Advances in proteomic profiling of pediatric kidney diseases.

Chronic kidney disease (CKD) can progress to kidney failure and require dialysis or transplantation, while early diagnosis can alter the course of disease and lead to better outcomes in both pediatric and adult patients. Significant CKD comorbidities include the manifestation of cardiovascular disease, heart failure, coronary disease, and hypertension. The pathogenesis of chronic kidney diseases can present as subtle and especially difficult to distinguish between different glomerular pathologies. Early detection of adult and pediatric CKD and detailed mechanistic understanding of the kidney damage can be helpful in delaying or curtailing disease progression via precise intervention toward diagnosis and prognosis. Clinically, serum creatinine and albumin levels can be indicative of CKD, but often are a lagging indicator only significantly affected once kidney function has severely diminished. The evolution of proteomics and mass spectrometry technologies has begun to provide a powerful research tool in defining these mechanisms and identifying novel biomarkers of CKD. Many of the same challenges and advances in proteomics apply to adult and pediatric patient populations. Additionally, proteomic analysis of adult CKD patients can be transferred directly toward advancing our knowledge of pediatric CKD as well. In this review, we highlight applications of proteomics that have yielded such biomarkers as PLA2R, SEMA3B, and other markers of membranous nephropathy as well as KIM-1, MCP-1, and NGAL in lupus nephritis among other potential diagnostic and prognostic markers. The potential for improving the clinical toolkit toward better treatment of pediatric kidney diseases is significantly aided by current and future development of proteomic applications.

Proteomic Analysis Identifies Distinct Glomerular Extracellular Matrix in Collapsing Focal Segmental Glomerulosclerosis.

BACKGROUND: The mechanisms leading to extracellular matrix (ECM) replacement of areas of glomerular capillaries in histologic variants of FSGS are unknown. This study used proteomics to test the hypothesis that glomerular ECM composition in collapsing FSGS (cFSGS) differs from that of other variants. METHODS: ECM proteins in glomeruli from biopsy specimens of patients with FSGS not otherwise specified (FSGS-NOS) or cFSGS and from normal controls were distinguished and quantified using mass spectrometry, verified and localized using immunohistochemistry (IHC) and confocal microscopy, and assessed for gene expression. The analysis also quantified urinary excretion of ECM proteins and peptides. RESULTS: Of 58 ECM proteins that differed in abundance between cFSGS and FSGS-NOS, 41 were more abundant in cFSGS and 17 in FSGS-NOS. IHC showed that glomerular tuft staining for cathepsin B, cathepsin C, and annexin A3 in cFSGS was significantly greater than in other FSGS variants, in minimal change disease, or in membranous nephropathy. Annexin A3 colocalized with cathepsin B and C, claudin-1, phosphorylated ERK1/2, and CD44, but not with synaptopodin, in parietal epithelial cells (PECs) infiltrating cFSGS glomeruli. Transcripts for cathepsins B and C were increased in FSGS glomeruli compared with normal controls, and urinary excretion of both cathepsins was significantly greater in cFSGS compared with FSGS-NOS. Urinary excretion of ECM-derived peptides was enhanced in cFSGS, although in silico analysis did not identify enhanced excretion of peptides derived from cathepsin B or C. CONCLUSIONS: ECM differences suggest that glomerular sclerosis in cFSGS differs from that in other FSGS variants. Infiltration of activated PECs may disrupt ECM remodeling in cFSGS. These cells and their cathepsins may be therapeutic targets.

CureGN Study Rationale, Design, and Methods: Establishing a Large Prospective Observational Study of Glomerular Disease.

RATIONALE & OBJECTIVES: Glomerular diseases, including minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and immunoglobulin A (IgA) nephropathy, share clinical presentations, yet result from multiple biological mechanisms. Challenges to identifying underlying mechanisms, biomarkers, and new therapies include the rarity of each diagnosis and slow progression, often requiring decades to measure the effectiveness of interventions to prevent end-stage kidney disease (ESKD) or death. STUDY DESIGN: Multicenter prospective cohort study. SETTING & PARTICIPANTS: Cure Glomerulonephropathy (CureGN) will enroll 2,400 children and adults with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, or IgA nephropathy (including IgA vasculitis) and a first diagnostic kidney biopsy within 5 years. Patients with ESKD and those with secondary causes of glomerular disease are excluded. EXPOSURES: Clinical data, including medical history, medications, family history, and patient-reported outcomes, are obtained, along with a digital archive of kidney biopsy images and blood and urine specimens at study visits aligned with clinical care 1 to 4 times per year. OUTCOMES: Patients are followed up for changes in estimated glomerular filtration rate, disease activity, ESKD, and death and for nonrenal complications of disease and treatment, including infection, malignancy, cardiovascular, and thromboembolic events. ANALYTICAL APPROACH: The study design supports multiple longitudinal analyses leveraging the diverse data domains of CureGN and its ancillary program. At 2,400 patients and an average of 2 years' initial follow-up, CureGN has 80% power to detect an HR of 1.4 to 1.9 for proteinuria remission and a mean difference of 2.1 to 3.0mL/min/1.73m2 in estimated glomerular filtration rate per year. LIMITATIONS: Current follow-up can only detect large differences in ESKD and death outcomes. CONCLUSIONS: Study infrastructure will support a broad range of scientific approaches to identify mechanistically distinct subgroups, identify accurate biomarkers of disease activity and progression, delineate disease-specific treatment targets, and inform future therapeutic trials. CureGN is expected to be among the largest prospective studies of children and adults with glomerular disease, with a broad goal to lessen disease burden and improve outcomes.

Compassion cultivation training promotes medical student wellness and enhanced clinical care.

BACKGROUND: Compassionate health care is associated with positive patient outcomes. Educational interventions for medical students that develop compassion may also increase wellness, decrease burnout, and improve provider-patient relationships. Research on compassion training in medical education is needed to determine how students learn and apply these skills. The authors evaluated an elective course for medical students modeled after the Compassion Cultivation Training course developed by the Stanford Center for Compassion and Altruism Research and Education. The elective goals were to strengthen student compassion, kindness, and wellness through compassion training and mindfulness meditation training modeled by a faculty instructor. The research objectives were to understand students' applications and perceptions of this training. METHODS: Over three years, 45 students participated in the elective at the University of Louisville School of Medicine. The course administered a pre/post Kentucky Inventory of Mindfulness Skills that measured observing, describing, acting with awareness, and accepting without judgment. Qualitative analyses of self-reported experiences were used to assess students' perceptions of compassion training and their application of skills learned through the elective. RESULTS: The mindfulness inventory showed significant improvements in observing (t = 3.62, p = 0.005) and accepting without judgment skills (t = 2.87, p = 0.017) for some elective cohorts. Qualitative data indicated that students across all cohorts found the elective rewarding, and they used mindfulness, meditation, and compassion skills broadly outside the course. Students described how the training helped them address major stressors associated with personal, academic, and clinical responsibilities. Students also reported that the skills strengthened interpersonal interactions, including with patients. CONCLUSIONS: These outcomes illuminate students' attitudes toward compassion training and suggest that among receptive students, a brief, student-focused intervention can be enthusiastically received and positively influence students' compassion toward oneself and others. To underscore the importance of interpersonal and cognitive skills such as compassion and mindfulness, faculty should consider purposefully modeling these skills to students. Modeling compassion cultivation and mindfulness skills in the context of patient interactions may address student empathy erosion more directly than stress management training alone. This pilot study shows compassion training could be an attractive, efficient option to address burnout by simultaneously promoting student wellness and enhanced patient interactions.

Urine inositol pentakisphosphate 2-kinase and changes in kidney structure in early diabetic kidney disease in type 1 diabetes.

We examined the association of urine inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IPP2K) with the presence and progression of diabetic kidney disease (DKD) lesions. Urine IPP2K was measured at baseline by quantitative liquid chromatography-mass spectrometry in 215 participants from the Renin-Angiotensin System Study who had type 1 diabetes and were normoalbuminuric and normotensive with normal or increased glomerular filtration rate (GFR). Urine IPP2K was detectable in 166 participants. Participants with IPP2K below the limit of quantification (LOQ) were assigned concentrations of LOQ/√2. All concentrations were then standardized to urine creatinine (Cr) concentration. Kidney morphometric data were available from biopsies at baseline and after 5 yr. Relationships of IPP2K/Cr with morphometric variables were assessed by linear regression after adjustment for age, sex, diabetes duration, hemoglobin A1c, mean arterial pressure, treatment assignment, and, for longitudinal analyses, baseline structure. Baseline mean age was 29.7 yr, mean diabetes duration 11.2 yr, median albumin excretion rate 5.0 µg/min, and mean iohexol GFR 129 ml·min-1·1.73m-2. Higher IPP2K/Cr was associated with higher baseline peripheral glomerular total filtration surface density [Sv(PGBM/glom), tertile 3 vs. tertile 1 ß = 0.527, P = 0.011] and with greater preservation of Sv(PGBM/glom) after 5 yr ( tertile 3 vs. tertile 1 ß = 0.317, P = 0.013). Smaller increases in mesangial fractional volume ( tertile 3 vs. tertile 1 ß = -0.578, P = 0.018) were observed after 5 yr in men with higher urine IPP2K/Cr concentrations. Higher urine IPP2K/Cr is associated with less severe kidney lesions at baseline and with preservation of kidney structure over 5 yr in individuals with type 1 diabetes and no clinical evidence of DKD at baseline.

Poly-ubiquitin profile in Alzheimer disease brain.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by progressive loss of memory, reasoning and other cognitive functions. Pathologically, patients with AD are characterized by deposition of senile plaques (SPs), formed by ß-amyloid (Aß), and neurofibrillary tangles (NTFs) that consist of aggregated hyperphosphorylated tau protein. The accumulation of insoluble protein aggregates in AD brain can be associated with an impairment of degradative systems. This current study investigated if the disturbance of protein polyubiquitination is associated with AD neurodegeneration. By using a novel proteomic approach, we found that 13 brain proteins are increasingly polyubiquitinated in AD human brain compared to age-matched controls. Moreover, the majority of the identified proteins were previously found to be oxidized in our prior proteomics, and these proteins are mainly involved in protein quality control and glucose metabolism. This is the first study showing alteration of the poly-ubiquitin profile in AD brain compared with healthy controls. Understanding the onset of the altered ubiquitin profile in AD brain may contribute to identification of key molecular regulators of cognitive decline. In AD, deficits of the proteolytic system may further exacerbate the accumulation of oxidized/misfolded/polyubiquitinated proteins that are not efficiently degraded and may become harmful to neurons and contribute to AD neuropathology and cognitive decline.

Biomarker enhanced risk prediction for development of AKI after cardiac surgery.

BACKGROUND: Acute kidney injury (AKI) is a common post-cardiac surgery complication and influences patient morbidity and prognosis. This study was designed to identify preoperative candidate urine biomarkers in patients undergoing cardiac surgery. METHODS: A prospective cohort study of adults undergoing cardiac surgery at increased risk for AKI at a single hospital between July 2010 and September 2012 was performed. The primary outcome was the development of AKI, defined as an absolute serum creatinine (SCr) level increase ≥ 0.5 mg/dL or a ≥ 50% relative increase within 72 h of surgery. A secondary outcome was development of AKI defined by Kidney Disease Improving Global Outcomes (KDIGO). Urine collected by voiding within 4 h prior to surgery was used for proteomic analysis and confirmatory enzyme linked immunosorbent assays (ELISAs) studies. Biomarkers were tested for AKI-prediction using Cox and Snell R2, area under the receiver operating curve (AUROC), and percent of corrected classifications. To evaluate the added effect of each candidate biomarker on AKI discrimination, receiver operator characteristic (ROC) curves, integrated discrimination improvement (IDI), and net reclassification improvement (NRI) were calculated. RESULTS: Forty-seven of 755 patients met screening criteria including 15 with AKI. Proteomic analysis identified 29 proteins with a significant ≥2-fold change. Confirmatory ELISA measurements of five candidate markers showed urinary complement factor B (CFB) and histidine rich glycoprotein (HRG) concentrations were significantly increased in patients with AKI. By multivariate analysis, NRI, and IDI the addition of CFB and HRG to the standard clinical assessment significantly improved risk prediction for the primary outcome. Only HRG was a significant predictor in the 21 patients with AKI defined by KDIGO criteria. CONCLUSIONS: Pre-operative urine measurement of CFB or HRG significantly enhanced the current post-surgery AKI risk stratification for more restrictive definition of AKI. HRG, but not CFB or clinical risk stratification, predicted AKI defined by KDIGO. The ability of these biomarkers to predict risk for dialysis-requiring AKI or death could not be reliably assessed in our study due to a small number of patients with either outcome.

Common Elements in Rare Kidney Diseases: Conclusions from a Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference.

Rare kidney diseases encompass at least 150 different conditions, most of which are inherited. Although individual rare kidney diseases raise specific issues, as a group these rare diseases can have overlapping challenges in diagnosis and treatment. These challenges include small numbers of affected patients, unidentified causes of disease, lack of biomarkers for monitoring disease progression, and need for complex care. To address common clinical and patient issues among rare kidney diseases, the KDIGO Controversies Conference entitled, Common Elements in Rare Kidney Diseases, brought together a panel of multidisciplinary clinical providers and patient advocates to address five central issues for rare kidney diseases. These issues encompassed diagnostic challenges, management of kidney functional decline and progression of chronic kidney disease, challenges in clinical study design, translation of advances in research to clinical care, and provision of practical and integrated patient support. Thus, by a process of consensus, guidance for addressing these challenges was developed and is presented here.

Biomarkers of lupus nephritis histology and flare: deciphering the relevant amidst the noise.

Biomarker development in lupus nephritis (LN) has traditionally relied on comparing the characteristics of candidate markers to clinical findings in patients and controls from cross-sectional cohorts. In this work, two additional strategies for LN biomarker development that are gaining ground will be discussed. One approach compares analytes directly to kidney histology. The second strategy utilizes longitudinal measurements of biomarker levels at regular intervals as patients move from disease quiescence to disease flare. These approaches have begun to empower biomarkers as diagnostic and prognostic tools in LN and have revealed novel and sometimes unexpected roles for these biomarkers in the pathogenesis and prediction of LN disease activity.

Age-related changes in the proteostasis network in the brain of the naked mole-rat: Implications promoting healthy longevity.

The naked mole-rat (NMR) is the longest-lived rodent and possesses several exceptional traits: marked cancer resistance, negligible senescence, prolonged genomic integrity, pronounced proteostasis, and a sustained health span. The underlying molecular mechanisms that contribute to these extraordinary attributes are currently under investigation to gain insights that may conceivably promote and extend human health span and lifespan. The ubiquitin-proteasome and autophagy-lysosomal systems play a vital role in eliminating cellular detritus to maintain proteostasis and have been previously shown to be more robust in NMRs when compared with shorter-lived rodents. Using a 2-D PAGE proteomics approach, differential expression and phosphorylation levels of proteins involved in proteostasis networks were evaluated in the brains of NMRs in an age-dependent manner. We identified 9 proteins with significantly altered levels and/or phosphorylation states that have key roles involved in proteostasis networks. To further investigate the possible role that autophagy may play in maintaining cellular proteostasis, we examined aspects of the PI3K/Akt/mammalian target of rapamycin (mTOR) axis as well as levels of Beclin-1, LC3-I, and LC3-II in the brain of the NMR as a function of age. Together, these results show that NMRs maintain high levels of autophagy throughout the majority of their lifespan and may contribute to the extraordinary health span of these rodents. The potential of augmenting human health span via activating the proteostasis network will require further studies.

Reaching Out to Send a Message: Proteins Associated with Neurite Outgrowth and Neurotransmission are Altered with Age in the Long-Lived Naked Mole-Rat.

Aging is the greatest risk factor for developing neurodegenerative diseases, which are associated with diminished neurotransmission as well as neuronal structure and function. However, several traits seemingly evolved to avert or delay age-related deterioration in the brain of the longest-lived rodent, the naked mole-rat (NMR). The NMR remarkably also exhibits negligible senescence, maintaining an extended healthspan for ~75 % of its life span. Using a proteomic approach, statistically significant changes with age in expression and/or phosphorylation levels of proteins associated with neurite outgrowth and neurotransmission were identified in the brain of the NMR and include: cofilin-1; collapsin response mediator protein 2; actin depolymerizing factor; spectrin alpha chain; septin-7; syntaxin-binding protein 1; synapsin-2 isoform IIB; and dynamin 1. We hypothesize that such changes may contribute to the extended lifespan and healthspan of the NMR.

Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease.

Parkinson's disease (PD) is an age-related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α-synuclein-containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN-induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early-onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy-lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2-D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6-month-old PINK1-deficient mice and wild-type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3ε, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70 kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2.

Metabolic clues to salubrious longevity in the brain of the longest-lived rodent: the naked mole-rat.

Naked mole-rats (NMRs) are the oldest-living rodent species. Living underground in a thermally stable ecological niche, NMRs have evolved certain exceptional traits, resulting in sustained health spans, negligible cognitive decline, and a pronounced resistance to age-related disease. Uncovering insights into mechanisms underlying these extraordinary traits involved in successful aging may conceivably provide crucial clues to extend the human life span and health span. One of the most fundamental processes inside the cell is the production of ATP, which is an essential fuel in driving all other energy-requiring cellular activities. Not surprisingly, a prominent hallmark in age-related diseases, such as neurodegeneration and cancer, is the impairment and dysregulation of metabolic pathways. Using a two-dimensional polyacrylamide gel electrophoresis proteomics approach, alterations in expression and phosphorylation levels of metabolic proteins in the brains of NMRs, aged 2-24 years, were evaluated in an age-dependent manner. We identified 13 proteins with altered levels and/or phosphorylation states that play key roles in various metabolic pathways including glycolysis, ß-oxidation, the malate-aspartate shuttle, the Tricarboxylic Acid Cycle (TCA) cycle, the electron transport chain, NADPH production, as well as the production of glutamate. New insights into potential pathways involved in metabolic aspects of successful aging have been obtained by the identification of key proteins through which the NMR brain responds and adapts to the aging process and how the NMR brain adapted to resist age-related degeneration. This study examines the changes in the proteome and phosphoproteome in the brain of the naked mole-rat aged 2-24 years. We identified 13 proteins (labeled in red) with altered expression and/or phosphorylation levels that are conceivably associated with sustained metabolic functions in the oldest NMRs that may promote a sustained health span and life span.

Proteomics and autoimmune kidney disease.

Proteomics has long been considered an ideal platform, and urine an ideal source for biomarker discovery in human autoimmune kidney diseases. A number of studies have examined the urine proteome to identify biomarkers of disease activity, kidney pathology, and response to therapy. Increasingly, proteomic studies of kidney disease have expanded to include blood, circulating cells and kidney tissue. Recently the clinical potential of renal proteomics has been realized through a handful of investigations whose results appear to be applicable to patient care. In this review, approaches to the proteomic evaluation of autoimmune kidney diseases will be considered in the context of developing clinically useful disease biomarkers.

Urinary monocyte chemoattractant protein-1 and hepcidin and early diabetic nephropathy lesions in type 1 diabetes mellitus.

BACKGROUND: Urinary monocyte chemoattractant protein-1 (MCP-1) and hepcidin are potential biomarkers of renal inflammation. We examined their association with development of diabetic nephropathy (DN) lesions in normotensive normoalbuminuric subjects with type 1 diabetes (T1D) from the Renin-Angiotensin System Study. METHODS: Biomarker concentrations were measured in baseline urine samples from 224 subjects who underwent kidney biopsies at baseline and after 5 years. Fifty-eight urine samples below the limit of quantitation (LOQ, 28.8 pg/mL) of the MCP-1 assay were assigned concentrations of LOQ/√2 for analysis. Relationships between ln(MCP-1/Cr) or ln(hepcidin/Cr) and morphometric variables were assessed by sex using multiple linear regression after adjustment for age, T1D duration, HbA1c, mean arterial pressure, albumin excretion rate (AER) and glomerular filtration rate (GFR). In models that examined changes in morphometric variables, the baseline morphometric value was also included. RESULTS: Baseline mean age was 24.6 years, mean duration of T1D 11.2 years, median AER 6.4 µg/min and mean iohexol GFR 129 mL/min/1.73 m(2). No associations were found between hepcidin/Cr and morphometric variables. Higher MCP-1/Cr was associated with higher interstitial fractional volume at baseline and after 5 years in women (baseline partial r = 0.244, P = 0.024; 5-year partial r = 0.299, P = 0.005), but not in men (baseline partial r = -0.049, P = 0.678; 5-year partial r = 0.026, P = 0.830). MCP-1 was not associated with glomerular lesions in either sex. CONCLUSIONS: Elevated urinary MCP-1 concentration measured before clinical findings of DN in women with T1D was associated with changes in kidney interstitial volume, suggesting that inflammatory processes may be involved in the pathogenesis of early interstitial changes in DN.

Role of aldose reductase in the metabolism and detoxification of carnosine-acrolein conjugates.

Oxidation of unsaturated lipids generates reactive aldehydes that accumulate in tissues during inflammation, ischemia, or aging. These aldehydes form covalent adducts with histidine-containing dipeptides such as carnosine and anserine, which are present in high concentration in skeletal muscle, heart, and brain. The metabolic pathways involved in the detoxification and elimination of these conjugates are, however, poorly defined, and their significance in regulating oxidative stress is unclear. Here we report that conjugates of carnosine with aldehydes such as acrolein are produced during normal metabolism and excreted in the urine of mice and adult human non-smokers as carnosine-propanols. Our studies show that the reduction of carnosine-propanals is catalyzed by the enzyme aldose reductase (AR). Carnosine-propanals were converted to carnosine-propanols in the lysates of heart, skeletal muscle, and brain tissue from wild-type (WT) but not AR-null mice. In comparison with WT mice, the urinary excretion of carnosine-propanols was decreased in AR-null mice. Carnosine-propanals formed covalent adducts with nucleophilic amino acids leading to the generation of carnosinylated proteins. Deletion of AR increased the abundance of proteins bound to carnosine in skeletal muscle, brain, and heart of aged mice and promoted the accumulation of carnosinylated proteins in hearts subjected to global ischemia ex vivo. Perfusion with carnosine promoted post-ischemic functional recovery in WT but not in AR-null mouse hearts. Collectively, these findings reveal a previously unknown metabolic pathway for the removal of carnosine-propanal conjugates and suggest a new role of AR as a critical regulator of protein carnosinylation and carnosine-mediated tissue protection.

Calorimetric analysis of the plasma proteome: identification of type 1 diabetes patients with early renal function decline.

BACKGROUND: Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome. METHODS: DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n=15) and matched control subjects (n=14). RESULTS: The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status. CONCLUSIONS: Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D. GENERAL SIGNIFICANCE: DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.

Plasma kininogen and kininogen fragments are biomarkers of progressive renal decline in type 1 diabetes.

The ability of microalbuminuria to predict early progressive renal function decline in type 1 diabetic patients has been questioned. To resolve this, we determined the plasma proteome differences between microalbuminuric patients with type 1 diabetes and stable renal function (controls) and patients at risk for early progressive renal function decline (cases) and asked whether these differences have value as surrogate biomarkers. Mass spectrometry was used to analyze small (<3 kDa) plasma peptides isolated from well-matched case and control plasma obtained at the beginning of an 8-12 year follow-up period. A Spearman analysis of plasma peptide abundance and the rate of renal function decline during follow-up identified seven masses with a significant negative correlation with early progressive renal function decline. Tandem mass spectrometry identified three fragments of high-molecular-weight kininogen. Increased plasma high-molecular-weight kininogen in the cases was confirmed by immunoblot. One peptide, des-Arg9-BK(1-8), induced Erk1/2 phosphorylation when added apically to two proximal tubular cell lines grown on permeable inserts. Thus, we have identified plasma protein fragments, some of which have biological activity with moderate to strong correlation, with early progressive renal function decline in microalbuminuric patients with type 1 diabetes. Other peptides are candidates for validation as candidate biomarkers of diabetes-associated renal dysfunction.