Papillary renal cell carcinomas rewire glutathione metabolism and are deficient in anabolic glucose synthesis

Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, rewiring of the main pathways involved in ATP and glucose synthesis was observed at the protein level. In contrast, transcripts encoding for the respiratory chain were not regulated, which prompts for non-genetic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies. SIGNIFICANCE STATEMENT We applied proteome- and metabolome profiling to elucidate molecular features in malign papillary renal cell carcinomas. By this characterization, a reprogramming of the main metabolic pathways, such as gluconeogenesis and fatty acid- and amino acid metabolism were identified. The proteins involved in the respiratory chain and the corresponding enzymatic activities were strongly reduced in pRCC, showing an anti-correlation compared with the transcriptome. Similar to renal oncocytomas, the ROS scavenger glutathione was identified as a hallmark in pRCC. Our results suggest that impaired metabolism and dysfunctional mitochondria determine the fate of pRCC. Furthermore, we propose that the specific regulation of the mitochondrial respiratory chain can differentiate highly similar malignant pRCCs from benign renal oncocytomas.


Abstract
Papillary renal cell carcinoma (pRCC) is a malignant kidney cancer with a prevalence of 7-20% of all renal tumors. Proteome and metabolome profiles of 19 pRCC and patient-matched healthy kidney controls were used to elucidate the regulation of metabolic pathways and the underlying molecular mechanisms. Glutathione (GSH), a main reactive oxygen species (ROS) scavenger, was highly increased and can be regarded as a new hallmark in this malignancy. Isotope tracing of pRCC derived cell lines revealed an increased de novo synthesis rate of GSH, based on glutamine consumption. Furthermore, rewiring of the main pathways involved in ATP and glucose synthesis was observed at the protein level. In contrast, transcripts encoding for the respiratory chain were not regulated, which prompts for non-genetic profiling. The molecular characteristics of pRCC are increased GSH synthesis to cope with ROS stress, deficient anabolic glucose synthesis, and compromised oxidative phosphorylation, which could potentially be exploited in innovative anti-cancer strategies.

SIGNIFICANCE STATEMENT
We applied proteome-and metabolome profiling to elucidate molecular features in malign papillary renal cell carcinomas. By this characterization, a reprogramming of the main metabolic pathways, such as gluconeogenesis and fatty acid-and amino acid metabolism were identified.
The proteins involved in the respiratory chain and the corresponding enzymatic activities were strongly reduced in pRCC, showing an anti-correlation compared with the transcriptome. Similar to renal oncocytomas, the ROS scavenger glutathione was identified as a hallmark in pRCC.
Our results suggest that impaired metabolism and dysfunctional mitochondria determine the fate of pRCC. Furthermore, we propose that the specific regulation of the mitochondrial respiratory chain can differentiate highly similar malignant pRCCs from benign renal oncocytomas.

INTRODUCTION
Papillary renal cell carcinoma (pRCC) is a heterogeneous disease, representing 7-20% of all renal cancers (1-3), subdivided into clinically and biologically distinct type I and type II entities (3). Type I pRCC tumors consist of basophilic cells with papillae and tubular structures and small nucleoli, whereas pRCC type II tumors exhibit large cells with abundant eosinophilic cells and prominent nucleoli (3). The cytogenetic differences are a trisomy 7 and 17 in pRCC type I and the loss of 1p and 3p in pRCC type II tumors (4,5). Intra-and interchromosomal rearrangements are significantly increased in pRCC type II, leading frequently to a gene fusion involving the transcription factor TFE3 (6), by which the promoter substitution appears to be the key molecular event, causing dysregulation of many signaling pathways already implicated in carcinogenesis (7).
Type I of this malignant tumor is characterized by frequent mutations in the MET oncogene, including alternative splice variants. Currently, the MET pathway is the most common target for developing new treatments for pRCC, such as the MET kinase inhibitor Savolitinib, which interrupts angiogenesis (8). Type II has more likely mutations such as SETD2, NF2, and the inactivation of CDKN2A by mutation, deletion, or CpG island hypermethylation (5,9). Structural variants were observed in sporadic events, including duplications in EGFR and HIF1A, and deletions in SDHB, DNMT3A, and STAG2 (10). These genes and several more, which can be found mutated in both tumor types, play a pivotal role in epigenetic regulation, signaling, and proliferation regulation, such as in PI3K/AKT/mTOR, NRF2-ARE, and the Hippo pathways.
Furthermore, type II pRCC was subdivided into three subtypes, based on distinct molecular and phenotypic features. pRCC type II tumors are more likely to metastasize (4), and FH mutations and DNA hypermethylation were found to be correlate with inferior prognosis (5,9). Hence, the hypermethylation group was termed "CpG island methylation phenotype" (CIMP), which additionally featured a metabolic shift known as the Warburg effect (5).
Many studies have been performed recently at the transcript level in pRCC to better understand its classification and subclassification and to elucidate pathway remodeling in these cancer (5,11) studies. Based on all these findings, a new classification system was proposed. Compared with the current model where the organ of origin determines the tumor type, a system was proposed based solely on molecular features which could be considered more relevant for tumor classification (12).
Nephrectomy or partial nephrectomy and in the presence of metastases, and treatment with VEGF and mTOR inhibitors are currently considered the standard treatments. Furthermore, resection or irradiation of metastases can be a useful palliative treatment for patients with brain metastases or osseous metastases that are painful or increase the risk of fracture (13).
Besides transcript data, little is known in pRCC about its regulation at the protein-and metabolome level, the underlying molecular mechanisms, the alterations of metabolic pathways, and how well these "omics" data correlate with each other. One study compared metabolomic/lipidomic profiles of clear cell RCC (ccRCC), chromophobe RCC (chRCC), and pRCC, and determined that RCC subtypes clustered into two groups separating ccRCC and pRCC from chRCC, which mainly reflected the different cells of origin (14).
To fill in the aforesaid knowledge gaps, we undertook a multi-'omics' survey to compare seven type I, seven type II, and five metastatic type II pRCCs with patient-matched adjacent healthy kidney tissues. To confirm the main findings obtained by profiling, the dysregulated pathways were validated by either enzymatic measurements or isotope tracing experiments.

Proteome Profiling of pRCC
Malignant and non-malignant tissues from 19 nephrectomies representing papillary RCC of type I, II, and IIM (with metastases) were investigated; clinical parameters are shown in Table 1.
Proteome profiling revealed a total of 8,554 protein groups, consisting of 1,330,129 identified peptides in all 19 pRCC samples and adjacent healthy kidney tissues, both at a false discovery rate (FDR) of 1% (Dataset S1). 3.785, 3.838, and 4.200 protein groups could be quantified by label-free quantification in type I, II, and IIM, respectively. Pearson correlation ranged from 0.597 to 0.951 for controls and 0.631 to 0.938 for all pRCC specimens for the least to the most similar individuals. Furthermore, proteome profiling revealed pRCC I, II, and IIM versus healthy adjacent kidney tissues as distinct groups in a principal component analysis ( Figure 1A-C). Significantly regulated proteins were identified by a t-test and volcano plots are shown for pRCC type I, II, and IIM (Dataset S2, Figure S1A-C).

mtDNA Mutations in pRCC did not Reveal any Major Impact on the Respiratory Chain
The assembly of mitochondrial whole exome sequencing (WES) reads derived from 19 patients with pRCC and matched with adjacent healthy kidney tissues showed adequate coverage and quality for reliable mtDNA reconstruction and variant calling (Dataset S3). Mitochondrial mean coverage read depth and mitochondrial assembled bases in the WES dataset ranged from 12.42X to 371.41X and from 91.21% to 100%, respectively. The mtDNA content was 46% reduced ( Figure 1D), based on log 2 ratios of the mtDNA WES read depths between pRCC and matching controls, which is in line with a previous observation in RCC by Southern blot (Meierhofer, Mayr et al. 2004). Furthermore, the abundance of proteins located within the mitochondrion versus all non-mitochondrial proteins showed no difference ( Figure 1D).
A total of 260 somatic mtDNA mutations were detected in pRCC samples. Altogether 86 mutations were located within the protein-coding genes, divided into 44 synonymous, 40 nonsynonymous and two nonsense mutations. Among the non-synonymous variants, 25 showed a disease score higher than the threshold (>0.7) and a nucleotide variability that was lower than the nucleotide variability cutoff (0.0026). A total of 197 germline mutations were detected, but only one of the seven non-synonymous germline variants were shown to be potentially pathogenic (Dataset S4).
Although 25 somatic non-synonymous and potentially pathogenic events were identified, it was not possible to infer a strong relationship with pRCC, considering that all mutations were found in only ten of the 19 tumors. None of the somatic mutations were shared between the different subjects and homoplasmic rates were generally very low (Dataset S4), however the number of studied cases was too low to draw any final conclusions.
An analysis of copy number variations (CNV) revealed a fragmented pattern of chromosomal gains and losses spread over all chromosomes in all pRCC types ( Figure S2), but no clear chromosomal patterns were identified.

Significantly Decreased Enzymatic Activity of the Respiratory Chain in pRCC
A gene set enrichment analysis (GSEA) was conducted to identify significantly rewired metabolic pathways in the tumor. Significant decreases in all three investigated pRCC types were found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for: oxidative phosphorylation, the TCA cycle, branched chain amino acids, cytochrome P450 drug metabolism, peroxisomes, fatty acid metabolism, and several amino acid metabolism pathways.
( Figure 2A, Dataset S5). The OXPHOS system was the most severely reduced in all pRCC types. Interestingly, there was no obviously different regulation between the three types of pRCC that was detectable at the protein pathway level. The three most significantly increased KEGG pathways in all pRCCs were the spliceosome, the ribosome and the cell cycle ( Figure 2A; Dataset S5). An aberrantly increased rate of ribosome biogenesis has been recognized as a hallmark of many cancers, caused by hyperactivation of RNA polymerase I transcription and ribosome biogenesis factors, reviewed in (15,16).
In order to investigate the regulation of the respiratory chain between pRCC and controls in more detail, separate gene sets for all five OXPHOS complexes were created, including the assembly factors. This revealed a reduction in protein abundance for all complexes with the highest observed for complex I (CI) in pRCC, exemplarily shown for type I tumors ( Figure 3A).
Only assembly factors, that weren't part of the final complexes, were not decreased.

Anti-correlation of Transcripts and Proteins of the Respiratory Chain in pRCC
Altogether, 291 existing pRCC and 32 control transcriptome data retrieved from TCGA (ID: KIRP) (5) were used to clarify the correlation between the abundance of proteins and the expression of transcripts in pRCC versus controls (Dataset S6). GSEA was performed and identified that most of the significantly regulated pathways ( Figure 2B, Dataset S7) were very similarly regulated for our proteome study. The ribosome and cell cycle were significantly increased in both omics datasets, whereas the TCA cycle, drug metabolism, fatty acid metabolism, and the pathways involved in amino acid metabolism were all significantly decreased (Figure 2A-B; Dataset S5 and S7). The only striking differences were the "spliceosome" pathway, which was significantly up-regulated only in the pRCC proteomes and the "oxidative phosphorylation" pathway, which was the most decreased pathway at the protein level ( Figure 2C), but that was unchanged at the transcript level ( Figure 2D). Since this KEGG pathway also includes V-ATPases, which are not part of the respiratory chain, we manually removed them from the analysis. The enzymatic activities of the individual respiratory chain complexes and citrate synthase (CS) were measured for pRCC and the adjacent matching healthy tissues. This revealed a significant reduction in all enzymatic activities of the respiratory chain, the F 0 F 1 ATPase, and CS in pRCC ( Figure 4A-F). Thus, the regulation of the respiratory chain was not determined by the abundance of the respective transcripts but was correlated with the abundance and enzymatic activity of the OXPHOS complexes.

Malignant pRCC and Benign Renal Oncocytomas
In our previous study on renal oncocytomas, we identified a coordinated up-regulation of proteins of the OXPHOS complexes II-V and the mtDNA, but a striking reduction in the abundance of CI proteins ( Figure 3A) (17). This was explained by several specific low-level heteroplasmic mtDNA mutations of the CI genes in renal oncocytomas. In contrast, all pRCC tumor types featured a general reduction in all OXPHOS complexes ( Figure 3B) where mtDNA mutations seemed to play no major role, but where the number of mtDNA molecules correlated with respiratory chain protein abundances between these tumor entities.

pRCCs have Significantly Decreased Levels of V-and P-ATPases
Vacuolar-type H + -ATPases (V-ATPase) acidify a wide array of intracellular organelles and pump protons via ATP hydrolysis across intracellular and plasma membranes. Acidity is one of the main features of tumors, V-ATPases control their microenvironment by proton extrusion into the extracellular medium (18). This allows secreted lysosomal enzymes to work more efficiently to degrade the extracellular matrix and promote cellular invasion. In contrast to almost all other cancer types, the V-ATPases were found to be down-regulated in pRCC: This specifically applies to the V-type proton ATPase subunit B, kidney isoform (ATP6V1B1), 21-, 14-, 13-fold (fold changes are shown sequentially for type I, II, IIM, respectively) and others such as ATP6V1H, 3-, 4-, 4-fold; ATP6F1, 3-, 3-, 2-fold; ATP6V1E1, 3-, 3-, 4-fold; and ATP6V1A, 2-, 2-, 2-fold; (Dataset S2). This seems to be a specific feature of pRCC and might play a key role in malignancy.
These ATPases are an integral part of the membrane proteins responsible for establishing and maintaining the electrochemical gradients of Na + and K + ions across the plasma membrane.
They are also important for osmoregulation, sodium-coupled transport of several organic and inorganic molecules, and the electrical excitability of nerve and muscle. The enzymatic activities of V-and P-ATPase types were evaluated and showed a significantly reduced activity in pRCC tissues ( Figure 4G-H), which correlated to the observed protein abundances.

Anabolic Glucose Synthesis was Abated in pRCC
The KEGG pathway "glycolysis and gluconeogenesis" was significantly reduced in pRCC (not detected (nd), nd, 53-fold) ( Figure 5A-C, Dataset S2). Interestingly, the two fructosebisphosphate aldolase isoforms A (2-, 2-, 5-fold) and C (1-, 1-, 4-fold) were instead increased or unchanged in pRCC ( Figure 5D). These isoforms have a high affinity for fructose-bisphosphate (FDP) to foster glycolysis, whereas the highly diminished isoform B has a low affinity for FDP and hence converts the back-reaction from glyceraldehyde-3-phosphate to FDP during gluconeogenesis (19,20). A similar decrease in the respective transcripts was found in the TCGA data, where ALDOB was 600-fold and ALDOC 2-fold decreased and ALDOA 2-fold increased. A specific and regulated interaction between ALDOB and the rate-limiting gluconeogenic enzyme fructose-1, 6-bisphosphatase 1 (FBP1) has been shown. This result confirms the view that ALDOA and ALDOB play different roles in glucose metabolism (21). The shut-down of the gluconeogenic pathway was thus one of the most relevant metabolic changes observed and can be regarded as a metabolic hallmark in pRCC.

Consumption
Metabolome profiling revealed a highly significant increase in reduced glutathione (GSH, 47-, 68-, 219-fold; fold changes are shown sequentially for type I, II, IIM, respectively) and oxidized glutathione (GSSG, 871-, 847-, 6,707-fold) in pRCC types, Figure 6A-F, Dataset S8). A case by case specific GSH/GSSG ratio was calculated ( Figure 6F) and revealed that there was a 10-fold average increase in oxidative stress burden in the tumor.
Glutathione functions as a cellular redox buffer for detoxification and can be either synthesized de-novo or imported via the glutathione salvage pathway, where extracellular GSH is cleaved by γ-glutamyltranspeptidases (GGTs) (22). Furthermore, ophthalmic acid, a tripeptide analog of glutathione, was increased (468-, 77-, 58-fold) in pRCC. It was described as a byproduct of glutathione synthetase (GS) and γ-glutamylcysteine synthetase (GCS) and as a new biomarker of oxidative stress (23).
Remarkably, even though metabolites involved in glutathione metabolism were significantly Glutathione peroxidase 3, which protects cells and enzymes from oxidative damage by catalyzing the reduction of hydrogen peroxide, lipid peroxides, and organic hydroperoxide was also significantly reduced in pRCC (GPX3, 6-, 5-, 6-fold).

Discussion
pRCCs are well characterized at the genomic level, with several driver mutations and chromosomal rearrangements having been identified (5). How these alterations translate to proteome-and metabolome regulation are not well understood, but they determine the fate and progression of tumors. Multi-omics profiling of pRCC was performed, revealing a fundamental reprogramming of the pathways for gluconeogenesis, the respiratory chain, and for glutathione metabolism. These can be regarded as a general hallmark of kidney tumors, as was previously observed in renal oncocytomas (17), chRCCs (27), and in ccRCCs at the transcript level (28).
The anti-correlations that have been identified between genetic and non-genetic profiling argue for focusing on these so far under-studied fields.
Gluconeogenesis, an anabolic and highly endergonic pathway, generates glucose from small carbohydrate precursors, for example from lactate during intense exercise, or over periods of fasting and starvation. This pathway is also regarded as an essential process for tumor cell growth (29), since biosynthetic reactions in cancer cells are highly dependent on glycolytic intermediates (30). The kidney may be nearly as important as the liver in gluconeogenesis (31) and pRCCs have been shown to moderately accumulated glucose, which further increases the already higher Fuhrman grades (32). This might be the reason why pRCCs reduce this endergonic pathway, since enough glucose can be imported. Blocking of mTOR activity was shown to augment shuttling of pyruvate into gluconeogenesis, which results in futile cycling of glucose that finally leads to a halt in cancer cell proliferation and ultimately to cell death (33).
Equal amounts of amino acid levels were detected in pRCC and kidney tissues in our study, supporting the idea of a sufficient nutrient supply.
The gluconeogenic gene FBP1 was previously found to be down-regulated in over 600 ccRCCs and was associated with a poor disease prognosis. Thus, FBP1 has been shown to fulfill two distinct functions, by antagonizing glycolytic flux and thus inhibiting the Warburg effect, and by inhibiting the nuclear function of HIF in a catalytic-activity-independent manner, leading to reduced expression of HIF targets such as VEGF, LDHA, and GLUT1 (28). This unique dual function of the FBP1 protein explains its ubiquitous loss in ccRCC, distinguishing it from other tumor suppressors that are not consistently mutated in all tumors (28).
Moreover, the inhibition of FBP1 leads to the activation of AMP-activated protein kinase (AMPK).
The aldolases (A-C) are required for the formation of a super lysosomal complex containing V-ATPase, ragulator, axin, liver kinase B1 (LKB1), and AMPK in its active form (34). AMPK activation plays a central role in glucose sensing at the lysosome and acts contrary to other regulatory systems such as the mammalian target of rapamycin (mTOR).
A general shut-down of the entire gluconeogenesis pathway in pRCC was also identified at the proteome level in our study, which has fundamental implications for the metabolic regulation of a cell and organ. Specifically, the two aldolase isoforms A and C, which foster glycolysis were unchanged, but the aldolase isoform B, necessary for gluconeogenesis was greatly diminished in pRCC. This again indicates a critical metabolic change, as previously observed in chRCC (27) and in gastric cancer (35). Conversely, an over-expression of gluconeogenic genes and proteins is frequently found in other tumor species, such as the over-expression of ALDOB in ccRCC (36) and colon cancer (37)(38)(39), which was also associated with tumor progression and poor prognosis.
Also the up-regulation of ALDOA has been reported in many cancer types, such as oral squamous cell carcinoma (40), osteosarcoma (41), lung cancer (42), and hepatocellular carcinoma (43). Specifically, an increase in ALDOA was shown for all RCC types and was associated with metastasis, histological differentiation, and poor prognosis. Furthermore, silencing ALDOA expression in ccRCC cell lines decreased their proliferative, migratory, and invasive abilities, while ALDOA overexpression increased these abilities (44).
In addition, the abundance and enzymatic activities of the P-and V-ATPases were found to be significantly reduced in our pRCC panel. A possible mechanism by which V-ATPases are thought to contribute to cancer cell migration and invasion is to acidify extracellular space to promote the activity of acid-dependent proteases that are involved in invasion (18,45,46).
Besides the classical role of regulating acidity within a cell, recent studies showed that V-ATPases, as part of the V-ATPase-Ragulator complex, serve as a dual sensor for energy/nutrient sufficiency and deficiency and they can initiate the metabolic switch between catalytic and anabolic pathways (47). It has been further shown and that glycolysis is directly coupled to the V-ATPases by protein-protein interactions (48,49).
The significant reduction of enzymes involved in gluconeogenesis thus has metabolic consequences on multiple layers and is a hallmark of all investigated kidney cancers, such as in pRCC (this study), ccRCC (28), chRCC (27), and renal oncocytomas (17). By abandoning this endergonic pathway in pRCC the tumor is able to simultaneously reduce other processes involved in the generation of ATP. Indeed, pathways involved in fatty acid metabolism, amino acid metabolism, as well as OXPHOS and the TCA cycle pathways were significantly downregulated in our pRCC specimen.
Another frequently observed phenomenon in cancer is the diminished oxidative phosphorylation capacity, known already for decades as the "Warburg effect" . The abundance of all proteins involved in oxidative phosphorylation and the F 0 F 1 ATPase as well as the corresponding enzymatic activities were significantly reduced in our pRCC panel. This was previously shown for chRCC (27) and only for OXPHOS enzymatic activities and the mtDNA content in ccRCC and pRCC (50). In contrast, the enhanced expression of LDH-A and lactic acid was observed in our study and this has been associated with aggressive and metastatic cancers in a variety of tumor types (51-53).
By comparing our proteome-with transcriptome data from TCGA (5), the main differentially regulated pathway was found to be the respiratory chain, which was the most highly decreased pathway on the protein level, but unchanged at the transcript level. A similar discrepancy between transcripts and proteins in the regulation of the respiratory chain was previously observed by us in benign renal oncocytomas (17) and malignant chRCC (27). Enzymatic activities of the respiratory chain in pRCC and in renal oncocytomas (54) and chRCC (27) matched with protein abundances rather than gene expression. The mechanism for this anticorrelation still remains elusive, but might be directly correlated to the decreased mtDNA level, or also caused by the interference of miRNAs and the stability of transcripts or proteins. This demonstrates the necessity of surveying multiple omics profiles.
The most strikingly increased set of metabolites in pRCC were those involved in glutathione metabolism (GSH, GSSG, cysteine-glutathione disulfide, ophthalmic acid). This is similar to those previously identified in renal oncocytomas (17,55) and chRCC (27,56). GSH is an important ROS scavenger (57) and frequently produced by several tumor types to withstand unusual levels of oxidative stress (22). Therefore increased GSH levels in pRCC may be considered as the main strategy for the tumor to overcome ROS stress originating from a dysregulated respiratory chain.
By probing the metabolic flux for GSH synthesis, a significant increase of the synthesis rate was observed in pRCC derived cell lines over kidney controls when using glutamate as a substrate.
This is in agreement with another study, which found that glutamine dependence in ccRCC suppresses oxidative stress (58). The inhibition of GSH synthesis by a specific glutaminase (GLS) inhibitor and the simultaneous treatment with hydrogen peroxide resulted in a high apoptosis rate in ccRCC (58). Hence, additional administrationof antioxidants during (chemotherapeutic) cancer treatment have been frequently shown to have no or even pro-tumor effects (59,60). High GSH levels in RCC, which protect the tumor from increased ROS stress, should therefore be therapeutically exploited by reducing the antioxidant levels, of GSH, and increasing ROS stress at the same time to a level where healthy cells can still survive, but tumorous cells are forced into apoptosis.

Conclusion
Key metabolic reprogramming processes, such those for gluconeogenesis, the respiratory chain, and glutathione metabolism are not only the main molecular characteristics for papillary RCC, but rather seem to be a general feature for other kidney tumors as well. Specifically, the reinforcement of glutathione metabolism, reflecting the increased burden of oxidative stress, and abandoning endergonic processes may hold key therapeutic implications as a future treatment option. The fragmented DNA (150-200 bp) was purified using AMPure XP beads and subjected to an end-repair reaction. Following another purification step, the DNA was 3'adenylated and furthermore purified. Paired-end adaptors were ligated and the afterward purified library was amplified with 10 amplification cycles. The amplified library was purified, quantified and hybridized to the probe library for exome capture. Captured fragments were purified using streptavidin-coated beads and eluted with 30 μl nuclease-free water. Using Herculase-enzyme, Genome Analyzer IIx platform were generated following the manufacturer's protocol. Images from the instrument were processed using the manufacturer's software to generate FASTQ sequence files.

Analysis of mtDNA Mutations
The FASTQ files were used as input for the MToolBox pipeline (61)  To determine the source for GSH de novo synthesis and potential differences between the pRCC cell lines Caki-2 and ACHN versus HK-2 kidney controls, two isotope tracing experiments were performed. The first experiment employed 13 C 6 labeled glucose, the second 13 C 5 15 N glutamic acid as probe, a scheme of GSH synthesis is outlined in figure 7. In addition, proline de novo synthesis was monitored simultaneously within the experimental setting of glutamate as a tracer ( Figure 6J).
GSH labeling dynamics were probed by sampling at time points 0, 12, and 24 hours, for proline 0 and 12 hours were taken in 6-well plate triplicates for all three cell lines.

Ethics Approval and Consent to Participate
The study was approved by the institutional Ethics Committee (no. EA1/134/12, Charité -Universitätsmedizin Berlin) and was carried out in accordance with the Declaration of Helsinki.
All participants gave informed consent.

Availability of Data and Materials
The datasets generated during the current study are available as supplementary files and in the