Identification of urinary proteins potentially associated with diabetic kidney disease

Abstract

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease. Although several parameters are used to evaluate renal damage, in many instances, there is no pathological change until damage is already advanced. Mass spectrometry-based proteomics is a novel tool to identify newer diagnostic markers. To identify urinary proteins associated with renal complications in diabetes, we collected urine samples from 10 type 2 diabetes patients each with normoalbuminuria, micro- and macro-albuminuria and compared their urinary proteome with that of 10 healthy individuals. Urinary proteins were concentrated, depleted of albumin and five other abundant plasma proteins and in-gel trypsin digested after prefractionation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The peptides were analyzed using a nanoflow reverse phase liquid chromatography system coupled to linear trap quadrupole-Orbitrap mass spectrometer. We identified large number of proteins in each group, of which many were exclusively present in individual patient groups. A total of 53 proteins were common in all patients but were absent in the controls. The majority of the proteins were functionally binding, biologically involved in metabolic processes, and showed enrichment of alternative complement and blood coagulation pathways. In addition to identifying reported proteins such as α2-HS-glycoprotein and Vitamin D binding protein, we detected novel proteins such as CD59, extracellular matrix protein 1 (ECM1), factor H, and myoglobin in the urine of macroalbuminuria patients. ECM1 and factor H are known to influence mesangial cell proliferation, and CD59 causes microvascular damage by influencing membrane attack complex deposition, suggestive their biological relevance to DN. Thus, we have developed a proteome database where various proteins exclusively present in the patients may be further investigated for their role as stage-specific markers and possible therapeutic targets.

Current Controversies

“…That said, various substances in the blood such as polyunsaturated fatty acids may alter the affinity of DBP for the vitamin D metabolites [50]. Moreover, as noted previously liver disease leads to reduced levels of DBP [32] as do protein losing nephropathy [51] and acute illness (DBP is an acute phase reactant) [52 53], whereas DBP levels are elevated during the latter stages of pregnancy and with oral contraceptive use [22 29]. …”

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“…The World Health Organization’s (WHO) latest estimate is that there were about 422 million people with diabetes worldwide in 2014, which is expected to increase to 600 million by 2040 (WHO 2016), and about 1/3 of them will develop DN (Marikanty et al. 2016). Podocytes, or glomerular visceral epithelial cells, together with the glomerular basement membrane and the glomerular endothelial cells, constitute the glomerular filtration barrier.…”

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“…Hence, early diagnosis, intervention, and treatment are critical for reversing or halting the progression of DN. Elevated proteinuria secretion (20 µg/min < urine albumin excretion rate < 200 µg/min) due to decreased glomerular filtration rate is the major clinical and recognized hallmark of early DN [7]. It results from damage to the filtration barrier by chronic hyperglycemia and the subsequent alteration of renal hemodynamics.…”