Because of its availability, ease of collection, and correlation with physiology and pathology, urine is an attractive source for clinical proteomics/peptidomics. However, the lack of comparable data sets from large cohorts has greatly hindered the development of clinical proteomics. Here, we report the establishment of a reproducible, high resolution method for peptidome analysis of naturally occurring human urinary peptides and proteins, ranging from 800 to 17,000 Da, using samples from 3,600 individuals analyzed by capillary electrophoresis coupled to MS. All processed data were deposited in an Structured Query Language (SQL) database. This database currently contains 5,010 relevant unique urinary peptides that serve as a pool of potential classifiers for diagnosis and monitoring of various diseases. As an example, by using this source of information, we were able to define urinary peptide biomarkers for chronic kidney diseases, allowing diagnosis of these diseases with high accuracy. Application of the chronic kidney disease-specific biomarker set to an independent test cohort in the subsequent replication phase resulted in 85.5% sensitivity and 100% specificity. These results indicate the potential usefulness of capillary electrophoresis coupled to MS for clinical applications in the analysis of naturally occurring urinary peptides. Molecular & Cellular Proteomics 9:2424 -2437, 2010.From the Departments of a Chemistry and

Our long-term prospective study of type 2 diabetic patients with nephropathy has revealed several modifiable risk factors of enhanced progression in kidney disease and increased mortality.

The aim of this manuscript is to initiate a constructive discussion about the definition of clinical proteomics, study requirements, pitfalls and (potential) use. Furthermore, we hope to stimulate proposals for the optimal use of future opportunities and seek unification of the approaches in clinical proteomic studies. We have outlined our collective views about the basic principles that should be considered in clinical proteomic studies, including sample selection, choice of technology and appropriate quality control, and the need for collaborative interdisciplinary efforts involving clinicians and scientists. Furthermore, we propose guidelines for the critical aspects that should be included in published reports. Our hope is that, as a result of stimulating discussion, a consensus will be reached amongst the scientific community leading to guidelines for the studies, similar to those already published for mass spectrometric sequencing data. We contend that clinical proteomics is not just a collection of studies dealing with analysis of clinical samples. Rather, the essence of clinical proteomics should be to address clinically relevant questions and to improve the state-of-the-art, both in diagnosis and in therapy of diseases.

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OBJECTIVE -Conflicting evidence of a decline in incidence of microvascular complications in type 1 diabetes during the last decades has been reported. To assess recent trends in the cumulative incidence of diabetic microangiopathy in type 1 diabetes, we analyzed data from long-term prospective observational studies lasting Ն20 years. RESULTS -In patients followed for Ն20 years, the cumulative incidence (95% CI) of diabetic nephropathy after 20 years of diabetes (urinary albumin excretion Ͼ300 mg/24 h) was reduced in patients with more recent diabetes onset (groups A-D): 31.1% (22.5-39.7) vs. 28.4% (19.8 -37.0) vs. 18.9% (10.9 -26.9) vs. 13.7% (6.2-21.2) (P ϭ 0.015). Similarly, the cumulative incidence of proliferative retinopathy was as follows: 31.2% (22.2-39.8) vs. 30.3% (22.2-38.4) vs. 19.3% (11.2-27.4) vs. 12.5% (5.2-19.8) (P Ͻ 0.01). In the latter groups, antihypertensive treatment was started earlier, blood pressure and HbA 1c were lower, and fewer patients smoked. RESEARCH DESIGN AND METHODSCONCLUSIONS -Our study demonstrates a decrease in the cumulative incidence of diabetic microangiopathy in type 1 diabetes over the past 35 years. Improved glycemic control, lower blood pressure (in part due to early aggressive antihypertensive treatment), and reduced prevalence of smoking rates were associated with the improved prognosis.

OBJECTIVE -The objective of this study was to evaluate the safety and short-term effect of adding spironolactone to conventional antihypertensive treatment including diuretics and maximally recommended doses of an ACE inhibitor or an angiotensin II receptor blocker (ARB) on albuminuria and blood pressure in type 2 diabetic patients with nephropathy.RESEARCH DESIGN AND METHODS -Twenty-one type 2 diabetic patients with nephropathy were enrolled in a randomized, double-masked, cross-over study. Patients were treated in random order with spironolactone 25 mg once daily and matched placebo for 8 weeks, respectively, in addition to ongoing antihypertensive treatment including diuretics and maximally recommended doses of an ACE inhibitor and/or an ARB. At the end of each treatment period, albuminuria, 24-h ambulatory blood pressure (ABP), and glomerular filtration rate (GFR) were determined.RESULTS -During the addition of placebo, values were as follows: albuminuria (geometric mean [range]) 1,566 762] mg/24 h, ABP (mean Ϯ SE) 138 Ϯ 3/71 Ϯ 1 mmHg, and GFR (mean Ϯ SE) 74 Ϯ 6 ml/min per 1.73 m 2 . During the addition of spironolactone, albuminuria was reduced by 33% (95% CI 25-41) (P Ͻ 0.001), fractional clearance of albumin by 40% (24 -53) (P Ͻ 0.001), and 24-h ABP by 6 mmHg (2-10) for systolic and 4 mmHg (2-6) for diastolic (P Ͻ 0.001 for both). The change in albuminuria did not correlate with the change in systolic 24-h ABP (r ϭ 0.19, P ϭ 0.42) or diastolic 24-h ABP (r ϭ 0.01, P ϭ 0.96). Spironolactone treatment induced an insignificant reversible reduction in GFR of 3 ml/min per 1.73 m 2 (Ϫ0.3 to 6) (P ϭ 0.08). One patient was excluded from the study due to hyperkalemia. Otherwise treatment was well tolerated.CONCLUSIONS -Our study suggests that spironolactone safely adds to the reno-and cardiovascular protective benefits of treatment with maximally recommended doses of ACE inhibitor and ARB by reducing albuminuria and blood pressure in type 2 diabetic patients with nephropathy. Diabetes Care 28:2106 -2112, 2005A ldosterone, the end product of the renin-angiotensin-aldosterone system (RAAS), has attracted renewed attention as an important mediator of both cardiovascular and renal disease (1,2). Accumulating experimental evidence suggests that circulating aldosterone per se contributes directly to renal and cardiovascular disease by inducing inflammation, fibrosis, and necrosis in end-organ tissues such as the heart, brain, and kidney (3). Moreover, aldosterone blockade has been shown to greatly improve survival in patients with chronic heart failure (4,5).Angiotensin II has long been considered the main mediator of the pathophysiological effects of the RAAS, and in diabetic nephropathy the renoprotective effects of treatment with an ACE inhibitor or an angiotensin receptor blocker (ARB) are well established (6 -9). Both ACE inhibitor and ARB treatments initially suppress plasma aldosterone. Eventually, however, plasma aldosterone may return to pretreatment levels, i.e., the aldosterone escape phenomenon. Aldosterone e...

Owing to its availability, ease of collection, and correlation with pathophysiology of diseases, urine is an attractive source for clinical proteomics. However, many proteomic studies have had only limited clinical impact, due to factors such as modest numbers of subjects, absence of disease controls, small numbers of defined biomarkers, and diversity of analytical platforms. Therefore, it is difficult to merge biomarkers from different studies into a broadly applicable human urinary proteome database. Ideally, the methodology for defining the biomarkers should combine a reasonable analysis time with high resolution, thereby enabling the profiling of adequate samples and recognition of sufficient features to yield robust diagnostic panels. Capillary electrophoresis coupled to mass spectrometry (CE-MS), which was used to analyze urine samples from healthy subjects and patients with various diseases, is a suitable approach for this task. The database of these datasets compiled from the urinary peptides enabled the diagnosis, classification, and monitoring of a wide range of diseases. CE-MS exhibits excellent performance for biomarker discovery and allows subsequent biomarker sequencing independent of the separation platform. This approach may elucidate the pathogenesis of many diseases, and better define especially renal and urological disorders at the molecular level.

A limitation of proteomic methods with respect to their clinical applicability is the lack of possibilities to directly deduce the amount of a protein or peptide from a particular mass spectrometry (MS) spectrum. For quantification of chronic kidney disease (CKD)-specific urinary polypeptides in capillary electrophoresis coupled with mass spectrometry (CE-MS), we compared signal intensity calibration methods based on either urinary creatinine or stable isotope labeled synthetic marker analogues (absolute quantification) with those based on ion counting using highly abundant collagen fragments as nonmarker references (relative quantification). Our results indicate that relative quantification of biomarker excretion based on ion counts in reference to endogenous "housekeeping" peptides is sufficient for the determination of urinary polypeptide levels. The calculation of absolute concentrations via exogenous stable isotope-labeled peptide standards is of no additional benefit.