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Microalbuminuria is an important clinical marker in patients with diabetes because of its well-established association with progressive renal disease. It is also becoming increasingly recognized as an independent risk factor for cardiovascular disease in patients with hypertension and diabetes, and in the general population. This review will discuss (i) the relationship between microalbuminuria, diabetes, hypertension, and cardiovascular disease, (ii) the mechanism by which albumin is processed by the kidney and enters the urine in health and disease, and (iii) the complex nature of urinary albumin and how it can be measured. [ABSTRACT FROM AUTHOR]

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 pep-tides 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. [ABSTRACT FROM AUTHOR]

SUMMARY: This study evaluated the potential toxicity of Fast green and Lissamine green, the two dyes commonly used for visualizing tubular fluid flow in micropuncture studies. In both the in vitro perfused kidney and cultured tubular epithelial cells, Fast green exhibited a profound, dose-dependent toxicity. In contrast, Lissamine green was non-toxic. In conclusion, we recommend that Fast green not be used in micropuncture studies. [ABSTRACT FROM AUTHOR]

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.Kidney International (2007) 72, 1195-1197; doi:10.1038/sj.ki.5002528; published online 12 September 2007. [ABSTRACT FROM AUTHOR]

The fractional clearance of proteins as measured in healthy human subjects increases 10,000-100,000- fold when studied in nephrotic patients. This remarkable increase cannot be accounted for by extracellular biophysical mechanisms centered at the glomerular filtration barrier. Rather, it is the nephron and its combination of filtration and cellular uptake that can provide a plausible explanation of these fractional clearance changes. The nephron has two regions that critically determine the level proteinuria/albuminuria. Glomerular filtration of plasma proteins is primarily a size selective event that is basically unchanged in acquired and genetic kidney disease. The glomerular concepts of 'charge selectivity' and of 'large pores', previously used to explain proteinuria, are now recognized to be flawed and non-existent. Filtered proteins then encounter downstream two protein receptors of the Park and Maack type associated with the proximal tubular cell. The high capacity receptor is thought to retrieve the majority of filtered proteins and return them to the blood supply. Inhibition/saturation of this pathway in kidney disease may create the nephrotic condition and hypoproteinemia/hypoalbuminemia. Inhibitors of this pathway (possibly podocyte derived) are still to be identified. A relatively small proportion of the filtered protein is directed towards a high affinity, low capacity receptor that guides the protein to undergo lysosomal degradation. Proteinuria in normoproteinemic states is derived by inhibition of this pathway, such as in diabetes. The combination of glomerular sieving, and the degradation and retrieval pathways can quantitatively account for the changes in fractional clearance of proteins in the nephrotic condition. Finally, the general retrieval of filtered protein by the proximal tubular cell focuses on the teleological importance of this cell as this retrieval represents the third pillar of retrieval that this cell participates in (it also retrieves water and salt)., Competing Interests: WDC is employed by Salaqua Diagnostics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Comper, Vuchkova and McCarthy.)

Competing Interests: WDC was employed by company Salaqua Diagnostics Inc.

Background: The most vexing problem in hyponatremic conditions is to differentiate the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) from cerebral/renal salt wasting (C-RSW). Both have identical clinical parameters but diametrically opposite therapeutic goals of water- restricting water-logged patients with SIADH or administering salt and water to dehydrated patients with C-RSW. While C-RSW is considered a rare condition, the report of a high prevalence of C-RSW in the general hospital wards creates an urgency to differentiate one syndrome from the other on first encounter. We decided to identify the natriuretic factor (NF) we previously demonstrated in plasma of neurosurgical and Alzheimer diseases (AD) who had findings consistent with C-RSW., Methods: We performed the same rat renal clearance studies to determine natriuretic activity (NA) in serum from a patient with a subarachnoid hemorrhage (SAH) and another with AD and demonstrated NA in their sera. The sera were subjected to proteomic and SWATH (Sequential Windowed Acquisition of All) analyses which identified increased levels of haptoglobin related protein (Hpr) without signal peptide (Hpr-WSP)., Results: Recombinant Hpr with His tag at the N terminus had no NA. Hpr-WSP had a robust NA in a dose-dependent manner when injected into rats. Serum after recovery from C-RSW in the SAH patient had no NA., Conclusions: Hpr-WSP may be the NF in C-RSW which should be developed as a biomarker to differentiate C-RSW from SIADH on first encounter, introduces a new syndrome of C-RSW in AD and can serve as a proximal diuretic to treat congestive heart failure., (Copyright © 2020. Published by Elsevier Inc.)

In order to understand the mechanism of albuminuria we have explored how other plasma proteins are processed by the kidney as compared to inert molecules like Ficolls. When fractional clearances are plotted versus protein radius there is a remarkable parallelism between protein (molecular weight range 30-150kDa) clearance in healthy controls, in Dent's disease, in nephrotic states and the clearance of Ficolls. Although there are significant differences in the levels of fractional clearances in these states. Dent's disease results in a 2-fold increase in the fractional clearance of proteins as compared to healthy controls whereas in nephrotic states there is a further 3-fold increase in fractional clearance. Previous thinking that albumin uptake was controlled primarily by the megalin/cubilin receptor does not explain the albumin urinary excretion data and is therefore an incorrect concept. Protein clearance in nephrotic states approach the fractional clearance of inert Ficolls for a given radius. It therefore appears that there are two pathways processing these proteins. A low capacity pathway associated with megalin/cubilin that degrades filtered protein (that is inhibited in Dent's disease) and a high capacity pathway that retrieves the filtered protein and returns it to the blood supply (without retrieval nephrotic protein excretion will occur and this will account for hypoproteinemia). On the other hand low molecular weight proteins (<20kDa) are processed entirely differently by the kidney. They are not retrieved but are comprehensively degraded in the kidney with the degradation products predominantly returned to the blood supply., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Purpose: The analysis of exosome/microvesicle (extracellular vesicles (EVs)) and the RNA packaged within them (exoRNA) has the potential to provide a non-invasive platform to detect and monitor disease related gene expression potentially in lieu of more invasive procedures such as biopsy. However, few studies have tested the diagnostic potential of EV analysis in humans., Experimental Design: The ability of EV analysis to accurately reflect prostate tissue mRNA expression was examined by comparing urinary EV TMPRSS2:ERG exoRNA from pre-radical prostatectomy (RP) patients versus corresponding RP tissue in 21 patients. To examine the differential expression of TMPRSS2:ERG across patient groups a random urine sample was taken without prostate massage from a cohort of 207 men including prostate biopsy negative (Bx Neg, n = 39), prostate biopsy positive (Bx Pos, n = 47), post-radical prostatectomy (post-RP, n = 37), un-biopsied healthy age-matched men (No Bx, n = 44), and young male controls (Cont, n = 40). The use of EVs was also examined as a potential platform to non-invasively differentiate Bx Pos versus Bx Neg patients via the detection of known prostate cancer genes TMPRSS2:ERG, BIRC5, ERG, PCA3 and TMPRSS2., Results: In this technical pilot study urinary EVs had a sensitivity: 81% (13/16), specificity: 80% (4/5) and an overall accuracy: 81% (17/21) for non-invasive detection of TMPRSS2:ERG versus RP tissue. The rate of TMPRSS2:ERG exoRNA detection was found to increase with age and the expression level correlated with Bx Pos status. Receiver operator characteristic analyses demonstrated that various cancer-related genes could differentiate Bx Pos from Bx Neg patients using exoRNA isolated from urinary EVs: BIRC5 (AUC 0.674 (CI:0.560-0.788), ERG (AUC 0.785 (CI:0.680-0.890), PCA3 (AUC 0.681 (CI:0.567-0.795), TMPRSS2:ERG (AUC 0.744 (CI:0.600-0.888), and TMPRSS2 (AUC 0.637 (CI:0.519-0.754)., Conclusion: This pilot study suggests that urinary EVs have the potential to be used as a platform to non-invasively differentiate patients with prostate cancer with very good accuracy. Larger studies are needed to confirm the potential for clinical utility.

Inhibition of the degradation of filtered albumin has been proposed as a widespread, benign form of albuminuria. There have however been recent reports that radiolabeled albumin fragments in urine are not exclusively generated by the kidney and that in albuminuric states albumin fragment excretion is not inhibited. In order to resolve this controversy we have examined the fate of various radiolabeled low molecular weight protein degradation products (LMWDPs) introduced into the circulation in rats. The influence of puromycin aminonucleoside nephrosis on the processing and excretion of LMWDPs is also examined. The status and destinies of radiolabeled LMWDPs in the circulation are complex. A major finding is that LMWDPs are rapidly eliminated from the circulation (>97% in 2 h) but only small quantities (<4%) are excreted in urine. Small (<4%) but significant amounts of LMWDPs may have prolonged elimination (>24 h) due to binding to high molecular weight components in the circulation. If LMWDPs of albumin seen in the urine are produced by extra renal degradation it would require the degradation to far exceed the known catabolic rate of albumin. Alternatively, if an estimate of the role of extra renal degradation is made from the limit of detection of LMWDPs in plasma, then extra renal degradation would only contribute <1% of the total excretion of LMWDPs of albumin. We confirm that the degradation process for albumin is specifically associated with filtered albumin and this is inhibited in albuminuric states. This inhibition is also the primary determinant of the massive change in intact albuminuria in nephrotic states.

Albuminuria in nephrotic states is thought to arise from the formation of large pores in the glomerular capillary wall as large hydrodynamic probes, like Ficoll, have increased fractional clearance. In the present study, we tested for large pore formation in a novel manner. We accounted for the rates of plasma elimination as determined for tritium-labeled tracers of uncharged polydisperse Ficoll (radii range: 35-85 Å) and two globular (14)C-labeled proteins, albumin (radius: 36 Å) and IgG (radius: 55 Å), in control and puromycin aminonucleoside nephrotic (PAN) Sprague-Dawley rats. The plasma elimination rates were then matched to the urinary excretion of these labeled materials (n = 7). Albumin and IgG plasma retention rates were identical and far enhanced compared with the retention rates of inert transport markers of equivalent hydrodynamic radius; their elimination rate corresponded to the elimination of a 75-Å radius Ficoll (n = 5) and >105-Å radius dextran (n = 5). In PAN, they were eliminated as ∼36- and ∼55-Å radii Ficoll, respectively, equivalent to their hydrodynamic radii. In contrast, there was no comparable increase in the elimination rate of Ficoll in PAN. The total plasma clearance of Ficoll in control and PAN rats and the urinary clearance in PAN rats were essentially the same for all radii. On the other hand, the urinary clearance of >45-Å radii Ficoll in controls was considerably lower with increasing radii, demonstrating a postfiltration cellular uptake in controls, which, when inhibited in nephrotic states, would account for apparent large pore formation., (Copyright © 2014 the American Physiological Society.)

For over 50 years, the glomerular filter has been thought to exert an uniquely significant barrier to the transport of albumin. The glomerular endothelial cell glycocalyx is considered to contribute to this important barrier restriction. In renal disease, structural alterations to this layer have been associated with albuminuria. It appears however the claims of the influence of this barrier have been overstated. The behaviour of albumin in systems that model the glycocalyx has been studied widely and the results demonstrate that the endothelial glycocalyx would offer only relatively small effective barrier to albumin. This has been confirmed in studies on macromolecular exchange in non-renal capillary beds with similar endothelial glycocalyx. The experimental perturbations to the glomerular endothelial glycocalyx (through enzyme treatments, saline washouts) also create only relatively small changes in the level of albuminuria as compared to changes in albumin excretion seen in renal disease and nephrotic states. Additionally, it is questionable how specific these perturbations are. Overall, the endothelial glycocalyx most likely has biological functions like it does in other extracellular regions involving hydration through osmotic pressure and offering charge-mediated binding of various molecules. This confirms work by Comper and colleagues that the glomerular sieving of albumin is not unique and is consistent with that of size selectivity that results in significant amounts of albumin being filtered normally, retrieved by proximal tubules and returned to the blood supply.

Here we address the assumption that the massive intact albuminuria accompanying mutations of structural components of the slit diaphragm is due to changes in glomerular permeability. The increase in intact albumin excretion rate in Cd2ap knockout mice by >100-fold was not accompanied by equivalent changes in urine flow rate, glomerular filtration rate or increases in dextran plasma clearance rate, which demonstrates that changes in glomerular permeability alone could not account for the increase in intact albumin excretion. The albuminuria could be accounted for by inhibition of the tubule degradation pathway associated with degrading filtered albumin. There are remarkable similarities between these results and all types of podocytopathies in acquired and toxin-induced renal disease, and nephrotic states seen in mice with podocyte mutations., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Understanding the pathogenesis of albuminuria in diabetic nephropathy is important to improve methods for early diagnosis and treatment. In this study, we addressed whether albuminuria in diabetes results from altered glomerular filtration and/or altered processing of filtered albumin by the proximal tubule. Type 1 diabetic Munich Wistar rats developed albuminuria after 12 wk of diabetes. Intravital two-photon microscopy revealed similar glomerular permeability in the diabetic and control animals, assessed using both albumin-Alexa568 and 69-kD FITC-dextran; however, diabetic animals demonstrated significantly less filtered fluorescent albumin in renal proximal tubule (PT) cells compared with control animals. We also observed increased albumin-derived urinary peptide excretion in diabetic animals, and hyperglycemia modulated this peptideuria. In conclusion, in the early stages of diabetic nephropathy, the PT plays a major role in the development of albuminuria, which may be preceded by peptideuria.

Background: Recent studies suggest that expression of the transforming growth factor-beta (TGF-beta)-inducible gene-h3 (betaig-h3) and its anti-lysosomal activity may be responsible for the development of albuminuria and cardiovascular disease associated with hypertension., Methods: We evaluated the proposed linkage using the spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat models. The kidney and left ventricular weight/body weight ratios were measured and cardiac collagen deposition was analyzed by Masson's trichrome stain. Renal and cardiac TGF-beta(1) and betaig-h3 expression were determined by real-time reverse transcription-polymerase chain reaction, and renal and cardiac cathepsin B and L activities were measured as an indicator of lysosomal proteolytic activity., Results: SHR exhibited increased levels of intact urinary albumin without significant change in total albumin (intact plus albumin-derived material) excretion. This was accompanied by renal hypertrophy, increased renal betaig-h3 expression, and reduced renal cathepsin B and L activities. At the same time, increased cardiac TGF-beta(1) and betaig-h3 expression and reduced cardiac cathepsin B activity was identified in SHR in addition to cardiac hypertrophy and increased collagen deposition. All these changes could be ameliorated with ramipril treatment., Conclusions: These findings implicate for the first time betaig-h3 expression and lysosomal activity as a key factor in the induction of albuminuria and cardiovascular disease associated with hypertension., (Copyright (c) 2008 S. Karger AG, Basel.)

The mechanism of albuminuria is perhaps one of the most complex yet important questions in renal physiology today. Recent studies have directly demonstrated that the normal glomerulus filters substantial amounts of albumin and that charge selectivity plays little or no role in preventing this process. This filtered albumin is then processed by proximal tubular cells by two distinct pathways; dysfunction in either one of these pathways gives rise to discrete forms of albuminuria. Most of the filtered albumin is returned to the peritubular blood supply by a retrieval pathway. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This degradation pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-beta1, whose production is stimulated by hyperglycemic and hypertensive environments. Dysfunction in this degradation pathway leads to albuminuria below the nephrotic range. These new insights into albumin filtration and processing argue for a reassessment of the role of podocytes and the slit diaphragm as major direct determinants governing albuminuria, provide information on how glomerular morphology and "tubular" albuminuria may be interrelated, and offer a new rationale for drug development.

The classic mechanism to explain albumin excretion in diabetes has been permeability defects in the glomerular filter. However, a new concept has emerged that albuminuria can be explained by the two major pathways the proximal tubular cell uses to process filtered albumin. Specifically, albumin permeability through the glomerular filter is only governed by size selectivity. Most of the filtered albumin is retrieved by the proximal tubular cell and returned to the peritubular blood supply. Albuminuria in the nephrotic range would arise from retrieval pathway dysfunction. The small quantities of filtered albumin that are not retrieved undergo obligatory lysosomal degradation before urinary excretion as small peptide fragments. This pathway is sensitive to metabolic factors responsible for hypertrophy and fibrosis, particularly molecules such as angiotensin II and transforming growth factor-beta1, whose production is stimulated by hyperglycemic environments. Dysfunction in this degradation pathway may lead to albuminuria below the nephrotic range.

The glomerular filtration barrier has long been thought largely impermeable to albumin. Startling new data suggest it may not be especially important in this process. Much of the argument hinges on whether charge selectivity really exists, how feasible it is for enormous quantities of albumin to instead be reclaimed by the proximal tubule, and the technical merits of previous experiments. Three experts in the field debate the merits of this argument.

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.

Objectives: To compare the analysis of different forms of intact albumin in urine from healthy volunteers. To determine contamination by common non-albumin proteins on HPLC analysis of urinary albumin and of purified immuno-unreactive albumin., Design and Methods: Overnight urine samples collected from healthy volunteers were analysed for total albumin (immunoreactive plus immuno-unreactive) by HPLC and densitometry following native PAGE separation and for immunoreactive albumin by RIA. The contamination by non-albumin proteins of the HPLC analysis of urinary albumin and of immuno-unreactive albumin preparations was determined by ELISA. Immuno-unreactive albumin was tested for Co2+-binding capacity., Results and Conclusions: HPLC analysis of healthy urine generates higher ACR values than immunological methods due to the presence of immuno-unreactive albumin. Immuno-unreactive albumin cannot be accounted for by the non-albumin urinary proteins tested. Isolated immuno-unreactive albumin is not recognised by antibodies to common urinary proteins or by an array of anti-albumin antibodies and behaves like serum albumin in terms of HPLC elution, native PAGE migration, and cobalt ion binding.

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

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.

Microalbuminuria is an important clinical marker in patients with diabetes and cardiovascular disease. The concentration of albumin in urine has traditionally been measured by semiquantitative dipsticks or by various quantitative immunochemical methods such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. However, until recently, urinary albumin not reabsorbed by proximal tubular cells was assumed to be excreted intact. Studies have now revealed that the nature of urinary albumin is complex and is excreted as a mixture of intact albumin, albumin-derived peptides that are not detected by routine dipstick and antibody-based tests, and a species of intact albumin (immunounreactive albumin), also not detected by dipstick and antibody-based tests. A new test, Accumin, based on high-performance liquid chromatography analysis, is able to detect all the immunoreactive intact albumin and immunounreactive intact albumin (total intact albumin) in urine. The advantage in the use of Accumin over a conventional dipstick test or antibody-based laboratory method for detecting microalbuminuria is that false negatives are reduced and a relatively earlier diagnosis of incipient kidney disease can be achieved. The introduction of Accumin has, therefore, highlighted the need for a global standard in the detection and measurement of microalbuminuria. By detecting all of the immunoreactive and immunounreactive intact albumin in urine, Accumin has virtually invalidated the use of dye and immunologically-based dipstick tests and immunologically-based laboratory methods in screening for microalbuminuria in diabetic patients and in identifying microalbuminuria as a risk factor for cardiovascular disease.

Background: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive., Methods: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70-80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis., Results: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r(2) = 0.83) than concentrations measured by native PAGE and RIA (r(2) = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r(2) = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r(2) = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles., Conclusions: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.

Recent studies have demonstrated the presence, in both rat and human urine, of a modified form of albumin not detected by conventional antibodies. This modified albumin behaves physicochemically as intact albumin under nondenaturing conditions. We have demonstrated this form of albumin to be disproportionately excreted in microalbuminuric states in diabetes. Quantitation of this modified form of albumin leads to the prediction of the onset of microalbuminuria in diabetic patients on average 3 to 4 years earlier than when measured by conventional immunoassays.

The major underlying factors associated with tissue damage and fibrosis in cardiovascular and kidney disease are the up-regulation and action of growth factors such as transforming growth factor-beta (TGF-beta) and cytokines produced in response to changes in systemic factors, particularly blood pressure or hyperglycemia. This study identifies the relationship of elevated levels of TGF-beta to increased levels of intact albumin in the urine (micro- and macroalbuminuria). This mechanism may be directly linked to the effect of TGF-beta on albumin uptake and the lysosomal breakdown of filtered albumin by proximal tubular cells prior to excretion.

Objective: To examine the role of renal processing in the turnover of circulating albumin in the model of reversible overload proteinuria. Specifically, this study determines whether proteinuria due to hyperalbuminemia may be attributed to saturation of renal cell processing of albumin or alterations in diffusive/convective transport of albumin across the glomerular capillary wall., Methods: Overload proteinuria was induced in male Wistar rats by daily intraperitoneal injections of 1.5 g bovine serum albumin (BSA; endotoxin free) for 3 days. Plasma concentration and urinary excretion of total protein and rat serum albumin (RSA) were determined by the biuret assay and radioimmunoassay, respectively. Glomerular size selectivity was assessed by examining the fractional clearance (FC) of neutral [3H]Ficoll using a short-term, steady-state approach. Cellular processing was examined by size exclusion chromatography analysis of urinary [14C]albumin and [14C]transferrin structural integrity., Results: Total protein excretion (n = 4-15) increased 7-fold in BSA-treated rats (58 +/- 26 mg/24 h at baseline vs. 417 +/- 259 mg/24 h at day 3), while intact RSA excretion (n = 4-6) increased 200-fold (0.27 +/- 0.15 mg/24 h vs. 53 +/- 26 mg/24 h) despite a significant decrease in its plasma concentration. Total protein and albumin excretion returned to basal levels by day 7 (82 +/- 16 mg/24 h and 0.93 +/- 0.42 mg/24 h, respectively). FC of [3H]Ficoll of equivalent size to albumin did not change in BSA-treated rats as compared to vehicle controls. Tubular lysosomal processing of proteins was altered at peak proteinuria, as determined by changes in the structural integrity of urinary [14C]albumin, as well as [14C]transferrin, but returned to normal by day 7., Conclusions: This study demonstrates the lack of proportionality between changes in plasma concentration and urinary excretion of albumin, as well as the lack of change in the glomerular size selectivity to albumin. The profound but reversible changes in the amount and integrity of excreted protein suggest that cell-mediated processes are saturated by albumin concentrations, leading to the development of proteinuria in this model.

Background: Microalbuminuria is regarded as the most important predictor of high risk for the development of diabetic nephropathy. Early detection may allow treatment to prevent progression to persistent albuminuria and renal failure. Recent studies have shown that conventional immunoassays underestimate urinary albumin concentration, as albumin in urine may exist in two forms, immuno-reactive and immuno-unreactive. The present study examines the differential lead-time for the development of microalbuminuria as measured by both conventional radioimmunoassay (RIA; measures immuno-reactive) and high-performance liquid chromatography (HPLC; measures total albumin = immuno-reactive plus immuno-unreactive) analysis in both type 1 and type 2 diabetic patients., Methods: Analysis was performed on 511 stored urine samples collected over the last 13 years from type 1 diabetic patients who either progressed from normo- to microalbuminuria (progressors, N= 17), or who remained normoalbuminuric (nonprogressors, N= 25) as defined by RIA, and on 634 urine samples collected from patients with type 2 diabetes defined as either progressors (N= 24) or nonprogressors (N= 25)., Results: For type 1 progressors, the mean lead-time for the HPLC assay versus the RIA was 3.9 years, with a 95% CI of 2.1 to 5.6 years. For type 2 progressors, the mean lead-time was 2.4 years with a 95% CI of 1.2 to 3.5 years. There was no significant difference between the lead-time analysis between type 1 and type 2 diabetic patients., Conclusion: These results demonstrate that measurement of total albumin may allow earlier detection of microalbuminuria associated with diabetic nephropathy.

Objectives: To compare the analysis of urinary albumin from diabetic patients by four conventional immunoassays including radioimmunoassay (RIA), immunonephelometry (IN), and two different methods of immunoturbidimetry (IT), as well as by high-performance liquid chromatography (HPLC)., Design and Methods: Urines were collected over a 24-h period and stored at -20 degrees C until assay. Urinary albumin concentration was determined by an in-house RIA, by IN using a Beckman Array Analyser with reagents from Beckman Diagnostics (Sydney, Australia), by IT using a Dade-Behring Turbitimer with reagents from Dade-Behring (Marburg, Germany), by IT using a Dade-Behring Dimension R x L Chemistry Analyser with reagents from DiaSorin (Stillwater, OK, USA), and by HPLC using a Zorbax Bio series preparative GF-250 column. Regression lines were calculated using a least squares method to determine the correlation between the assays studied. Bland-Altman bias plots including limits of agreement were also calculated., Results: The correlation coefficients calculated were high (>0.85) indicating a strong linear relationship between all assays studied. The slopes calculated for the comparisons demonstrate that each assay can vary from one another (up to threefold) and have a slope significantly different from an ideal slope of 1 (P < 0.001). These results were confirmed by Bland-Altman bias plots and calculation of the limits of agreement that were all large., Conclusions: At this time, there is no global standard by which urinary albumin assays may be standardized. This study suggests the need for such standards.

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.

Recent studies, using low-temperature perfusion of rat kidneys, have claimed the existence of renal charge selectivity simply on the basis of the differential excretion rates of uncharged Ficoll and charged proteins. To test for the existence of charge selectivity in vivo, we examined the clearance of negatively charged Ficoll compared with uncharged Ficoll. A short-term approach to steady state was used to study the fractional clearances. Relative clearances were also examined using an osmotic pump technique where the tracers reach a steady-state value in conscious rats after 7 days. Carboxymethyl Ficoll was stable during filtration and renal passage, was not taken up by the kidneys, and did not bind to plasma proteins. There was no significant difference in the fractional clearance of molecules with radius of 36 A for Ficoll (fractional clearance = 0.048 +/- 0.038, n = 5) and negatively charged carboxymethyl Ficoll (fractional clearance = 0.028 +/- 0.019, n = 5). For molecules with radii greater than 36 A, carboxymethyl Ficoll had facilitated clearance with respect to uncharged Ficoll [for example, at a radius of 60 A fractional clearance for Ficoll = 0.0012 +/- 0.0005 (n = 5), whereas that for carboxymethyl Ficoll = 0.015 +/- 0.005 (n = 5)]. Renal function was not compromised by carboxymethyl Ficoll as uncharged Ficoll in urine exhibited similar hydrodynamic size profiles when studied in the presence of excess unlabeled carboxymethyl Ficoll. The facilitated clearance of negatively charged Ficoll with respect to uncharged Ficoll reveals a property of the capillary wall, which has been previously observed with other nonproteinaceous polyanions. This study demonstrates that the glomerular capillary wall is not charge selective in the form of excluding negatively charged Ficoll. However, the charge properties of the capillary wall may influence the facilitated transport of charged Ficoll compared with uncharged Ficoll.

The excretion of serum albumin in the urine is considered the net result of renal glomerular filtration and tubular uptake. During routine experiments, we observed that a batch of tritium-labeled albumin yielded anomalous results, being excreted in the urine of isolated perfused kidneys at 10 times the rate of normal tritiated albumin. This anomalous albumin, when simultaneously studied with normal carbon 14-labeled albumin, exhibited 10 times greater excretion than normal [(14)C]albumin. Anomalous albumin could not be reversed to normal albumin by means of conditioning with blood. In vivo clearances of anomalous albumin could not be quantitated because anomalous albumin is degraded during circulation. Anomalous albumin appeared to have the same molecular size (as determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, capillary electrophoresis, and gel chromatography) and isoelectric-point profile (2-dimensional electrophresis) as normal albumin. Normal albumin could be transformed to anomalous albumin with alkali/heat treatment. Reverse-phase high-pressure liquid chromatography analysis of fragments from tryptic digests of anomalous albumin, alkali/heat-treated albumin, and normal albumin suggest that anomalous albumin and alkali/heat-treated albumin have altered tertiary structure, possibly as a result of denaturation and disulfide exchange. These studies show that the tertiary structure of albumin, beyond simple size and charge, is a critical determinant for albumin processing by the kidney and suggest that a specific albumin-recognition event by the kidneys is critical to normal renal handling of albumin.

Objective: This study examined the separate and combined effects of hypertension and diabetes on renal cortical expression of protein kinase C (PKC) isoforms -beta 1, -beta 2, -alpha and -epsilon, to determine whether albuminuria is the result of an increase in the expression of one or a combination of PKC isoforms. Corresponding changes in renal microtubules were also assessed., Methods: Diabetes (D) was induced in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) by streptozotocin. After 24 weeks, PKC expression was determined by Western blot and microtubules were assessed by immunohistochemistry for alpha-tubulin protein., Results: Diabetes was characterized by significant increases in glycated haemoglobin (HbA1c) as compared to controls (C). There was a significant increase of three- to four-fold in PKC protein content for all four isoforms in renal cortex from SHR-C and WKY-D, and similar and significant levels of albuminuria (approximately 10 mg/24 h) observed in these groups in comparison to WKY-C (approximately 1 mg/24 h). Interestingly, PKC-alpha and -epsilon but not PKC-beta 1 and -beta 2 protein content was doubled in SHR-D, and albuminuria increased tenfold (approximately 100 mg/24 h) in comparison to SHR-C and WKY-D. These changes were paralleled by a significant decrease in alpha-tubulin protein content of approximately 50% in SHR-C and approximately 33% in WKY-D compared to WKY-C, with a further decrease of approximately 67% in SHR-D compared to WKY-C., Conclusion: These findings indicate that PKC expression can be increased by either diabetes or hypertension, and that there are further specific increases in the expression of PKC isoforms -alpha and -epsilon in the model of combined diabetes and hypertension. In addition, the degree of disruption in microtubular cytoskeleton appears to be correlated with PKC activation and levels of albuminuria.

This study has investigated the microtubular cytoskeleton in rat glomerular and proximal tubule cells in experimental diabetes. The effect of treatment with ramipril on the relationship between microtubule organization and albuminuria in diabetes has also been examined. Diabetes was induced in male Sprague-Dawley rats by administration of streptozotocin (50 mg/kg, i.v.). Rats were treated with or without ramipril in their drinking water for 12 weeks. Diabetes was characterized by an increase in blood glucose level, glomerular filtration rate, and albumin excretion rate. Treatment of diabetic rats with ramipril did not affect glycaemic control, but reduced systolic blood pressure and prevented the rise in albuminuria and glomerular filtration rate. Immunohistochemistry was performed by using the ARK Peroxidase method with alpha-tubulin antibody. The regular, grainy staining pattern of the microtubules present in the renal proximal tubules from control kidneys was altered in diabetic animals, and appeared fragmented and striated. This was prevented by treatment with ramipril. Quantitative morphometric analysis revealed an increase in the percent proportional staining for alpha-tubulin in the proximal tubules of untreated diabetic rats (33.3 +/- 3.3%, n = 8, P < 0.05 vs control) compared with control rats (11.7 +/- 1.7%, n = 6), which was reduced by ramipril treatment (26.7 +/- 2.1%, n = 6, P < 0.05 vs untreated diabetic). Staining for alpha-tubulin in glomerular cells was unchanged in all groups. There was no significant difference in renal alpha-tubulin expression among all groups, as determined by real-time reverse transcription-polymerase chain reaction. These results raise the possibility that diabetes-induced changes in microtubules in the renal proximal tubules may contribute, in part, to the increase in albuminuria observed in diabetes.

Filtered albumin is excreted as a heterogeneous population of albumin-derived molecules resulting from degradation during renal passage. In order to understand the dynamics of this degradation process, albumin clearance was studied over a short-term (minutes) and a long-term (7 days) by both radioactivity and radioimmunoassay. The radiolabelled material in the urine was also analysed extensively by using size exclusion chromatography, size selective filtration and high performance liquid chromatography. These studies demonstrate that during renal passage, albumin degradation to fragments in the size range of 500-10,000 occurs in a matter of minutes. The fragments are not detected by using radioimmunoassay. Steady state excretion rates or fractional clearance of radiolabelled albumin occur over a similar time period. Both rates of degradation and approach to steady-state clearance, while rapid, were considerably slower than the transit time for molecules in the Bowman's capsule and early tubular lumen. The results are consistent with an extremely rapid lysosomal uptake of filtered albumin, and degradation and regurgitation of the albumin-derived peptide fragments into the tubular lumen prior to excretion.

Background/aims: Recent studies show that albuminuria may be the result of changes in post-glomerular cellular uptake and processing of albumin. This study aims to determine whether this processing is disrupted in diabetes and/or hypertension., Methods: Diabetes (d) was induced using streptozotocin in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) and studied after 8, 16 and 24 weeks of disease. Intact albumin excretion was determined by radioimmunoassay. Total albumin was determined by [(14)C]albumin. Lysosomal activity was determined by dextran sulfate desulfation. Renal TGF-beta1 and transforming growth factor-beta1 inducible gene-h3 mRNA (betaig-h3) expression was determined by real time RT-PCR., Results: SHR-c rats exhibited an increase in intact albuminuria without significant change in total albumin excretion (intact plus albumin-derived peptides). For WKY-d rats, intact albuminuria developed initially, followed by an increase in total albumin excretion primarily in the form of albumin peptides (peptiduria). SHR-d rats exhibited both increases in peptiduria and intact albuminuria. There was no increase in glomerular permeability at 24 weeks for polydisperse [(3)H]Ficoll in all groups. Increased renal TGF-beta1 and betaig-h3 expression was correlated with a decrease in dextran sulfate desulfation and increased intact albuminuria independent of peptiduria., Conclusion: Increased albumin excretion in hypertension and/or diabetes is manifested in different forms independent of glomerular permeability., (Copyright 2003 S. Karger AG, Basel)

Objectives: Albumin excretion is modulated post-filtration by lysosomal processing that produces a spectrum of albumin-derived material in urine, much of which is not detected by conventional immunoassays. This study aimed to determine the efficacy of ramipril treatment (+ RAM) after 24 weeks on total albumin excretion (intact plus albumin-derived peptides) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats with (d) and without (c) diabetes., Methods: Intact albumin excretion was analysed by radioimmunoassay and total albumin excretion was analysed by measuring radioactivity derived from circulating [ C]albumin. Renal lysosomal activity was determined by urinary [ H]dextran sulphate desulphation. Renal transforming growth factor-beta 1 (TGF-beta 1), TGF-beta inducible gene-h3 (beta ig-h3) and angiotensinogen mRNA production were analysed by real time reverse transcriptase-polymerase chain reaction., Results: Hypertension (SHR-c and SHR-d) resulted in a significant increase in intact albumin excretion, which was significantly reduced by ramipril treatment (P < 0.05 for SHR-c + RAM and 0.001 for SHR-d + RAM compared to non-treated). This was accompanied by a significant decrease in blood pressure (P < 0.001 for SHR-c + RAM and SHR-d + RAM), renal beta ig-h3 mRNA production (P < 0.05 for SHR-c + RAM and SHR-d + RAM), and an increase in lysosomal activity. Diabetes (WKY-d and SHR-d) primarily caused a significant increase in total albumin excretion, predominantly in the form of albumin-derived fragments in the WKY-d group and intact albumin in the SHR-d group. Ramipril treatment reduced total albumin excretion in the WKY-d + RAM group (P < 0.001)., Conclusions: Ramipril prevents increases in both intact albumin and total albumin excretion in hypertensive and diabetic states, respectively.

Background: Intact albumin in urine may exist in two forms, immunoreactive and immuno-unreactive. Previous estimates of albuminuria in diabetic urine have only detected immunoreactive forms., Methods: High performance liquid chromatography (HPLC) was used in this study to measure both forms of intact albumin (termed total intact albumin) to provide a more accurate measurement of albuminuria compared with radioimmunoassay (RIA) on 97 fresh urine samples from patients with diabetes. Eighty-six control urine samples from volunteers without diabetes were also tested., Results: There was no significant difference between the two methods for nondiabetic controls. For diabetic urine samples, 91.6% of samples showed a greater concentration of albumin measured by HPLC than RIA. For normoalbuminuric diabetic samples, HPLC gave a mean albumin excretion rate of 12.5 +/- 4.4 microgram/min (SD), whereas RIA gave a rate of 8.0 +/- 6.7 microgram/min (P = 0.004; N = 28). For microalbuminuric samples, there also was a statistically significant difference: HPLC albumin excretion rate, 82.0 +/- 49.9 microgram/min, and RIA, 49.0 +/- 34.6 microgram/min (P = 0.004; N = 30). Thirty-two urine samples were normoalbuminuric by RIA (albumin, 11.4 +/- 3.9 microgram/min), but in the microalbuminuric range as determined by HPLC (albumin, 38.5 +/- 14.4 microgram/min). For urine samples in the macroalbuminuric range, there was no statistically significant difference between HPLC and RIA. Immuno-unreactive albumin was confirmed as albumin, analyzed by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry., Conclusion: These studies show that to determine microalbuminuria accurately, there is a need to assess urinary total intact albumin, rather than simply immunoreactive albumin. Am J Kidney Dis 41:336-342., (Copyright 2003 by the National Kidney Foundation, Inc.)

Aims/hypothesis: Streptozotocin (STZ) diabetic rats are characterized by the development of albuminuria. It is not known, however, whether the excess excretion of protein is primarily due to intact protein or protein fragments or whether it is specific for albumin or occurs for all high-molecular-weight plasma proteins. To test this we have measured the excretion rates and fractional clearances of [(14)C]albumin, [(3)H]immunoglobulin G and [(3)H]transferrin in diabetic rats., Methods: The radiolabeled proteins were delivered to the circulation of conscious diabetic (STZ induced for 6 weeks) and control rats by ALZET osmotic pumps. The plasma level of the radiolabeled proteins reached steady-state levels by day 7. Urine and plasma samples from day 7 were used to determine the excretion rates of the proteins by radioactivity and radioimmunoassay., Results: When excretion rates were determined by radioactivity it was apparent that only the albumin excretion rate increased significantly with STZ diabetes to a value of 354 +/- 166 microg/min which agrees with proteinuria determined by Biuret assay of 299.9 +/- 52.4 microg/min. The major proportion of protein being excreted was in the form of protein fragments which are not detected by conventional immmunochemical assays., Conclusion: The previously unrecognized nephrotic-like levels of proteinuria in experimental diabetes appears to be associated with an albumin-specific mechanism responsible for the increase in albumin peptides in urine. There was significant lowering of plasma albumin concentration but plasma concentrations of transferrin and immunoglobulin G remained unchanged. There was also no significant appearance of intact protein in urine that is normally found in nephrotic states., (Copyright 2003 S. Karger AG, Basel)