Kasper Rossing

Clinical proteomics: A need to define the field and to begin to set adequate standards

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.

Naturally occurring human urinary peptides for use in diagnosis of chronic kidney disease

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.

Urinary Proteomics in Diabetes and CKD

Urinary biomarkers for diabetes, diabetic nephropathy, and nondiabetic proteinuric renal diseases were sought. For 305 individuals, biomarkers were defined and validated in blinded data sets using high-resolution capillary electrophoresis coupled with electrospray-ionization mass spectrometry. A panel of 40 biomarkers distinguished patients with diabetes from healthy individuals with 89% sensitivity and 91% specificity. Among patients with diabetes, 102 urinary biomarkers differed significantly between patients with normoalbuminuria and nephropathy, and a model that included 65 of these correctly identified diabetic nephropathy with 97% sensitivity and specificity. Furthermore, this panel of biomarkers identified patients who had microalbuminuria and diabetes and progressed toward overt diabetic nephropathy over 3 yr. Differentiation between diabetic nephropathy and other chronic renal diseases reached 81% sensitivity and 91% specificity. Many of the biomarkers were fragments of collagen type I, and quantities were reduced in patients with diabetes or diabetic nephropathy. In conclusion, this study shows that analysis of the urinary proteome may allow early detection of diabetic nephropathy and may provide prognostic information.

Unchanged Incidence of Diabetic Nephropathy in IDDM Patients

Recently, a dramatic decline in the cumulative incidence of diabetic nephropathy (<10% after 25 years of diabetes) has been reported in insulin-dependent diabetes mellitus (IDDM) patients diagnosed before the age of 15 years between 1961 and 1980. In a clinic-based study, we assessed recent trends in the incidence of diabetic nephropathy. All 356 patients in whom IDDM was diagnosed before the age of 41 years between 1965 and 1979, identified in 1984, were followed until 1991 or until death. All patients were Caucasians and resided in Copenhagen. The cumulative incidences (life-table method) of diabetic nephropathy (urinary albumin excretion ≥300 mg/24 h in two out of three consecutive samples) after 15 years of diabetes and in 1991 were 18 ± 4 and 35 ± 5% (cumulative incidence ± SE; onset of diabetes 1965–1969, n = 113), 20 ± 4 and 35 ± 5% (onset of diabetes 1970–1974, n = 130), and 16 ± 5& (onset of diabetes 1975–1979, n = 113), respectively (NS at 15 years). The prevalence of persistent microalbuminuria (31–299 mg/24 h) at time of follow-up was 24% (95% confidence interval: 16–33) in the group with onset of diabetes in 1965–1969, 28% (20–36) with onset of diabetes in 1970–1974, and 19% (13–28) with onset of diabetes in 1975–1979 (NS). The mean ± SE HbAlc measured yearly beginning in 1984 was higher in patients with nephropathy (9.4 ± 0.1%) and persistent microalbuminuria (8.9 ± 0.1%) than in patients with normoalbuminuria (8.5 ± 0.1%; P < 0.001). Our study revealed no evidence suggesting a so-called calendar effect, i.e., declining cumulative incidence of diabetic nephropathy with increasing calendar year of diabetes onset. The lack of calendar effect may in part be explained by poor metabolic control and a high and unchanged prevalence of smoking (58–71%).

Multicentric Validation of Proteomic Biomarkers in Urine Specific for Diabetic Nephropathy

Background Urine proteome analysis is rapidly emerging as a tool for diagnosis and prognosis in disease states. For diagnosis of diabetic nephropathy (DN), urinary proteome analysis was successfully applied in a pilot study. The validity of the previously established proteomic biomarkers with respect to the diagnostic and prognostic potential was assessed on a separate set of patients recruited at three different European centers. In this case-control study of 148 Caucasian patients with diabetes mellitus type 2 and duration ≥5 years, cases of DN were defined as albuminuria >300 mg/d and diabetic retinopathy (n = 66). Controls were matched for gender and diabetes duration (n = 82). Methodology/Principal Findings Proteome analysis was performed blinded using high-resolution capillary electrophoresis coupled with mass spectrometry (CE-MS). Data were evaluated employing the previously developed model for DN. Upon unblinding, the model for DN showed 93.8% sensitivity and 91.4% specificity, with an AUC of 0.948 (95% CI 0.898-0.978). Of 65 previously identified peptides, 60 were significantly different between cases and controls of this study. In <10% of cases and controls classification by proteome analysis not entirely resulted in the expected clinical outcome. Analysis of patients subsequent clinical course revealed later progression to DN in some of the false positive classified DN control patients. Conclusions These data provide the first independent confirmation that profiling of the urinary proteome by CE-MS can adequately identify subjects with DN, supporting the generalizability of this approach. The data further establish urinary collagen fragments as biomarkers for diabetes-induced renal damage that may serve as earlier and more specific biomarkers than the currently used urinary albumin.

Pregnancy and progression of diabetic nephropathy.

Abstract.Aims/hypothesis: Pregnancy could damage kidney function in diabetic nephropathy. We investigated the long-term impact of pregnancy on the progression of diabetic nephropathy. Methods: Our observational follow-up study included all women patients with Type I (insulin-dependent) diabetes mellitus who developed diabetic nephropathy between 1970 and 1989 at Steno Diabetes Center (n = 93). Follow-up lasted 16 years (range 3–28) from the onset of diabetic nephropathy until death or the year 2000. A total of 26 women became pregnant after the onset of diabetic nephropathy (group A). The remaining 67 served as control subjects (group B). All patients received aggressive antihypertensive treatment (blood pressure goal < 140/90 mmHg). Results: The two groups were comparable at onset of diabetic nephropathy regarding blood pressure, albuminuria, s-cholesterol, smoking, retinopathy and s-creatinine (mean 79(SD 23) μmol/l). The slopes of 1/s-creatinine (1000 · l ·μmol–1· year–1) during the whole observation period were –0.39(0.40) compared with –0.41(0.70) (group A vs B – NS). The slopes of 1/s-creatinine before and after pregnancy were similar. Decline in creatinine clearance (ml/min/yr) was 3.2 (3.4) compared with 3.2 (5.1) (group A vs B -NS). At the end of follow-up, 35 % (95 %-CI:17–53) of the pregnant women had died and 19 % (7–39) had reached end stage renal disease compared to 34 % (23–45) and 24 %(14–34) of the control subjects, respectively(NS). Group A and B had similar blood pressure levels during the whole observation period: 136(13)/83(7) vs 139 (14)/85(7) mmHg (NS). Conclusion/interpretation: Pregnancy has no adverse long-term impact on kidney function and survival in Type I diabetic patients with well-preserved kidney function (normal serum creatinine) suffering from diabetic nephropathy. [Diabetologia (2002) 45: 36–41]

Evolving strategies for renoprotection: diabetic nephropathy.

A cumulative incidence of diabetic nephropathy of 25-40% has been documented after duration of diabetes of at least 25 years in both type 1 and type 2 diabetic patients. Diabetic nephropathy has become the leading cause (25-44%) of end-stage renal failure in Europe, the United States and Japan. Until the early 1980s, no renoprotective treatment was available for use in diabetic nephropathy. Death occurred on average 5-7 years after the onset of persistent proteinuria. It should be recalled that development of treatment modalities occurred in reverse order: in the early 1980s, antihypertensive treatment of diabetic nephropathy was introduced, and in the early 1990s, primary and secondary prevention with improved glycaemic control and angiotensin-converting enzyme inhibition. The two main treatment strategies for primary prevention of diabetic nephropathy are improved glycaemic control and blood pressure lowering, particularly using drugs such as angiotensin-converting enzyme inhibitors. Megatrials and meta-analyses have clearly demonstrated the beneficial effect of both the above-mentioned treatment modalities. Secondary prevention, that is, treatment modalities applied to diabetic patients with high risk of development of diabetic nephropathy (e.g. those with microalbuminuria) has been documented, applying angiotensin-converting enzyme inhibitors in both type 1 and type 2 diabetic patients. Furthermore, improved metabolic control reduces the risk of progression. In special cases (such as pancreas transplantation) even reversal of diabetic glomerular lesions has been documented. Antihypertensive treatment of patients with overt nephropathy induces a reduction in albuminuria, a reduction in the rate of decline of glomerular filtration rate, delays development of end-stage renal failure and improves survival. Many potential treatment modalities in preventing and treating diabetic nephropathy are presently being evaluated.

The urinary proteome in diabetes and diabetes-associated complications: New ways to assess disease progression and evaluate therapy.

Diabetes represents one of the main chronic diseases worldwide. Diabetes and its associated complications may be detectable even at early stages in the urinary proteome. In this article we review the current literature on urinary proteomics applied to the study of diabetes and diabetic complications. Further, we present recent data that strongly indicate urinary proteome analysis may be a valuable tool in detecting diabetes‐associated pathophysiological changes at an early stage, and also may enable assessment of disease progression and efficacy of therapy. Current data indicate that collagen‐derived peptides represent one of the main peptidic components in urine, which are consistently found at reduced levels in diabetes. It is tempting to speculate that this decrease in urinary collagen‐derived peptides is related to an increase in extracellular matrix deposition which is a major complication in diabetes. Therefore, urinary proteome analysis might enable noninvasive assessment of this process at an early stage via determination of specific collagen fragments. This may open an avenue towards targeted therapeutic intervention.

Anaemia in diabetes: is there a rationale to TREAT?

Background Anaemia is a common finding in patients with diabetes, particularly in those with overt nephropathy or renal impairment. In tertiary clinics, at least one outpatient in five with diabetes has anaemia, for whom it constitutes a significant additional burden.DiscussionAnaemia is associated with an increased risk of diabetic complications including nephropathy, retinopathy and macrovascular disease. Anaemia may also be significant in determining the outcome of heart failure and hypoxia-induced organ damage in diabetes. While several factors contribute to the increased prevalence of anaemia in diabetes, the failure of the kidney to increase erythropoietin in response to falling haemoglobin appears to be the dominant factor. Although there is a clear rationale for correcting anaemia in people with diabetes, it remains to be established whether this will lead to improved outcomes. Moreover, the balance of risks, costs, and benefits remains to be established in patients with diabetes. The Trial to Reduce Cardiovascular Events with Aranesp (darbepoetin alpha) Therapy (TREAT) is a randomised controlled trial designed to determine the impact of anaemia correction on mortality and non-fatal cardiovascular events in patients with type 2 diabetes and stage 3–4 nephropathy.Conclusion It is anticipated that TREAT will help to define the optimal approach to the management of anaemia in diabetes.

Urinary collagen fragments are significantly altered in diabetes: a link to pathophysiology.

Background The pathogenesis of diabetes mellitus (DM) is variable, comprising different inflammatory and immune responses. Proteome analysis holds the promise of delivering insight into the pathophysiological changes associated with diabetes. Recently, we identified and validated urinary proteomics biomarkers for diabetes. Based on these initial findings, we aimed to further validate urinary proteomics biomarkers specific for diabetes in general, and particularity associated with either type 1 (T1D) or type 2 diabetes (T2D). Methodology/Principal Findings Therefore, the low-molecular-weight urinary proteome of 902 subjects from 10 different centers, 315 controls and 587 patients with T1D (n = 299) or T2D (n = 288), was analyzed using capillary-electrophoresis mass-spectrometry. The 261 urinary biomarkers (100 were sequenced) previously discovered in 205 subjects were validated in an additional 697 subjects to distinguish DM subjects (n = 382) from control subjects (n = 315) with 94% (95% CI: 92–95) accuracy in this study. To identify biomarkers that differentiate T1D from T2D, a subset of normoalbuminuric patients with T1D (n = 68) and T2D (n = 42) was employed, enabling identification of 131 biomarker candidates (40 were sequenced) differentially regulated between T1D and T2D. These biomarkers distinguished T1D from T2D in an independent validation set of normoalbuminuric patients (n = 108) with 88% (95% CI: 81–94%) accuracy, and in patients with impaired renal function (n = 369) with 85% (95% CI: 81–88%) accuracy. Specific collagen fragments were associated with diabetes and type of diabetes indicating changes in collagen turnover and extracellular matrix as one hallmark of the molecular pathophysiology of diabetes. Additional biomarkers including inflammatory processes and pro-thrombotic alterations were observed. Conclusions/Significance These findings, based on the largest proteomic study performed to date on subjects with DM, validate the previously described biomarkers for DM, and pinpoint differences in the urinary proteome of T1D and T2D, indicating significant differences in extracellular matrix remodeling.