High Molecular Weight Proteinuria Research Articles

Electrophoretic patterns of proteinuria in feline spontaneous chronic kidney disease.

The purpose of this study was to describe the electrophoretic patterns of proteinuria in cats at risk of and cats with chronic kidney disease (CKD), and to investigate whether the presence of high-molecular-weight (HMW) and low-molecular-weight (LMW) proteins were associated with CKD, proteinuria and/or disease progression. Healthy cats at risk of developing renal disease (n = 17) and cats affected with CKD at different stages (n = 22) were prospectively enrolled and sampled over time. Seventy urine samples were included and assayed with a commercially available sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) method. Each sample (gel lane) was inspected to identify albumin, HMW and LMW proteins, and an electrophoretic pattern (albuminuria, glomerular, tubular, mixed or negative) was assigned accordingly. Fisher's exact test was used to assess the distribution of HMW and LMW proteins in cats grouped according to International Renal Interest Society stage and to the magnitude of proteinuria, and to assess if HMW and LMW proteins at the time of inclusion were associated with the development and progression of CKD. In samples of cats at risk, the most common pattern was glomerular (84.6%); glomerular pattern was also common in cats with CKD (54.2%), although mixed proteinuria and tubular proteinuria were also present (29.5% and 11.4%, respectively). The presence of LMW proteins was associated with CKD (P <0.0001) and to a urine protein:creatinine ratio >0.2 (P = 0.025). Both HMW and LMW proteins were not associated with progression of CKD within 6 months (n = 14). Our results showed that HMW proteinuria is common in healthy cats at risk of developing CKD, although the pathological significance needs to be confirmed. The detection of LMW proteins in urine of cats suspected to be affected by CKD, especially in non-azotaemic, non-proteinuric or borderline proteinuric cats, suggests the presence of kidney damage.

Poster #M134 ASSOCIATION OF IMMUNE GENE POLYMORPHISMS WITH SCHIZOPHRENIA IN A NORTH INDIAN COHORT

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3−/−/ITGA2−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3−/−/ITGA2+/− and COL4A3−/−/ITGA2+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3−/−/ITGA2+/+ Alport mice, but not in COL4A3+/+/ITGA2−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.

Collagen receptors integrin alpha2beta1 and discoidin domain receptor 1 regulate maturation of the glomerular basement membrane and loss of integrin alpha2beta1 delays kidney fibrosis in COL4A3 knockout mice

Maturation of the glomerular basement membrane (GBM) is essential for maintaining the integrity of the renal filtration barrier. Impaired maturation causes proteinuria and renal fibrosis in the type IV collagen disease Alport syndrome. This study evaluates the role of collagen receptors in maturation of the GBM, matrix accumulation and renal fibrosis by using mice deficient for discoidin domain receptor 1 (DDR1), integrin subunit α2 (ITGA2), and type IV collagen α3 (COL4A3). Loss of both collagen receptors DDR1 and integrin α2β1 delays maturation of the GBM: due to a porous GBM filtration barrier high molecular weight proteinuria that more than doubles between day 60 and day 100. Thereafter, maturation of the GBM causes proteinuria to drop down to one tenth until day 200. Proteinuria and the porous GBM cause accumulation of glomerular and tubulointerstitial matrix, which both decrease significantly after GBM-maturation until day 250. In parallel, in a disease with impaired GBM-maturation such as Alport syndrome, loss of integrin α2β1 positively delays renal fibrosis: COL4A3−/−/ITGA2−/− double knockouts exhibited reduced proteinuria and urea nitrogen compared to COL4A3−/−/ITGA2+/− and COL4A3−/−/ITGA2+/+ mice. The double knockouts lived 20% longer and showed less glomerular and tubulointerstitial extracellular matrix deposition than the COL4A3−/− Alport mice with normal integrin α2β1 expression. Electron microscopy illustrated improvements in the glomerular basement membrane structure. MMP2, MMP9, MMP12 and TIMP1 were expressed at significantly higher levels (compared to wild-type mice) in COL4A3−/−/ITGA2+/+ Alport mice, but not in COL4A3+/+/ITGA2−/− mice. In conclusion, the collagen receptors DDR1 and integrin α2β1 contribute to regulate GBM-maturation and to control matrix accumulation. As demonstrated in the type IV collagen disease Alport syndrome, glomerular cell–matrix interactions via collagen receptors play an important role in the progression of renal fibrosis.

Proteinuria Is Reduced by Inhibition of Inducible Nitric Oxide Synthase in Rat Renal Ischemia-Reperfusion Injury

Ischemia-reperfusion (IR)-induced nephrotoxicity is associated with proteinuria. There are reports on the involvement of inducible nitric oxide synthase (iNOS) in proteinuria in conjunction with renal disease. This study was designed to investigate the effect of N6-(1-iminoethyl)-L-lysine hydrochloride (L-Nil), a selective inhibitor of iNOS, to prevent proteinuria in IR injury. Ischemia was induced by 40-minute clamping of the renal arteries followed by 6-hour reperfusion. Rats were administered either L-Nil (3 mg/kg intravenous bolus followed by infusion of 1 mg/kg/h) or saline. To monitor glomerular and tubular functional changes before and after treatment, we measured blood urea nitrogen, plasma creatinine, and urinary N-acetyl-beta-D-glucosaminidase activity. Total protein (TP), albumin, and low- (LMW) and high-molecular-weight (HMW) protein excretion rates were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis of urine samples. Kidney ultrastructure was examined through a transmission electron microscope (TEM). IR resulted in significant LMW and HMW proteinuria. L-Nil significantly prevented the IR-induced increases in TP, albumin, and alpha1-microglobulin excretion. TEM showed loss of microvilli of the proximal tubule cells, injured mitochondria, and foamy changes in the structure of nuclear and cytoplasm in IR group. L-Nil reduced IR-mediated renal ultrastructural changes and tubular proteinuria. This study suggested possible differences in the mechanism(s) of nephrotoxicity induced by iNOS in the glomeruli and tubular cells. The types of proteins excreted in the urine should be considered in the treatment strategy. In conclusion, this study suggested the involvement of iNOS in IR-induced tubular proteinuria.

Time course of proteinuria after living-donor kidney transplantation.

After cadaveric kidney transplantation, preservation-reperfusion damage results in glomerular and tubular proteinuria. There are no data on the time course of proteinuria after living-donor (LD) transplantation. In 10 patients receiving a kidney graft from an LD, the excretion of high molecular weight proteins (albumin, transferrin, and immunoglobulin G) and low molecular weight proteins (beta2-microglobulin and alpha1-microglobulin) was measured at various time points during the first 5 days after transplantation. Immediately after restoration of the circulation, we observed a massive nonselective high molecular weight proteinuria, indicative of glomerular damage. This proteinuria rapidly decreased to slightly elevated values beyond 24 hr after transplantation. Low molecular weight proteinuria, reflecting tubular damage, was also prominent and remained grossly abnormal even at day 5. After LD transplantation, preservation-reperfusion injury causes massive proteinuria during the first 24 hr. Thereafter proteinuria rarely exceeds 1 g per day.

Organic Solvents and the Kidney

Organic Solvents and the Kidney: Antonio Mum. Laboratory of industrial Toxicology, University of Parma Medical School—The association between solvent exposure and chronic glomerulonephritis is still a controversial scientific issue. Almost all published case‐referent studies consistently show an association between chronic glomerulonephritis and exposure to volatile hydrocarbons. Two nested prospective studies and a recent case‐referent study also suggest a role of solvent exposure in the progression of renal changes towards chronic renal failure. Cross‐sectional studies carried out in groups of workers exposed to relatively low concentrations of single solvents or mixtures demonstrate tubular damage frequently associated with high molecular weight proteinuria and changes in the glomerular basal membrane. In some studies, immunological disturbances (increased titer of antilaminin and/or anti‐glomerular basement membrane antibodies) have been found. Whereas unequivocal evidence of solvent‐induced renal disturbances is apparent on a group basis, the health significance of such changes is difficult to assess at the individual level. Although renal cancer in the male rat chronically exposed to a variety of hydrocarbons including several organic solvents is not a good model for assessing human risk, experimental studies may not be used as a proof to exclude the cancerogenic properties of all organic solvents. Indeed, epidemiological investigations suggest that a small excess mortality from renal‐cell cancer may occur as a consequence of exposure to some organic solvents, including dry‐cleaning products and petroleum‐derived hydrocarbons. Despite consistent results indicating renal effects of solvent exposure, no specific agents appeared to be implied more frequently, neither dose‐ effect nor dose response relationships were found and, finally, possible risk factors accounting for an increased individual susceptibility were not identified. Early markers of renal damage are very useful to identify groups at risk, since these subtle abnormalities may represent an early stage of clinically silent but potentially progressive renal diseases. Follow‐up should confirm the observed changes and assess their course and prognostic meaning. Repeatedly positive markers of renal damage also indicate the need for intervention, including job counseling and removal from exposure.

Nephropathies and exposure to perchloroethylene in dry-cleaners

Even in specific risk groups, the relation between exposure to organic solvents and chronic renal diseases remains controversial. Thus, in a collaborative European study, we assessed the renal effects of occupational exposure to perchloroethylene (PCE) in dry-cleaners compared with matched controls who were simultaneously examined. Single high and low molecular weight proteins, kidney-derived antigens and enzymes, and prostanoids were measured in urine. β 2-microglobulin, creatinine, laminin fragments, and anti-glomerular basement membrane antibodies were also measured in serum. A canonical function based on 23 such variables correctly classified 93% of individuals as either PCE-exposed or controls; with 13 markers, group membership was identified in 87% of subjects. Increased high molecular weight protein in urine was frequently (17/50 vs 1/50, p<0·0001) associated with tubular alterations. Changes were consistent with diffuse abnormalities along the nephron in workers exposed to low levels of PCE (median 15 parts per million). Generalised membrane disturbances might account for the increased release of laminin fragments, fibronectin, and glycosaminoglycans, for high molecular weight proteinuria, and for the increased shedding of epithelial membrane components from tubular cells with different location along the nephron (brush-border antigens and Tamm-Horsfall glycoprotein). These findings of early renal changes indicate that solvent-exposed subjects, especially dry-cleaners, need to be monitored for the possible development of chronic renal diseases.

Early detection of the nephrotoxic effects of industrial chemicals: state of the art and future prospects.

This paper discusses several tests that may permit the early detection of renal changes induced by long-term exposure to nephrotoxic industrial chemicals and may possibly serve as advance warning of pending renal damage. Some tests mainly attempt to assess the integrity of the glomerulus: high molecular weight proteinuria, glomerular basement membrane (GBM) antigens in blood and in urine, circulating anti-GBM antibodies, glomerular filtration rate after an acute oral load of proteins, and estimation of membrane negative charges (ie, glomerular polyanion). Others mainly attempt to identify functional and/or morphological changes at the tubular level: low molecular weight proteinuria, aminoaciduria, glucosuria, hyperphosphaturia, hypercalciuria, enzymuria, tubular antigen excretion, kallikrein, and prostaglandin excretion. Some of these tests are already routinely used, although controversy may still persist with regard to their clinical significance. Recently, new tests have been developed that may open new perspectives for assessing the significance of the early renal changes induced by chemicals.

An evaluation of renal function in workers occupationally exposed to mercury vapour.

The renal function of a population of workers occupationally exposed to mercury in the chlor-alkali industry has been examined and compared to that of a population of workers with no occupational exposure to mercury. Measurement of specific urinary proteins and enzymes have been carried out on each individual on three separate occasions and have been complemented by blood plasma measurements at the final visit. Under the conditions of exposure to mercury sustained in this study, there is no evidence of an increased prevalence of renal dysfunction as indicated by enzyme and protein measurements. The urinary concentration of the low molecular weight protein, beta 2-microglobulin, is significantly lower in the mercury-exposed group than in the control group. In contrast to recently published literature, no relationship is seen between urinary mercury concentration and the appearance of high molecular weight protein in urine. A small increase in the prevalence of higher activities of the urinary enzyme N-acetyl-beta-glucosaminidase and gamma glutamyl transferase is observed when the urinary mercury concentration exceeds 100 micrograms/g creatinine. A small increase in the prevalence of raised urinary N-acetyl-beta-glucosaminidase activity is observed when the duration of exposure to mercury exceeds ten years. The pattern of proteinuria has been characterised in a total of sixteen individuals from both populations; a low molecular weight proteinuria is seen in three individuals from the control group whilst a high molecular weight proteinuria is seen in the remainder (seven in the control and six in the mercury group).

Further views on the basement membrane controversy.

Although it has been claimed often that increased basement membrane thickness is an important factor in diabetic nephropathy, there is no direct evidence that it is associated necessarily with increased permeability or elevated glomerular filtration rate [1] and it should be noted that both changes could follow increased hydrostatic pressure. Hydrostatic pressure and capillary wall structure were thought to be involved in the extravasation of plasma proteins in diabetic microangiopathy [2]. The pressure dependence of human skin capillary permeability was demonstrated by Landis more than 40 years ago [3]. More recently, increased filtration pressure was linked causally with the increased glomerular filtration rate and elevated filtration fraction of diabetic nephropathy [4]. On the other hand, Viberti [5] expressed the view that basement membrane thickness and permeability to plasma proteins were 'somewhat independent'. Recent accepted concepts of basement membrane permeability are based upon a pore theory [6] in which the basement membrane is considered as a semi-permeable membrane containing cylindrical pores of 3.54.2 nm radius, or rectilinear slits of 3.6 nm half width [7]. The permeability of the membrane is believed to be determined by the interaction of particles with the anionic charge barrier on the basement membrane and endothelium [8, 9]. Alternative concepts of basement membrane structure treat it either as a hydrated gel through which filtration occurs by diffusion [10]: or a 'felted fibril' concept in which increased permeability was explained by the slackening of the fibrils when hydrostatic pressure fell [11]. It is noteworthy that these concepts provide no explanation of how cells or larger motile parasite larvae pass through basement membranes without leaving evidence of their passage. In 1980 a new concept was put forward that basement membranes are thixotropic gels which are capable of undergoing localised, reversible deformation under the effect of pressure [12]. Such gels would have the dispersed phase disposed as an irregular lattice which contained the dispersion medium (water). Solutions and small particles of about 2 nm radius or less would pass freely through the lattice, but larger particles would be able to traverse the membrane only if they had sufficient energy to deform the lattice, which reformed after the passage of the particles. In a recent study by Ota et al. [13] ultra-high magnification electron microscopy of unfixed glomerular basement membrane has revealed a sponge-like lattice without large pores or fibrils but which had interstices of similar size to that proposed in the thixotropic gel model [12]. Technical difficulties have prevented direct assessment of pressure-permeability relationships of basement membranes although indirect observations provide support for the idea that basement membranes are pressure dependent. Stolte et al. showed a straight line relationship between pressure and the amount of albumin filtered in perfused rat kidneys [14]. Mice with spontaneous lupus nephritis developed high molecular weight proteinuria in association with elevated systolic blood pressure [15]. Mogensen found that the use of antihypertensive drugs inhibited the progression of diabetic nephropathy as interpreted by altered glomerular filtration rate in five out of six patients [16]. In serial studies of diabetic urines, we have observed that some samples contained a range of plasma proteins, demonstrable by the use of sodium dodecyl sulphate polyacrylamide gel electrophoresis. Similar urinary protein profiles have been found in other types of human nephropathy, in murine lupus nephritis and in exercise proteinuria induced in subjects with normal renal function [unpublished observations]. The similarity of the protein profiles may be