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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]

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.

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.)

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.

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 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.

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.

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.

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.

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.

Increased intraglomerular pressure is considered a major factor for increased albumin excretion in hypertension. However, other factors should also be considered because recent studies in both humans and rats have demonstrated that proteins undergoing filtration and renal passage are extensively modified by renal cell lysosomal processing; >95% of albumin is degraded to peptides that are not detected by routine immunochemical assays. Changes in postglomerular lysosomal processing may therefore be responsible for the increased intact albumin excreted in hypertension-related kidney disease. We hypothesize that transforming growth factor-beta, which is known to decrease lysosomal activity, may be upregulated in hypertension and may play a role in increased intact albumin excretion. The aims of this study were to determine the effect that hypertension has on (1) renal cell lysosomal processing of albumin and dextran sulfate, (2) glomerular permeability, and (3) renal transforming growth factor-beta(1) expression. Spontaneously hypertensive rats and Wistar-Kyoto rats were used at 8, 16, and 24 weeks. We demonstrate that albuminuria in hypertension is linked to an inhibition of lysosomal processing as determined by (1) size exclusion chromatography analysis of urinary [(14)C]albumin structural integrity and (2) ion exchange analysis of urinary [(3)H]dextran sulfate. This inhibition gives rise to an increased proportion of radioimmunoassay detectable (intact) albumin and intact dextran sulfate independent of changes in glomerular capillary wall permeability as determined by the fractional clearance of [(3)H]Ficolls of various radii. These changes may be correlated with increased renal transforming growth factor-beta(1) expression.

The increased fractional clearance of albumin in nephrotic states has long been attributed to glomerular permselectivity dysfunction. Using radiolabeled rat serum albumin, transferrin, IgG, and polydisperse Ficoll, this study investigated the changes in their in vivo fractional clearance in puromycin aminonucleoside nephrosis and anti-glomerular basement membrane glomerulonephritis. In control rats the lack of charge selectivity was confirmed by the demonstration that carboxymethyl Ficoll (valence approximately -39) had the same fractional clearance as uncharged Ficoll. Both diseases exhibited similar effects on fractional clearance measurements suggesting an underlying common mechanism. In disease, there was good agreement between the fractional clearance of proteins determined by radioactivity as compared to those determined by radioimmunoassay. A small increase in the fractional clearance for IgG was evident in disease as compared to controls, which mirrored the change in the equivalent size Ficoll, suggesting that the increase is because of the development of a small proportion of large pores in the glomerular capillary wall. There was no increase, however, in the fractional clearance of Ficoll of equivalent size to albumin in either disease, yet the fractional clearance of the albumin increased by 12 to 14 times as determined by radioactivity and 4500 to 6600 times as determined by radioimmunoassay. This study demonstrates that glomerulonephritis is not a disease associated with changes in glomerular permeability to albumin but is because of alterations in albumin processing by cells distal to the glomerular basement membrane. It is also apparent that approaches to glomerular pathology and proteinuria as risk factors in renal disease must be reassessed.

Aims/hypothesis: We aimed to examine the time course for the diabetes-related changes in renal lysosomal processing and to determine whether these changes can be prevented by aminoguanidine or ramipril treatment., Methods: The percentage desulphation of intravenously injected tritium labelled dextran sulphate ([3H]DSO4) in the urine, as determined by ion-exchange chromatography, was used as a marker of lysosomal sulphatase activity. Sulphatase activity was determined 1, 2, 3 and 4 weeks after the onset of diabetes in rats as well as in rats treated with either aminoguanidine or ramipril for twelve weeks., Results: The amount of totally desulphated [3H]DSO4 in urine collected from control rats was 65.6 +/- 0.8%. This was significantly reduced in diabetic rats two (57.4 +/- 1.4% desulphated), three (56.8 +/- 1.3 % desulphated) and four (52.9 +/- 2.2% desulphated) weeks after the onset of diabetes. The significant decrease in the amount of totally desulphated [3H]DSO4 in the urine also found at 12 weeks after the onset of diabetes was not affected by drug treatment. There was no significant difference in the amount of partially desulphated [3H]DSO4 in the urine between all the study groups. However, the increase in totally sulphated [3H]DSO4 in the urine collected from diabetic rats (8.7 +/- 1.7 % sulphated) compared with that of control rats (2.2 +/- 0.5% sulphated) was normalised by treatment with both aminoguanidine (4.8 +/- 1.6% sulphated) or ramipril (4.5 +/- 0.8% sulphated)., Conclusions/interpretation: These results raise the possibility that the diabetes-induced changes in renal lysosomal processing may be one of the initial events in the development of diabetic nephropathy. Aminoguanidine and ramipril, known for their different mechanism of action, seem to prevent diabetes-induced changes in lysosomal processing either through their effects on enzyme activity within the lysosome or through their effects on the trafficking of molecules to and from the lysosome.

Previous studies by our group have shown that albumin is metabolized in rodents during renal passage and excreted in the urine as a mixture of intact protein and albumin-derived fragments. The aim of this study was to examine whether albumin is metabolized during renal passage in nondiabetic volunteers and in type 1 diabetic patients with varying levels of albuminuria. Nine nondiabetic normoalbuminuric volunteers and 11 type 1 diabetic patients with albumin excretion rates varying from normoalbuminuria to macroalbuminuria were studied. Each subject received an intravenous injection of tritium-labeled albumin ([3H]-albumin). Urine was collected at 4 h and 24 h after injection and analyzed by size exclusion chromatography. The amount of intact and fragmented albumin was quantified, and each fraction was analyzed by radioimmunoassay (RIA) for albumin. [3H]-albumin in nondiabetic volunteers was metabolized during renal passage to small peptide fragments not detectable by conventional RIA (only 0.05-3.8% of the total urinary radioactivity was associated with intact albumin). The process responsible for albumin fragmentation was similar in diabetic patients with normoalbuminuria (intact albumin represented 0.01-4.0% of total urinary radioactivity). However, there was a reduction in the fragmentation ratio (fragmented:intact) in diabetic patients with micro- or macroalbuminuria (intact albumin represented 2.7-55.5%, P = 0.048). This change in the fragmentation ratio was directly related to the degree of albuminuria. These results have important implications for understanding the mechanisms underlying albuminuria in nondiabetic volunteers and type 1 diabetic patients. In nondiabetic volunteers, the renal processing of albumin involves a relatively rapid and comprehensive degradation of albumin to small fragments (range 1-15 kDa). The degradation process is inhibited in diabetic nephropathy in proportion to the level of albuminuria detected by RIA.

This study examined whether the prevention of diabetes-related albuminuria by aminoguanidine (AG) or ramipril (RAM) may be mediated by a common post-glomerular basement membrane renal intracellular mechanism involving protein kinase C (PKC). The renal handling of albumin was examined over 24 weeks in control and streptozotocin (STZ)-induced diabetic rats. A radioimmunoassay (RIA) that measures intact albumin, and intravenously injected tritium-labeled rat serum albumin, was used to assess the proportion of intact albumin and albumin fragments in urine. Diabetes was induced in male Sprague-Dawley rats by the intravenous administration of STZ at a dose of 50 mg/kg. Age-matched control rats received buffer alone. Diabetes was characterized by an increase in blood glucose (>15 mmol/l), an increase in GHb (means at 24 weeks 29.3+/-1.1%; control 6.1+/-0.1%, P<0.005), an increase in glomerular filtration rate (GFR) (4.13+/-0.15 ml/min; control 3.54+/-0.19 ml/min, P<0.005), an increase in intact albumin excretion rate (expressed as geometric mean 11.64 times/divided by 2.11 mg/24 h; control 0.74 times/divided by 1.57 mg/24 h, P<0.005) as measured by RIA, and an increase in glomerular PKC activity (26.83+/-2.38 pmol x mg(-1) x min(-1); control 14.6+/-2.99 pmol x mg(-1) x min(-1), P<0.005). Treatment of diabetic rats with either AG or RAM prevented the rise in intact albuminuria and glomerular PKC activity. Renal lysosomal cathepsin activity decreased in diabetic rats and this was not prevented by AG or RAM. Neither drug affected glycemic control or GFR, but RAM reduced systolic blood pressure (BP), whereas AG did not. These data indicate that urinary excretion of intact albumin and albumin-derived fragments in diabetes may be modulated independently of glycemic control (AG and RAM) and systolic BP (RAM). While both drugs are known for their different mechanisms of action, the fact that both prevent diabetes-related increases in glomerular PKC activity and albuminuria supports the hypothesis that PKC plays a central role in the development of diabetic nephropathy.

Background: Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high, and this has been confirmed in studies in which albumin uptake by the tubules has been inhibited. Therefore, there must be a high-capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply., Methods: This study identifies the presence of glomerular-filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium-labeled albumin into the renal artery in vivo and in the isolated perfused kidney., Results: The postglomerular filtered albumin is returned to the blood supply by a high-capacity pathway that transports this albumin at a rate of 1830 +/- 292 micrograms/min.rat kidney (N = 14, mean +/- SEM). This pathway has been identified under physiological conditions in vivo and in the isolated perfused kidney. The pathway is specific for albumin, as it does not occur for horseradish peroxidase. The pathway is inhibited in a nonfiltering kidney. The pathway is also inhibited by ammonium chloride (an agent that inhibits tubular protein uptake but does not alter glomerular size selectivity) and by albumin peptides (which compete for the tubular albumin receptor)., Conclusions: The high-capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Background: The purported existence of "large pores" in the glomerular capillary wall has been derived primarily from studies using dextrans and Ficolls. Systematic studies using high molecular weight proteins have not been performed. One of the difficulties is that recent studies have demonstrated that albumin and other proteins undergo degradation during renal passage. Our study took into account this renal degradation in measuring the fractional clearance of various high molecular weight proteins (the hydrodynamic radii range was between 48 to 70 A)., Methods: Fractional clearances of tritium-labeled proteins were measured using ALZET osmotic pumps, which are designed to release a slow continuous infusion of tracer. Blood and urine collections were taken at 24-hour intervals over seven days and were counted for radioactivity, and glomerular filtration rate was measured by a creatinine assay., Results: Steady-state levels of [3H]protein in plasma were obtained by day 6. The [3H]proteins in the plasma showed no degradation. The fractional clearances (mean +/- sd, N = 5) of the various proteins were albumin (radius = 36 A; 0.0023 +/- 0.0009), transferrin (48 A; 0.0046 +/- 0.0007), lactoperoxidase (58 A; 0. 0045 +/- 0.0005), immunoglobulin G (62 A; 0.0043 +/- 0.0009), lactate dehydrogenase (64 A; 0.0041 +/- 0.0009), and glucose oxidase (70 A; 0.0036 +/- 0.0011)., Conclusions: These values suggest a weak dependence of fractional clearance on size-selective filtration, except for albumin, which undergoes a specific type of postglomerular processing. The fractional clearances were higher than expected from previous data on dextrans and Ficolls of equivalent hydrodynamic radius, and thus demonstrate that "large pores" may already exist in normal glomerular capillary walls.

Studies in isolated perfused rat kidney have demonstrated that it exhibits apparently normal charge selectivity and tubular uptake of anionic horseradish peroxidase (aHRP; pI < 4.0) and neutral horseradish peroxidase (nHRP; pI = 7.5) when these proteins are measured for their enzyme activity. The absolute fractional clearance values for aHRP and nHRP were 0.006 +/- 0.002 and 0.041 +/- 0.007, respectively. It is evident, however, that the enzyme assay for horseradish peroxidase severely underestimates the quantity of protein in urine as compared to measurement of its tritium labeled form through radioactivity. Fractional clearances estimated by radioactivity and corrected for tubular reabsorption for [3H]aHRP and [3H]nHRP were 0.040 +/- 0.029 and 0.099 +/- 0.043, respectively, compared to those estimated by enzyme activity which were 0.012 +/- 0.004 and 0.070 +/- 0.037, respectively. While charge selectivity between the anionic and neutral forms of HRP was still evident, albeit significantly reduced, the major feature of this type of analysis is that the clearance of the aHRP protein is significantly increased compared to that determined by enzyme assay. This difference correlates with the observation that the aHRP protein is markedly degraded (61 to 65%), as determined by gel chromatography, during filtration. Similar degradation was seen in urine fractions collected after the aHRP protein was administered in vivo. Degradation also occurred for the nHRP protein in both the perfused kidney and in vivo but to a far lesser extent (approximately 14 to 21%). These studies demonstrate that the anionic form of HRP was preferentially degraded during filtration and that charge selectivity for differently charged proteins is not as marked as originally thought.

The binding of albumin to the glomerular capillary wall was studied using albumin-gold in perfused kidneys, the interaction of [3H]albumin with isolated glomeruli at 37 degrees C and 4 degrees C and the interaction at [3H]albumin with purified basement membrane. The albumin-gold was found to bind predominantly to the basement membrane and this interaction could be dissociated with high concentrations of albumin. There was binding of albumin to isolated rat glomeruli which exhibited temperature dependence. Glomeruli exhibited a binding site at both 37 degrees C and 4 degrees C with an association constant in the range of 1 to 3 x 10(4) M-1 that bound 7 x 10(13) molecules/glomerulus. At 37 degrees C, however, there was anomalous Scatchard binding behaviour at relatively higher concentrations of albumin (30 to 50 mg/ml) which could be due to either glomerular cell uptake or the appearance of multiple binding sites or both. The binding of albumin to isolated glomeruli and the glomerular albumin levels in isolated kidney perfusion could largely be accounted for by the binding of albumin to the glomerular basement membrane. The albumin binding to glomeruli at 37 degrees C was enhanced by Pronase digestion and heparinase digestion, but remained unchanged following trypsin treatment or neuraminidase treatment. Similarly, albumin was shown to bind to purified basement membrane preparations. This binding was also enhanced (approximately 80 times) by heparinase digestion but remained unchanged after digestion with chondroitinase ABC or hyaluronidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Dextran sulphate is often used as a model for albumin in understanding capillary-tissue exchange and the charge selective nature of the capillary wall, particularly in kidney ultrafiltration. In investigating the mechanism of transport in the kidney, autoradiographic analysis of the distribution of iodinated dextran sulphate in perfused rat kidneys demonstrates the preferential accumulation of the probe in the glomerular capillary wall. Tritium-labeled dextran sulphate is found to be specifically taken up by intracellular 20 to 40 nm vesicles that can be isolated post-perfusion. The molecular weight profile of vesicular dextran sulphate demonstrates that the dextran sulphate containing vesicles are from vascular glomerular cells. Dextran is not taken up by the vesicles. These results suggest that the apparent charge selectivity associated with the transglomerular transport of the dextran sulphate is associated, in part, with cell-mediated processes at the glomerular level.

The renal clearance of [3H]dextran sulphate by the isolated perfused rat kidney was associated with desulphation of the molecule, as demonstrated by ion-exchange and affinity chromatography of material resident in both glomeruli and urine samples. This process also occurred in vivo. The molecular size distribution of glomerular dextran sulphate in the perfused kidney was indistinguishable from that in the perfusate, and although urinary material was smaller it remained macromolecular. Sulphatase activity was not detected in urine or in the perfusate of perfused kidneys, but was detected in glomerular and non-glomerular cortex fractions isolated by a sieving procedure. The identification of significant biochemical changes to dextran sulphate demonstrates that it does not function as an inert transport probe, and supports the concept of cellular involvement in the process of renal charge selectivity.

Estimates of levels of glomerular and glomerular-basement-membrane anion charge should serve as useful quantitative markers for the integrity of the tissues in health and disease. We have developed a simple, rapid, technique to measure this charge through the use of ion exchange with radioisotopes 22Na+ and 36Cl- at low ionic strengths in phosphate buffer. When this technique is used, normal glomeruli isolated from rat have a measured net anion charge concentration of 17.4 +/- 3.7 p-equiv. per glomerulus (n = 20). Perfused rat kidneys that lose approximately half of their glomerular heparan [35S]sulphate content (owing to oxygen-radical damage) exhibited a lower anion charge, of 7.5 +/- 1.6 p-equiv. per glomerulus (n = 5). Glomerular basement membranes prepared from rat glomeruli by a sonication-centrifugation procedure in the presence of enzyme inhibitors had a charge concentration of 6.3 +/- 0.7 mu-equiv./g wet wt. of tissue (n = 4), whereas membranes prepared by sonication, centrifugation, DNAse and detergent treatment had a charge concentration of 7.1 +/- 1.6 mu-equiv./g wet wt. (n = 4). Isotope-dilution experiments with 3H2O on these detergent-prepared glomerular basement membranes demonstrated that they had a water content of approx. 93%, which would then give a net anion charge concentration of 7.6 +/- 1.7 m-equiv./l (n = 4). These values are in good agreement with those obtained by others using titration techniques [Bray and Robinson (1984) Kidney Int. 25, 527-533]. The relatively low magnitude of glomerular anion charge in normal kidneys is consistent with other recent findings that glomerular anion charge is too low to affect the glomerular transport of charged molecules in a direct, passive, biophysical manner through electrostatic interactions.

The hydrodynamic frictional resistance to water flow exerted by articular cartilage proteoglycan is shown to be similar to that of proteoglycan isolated from Swarm rat chondrosarcoma, and independent of the state of aggregation of the proteoglycan. Frictional resistance is dependent, however, on the chain segments of the constituent chondroitin-sulphate and keratan-sulphate chains of the proteoglycan. Frictional resistance offered by chondroitin sulphate was independent of pH over the range 3.2-8.7. This confirms previous studies, associated with varying ionic strength and chemical modification of ionic groups of chondroitin sulphate, which showed that the frictional resistance offered by this molecule is independent of electrostatic factors. Water-structure-breaking and hydrogen-bond-breaking solvents were also without major effects on the flow resistance offered by chondroitin sulphate. An overall secondary structure of chondroitin sulphate was not evident, as it showed no significant difference to dextran in terms of its temperature dependence of relative viscosity. Local regions of rigid secondary structure, as manifested through inter-residue hydrogen bonding between sugar residues, is likely to control flow resistance as periodate-oxidized chondroitin sulphate and periodate-oxidized and reduced preparations showed a significant decrease in their frictional resistance to water.

The role of 'generic' physical mechanisms in morphogenesis and pattern formation of tissues is considered. Generic mechanisms are defined as those physical processes that are broadly applicable to living and non-living systems, such as adhesion, surface tension and gravitational effects, viscosity, phase separation, convection and reaction-diffusion coupling. They are contrasted with 'genetic' mechanisms, a term reserved for highly evolved, machine-like, biomolecular processes. Generic mechanisms acting upon living tissues are capable of giving rise to morphogenetic rearrangements of cytoplasmic, tissue and extracellular matrix components, sometimes leading to 'microfingers', and to chemical waves or stripes. We suggest that many morphogenetic and patterning effects are the inevitable outcome of recognized physical properties of tissues, and that generic physical mechanisms that act on these properties are complementary to, and interdependent with genetic mechanisms. We also suggest that major morphological reorganizations in phylogenetic lineages may arise by the action of generic physical mechanisms on developing embryos. Subsequent evolution of genetic mechanisms could stabilize and refine developmental outcomes originally guided by generic effects.

The major hydrodynamic properties of the connective-tissue polysaccharides are those that describe polysaccharide-water interaction as embodied in their osmotic-pressure and hydraulic-conductivity properties. This study shows that, for polysaccharides such as chondroitin sulphate, hyaluronate and the heparin-like polysaccharides, their hydrodynamic properties depend primarily on the presence of the uronic residue and the nature of the glycosidic linkage. Other parameters such as the degree of N-acetylation and sulphation were found not to influence these properties to any great extent. These studies particularly delineate structural-functional aspects of the connective-tissue polysaccharides in terms of their primary structure.

Diffusion in multicomponent solutions containing proteoglycan is shown to result in the formation of coherent, fluid structures (known as dissipative structures) and induction of rapid polymer transport. These phenomena occur over a wide range of conditions (i.e. varying solute distribution, concentration, size, and chemical composition) which are envisaged to occur in the extracellular matrix of connective tissues. A concentration gradient of chondroitin sulfate in a proteoglycan matrix of uniform concentration yields dissipative structures which transport the proteoglycan up to 300-fold faster than its transport in the absence of the gradient component. Similar behavior was observed with other polysaccharide and monosaccharide concentration gradient components. Amplification of structure formation and rapid transport was achieved by 1) increasing the concentration of proteoglycan matrix, 2) increasing the magnitude of the concentration gradient, 3) decreasing the molecular weight of the gradient-forming component, and 4) decreasing the concentration gradient of proteoglycan in the matrix. Dissipative structure morphology exhibits a marked dependence on the initial component distribution. Non-specific, excluded volume interactions between the proteoglycan and the gradient component are believed to induce coupled diffusive transport of the proteoglycan. This leads to microscopic density inversions which nucleate and develop into macroscopic convective flows. These results are similar to those previously observed in ternary solutions of uncharged polymers (i.e. dextran/polyvinylpyrrolidone). We have demonstrated that dissipative structures may transport Micrococcus luteus cells as well as various solutes. Flows were also observed in proteoglycan solutions after localized addition of small amounts of either a proteolytic enzyme or hyaluronic acid. It is likely that the prerequisites for this spontaneous macroscopic self-organization, as manifested by the flow phenomenon, are present in the extracellular matrix of connective tissues.

The dynamics of water transport in proteoglycan compartments has been studied in relation to osmotic flow (proteoglycan diffusion) and hydraulic permeability (proteoglycan sedimentation) in concentrated solutions of proteoglycan subunit and native proteoglycan aggregate isolated from Swarm rat chondrosarcoma. A central parameter that describes the kinetics of both types of water movement is the hydrodynamic frictional coefficient of water with proteoglycan. The frictional coefficient is markedly concentration dependent, increasing with increasing concentration, and highlights important structural features and types of organization of the proteoglycans in concentrated solutions. These include the requirements that proteoglycans in the extracellular matrix not to be immobilized but to have translational diffusive mobility and concentration gradients to be osmotically active, that chondroitin sulfate segmental mobility describing translational motion largely determines osmotic flow and hydraulic permeability of the proteoglycans, and that the proteoglycans exhibit an enhanced ability to resist flow as compared to other macromolecules. Additional dynamic studies suggest the formation of transient super-aggregate structures may occur at high concentrations which endows the proteoglycan subunit hydrodynamic properties similar to proteoglycan aggregate.

It is demonstrated that exclusion phenomena appear to dominate the interaction of dextran with albumin in aqueous solution. The enthalpic contribution to the interaction coefficient describing dextran/albumin mixtures is small, although its determination was subject to considerable error. These results support the earlier assumptions of the type of interaction between the two polymers. The conclusions are primarily based on the interpretation of the temperature-dependence of the interaction coefficient, as measured by light-scattering in the temperature range 6--33 degrees C. Enthalpy of dilution measurements of dextran/albumin mixtures by microcalorimetry were in qualitative agreement with the light-scattering data.

The osmotic pressure of solutions of sulphated proteoglycans isolated from the intervertebral discs of animals of various ages was determined. The behaviour of the solutions in salt-added systems was investigated in terms of the Donnan distribution of the mobile ions. It is evident that this effect is the dominating factor in explaining the observed nonidealities. Although marked variations in the compositions of the proteoglycan, with regard to their chondroitin sulphate and keratan sulphate content and hence charge content, occur with increasing age of parent tissue, the osmotic activities of the various preparations are very similar. This is explained by the ;fixation' of the counterions in such a way as to counteract any change in the charge content of the polyion; an ;osmotic buffering' effect. The swelling behaviour of gelatin gels containing the proteoglycan preparations has been measured. In all cases pressures in excess of the sum of the osmotic pressures of the individual components are observed. However, the magnitude of the excess decreases with increasing age of the parent tissue. It is suggested that the age changes, as reflected by a decrease in water content of the gel system, are not the result of changes in the osmotic properties of the individual components but rather reflect changes in the entropic interaction of the proteoglycan with the gelatin matrix. The relevance of this observation to the situation in vivo is discussed.