Renal Cell Carcinoma: A Study through NMR-Based Metabolomics Combined with Transcriptomics

Abstract

Renal cell carcinoma (RCC) is a heterogeneous cancer often showing late symptoms. Until now, some candidate protein markers have been proposed for its diagnosis. Metabolomics approaches have been applied, predominantly using Mass Spectrometry (MS), while Nuclear Magnetic Resonance (NMR)-based studies remain limited. There is no study about RCC integrating NMR-based metabolomics with transcriptomics. In this work, 1H-NMR spectroscopy combined with multivariate statistics was applied on urine samples, collected from 40 patients with clear cell RCC (ccRCC) before nephrectomy and 29 healthy controls; nine out of 40 patients also provided samples one-month after nephrectomy. We observed increases of creatine, alanine, lactate and pyruvate, and decreases of hippurate, citrate, and betaine in all ccRCC patients. A network analysis connected most of these metabolites with glomerular injury, renal inflammation and renal necrosis/cell death. Interestingly, intersecting metabolites with transcriptomic data from CD133+/CD24+ tumoral renal stem cells isolated from ccRCC patients, we found that both genes and metabolites differentially regulated in ccRCC patients belonged to HIF-α signaling, methionine and choline degradation, and acetyl-CoA biosynthesis. Moreover, when comparing urinary metabolome of ccRCC patients after nephrectomy, some processes, such as the glomerular injury, renal hypertrophy, renal necrosis/cell death and renal proliferation, were no more represented.

Nuclear Magnetic Resonance metabolomics reveals an excretory metabolic signature of renal cell carcinoma

“…These results partially confirm previous suggestions of a 7- metabolite urinary signature of early RCC42, particularly regarding the increases in lactate (not statistically relevant here) and pyruvate (here with p -value 5.43 × 10 −7 , compared to 0.010 in ref. 42) and the decrease in hippurate (here with p -value 4.60 × 10 −6 , compared to 0.023 in ref. 42).…”

“…42) and the decrease in hippurate (here with p -value 4.60 × 10 −6 , compared to 0.023 in ref. 42). Previously observed variations in creatine, alanine, betaine and citrate were either not confirmed in this cohort or found to have a confounder contribution (namely, decreased citrate was here related to higher BMI, Table 2).…”

“…4). Changes in hippurate levels may arise from sources as varied as diet, oxidative stressors and gut microflora and, while hippurate decreases have been reported in a recent account of RCC urine metabolomics42 and lung cancer27, some inconsistency is found in other cancer studies525354. This indicates the importance of diet/lifestyle parameters in determining the relationship of hippurate and cancer.…”

“…Notwithstanding the important developments described above, NMR-based metabolomics studies of RCC urine has been relatively underexplored4142 NMR metabolomics importantly complements MS-based methodologies since, in spite of its lower sensitivity, it enables a wide range of compound families to be detected, with high reproducibility, in a single experiment, thus being particularly suited to untargeted analysis.…”

“…This paper reports a NMR profiling study of human urine from a cohort of RCC patients ( n  = 42) and controls ( n  = 49) building on previous work42 by adding a systematic evaluation of the impacts of subject age, gender, body mass index (BMI) and smoking habits, to be later considered when defining a RCC metabolic signature. Furthermore, our methodology entailed variable selection of the 1 H NMR spectra43, in tandem with multivariate analysis and validation procedures, to more efficiently retrieve meaningful profiles of metabolite variations related to confounders and disease.…”