Tumors were measured and then removed at animal euthanasia for subsequent studies. RESULTS: We demonstrate a significant decrease in allograft growth with the combination treatment of KPT-9274 and anti-PD1 antibody without significant weight loss by the animals. This is associated with decreased (MOUSE) Naprt expression, indicating dependence of these tumors on NAMPT in parallel to what we have observed in human RCC. Histology of the tumors showed substantial necrosis regardless of treatment condition, and flow cytometry of antibody-stained tumor cells revealed that the enhanced therapeutic effect of KPT-9274 and anti-PD1 antibody was not driven by infiltration of T cells into tumors. CONCLUSIONS: This study highlights the potential of the RENCA model for evaluating immunologic responses to KPT-9274 and checkpoint inhibitor (CPI) and suggests that therapy with this combination could improve efficacy in RCC beyond what is achievable with CPI alone.

The innate and adaptive immunities have been documented to participate in the pathogenesis of nephrotoxic acute kidney injury (AKI); however, the mechanisms controlling these processes have yet to be established. In our cisplatin-induced AKI mouse model, we show pathological damage to the kidneys, with the classical markers elevated, consistent with the response to cisplatin treatment. Through assessments of the components of the immune system, both locally and globally, we demonstrate that the immune microenvironment of injured kidneys was associated with an increased infiltration of CD4+ T cells and macrophages concomitant with decreased Treg cell populations. Our cell-based assays and animal studies further show that cisplatin exposure downregulated the protein levels of programmed death-ligand 1 (PD-L1), an immune checkpoint protein, in primary renal proximal tubular epithelial cells, and that these inhibitions were dose-dependent. After orthotopic delivery of PD-L1 gene into the kidneys, cisplatin-exposed mice displayed lower levels of both serum urea nitrogen and creatinine upon PD-L1 expression. Our data suggest a renoprotective effect of the immune checkpoint protein, and thereby provide a novel therapeutic strategy for cisplatin-induced AKI.

Arginine vasopressin (AVP) and its type-2 receptor (V2R) play an essential role in the regulation of salt and water homeostasis by the kidneys. V2R activation also stimulates proliferation of renal cell carcinoma (RCC) cell lines in vitro. The current studies investigated V2R expression and activity in human RCC tumors, and its role in RCC tumor growth. Examination of the cancer genome atlas (TCGA) database, and analysis of human RCC tumor tissue microarrays, cDNA arrays and tumor biopsy samples demonstrated V2R expression and activity in clear cell RCC (ccRCC). In vitro, V2R antagonists OPC31260 and Tolvaptan, or V2R gene silencing reduced wound closure and cell viability of 786-O and Caki-1 human ccRCC cell lines. Similarly in mouse xenograft models, Tolvaptan and OPC31260 decreased RCC tumor growth by reducing cell proliferation and angiogenesis, while increasing apoptosis. In contrast, the V2R agonist dDAVP significantly increased tumor growth. High intracellular cAMP levels and ERK1/2 activation were observed in human ccRCC tumors. In mouse tumors and Caki-1 cells, V2R agonists reduced cAMP and ERK1/2 activation, while dDAVP treatment had the reverse effect. V2R gene silencing in Caki-1 cells also reduced cAMP and ERK1/2 activation. These results provide novel evidence for a pathogenic role of V2R signaling in ccRCC, and suggest that inhibitors of the AVP-V2R pathway, including the FDA-approved drug Tolvaptan, could be utilized as novel ccRCC therapeutics.

INTRODUCTION AND OBJECTIVE:Nephrectomy, the standard of care for localized renal cell carcinoma (RCC), may lead to kidney function loss. To identify prognostic biomarkers of postoperative renal fun...

Treatment options for high grade urothelial cancers are limited and have remained largely unchanged for several decades. Selinexor (KPT-330), a first in class small molecule that inhibits the nuclear export protein XPO1, has shown efficacy as a single agent treatment for numerous different malignancies, but its efficacy in limiting bladder malignancies has not been tested. In this study we assessed selinexor-dependent cytotoxicity in several bladder tumor cells and report that selinexor effectively reduced XPO1 expression and limited cell viability in a dose dependent manner. The decrease in cell viability was due to an induction of apoptosis and cell cycle arrest. These results were recapitulated in in vivo studies where selinexor decreased tumor growth. Tumors treated with selinexor expressed lower levels of XPO1, cyclin A, cyclin B, and CDK2 and increased levels of RB and CDK inhibitor p27, a result that is consistent with growth arrest. Cells expressing wildtype RB, a potent tumor suppressor that promotes growth arrest and apoptosis, were most susceptible to selinexor. Cell fractionation and immunofluorescence studies showed that selinexor treatment increased nuclear RB levels and mechanistic studies revealed that RB ablation curtailed the response to the drug. Conversely, limiting CDK4/6 dependent RB phosphorylation by palbociclib was additive with selinexor in reducing bladder tumor cell viability, confirming that RB activity has a role in the response to XPO1 inhibition. These results provide a rationale for XPO1 inhibition as a novel strategy for the treatment of bladder malignancies.

In the age of bioinformatics and with the advent of high-powered computation over the past decade or so the landscape of biomedical research has become radically altered. Whereas a generation ago, investigators would study their “favorite” protein or gene and exhaustively catalog the role of this compound in their disease of interest, the appearance of omics has changed the face of medicine such that much of the cutting edge (and fundable!) medical research now evaluates the biology of the disease nearly in its entirety. Couple this with the realization that kidney cancer is a “metabolic disease” due to its multiple derangements in biochemical pathways [1, 2], and clear cell renal cell carcinoma (ccRCC) becomes ripe for data mining using multiple omics approaches.

Research in many cancers has uncovered changes in metabolic pathways that control tumour energetics and biosynthesis, so-called metabolic reprogramming. Studies in clear cell renal cell carcinoma (ccRCC) have been particularly revealing, leading to the concept that ccRCC is a metabolic disease. ccRCC is generally accompanied by reprogramming of glucose and fatty acid metabolism and of the tricarboxylic acid cycle. Metabolism of tryptophan, arginine and glutamine is also reprogrammed in many ccRCCs, and these changes provide opportunities for new therapeutic strategies, biomarkers and imaging modalities. In particular, metabolic reprogramming facilitates the identification of novel and repurposed drugs that could potentially be used to treat ccRCC, which when metastatic has currently limited long-term treatment options. Further research and dissemination of these concepts to nephrologists and oncologists will lead to clinical trials of therapeutics specifically targeted to tumour metabolism, rather than generally toxic to all proliferating cells. Such novel agents are highly likely to be more effective and to have far fewer adverse effects than existing drugs.

Renal cell carcinoma (RCC) is the malignancy seen most commonly in the clinical practice of nephrology. The most common subtype by far, clear cell RCC (ccRCC), is one of the relatively few malignancies that is increasing in incidence, and RCC is one of the cancers which is characterized by metabolic reprogramming, hence it is known as a metabolic disease. RCC is often asymptomatic at presentation and is commonly detected by nephrologists in the process of working up other diseases, such as acute kidney injury or obstruction, often by the observation of paraneoplastic phenomena. In addition, RCC is frequently discovered in an advanced form, such that many patients have metastases at the time of diagnosis, a finding which is associated with a markedly poor prognosis. Thus the practicing nephrologist needs at least a rudimentary understanding of this disease. In this chapter, we consider surgical, as well as medical treatment, with an emphasis on the very recent and evolving approaches from a medical, as well as surgical standpoint, including novel treatments related to immune modulation, which may soon change the landscape of treatment of this disease.

The most common urologic malignancy seen in the nephrology clinic is clear cell renal cell carcinoma (RCC), which is generally asymptomatic and often found incidentally during imaging. Greater than 25% of patients with RCC have concomitant CKD at diagnosis, presumably because of overlapping risk

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.

Targeted therapeutics, such as those abrogating hypoxia inducible factor (HIF)/vascular endothelial growth factor signaling, are initially effective against kidney cancer (or renal cell carcinoma, RCC); however, drug resistance frequently occurs via subsequent activation of alternative pathways. Through genome-scale integrated analysis of the HIF-α network, we identified the major protein kinase C substrate MARCKS (myristoylated alanine-rich C kinase substrate) as a potential target molecule for kidney cancer. In a screen of nephrectomy samples from 56 patients with RCC, we found that MARCKS expression and its phosphorylation are increased and positively correlate with tumor grade. Genetic and pharmacologic suppression of MARCKS in high-grade RCC cell lines in vitro led to a decrease in cell proliferation and migration. We further demonstrated that higher MARCKS expression promotes growth and angiogenesis in vivo in an RCC xenograft tumor. MARCKS acted upstream of the AKT/mTOR pathway, activating HIF-target genes, notably vascular endothelial growth factor-A. Following knockdown of MARCKS in RCC cells, the IC50 of the multikinase inhibitor regorafenib was reduced. Surprisingly, attenuation of MARCKS using the MPS (MARCKS phosphorylation site domain) peptide synergistically interacted with regorafenib treatment and decreased survival of kidney cancer cells through inactivation of AKT and mTOR. Our data suggest a major contribution of MARCKS to kidney cancer growth and provide an alternative therapeutic strategy of improving the efficacy of multikinase inhibitors.

Examine whether the acute kidney injury (AKI) commonly observed among ultramarathon participants places the individual at risk for subsequent AKI of worse magnitude.Observational.Western States Endurance Run.Race finishers with postrace blood studies.Acute kidney injury after 1 race.Extent of AKI in subsequent race.Among 627 finishes in which serum creatinine values were known, 36.2% met "risk" or "injury" criterion with this group characterized by having faster finish times, greater body weight loss during the race, and higher postrace serum creatine kinase and urea nitrogen concentrations when compared with those not meeting the criteria. We identified 38 runners who had undergone postrace blood analyses at multiple races among which 16 (42.1%) met the risk or injury criterion at the first race. Of those 16 runners, 12 (75%) met the criteria at a subsequent race, an incidence that was higher (P = 0.0026) than the overall 36.2% incidence. For most (56.2%) of the 16 runners meeting the criteria at the first race, the subsequent race caused less increase in serum creatinine concentration and decrement in estimated glomerular filtration rate than the first race.Mild AKI is common in 161-km ultramarathons, but there was no evidence that previous AKI caused greater renal dysfunction from a subsequent exercise stimulus of similar magnitude. This offers some reassurance to runners and their physicians that mild to moderate AKI in the setting of an ultramarathon is not cumulative or without complete recovery of kidney function when stressed.

Background Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity. Methods To investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey’s false discovery rate (FDR) q-values to correct for multiple testing. Results Twelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value

Renal cell carcinoma (RCC) has emerged as a metabolic disease characterized by dysregulated expression of metabolic enzymes. Patients with metastatic RCC have an unusually poor prognosis and near-universal resistance to all current therapies. To improve RCC treatment and the survival rate of patients with RCC, there is an urgent need to reveal the mechanisms by which metabolic reprogramming regulates aberrant signaling and oncogenic progression. Through an integrated analysis of RCC metabolic pathways, we showed that methylthioadenosine phosphorylase (MTAP) and its substrate methylthioadenosine (MTA) are dysregulated in aggressive RCC. A decrease in MTAP expression was observed in RCC tissues and correlated with higher tumor grade and shorter overall survival. Genetic manipulation of MTAP demonstrated that MTAP expression inhibits the epithelial-mesenchymal transition, invasion and migration of RCC cells. Interestingly, we found a decrease in the protein methylation level with a concomitant increase in tyrosine phosphorylation after MTAP knockout. A phospho-kinase array screen identified the type 1 insulin-like growth factor-1 receptor (IGF1R) as the candidate with the highest upregulation in tyrosine phosphorylation in response to MTAP loss. We further demonstrated that IGF1R phosphorylation acts upstream of Src and STAT3 signaling in MTAP-knockout RCC cells. IGF1R suppression by a selective inhibitor of IGF1R, linsitinib, impaired the cell migration and invasion capability of MTAP-deleted cells. Surprisingly, an increase in linsitinib-mediated cytotoxicity occurred in RCC cells with MTAP deficiency. Our data suggest that IGF1R signaling is a driver pathway that contributes to the aggressive nature of MTAP-deleted RCC., Uncovering metabolic changes that underlie kidney cancer A receptor that is triggered by an enzyme deficiency in kidney cancer could act as an anticancer drug target. Ching-Hsien Chen of the University of California Davis and colleagues in the USA and Taiwan found that renal cell carcinomas are deficient in the enzyme methylthioadenosine phosphorylase (MTAP). This deficiency, which correlates with higher tumour grade and shorter overall survival, leads to the activation of type 1 insulin-like growth factor-1 receptor (IGF1R). This in turn activates signaling pathways that support cancer cell survival, growth, and invasiveness. The team found that a selective IGF1R inhibitor, called linsitinib, suppressed colony-forming ability and reduced cell motility in renal carcinoma cells. The findings suggest that IGF1R signaling drives pathways that contribute to the aggressive nature of renal carcinoma cells lacking MTAP.

Research into metabolic reprogramming in cancer has become commonplace, yet this area of research has only recently come of age in nephrology. In light of the parallels between cancer and autosomal dominant polycystic kidney disease (ADPKD), the latter is currently being studied as a metabolic disease. In clear cell renal cell carcinoma (RCC), which is now considered a metabolic disease, we and others have shown derangements in the enzyme arginosuccinate synthase 1 (ASS1), resulting in RCC cells becoming auxotrophic for arginine and leading to a new therapeutic paradigm involving reducing extracellular arginine. Based on our earlier finding that glutamine pathways are reprogrammed in ARPKD, and given the connection between arginine and glutamine synthetic pathways via citrulline, we investigated the possibility of arginine reprogramming in ADPKD. We now show that, in a remarkable parallel to RCC, ASS1 expression is reduced in murine and human ADPKD, and arginine depletion results in a dose-dependent compensatory increase in ASS1 levels as well as decreased cystogenesis in vitro and ex vivo with minimal toxicity to normal cells. Nontargeted metabolomics analysis of mouse kidney cell lines grown in arginine-deficient versus arginine-replete media suggests arginine-dependent alterations in the glutamine and proline pathways. Thus, depletion of this conditionally essential amino acid by dietary or pharmacological means, such as with arginine-degrading enzymes, may be a novel treatment for this disease.

Kidney cancer, or renal cell carcinoma (RCC), is a disease of increasing incidence that is commonly seen in the general practice of nephrology. However, RCC is under-recognized by the nephrology community, such that its presence in curricula and research by this group is lacking. In the most common form of RCC, clear cell renal cell carcinoma (ccRCC), inactivation of the von Hippel–Lindau tumor suppressor is nearly universal; thus, the biology of ccRCC is characterized by activation of hypoxia-relevant pathways that lead to the associated paraneoplastic syndromes. Therefore, RCC is labeled the internist’s tumor. In light of this characterization and multiple other metabolic abnormalities recently associated with ccRCC, it can now be viewed as a metabolic disease. In this review, we discuss the basic biology, pathology, and approaches for treatment of RCC. It is important to distinguish between kidney confinement and distant spread of RCC, because this difference affects diagnostic and therapeutic approaches and patient survival, and it is important to recognize the key interplay between RCC, RCC therapy, and CKD. Better understanding of all aspects of this disease will lead to optimal patient care and more recognition of an increasingly prevalent nephrologic disease, which we now appropriately label the nephrologist’s tumor.

Antigen persistence due to HIV is a major source of inflammation and substantial immune activation, both of which are linked to accelerated aging. This illustrates the need to reduce immune activation in these patients and subsequently decrease the risk of cardiovascular diseases and other non-AIDS-defining comorbidities.CD4 T cells were infected with HIV-1 isolates in the presence or absence of atorvastatin (0.25 to 1 μg/ml) for 24-48 h. Atorvastatin-induced anti-inflammatory functions and anti-viral replication were measured in vitro.Atorvastatin, a lipid-lowering medication, exerted a broad spectrum of anti-inflammatory functions by reducing T-cell immune activation markers (e.g. CD38, HLA-DR and Ki67), lowering HIV-1 co-receptor CCR-5, and decreasing proliferative capabilities of CD4 T cells in vitro. In contrast, atorvastatin expanded regulatory T cells (Tregs) and upregulated the expression of T-cell immunoglobulin and ITIM domain (TIGIT), which enhanced the suppressive activity of Tregs. Furthermore, atorvastatin upregulated the cyclin-dependent kinase inhibitor p21, which is also known as cip-1 and waf-1, in the CD4 T cells. Upregulation of p21 in CD4 T cells rendered them less susceptible to HIV-1 infection and replication whereas siRNA-mediated p21 depletion and/or p21 selective inhibitor rescued viral replication. Interestingly, atorvastatin reduced HIV infection in both rested and phytohemagglutinin-activated CD4 T cells in vitro. Finally, atorvastatin mediated p21 upregulation occurred via mevalonate pathway, but independent of p53.The results demonstrate a novel mechanism by which atorvastatin induced resistance of CD4 T cells to HIV-1 infection via p21 upregulation and suggest that statins may hold particular promise for some HIV-infected individuals.

Since polycystic kidney disease (PKD) was first noted over 30 years ago to have neoplastic parallels, there has been a resurgent interest in elucidating neoplasia-relevant pathways in PKD. Taking a nontargeted metabolomics approach in the B6(Cg)- Cys1 cpk/J ( cpk) mouse model of recessive PKD, we have now characterized metabolic reprogramming in these tissues, leading to a glutamine-dependent TCA cycle shunt toward total 2-hydroxyglutarate (2-HG) production in cpk compared with B6 wild-type kidney tissue. After confirmation of increased 2-HG expression in immortalized collecting duct cpk cells as well as in human autosomal recessive PKD tissue using targeted analysis, we show that the increase in 2-HG is likely due to glutamine-sourced α-ketoglutarate. In addition, cpk cells require exogenous glutamine for growth such that inhibition of glutaminase-1 decreases cell viability as well as proliferation. This study is a demonstration of the striking parallels between recessive PKD and cancer metabolism. Our data, once confirmed in other PKD models, suggest that future therapeutic approaches targeting this pathway, such as using glutaminase inhibitors, have the potential to open novel treatment options for renal cystic disease.

Phosphorylation of H2AX on Ser139 (γH2AX) after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s) for this response remains elusive. Here we show that cancer cells (e.g., HCT116, MCF7) that constitutively express detectable levels of p21WAF1 (p21) exhibit low numbers of γH2AX foci (&lt, 3/nucleus), whereas p21 knockout cells (HCT116p21−/−) and constitutively low p21-expressing cells (e.g., MDA-MB-231) exhibit high numbers of foci (e.g., &gt, 50/nucleus), and that these foci are not associated with apoptosis. The majority (&gt, 95%) of cells within HCT116p21−/− and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i) p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53, and (ii) p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX.

Kidney cancer, or renal cell carcinoma (RCC), is a disease of increasing incidence that commonly is seen in the general practice of nephrology. Despite this state of affairs, this fascinating and highly morbid disease frequently is under-represented, or even absent, from the curriculum of nephrologists in training and generally is underemphasized in national nephrology meetings, both scientific as well as clinical. Although classic concepts in cancer research in general had led to the concept that cancer is a disease resulting from mutations in the control of growth-regulating pathways, reinforced by the discovery of oncogenes, more contemporary research, particularly in kidney cancer, has uncovered changes in metabolic pathways mediated by those same genes that control tumor energetics and biosynthesis. This adaptation of classic biochemical pathways to the tumor's advantage has been labeled metabolic reprogramming. For example, in the case of kidney cancer there exists a near-universal presence of von Hippel-Lindau tumor suppressor (pVHL) inactivation in the most common form, clear cell RCC (ccRCC), leading to activation of hypoxia-relevant and other metabolic pathways. Studies of this and other pathways in clear cell RCC (ccRCC) have been particularly revealing, leading to the concept that ccRCC can itself be considered a metabolic disease. For this reason, the relatively new method of metabolomics has become a useful technique in the study of ccRCC to tease out those pathways that have been reprogrammed by the tumor to its maximum survival advantage. Furthermore, identification of the nodes of such pathways can lead to novel areas for drug intervention in a disease for which such targets are seriously lacking. Further research and dissemination of these concepts, likely using omics techniques, will lead to clinical trials of therapeutics specifically targeted to tumor metabolism, rather than those generally toxic to all proliferating cells. Such novel agents are highly likely to be more effective than existing drugs and to have far fewer adverse effects. This review provides a general overview of the technique of metabolomics and then discusses how it and other omics techniques have been used to further our understanding of the basic biology of kidney cancer as well as to identify new therapeutic approaches.

INTRODUCTION:Sustained proliferative signaling and de-regulated cellular bioenergetics are two of the chief hallmarks of cancer. Alterations in the Ras pathway and its downstream effectors are among the major drivers for uncontrolled cell growth in many cancers. The GTPases are one of the signaling molecules that activate crucial signal transducing pathways downstream of Ras through several effector proteins. The GTPases (GTP bound) interact with several effectors and modulate a number of different biological pathways including those that regulate cytoskeleton, cellular motility, cytokinesis, proliferation, apoptosis, transcription and nuclear signaling. Similarly, the altered glycolytic pathway, the so-called 'Warburg effect', rewires tumor cell metabolism to support the biosynthetic requirements of uncontrolled proliferation. There exists strong evidence for the critical role of the glycolytic pathway's rate limiting enzymes in promoting immunosuppression. Areas covered: We review the emerging roles of GTPase effector proteins particularly the p21 activated kinase 4 (PAK4) and nicotinamide biosynthetic pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) as signaling molecules in immune surveillance and the immune response. Expert opinion: In this expert opinion article we highlight the recent information on the role of GTPases and the metabolic enzymes on the immune microenvironment and propose some unique immune therapeutic opportunities.

Many cancers appear to activate intrinsic antioxidant systems as a means to counteract oxidative stress. Some cancers, such as clear cell renal cell carcinoma (ccRCC), require exogenous glutamine for growth and exhibit reprogrammed glutamine metabolism, at least in part due to the glutathione pathway, an efficient cellular buffering system that counteracts reactive oxygen species and other oxidants. We show here that ccRCC xenograft tumors under the renal capsule exhibit enhanced oxidative stress compared with adjacent normal tissue and the contralateral kidney. Upon glutaminase inhibition with CB-839 or BPTES, the RCC cell lines SN12PM-6-1 (SN12) and 786-O exhibited decreased survival and pronounced apoptosis associated with a decreased GSH/GSSG ratio, augmented nuclear factor erythroid-related factor 2, and increased 8-oxo-7,8-dihydro-2'-deoxyguanosine, a marker of DNA damage. SN12 tumor xenografts showed decreased growth when treated with CB-839. Furthermore, PET imaging confirmed that ccRCC tumors exhibited increased tumoral uptake of 18F-(2S,4R)4-fluoroglutamine compared with the kidney in the orthotopic mouse model. This technique can be utilized to follow changes in ccRCC metabolism in vivo. Further development of these paradigms will lead to new treatment options with glutaminase inhibitors and the utility of PET to identify and manage patients with ccRCC who are likely to respond to glutaminase inhibitors in the clinic. Cancer Res; 77(23); 6746–58. ©2017 AACR.

Autosomal-dominant polycystic kidney disease (ADPKD) is a progressive, proliferative renal disease. Kidneys from ADPKD patients are characterized by the presence of cysts that are marked by enhanced proliferation and apoptosis of renal tubular epithelial cells. Current treatment of this disease is supportive, as there are few if any clinically validated targeted therapeutics. Given the parallels between cystic disease and cancer, and in light of our findings of the efficacy of the nuclear transport inhibitors in kidney cancer, which has similarities to ADPKD, we asked whether such inhibitors show utility in ADPKD. In this study, we tested selective inhibitors of nuclear export (SINE) in two human ADPKD cell lines and in an in vivo mouse model of ADPKD. After effective downregulation of a nuclear exporter, exportin 1 (XPO1), with KPT-330, both cell lines showed dose-dependent inhibition of cell proliferation through G0/G1 arrest associated with downregulation of CDK4, with minimal apoptosis. To analyze mechanisms of CDK4 decrease by XPO1 inhibition, localization of various XPO1 target proteins was examined, and C/EBPβ was found to be localized in the nucleus by XPO1 inhibition, resulting in an increase of C/EBPα, which activates degradation of CDK4. Furthermore, inhibition of XPO1 with the parallel inhibitor KPT-335 attenuated cyst growth in vivo in the PKD1 mutant mouse model Pkd1v/v. Thus, inhibition of nuclear export by KPT-330, which has shown no adverse effects in renal serum chemistries and urinalyses in animal models, and which is already in phase 1 trials for cancers, will be rapidly translatable to human ADPKD.

Sorafenib (Nexavar®) is currently the only FDA-approved small molecule targeted therapy for advanced hepatocellular carcinoma. The use of structural analogues and derivatives of sorafenib has enabled the elucidation of critical targets and mechanism(s) of cell death for human cancer lines. We previously performed a structure-activity relationship study on a series of sorafenib analogues designed to investigate the inhibition overlap between the major targets of sorafenib Raf-1 kinase and VEGFR-2, and an enzyme shown to be a potent off-target of sorafenib, soluble epoxide hydrolase. In the current work, we present the biological data on our lead sorafenib analogue, t-CUPM, demonstrating that this analogue retains cytotoxicity similar to sorafenib in various human cancer cell lines and strongly inhibits growth in the NCI-60 cell line panel. Co-treatment with the pan-caspase inhibitor, Z-VAD-FMK, failed to rescue the cell viability responses of both sorafenib and t-CUPM, and immunofluorescence microscopy shows similar mitochondrial depolarization and apoptosis-inducing factor release for both compounds. These data suggest that both compounds induce a similar mechanism of caspase-independent apoptosis in hepatoma cells. In addition, t-CUPM displays anti-proliferative effects comparable to sorafenib as seen by a halt in G0/G1 in cell cycle progression. The structural difference between sorafenib and t-CUPM significantly reduces inhibitory spectrum of kinases by this analogue, and pharmacokinetic characterization demonstrates a 20-fold better oral bioavailability of t-CUPM than sorafenib in mice. Thus, t-CUPM may have the potential to reduce the adverse events observed from the multikinase inhibitory properties and the large dosing regimens of sorafenib.