Sex differences in kidney metabolism may reflect sex-dependent outcomes in human diabetic kidney disease.

Diabetic kidney disease (DKD) is the main cause of chronic kidney disease (CKD) and progresses faster in males than in females. We identify sex-based differences in kidney metabolism and in the blood metabolome of male and female individuals with diabetes. Primary human proximal tubular epithelial cells (PTECs) from healthy males displayed increased mitochondrial respiration, oxidative stress, apoptosis, and greater injury when exposed to high glucose compared with PTECs from healthy females. Male human PTECs showed increased glucose and glutamine fluxes to the TCA cycle, whereas female human PTECs showed increased pyruvate content. The male human PTEC phenotype was enhanced by dihydrotestosterone and mediated by the transcription factor HNF4A and histone demethylase KDM6A. In mice where sex chromosomes either matched or did not match gonadal sex, male gonadal sex contributed to the kidney metabolism differences between males and females. A blood metabolomics analysis in a cohort of adolescents with or without diabetes showed increased TCA cycle metabolites in males. In a second cohort of adults with diabetes, females without DKD had higher serum pyruvate concentrations than did males with or without DKD. Serum pyruvate concentrations positively correlated with the estimated glomerular filtration rate, a measure of kidney function, and negatively correlated with all-cause mortality in this cohort. In a third cohort of adults with CKD, male sex and diabetes were associated with increased plasma TCA cycle metabolites, which correlated with all-cause mortality. These findings suggest that differences in male and female kidney metabolism may contribute to sex-dependent outcomes in DKD.

Proteomics: Its Promise and Pitfalls in Shaping Precision Medicine in Solid Organ Transplantation.

Solid organ transplantation is an established treatment of choice for end-stage organ failure. However, all transplant patients are at risk of developing complications, including allograft rejection and death. Histological analysis of graft biopsy is still the gold standard for evaluation of allograft injury, but it is an invasive procedure and prone to sampling errors. The past decade has seen an increased number of efforts to develop minimally invasive procedures for monitoring allograft injury. Despite the recent progress, limitations such as the complexity of proteomics-based technology, the lack of standardization, and the heterogeneity of populations that have been included in different studies have hindered proteomic tools from reaching clinical transplantation. This review focuses on the role of proteomics-based platforms in biomarker discovery and validation in solid organ transplantation. We also emphasize the value of biomarkers that provide potential mechanistic insights into the pathophysiology of allograft injury, dysfunction, or rejection. Additionally, we forecast that the growth of publicly available data sets, combined with computational methods that effectively integrate them, will facilitate a generation of more informed hypotheses for potential subsequent evaluation in preclinical and clinical studies. Finally, we illustrate the value of combining data sets through the integration of 2 independent data sets that pinpointed hub proteins in antibody-mediated rejection.

Increased Autoantibodies Against Ro/SS-A, CENP-B, and La/SS-B in Patients With Kidney Allograft Antibody-mediated Rejection.

Antibody-mediated rejection (AMR) causes more than 50% of late kidney graft losses. In addition to anti-human leukocyte antigen (HLA) donor-specific antibodies, antibodies against non-HLA antigens are also linked to AMR. Identifying key non-HLA antibodies will improve our understanding of AMR. METHODS: We analyzed non-HLA antibodies in sera from 80 kidney transplant patients with AMR, mixed rejection, acute cellular rejection (ACR), or acute tubular necrosis. IgM and IgG antibodies against 134 non-HLA antigens were measured in serum samples collected pretransplant or at the time of diagnosis. RESULTS: Fifteen non-HLA antibodies were significantly increased (P < 0.05) in AMR and mixed rejection compared with ACR or acute tubular necrosis pretransplant, and 7 at diagnosis. AMR and mixed cases showed significantly increased pretransplant levels of IgG anti-Ro/Sjögren syndrome-antigen A (SS-A) and anti-major centromere autoantigen (CENP)-B, compared with ACR. Together with IgM anti-CENP-B and anti-La/SS-B, these antibodies were significantly increased in AMR/mixed rejection at diagnosis. Increased IgG anti-Ro/SS-A, IgG anti-CENP-B, and IgM anti-La/SS-B were associated with the presence of microvascular lesions and class-II donor-specific antibodies (P < 0.05). Significant increases in IgG anti-Ro/SS-A and IgM anti-CENP-B antibodies in AMR/mixed rejection compared with ACR were reproduced in an external cohort of 60 kidney transplant patients. CONCLUSIONS: This is the first study implicating autoantibodies anti-Ro/SS-A and anti-CENP-B in AMR. These antibodies may participate in the crosstalk between autoimmunity and alloimmunity in kidney AMR.

Normothermic Ex-vivo Kidney Perfusion in a Porcine Auto-Transplantation Model Preserves the Expression of Key Mitochondrial Proteins: An Unbiased Proteomics Analysis.

Normothermic ex-vivo kidney perfusion (NEVKP) results in significantly improved graft function in porcine auto-transplant models of donation after circulatory death injury compared with static cold storage (SCS); however, the molecular mechanisms underlying these beneficial effects remain unclear. We performed an unbiased proteomics analysis of 28 kidney biopsies obtained at three time points from pig kidneys subjected to 30 min of warm ischemia, followed by 8 h of NEVKP or SCS, and auto-transplantation. 70/6593 proteins quantified were differentially expressed between NEVKP and SCS groups (false discovery rate < 0.05). Proteins increased in NEVKP mediated key metabolic processes including fatty acid ß-oxidation, the tricarboxylic acid cycle, and oxidative phosphorylation. Comparison of our findings with external datasets of ischemia-reperfusion and other models of kidney injury confirmed that 47 of our proteins represent a common signature of kidney injury reversed or attenuated by NEVKP. We validated key metabolic proteins (electron transfer flavoprotein subunit beta and carnitine O-palmitoyltransferase 2, mitochondrial) by immunoblotting. Transcription factor databases identified members of the peroxisome proliferator-activated receptors (PPAR) family of transcription factors as the upstream regulators of our dataset, and we confirmed increased expression of PPARA, PPARD, and RXRA in NEVKP with reverse transcription polymerase chain reaction. The proteome-level changes observed in NEVKP mediate critical metabolic pathways. These effects may be coordinated by PPAR-family transcription factors and may represent novel therapeutic targets in ischemia-reperfusion injury.

Association between the renin-angiotensin system and chronic lung allograft dysfunction.

Chronic lung allograft dysfunction (CLAD) is the major cause of death after lung transplantation. Angiotensin II (AngII), the main effector of the renin-angiotensin system, elicits fibrosis in both kidney and lung. We identified six AngII-regulated proteins (Ras homolog family member B (RHOB), bone marrow stromal cell antigen 1 (BST1), lysophospholipase 1 (LYPA1), glutamine synthetase (GLNA), thrombospondin 1 (TSP1) and laminin subunit ß2 (LAMB2)) that were increased in urine of patients with kidney allograft fibrosis. We hypothesised that the renin-angiotensin system is active in CLAD and that AngII-regulated proteins are increased in bronchoalveolar lavage fluid (BAL) of CLAD patients.We performed immunostaining of AngII receptors (AGTR1 and AGTR2), TSP1 and GLNA in 10 CLAD lungs and five controls. Using mass spectrometry, we quantified peptides corresponding to AngII-regulated proteins in BAL of 40 lung transplant recipients (stable, acute lung allograft dysfunction (ALAD) and CLAD). Machine learning algorithms were developed to predict CLAD based on BAL peptide concentrations.Immunostaining demonstrated significantly more AGTR1+ cells in CLAD versus control lungs (p=0.02). TSP1 and GLNA immunostaining positively correlated with the degree of lung fibrosis (R2=0.42 and 0.57, respectively). In BAL, we noted a trend towards higher concentrations of AngII-regulated peptides in patients with CLAD at the time of bronchoscopy, and significantly higher concentrations of BST1, GLNA and RHOB peptides in patients that developed CLAD at follow-up (p<0.05). The support vector machine classifier discriminated CLAD from stable and ALAD patients at the time of bronchoscopy (area under the curve (AUC) 0.86) and accurately predicted subsequent CLAD development (AUC 0.97).Proteins involved in the renin-angiotensin system are increased in CLAD lungs and BAL. AngII-regulated peptides measured in BAL may accurately identify patients with CLAD and predict subsequent CLAD development.

Distinct roles of UVRAG and EGFR signaling in skeletal muscle homeostasis.

OBJECTIVE: Autophagy is a physiological self-eating process that can promote cell survival or activate cell death in eukaryotic cells. In skeletal muscle, it is important for maintaining muscle mass and function that is critical to sustain mobility and regulate metabolism. The UV radiation resistance-associated gene (UVRAG) regulates the early stages of autophagy and autophagosome maturation and plays a key role in endosomal trafficking. This study investigated the essential in vivo role of UVRAG in skeletal muscle biology. METHODS: To determine the role of UVRAG in skeletal muscle in vivo, we generated muscle-specific UVRAG knockout mice using the Cre-loxP system driven by Myf6 promoter that is exclusively expressed in skeletal muscle. Myf6-Cre+ UVRAGfl/fl (M-UVRAG-/-) mice were compared to littermate Myf6-Cre+ UVRAG+/+ (M-UVRAG+/+) controls under basal conditions on a normal chow diet. Body composition, muscle function, and mitochondria morphology were assessed in muscles of the WT and KO mice at 24 weeks of age. RESULTS: M-UVRAG-/- mice developed accelerated sarcopenia and impaired muscle function compared to M-UVRAG+/+ littermates at 24 weeks of age. Interestingly, these mice displayed improved glucose tolerance and increased energy expenditure likely related to upregulated Fgf21, a marker of muscle dysfunction. Skeletal muscle of the M-UVRAG-/- mice showed altered mitochondrial morphology with increased mitochondrial fission and EGFR accumulation reflecting defects in endosomal trafficking. To determine whether increased EGFR signaling had a causal role in muscle dysfunction, the mice were treated with an EGFR inhibitor, gefitinib, which partially restored markers of muscle and mitochondrial deregulation. Conversely, constitutively active EGFR transgenic expression in UVRAG-deficient muscle led to further detrimental effects with non-overlapping distinct defects in muscle function, with EGFR activation affecting the muscle fiber type whereas UVRAG deficiency impaired mitochondrial homeostasis. CONCLUSIONS: Our results show that both UVRAG and EGFR signaling are critical for maintaining muscle mass and function with distinct mechanisms in the differentiation pathway.

Extracellular Matrix Injury of Kidney Allografts in Antibody-Mediated Rejection: A Proteomics Study.

BACKGROUND: Antibody-mediated rejection (AMR) accounts for >50% of kidney allograft loss. Donor-specific antibodies (DSA) against HLA and non-HLA antigens in the glomeruli and the tubulointerstitium cause AMR while inflammatory cytokines such as TNFα trigger graft injury. The mechanisms governing cell-specific injury in AMR remain unclear. METHODS: Unbiased proteomic analysis of laser-captured and microdissected glomeruli and tubulointerstitium was performed on 30 for-cause kidney biopsy specimens with early AMR, acute cellular rejection (ACR), or acute tubular necrosis (ATN). RESULTS: A total of 107 of 2026 glomerular and 112 of 2399 tubulointerstitial proteins was significantly differentially expressed in AMR versus ACR; 112 of 2026 glomerular and 181 of 2399 tubulointerstitial proteins were significantly dysregulated in AMR versus ATN (P<0.05). Basement membrane and extracellular matrix (ECM) proteins were significantly decreased in both AMR compartments. Glomerular and tubulointerstitial laminin subunit γ-1 (LAMC1) expression decreased in AMR, as did glomerular nephrin (NPHS1) and receptor-type tyrosine-phosphatase O (PTPRO). The proteomic analysis revealed upregulated galectin-1, which is an immunomodulatory protein linked to the ECM, in AMR glomeruli. Anti-HLA class I antibodies significantly increased cathepsin-V (CTSV) expression and galectin-1 expression and secretion in human glomerular endothelial cells. CTSV had been predicted to cleave ECM proteins in the AMR glomeruli. Glutathione S-transferase ω-1, an ECM-modifying enzyme, was significantly increased in the AMR tubulointerstitium and in TNFα-treated proximal tubular epithelial cells. CONCLUSIONS: Basement membranes are often remodeled in chronic AMR. Proteomic analysis performed on laser-captured and microdissected glomeruli and tubulointerstitium identified early ECM remodeling, which may represent a new therapeutic opportunity.

Generation of monoclonal antibodies and development of an immunofluorometric assay for the detection of CUZD1 in tissues and biological fluids.

BACKGROUND: CUB and zona pellucida-like domain-containing protein 1 (CUZD1) was identified as a pancreas-specific protein and was proposed as a candidate biomarker for pancreatic related disorders. CUZD1 protein levels in tissues and biological fluids have not been extensively examined. The purpose of the present study was to generate specific antibodies targeting CUZD1 to assess CUZD1 expression within tissues and biological fluids. METHODS: Mouse monoclonal antibodies against CUZD1 were generated and used to perform immunohistochemical analyses and to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA). CUZD1 protein expression was assessed in various human tissue extracts and biological fluids and in gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant protein. RESULTS: Immunohistochemical staining of CUZD1 in pancreatic tissue showed that the protein is localized to the acinar cells and the lumen of the acini. Western blot analysis detected the protein in pancreatic tissue extract and pancreatic juice. The newly developed ELISA measured CUZD1 in high levels in pancreas and in much lower but detectable levels in several other tissues. In the biological fluids tested, CUZD1 expression was detected exclusively in pancreatic juice. The analysis of gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant CUZD1 suggested that the protein exists in high molecular weight protein complexes. CONCLUSION: This study describes the development of tools targeting CUZD1 protein, its tissue expression pattern and levels in several biological fluids. These new tools will facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology.

Biochemical and functional characterization of the human tissue kallikrein 9.

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+ KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

Novel immunoassays for detection of CUZD1 autoantibodies in serum of patients with inflammatory bowel diseases.

BACKGROUND: Pancreatic autoantibodies (PABs) are detected in patients with inflammatory bowel disease (IBD). Their prevalence is higher in Crohn's disease (CrD) than in ulcerative colitis (UC). Glycoprotein 2 (GP2) and, more recently, CUB and zona pellucida-like domain-containing protein 1 (CUZD1) have been identified as target autoantigens of PAB. The clinical utility of CUZD1 autoantibodies has only recently been assessed by indirect immunofluorescence (IIF) assays. In this study, we developed and validated novel immunoassays for the detection of CUZD1 autoantibodies. METHODS: Recombinant CUZD1 protein was utilized as a solid-phase antigen for the development of two immunoassays for the detection of IgG and IgA CUZD1 autoantibodies. Serum samples from 100 patients with CrD, 100 patients with UC, 129 patients assessed for various autoimmune diseases (vADs) and 50 control individuals were analyzed. RESULTS: Two immunofluorometric assays for the detection of IgG and IgA CUZD1-specific antibodies were developed. CUZD1 autoantibodies were detected in 12.5% (25/200) IBD patients, including 16% of patients with CrD and in 9% of patients with UC (CrD vs. UC, p<0.05), compared with 3.1% (4/129) patients suspected of having vADs (CrD vs. ADs, p<0.05; UC vs. ADs, p=0.08). CUZD1 autoantibody positivity was not found to be related to disease location, age of disease onset or disease phenotype. CONCLUSIONS: This is the first study to describe novel IgA and IgG CUZD1 autoantibody enzyme-linked immunosorbent assay. These immunoassays agree well with standard IIF techniques and can be utilized in multicenter studies to investigate the diagnostic and clinical utility of CUZD1 autoantibodies.

A new enzyme-linked immunosorbent assay (ELISA) for human free and bound kallikrein 9.

BACKGROUND: Kallikrein 9 (KLK9) is a member of the human kallikrein-related peptidases family, whose physiological role and implications in disease processes remain unclear. The active form of the enzyme is predicted to have chymotryptic activity. In the present study, we produced for the first time the active recombinant protein and monoclonal antibodies, and developed novel immunoassays for the quantification of free and bound KLK9 in biological samples. METHODS: The coding sequence of mature KLK9 isoform (mat-KLK9) was expressed in an Expi293F mammalian system and the synthesized polypeptide was purified through a two-step protocol. The purified protein was used as an immunogen for production of monoclonal antibodies in mice. Hybridomas were further expanded and antibodies were purified. Newly-produced monoclonal antibodies were screened for reaction with the KLK9 recombinant protein by a state-of-the-art immunocapture/parallel reaction monitoring mass spectrometry-based methodology. RESULTS: Anti-KLK9 antibodies were combined in pairs, resulting in the development of a highly sensitive (limit of detection: 15 pg/mL) and specific (no cross-reactivity with other KLKs) sandwich-type ELISA. Highest KLK9 protein levels were found in tonsil and sweat and lower levels in the heart, kidney and liver. Hybrid immunoassays using an anti-KLK9 antibody for antigen capture and various anti-serine protease inhibitor polyclonal antibodies, revealed the presence of an a1-antichymotrypsin-bound KLK9 isoform in biological samples. CONCLUSIONS: The ELISAs for free and bound forms of KLK9 may be highly useful for the detection of KLK9 in a broad range of biological samples, thus enabling the clarification of KLK9 function and use as a potential disease biomarker.

Cancer immunotherapy: the beginning of the end of cancer?

These are exciting times for cancer immunotherapy. After many years of disappointing results, the tide has finally changed and immunotherapy has become a clinically validated treatment for many cancers. Immunotherapeutic strategies include cancer vaccines, oncolytic viruses, adoptive transfer of ex vivo activated T and natural killer cells, and administration of antibodies or recombinant proteins that either costimulate cells or block the so-called immune checkpoint pathways. The recent success of several immunotherapeutic regimes, such as monoclonal antibody blocking of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD1), has boosted the development of this treatment modality, with the consequence that new therapeutic targets and schemes which combine various immunological agents are now being described at a breathtaking pace. In this review, we outline some of the main strategies in cancer immunotherapy (cancer vaccines, adoptive cellular immunotherapy, immune checkpoint blockade, and oncolytic viruses) and discuss the progress in the synergistic design of immune-targeting combination therapies.

PIK3CA mutational status in circulating tumor cells can change during disease recurrence or progression in patients with breast cancer.

PURPOSE: Molecular characterization of circulating tumor cells (CTC) is crucial for the investigation of molecular-targeted therapies while PIK3CA somatic mutations play a crucial role in therapy response. We investigated the presence of PIK3CA mutations in CTC and whether this is associated with clinical outcome. EXPERIMENTAL DESIGN: We developed and validated an ultrasensitive methodology for the detection of PIK3CA mutations that is based on a combination of allele-specific, asymmetric rapid PCR and melting analysis. We analyzed PIK3CA hotspot mutations in: (i) a training group consisting of EpCAM-positive CTC fraction from 37 patients with clinically confirmed metastasis, and 26 healthy female volunteers and 15 primary breast tumor tissues and (ii) an independent group consisting of EpCAM-positive CTC fraction from 57 metastatic and 118 operable breast cancer patients and 76 corresponding primary tumors. RESULTS: The assay could detect 0.05% of mutated dsDNA in the presence of 99.95% wtDNA for both exons (9 and 20) and was highly specific (0/26 healthy donors). PIK3CA mutations were identified in EpCAM-positive CTC in 20 of 57(35.1%) and in 23 of 118 (19.5%) patients with metastatic and operable breast cancer, and in 45 of 76(59.2%) corresponding FFPEs. Our data indicate that PIK3CA mutational status in CTCs can change during disease progression and is associated with worse survival (P = 0.047). CONCLUSIONS: PIK3CA hotspot mutations are present at a relatively high frequency in CTCs and their presence is associated with worse survival in patients with breast cancer with metastasis. Evaluation of PIK3CA mutational status in CTCs is a strategy with potential clinical application.

False biomarker discovery due to reactivity of a commercial ELISA for CUZD1 with cancer antigen CA125.

BACKGROUND: By using proteomics and bioinformatics, we have previously identified a group of highly pancreas-specific proteins as candidate pancreatic ductal adenocarcinoma (PDAC) biomarkers. With the use of commercially available ELISAs, the performance of some of these candidates was initially evaluated in a relatively small serum cohort (n = 100 samples). This phase revealed that CUB and zona pellucida-like domains protein 1 (CUZD1) may represent a new, promising PDAC biomarker. METHODS: We performed detailed experiments to investigate the specificity of the commercial CUZD1 ELISA assay. CUZD1 was expressed in house in both bacteria and yeast expression systems. Recombinant CUZD1 and biological samples containing CUZD1, as well as commercial CUZD1 ELISA standards, were analyzed by Western blot, size exclusion HPLC, and mass spectrometry (LC-MS Orbitrap). RESULTS: We confirmed that instead of CUZD1, the commercial assay is recognizing a nonhomologous, known cancer antigen [cancer antigen 125 (CA125)]. CONCLUSIONS: We conclude that poor characterization of commercial ELISA assays is a factor that could lead to false biomarker discovery. To our knowledge, this is the first report documenting that a commercial ELISA marketed for one analyte (CUZD1) may, in fact, recognize a different, nonhomologous antigen (CA125).