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

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 -70 and -20 degrees C, 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 -20 degrees C. 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.

Urinary-peptide excretion by patients with and volunteers without diabetes.

Large quantities of peptides (1-4 g) are excreted in human urine each day. In this study we sought to analyze how peptide excretion varies with increasing albuminuria associated with diabetes, as well as to characterize the size distribution of albumin-derived peptides in urine from volunteers without diabetes and from patients with macroalbuminuria and diabetes. We detected albumin-derived peptides by injecting tritiated albumin intravenously into human volunteers and patients with diabetes. Urine was collected after 24 hours and fractionated on a size-exclusion column. This fractionation revealed peptides with molecular weights ranging from 300 to 500 Da in volunteers without diabetes. The albumin-derived peptides were of higher molecular weight in the urine of a patient with macroalbuminuria and diabetes. The molecular-weight distribution of the peptides derived from tritiated albumin peptides was paralleled by the distribution of all protein peptides (including albumin) as determined with the use of the Biuret protein assay or absorbance at 214 nm. We determined peptide-excretion rates by filtering urine from patients with diabetes through a 10,000 Da molecular-weight-cutoff membrane and then measuring the filtrate with the use of the Biuret assay. This analysis revealed that the peptide-excretion rate increased with increasing total protein excretion, regardless of whether the patient demonstrated normoalbuminuria or microalbuminuria. Among patients with macroalbuminuria, the peptide-excretion rate leveled off and even decreased in the face of an increasing albumin concentration or protein-excretion rate. This study confirms that albumin-derived and protein-derived peptides exist at high concentrations in urine. Although peptide-excretion rates are maintained at similar levels up to macroalbuminuric states, the relative proportion of peptide excretion is significantly reduced compared with total protein.

Mechanism of hypoalbuminemia in rodents.

Normal albumin loss from the plasma is thought to be minimized by a number of mechanisms, including charge repulsion with the capillary wall and an intracellular rescue pathway involving the major histocompatibility complex-related Fc receptor (FcRn)-mediated mechanism. This study investigates how these factors may influence the mechanism of hypoalbuminemia. Hypoalbuminemia in rats was induced by treatment with puromycin aminonucleoside (PA). To test the effects of PA on capillary wall permeability, plasma elimination rates were determined for tritium-labeled tracers of different-sized Ficolls, negatively charged Ficolls, and (14)C-labeled tracer of albumin in control and PA-treated Sprague-Dawley rats. Urinary excretion and tissue uptake were also measured. Hypoalbuminemia was also examined in two strains of FcRn-deficient mice: beta(2)-microglobulin (beta(2)M) knockout (KO) mice and FcRn alpha-chain KO mice. The excretion rates of albumin and albumin-derived fragments were measured. PA-induced hypoalbuminemia was associated with a 2.5-fold increase in the plasma elimination rate of albumin. This increase could be completely accounted for by the increase in urinary albumin excretion. Changes in the permeability of the capillary wall were not apparent, inasmuch as there was no comparable increase in the plasma elimination rate of 36- to 85-A Ficoll or negatively charged 50- to 80-A Ficoll. In contrast, hypoalbuminemic states in beta(2)M and FcRn KO mice were associated with decreases in excretion of albumin and albumin-derived fragments. This demonstrates that the mechanism of hypoalbuminemia consists of at least two distinct forms: one specifically associated with the renal handling of albumin and the other mediated by systemic processes.

Renal processing of serum proteins in an albumin-deficient environment: an in vivo study of glomerulonephritis in the Nagase analbuminaemic rat.

BACKGROUND: Plasma albumin has been considered as important for governing glomerular permselectivity as well as being tubulotoxic in proteinuric states. The purpose of this study was to examine glomerular permselectivity and protein clearance of plasma albumin-deficient Nagase analbuminaemic rats (NAR) in normal and proteinuric states associated with puromycin aminonucleoside nephrosis (PAN) and anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) and to compare the results with those of previous studies using Sprague-Dawley rats. METHODS: Glomerular permselectivity was measured using tritium-labelled polydisperse Ficoll. In vivo fractional clearance (FC) of albumin, transferrin and immunoglobulin G was measured to include both intact and degraded forms of filtered material. Endogenous protein clearance was analysed using two-dimensional electrophoresis in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. RESULTS: FCs of proteins and Ficoll in control NAR were similar to those found in Sprague-Dawley rats. Despite the lack of serum albumin in NAR, proteinuria and morphological changes observed were also similar to those found in Sprague-Dawley rats, with total protein excretion increasing 6-fold in PAN rats and 4-fold in anti-GBM GN rats with respect to controls. Two-dimensional electrophoresis in combination with MALDI mass spectrometry identified the major proteins being excreted as transferrin and a group of mildly acidic proteins in the MW range 40-50 kDa, namely antithrombin III, kininogen, alpha-1-antiproteinase, haemopexin and vitamin D-binding protein. CONCLUSIONS: Both diseases exhibited similar effects to those observed in Sprague-Dawley rats despite the lack of serum albumin, including inhibition of renal protein degradation. The net changes in protein FC, particularly in the range of radii of 36-55 A, could not be accounted for by changes in size selectivity as Ficoll FC was little affected by the disease states. This emphasizes the need to reassess the relative importance of changes in renal tubular handling vs changes in glomerular capillary barrier in proteinuric states. These studies also demonstrate that albumin is not a critical factor in governing glomerular permselectivity or proteinuria.