Urinary proteases degrade albumin: implications for measurement of albuminuria in stored samples

Abstract

Background: Previous studies have shown that albumin in stored urine samples degrades over time, and that albumin losses are greatest in samples with low pH conditions ( pH , 5). Furthermore, the high-performance liquid chromatography (HPLC) assay for urinary albumin has been shown to be particularly susceptible to the effects of prolonged storage. Methods: Frozen urine samples, stored for 12 months at 270 and 2208C, were analysed for albumin fragmentation. Urinary protease activity was investigated in vitro in urine adjusted to pH 2.3 -2.5. Albumin was measured by nephelometry, HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Results: In the unadjusted samples, albumin was degraded in 11 out of 40 samples stored at 2208C. In the in vitro experiments, both endogenous albumin and exogenous albumin added to urine were rapidly degraded into large fragments within minutes after adjustment to low pH. The fragments produced were consistent with those produced during digestion with pepsin and urinary degradation was completely inhibited by pepstatin. Albumin concentration measured by HPLC was most dramatically affected, with near-complete loss of albumin-sized material within one hour of incubation at pH 2.3 -2.5. Sample reactivity with antiserum in a nephelometry assay initially declined then increased, possibly due to exposure of internal epitopes during albumin digestion. Conclusions: This study demonstrated that proteases are present and active in stored human urine samples. Urinary albumin digestion occurred in a manner consistent with activity of endogenous urinary proteases. Adjustment to neutral pH or addition of protease inhibitors may be useful techniques for sample preservation.

Angiotensin II Type 2–Receptor Agonist C21 Reduces Proteinuria and Oxidative Stress in Kidney of High-Salt–Fed Obese Zucker Rats

“…HSD intake caused additional tubular proteinuria without an increase in albuminuria as determined by decreased uAPr in HSD fed OZR in current study and Dahl S rats. 46 HSD-induced tubular proteinuria may have been due to (a) altered retrieval of excess filtered albumin by endocytosis as demonstrated by loss of megalin-cubilin and/or (b) abnormal protein degradation by lysosomes and urine proteases, 53 which return non-immunoreactive peptide fragments of albumin to tubular lumen for excretion that remained undetected by antibodies specific to intact albumin. 54,55 Other tubular protein retrieval mechanisms exist, 56 but how exactly HSD intake in obesity affects these mechanisms is not known.…”

The Proximal Tubule and Albuminuria

“…71 Urine proteases at the apical brush border can likewise hydrolyze and degrade tubular albumin into urinary fragments. 72 However, a note of caution is necessary. Modeling in vivo endocytosis to that occurring in cultured cells can be very revealing but also misleading.…”

Serum Cystatin C Reflects the Progress of Albuminuria

“…However, urinary albumin excretion can be affected by several factors including plasma concentrations of atrial natriuretic peptide, arginine vasopressin, angiotensin II, aldosterone and fasting blood glucose, glycated hemoglobin, and mean arterial blood pressure [16] and albumin can be degraded in a manner consistent with the activity of endogenous urinary proteases [17]. Furthermore, the intraindividual variation is as high as 47%.…”