The binding of beta-2-microglobulin to renal brush-border membrane: affinity measurement, inhibition by serum albumin

In the kidney, filtered proteins are rapidly reabsorbed so that the final excretion is less than 0.1% of the filtered amount for low molecular weight proteins such as beta 2-microglobulin and a few percent for albumin. In order to investigate the affinity of proteins for luminal membranes, rat renal brush-border membranes were incubated with 125I-labelled human beta 2-microglobulin and the initial binding rate determined by the filtration method. Scatchard plot analysis of binding rate revealed two types of binding sites: one with Km = 0.25.10(-6) M and Vmax = 0.1 nmol/min per mg protein and another with Km = 1.10(-5) M and Vmax = 1.3 nmol/min per mg protein. The lower affinity type is likely to represent non-specific binding the physiological role of which is to be discussed. The higher affinity sites seem to play the major role in binding rate. beta 2-Microglobulin initial binding is reversible, and inhibited by bovine serum albumin. Comparison of the time course of bound beta 2-microglobulin removal by unlabelled beta 2-microglobulin and by albumin suggests that these two proteins have a different internalization mechanism.     

Evaluation of selected indicators of the renal proximal tubule function in patients treated with gentamicin]

The study aimed at evaluating proximal renal tubule function in patients with nephrolithiasis and chronic pyelonephritis, and in patients with infectious diseases treated with gentamicin. The study involved 2 groups of patients: group A--17 patients with nephrolithiasis and chronic pyelonephritis and group B--30 patients with other infectious diseases (pneumonia, biliary tract infections) but with normal glomerular filtration rate. Patients from both groups were treated with gentamicin in a daily dose of 2-3 mg/kg for 7-10 days. Serum and urine creatinine levels were assayed in all patients prior to, 2-3, 7, 10 days, and after the treatment. Patients assigned to group B were divided into two subgroups: B1 included 15 patients with normal beta 2-microglobulinuria, and B2 15 patients with increased renal loss of beta 2-microglobulin and decreased tubular reabsorption of this protein. Significant increase in beta 2-microglobulinuria was seen on the third day of therapy, the decrease in the tubular reabsorption and glomerular filtration rate were noted in all patients on the seventh day of gentamicin administration. Beta 2-microglobulinuria was significantly higher in patients from groups A and B2 in comparison with group B1 in which no dysfunction of the proximal renal tubule was present before gentamicin therapy. A degree of beta 2-microglobulinuria is an early and sensitive indicator of gentamicin nephrotoxicity. The risk of nephrotixic symptoms is particularly obvious in patients with deteriorated function of renal proximal tubuli before the treatment with gentamicin.     

Diurnal variations of serum beta 2-microglobulin in multiple myeloma

The chronobiological circadian behaviour in serum levels of beta 2-microglobulin has been investigated in three groups of subjects: (A) 6 healthy controls; (B) 6 patients with untreated multiple myeloma; (C) 6 patients with multiple myeloma in complete remission after polychemotherapy. From all subjects, under the same standard life conditions, venous blood samples were drawn at 4-hour intervals starting from midnight during the span of a whole day. Circulating serum beta 2-microglobulin levels were determined by RIA method. The time-related data were analyzed by chronograms and the "mean-group cosinor" method. A significant circadian rhythm for serum beta 2-microglobulin was detected in the control group, with a peak in the morning hours, and in untreated patients, with a peak in the afternoon hours. No significant rhythm was found in treated patients with multiple myeloma. A significant mesor reduction was noted in patients with complete remission, correlated with the absence of circadian rhythm, in respect to untreated patients. These data suggest that serum levels of beta 2-microglobulin could be related to the neoplastic plasma cell proliferation and to the effect of therapy, and that the circadian evaluation could be used as a guide in monitoring myeloma patients.     

Beta 2-microglobulinaemia: a sensitive index of diminishing renal function in diabetics.

A sensitive single measure of diminishing renal function is of importance in attempts to modify the progression of diabetic nephropathy. In 12 insulin-dependent diabetics with proteinuria plasma concentrations of beta 2-microglobulin were found to correlate more closely than plasma creatinine concentrations or creatinine clearance with glomerular function as measured by clearance of 52Cr-EDTA. The plasma beta 2-microglobulin concentration was raised in all patients with diminished glomerular filtration rate (below 80 ml/min/1.73 m2). By contrast, in two of these patients plasma creatinine concentration was normal. Plasma beta 2-microglobulin concentrations were stable throughout the day and not affected by food intake, unlike plasma creatinine concentrations, which rose in the afternoon and evening and after a meat meal. Plasma beta 2-microglobulin concentrations were the same in venous and capillary blood, the capillary blood being readily self-collected. Concentrations of beta 2-microglobulin were stable for up to 24 hours when whole blood was stored at 4 degrees C; adding aprotinin inhibited loss of beta 2-microglobulin for up to seven days. The results of this study suggest, therefore, that measuring beta 2-microglobulin concentrations is a simple and accurate method of detecting minor degrees of renal impairment and monitoring the effects of treatment.     

Measurement of thyroid stimulating hormone (TSH) in human urine

Using a highly sensitive and specific immunoradiometric assay kit for human TSH, we measured TSH concentrations in unprocessed urines in normal subjects, in patients with primary hypothyroidism, and patients with renal disease. In five of ten normal subjects TSH was detectable in urine samples (less than 20-69 microU/day). In five patients with hypothyroidism, the urinary TSH excretion was increased. In seven out of ten patients with nephrotic syndrome, eight out of nine patients with chronic renal failure and two patients with tubular dysfunction, the urinary TSH excretion was increased. The urinary TSH excretion correlated significantly with both urinary protein excretion and urinary beta 2-microglobulin excretion. These results suggest that the renal handling of TSH involves both glomerular filtration and tubular re-absorption, and that urinary TSH excretion is increased when serum TSH is increased and either glomerular or tubular function is impaired.     

Evidence for a secretory component in the handling of unconjugated bilirubin by the isolated perfused rat kidney

Previous studies in rats have suggested that the urinary excretion of unconjugated bilirubin (UB) comprises only a small fraction of the pigment that reaches the tubular lumen by glomerular filtration and escapes from tubular cell reabsorption. However, additional data also indicated that UB interacts with renal peritubular cell membranes impairing the secretion of p-aminohippurate (PAH). In this study we examined the possibility of a secretory step which could also be involved in the renal excretory mechanism for UB. An isolated rat kidney preparation was used, and the uptake of UB by renal tissue, the UB appearance in the urine, and the secretion of PAH were analyzed throughout the perfusion. The results indicated that the UB urinary excretion rate changed independently of UB filtered load. The latter remained almost unchanged during the perfusion, whereas the excretion rate of UB and the UB-to-creatinine (Cr) clearance ratio increased significantly. Furthermore, a relationship between the uptake of UB by the kidney, the UB-to-Cr clearance ratio, and the decrease in PAH secretion rate, was proved. In addition, when probenecid was added to the perfusate solution the cumulative uptake of UB by the kidney and the rate of excretion of UB in the urine were diminished. We conclude that the mechanism of UB excretion by the kidney may be considered as the result of a process involving glomerular filtration plus tubular secretion followed by a back diffusion step from the lumen in a similar way to other endogenous compounds, thus explaining the virtual absence of UB from the normal urine.     

Reversibility of renal tubular dysfunction in streptozotocin-induced diabetes in the rat.

Enzymuria and specific proteinuria were examined over a period of 19 days in 4 groups of 5 rats: a control group, a nondiabetic polyuric group, a group of streptozotocin-induced diabetic rats treated with insulin as of the 10th day after the injection of the drug, and a similar group of untreated diabetic rats. Increased urinary excretion of beta-N-acetyl-D-glucosaminidase, lactate dehydrogenase, and alanine aminopeptidase was observed shortly after the induction of diabetes. It was partly or totally reversible following insulin treatment. Nondiabetic polyuria had a slight effect on the excretion of alanine aminopeptidase only. The urinary excretion of beta 2-microglobulin also rapidly increased after the onset of diabetes to a level approximately 50 times the control values. This effect was largely reversible with insulin treatment and was absent in the nondiabetic polyuric group. A small but significant 3-fold increase in albumin excretion was also noted but was not affected by insulin treatment. We conclude that streptozotocin-induced diabetes causes an early tubular dysfunction that is unrelated to polyuria and is reversible upon insulin treatment. This tubular dysfunction is best revealed by the urinary excretion of the low molecular weight protein beta 2-microglobulin. Our results suggest that it would be of interest to further examine the usefulness of sensitive markers of tubular dysfunction, especially low molecular weight proteinuria, in the detection of early stages of diabetic nephropathy.     

Beta(2)-microglobulin removal by extracorporeal renal replacement therapies

There is increasing evidence that end-stage renal disease patients with lower beta(2)-microglobulin plasma levels and patients on convective renal replacement therapy are at lower mortality risk. Therefore, an enhanced beta(2)-microglobulin removal by renal replacement procedures has to be regarded as a contribution to a more adequate dialysis therapy. In contrast to high-flux dialysis, low-flux hemodialysis is not qualified to eliminate substantial amounts of beta(2)-microglobulin. In hemodialysis using modern high-flux dialysis membranes, a beta(2)-microglobulin removal similar to that obtained in hemofiltration or hemodiafiltration can be achieved. Several of these high-flux membranes are protein-leaking, making them suitable only for hemodialysis due to a high albumin loss when used in more convective therapy procedures. On-line hemodiafiltration infusing large substitution fluid volumes represents the most efficient and innovative renal replacement therapy form. To maximize beta(2)-microglobulin removal, modifications of this procedure have been proposed. These modifications ensure safer operating conditions, such as mixed hemodiafiltration, or control albumin loss at maximum purification from beta(2)-microglobulin, such as mid-dilution hemodiafiltration, push/pull hemodiafiltration or programmed filtration. Whether these innovative hemodiafiltration options will become accepted in clinical routine use needs to be proven in future.     

Involvement of laminin binding integrins and laminin-5 in branching morphogenesis of the ureteric bud during kidney development.

Branching morphogenesis of the ureteric bud (UB) [induced by the metanephric mesenchyme (MM)] is necessary for normal kidney development. The role of integrins in this complex developmental process is not well understood. However, the recent advent of in vitro model systems to study branching of UB cells and isolated UB tissue makes possible a more detailed analysis of the integrins involved. We detected integrin subunits alpha3, alpha6, beta1, and beta4 in both the UB and cells derived from the early UB. Blocking the function of each of these integrin subunits individually markedly inhibited branching morphogenesis in cell culture models. However, inhibiting individual integrin function with blocking antibodies in whole kidney and isolated UB culture only partially inhibited UB branching morphogenesis, suggesting that, in these more complex in vitro systems, multiple integrins are involved in the branching program. In whole organ and isolated bud culture, marked retardation of UB branching was observed only when both alpha3 and alpha6 integrin subunits were inhibited. The alpha6 integrin subunit can be expressed as both alpha6beta1 and alpha6beta4, and both of these beta subunits are important for UB branching morphogenesis in both cell and organ culture. Furthermore, laminin-5, a common ligand for integrins alpha3beta1 and alpha6beta4, was detected in the developing UB and shown to be required for normal UB branching morphogenesis in whole embryonic kidney organ culture as well as isolated UB culture. Together, these data from UB cell culture, organ culture, and isolated UB culture models indicate that both integrin alpha3 and alpha6 subunits play a direct role in UB branching morphogenesis, as opposed to being modulators of the inductive effects of mesenchyme on UB development. Furthermore the data are consistent with a role for laminin-5, acting through its alpha3beta1 and/or alpha6beta4 integrin receptors, in UB branching during nephrogenesis. These data may help to partially explain the renal phenotype seen in integrin alpha3 and alpha3/alpha6 subunit-deficient animals.     

Mechanism of hypoalbuminemia in rodents

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

beta2-Microglobulins: isolation, properties, and distribution.

beta2-Microglobulin is structurally related to immunoglobulin domains and is identical to the light chain of histocompatibility (HL-A) antigens. Similar to free light chains of immunoglobulins, beta2-microglobulin is most easily isolated from urine. We have previously purified human beta2-microglobulin from urine of patients with renal tubular resorption defects. Corresponding proteins have now been obtained from urine of rabbits and guinea pigs treated with sodium chromate. Sequence studies have established that the rabbit protein is rabbit beta2-microglobulin. The guinea pig protein closely resembles the human and rabbit beta2-microglobulins in amino acid composition, charge, molecular size, and also in the presence of an apparently analogous disulfide loop. These findings indicate that this protein is the guinea pig homologue of beta2-microglobulin. Physical-chemical studies suggest that human beta2-microglobulin and isolated immunoglobulin domains are similar not only in amino acid sequence but also in three-dimensional structure. Both types of molecules are compact and globular in shape and apparently contain beta-pleated sheet conformation. beta2-Microglobulin is present in free form in various body fluids and as a subunit of histocompatibility antigens on cell surfaces. Current estimates suggest that the number of beta2-microglobulin molecules on cell surfaces is higher than the number of histocompatibility (HL-A) antigens. Accordingly, beta2-microglobulin is possibly a subunit of additional cellular antigens or receptors.     

Suppression of transforming growth factor-beta1 gene expression by Danggui buxue tang, a traditional Chinese herbal preparation, in retarding the progress of renal damage in streptozotocin-induced diabetic rats.

Danggui buxue tang (DBT), a preparation containing Angelica sinensis (danggui) and Astragalus membranaceus (huangqi) at a ratio of 1 : 5, is used widely in China for stimulating red blood cell production and enhancing cardiovascular function. The present study was undertaken to characterize the effects of this preparation on diabetic nephropathy using streptozotocin-diabetic rats as a model. Streptozotocin-dependent alterations in renal weight/body weight ratio, urinary albumin and beta (2)-microglobulin concentrations, urinary albumin excretion rate, and creatinine clearance were ameliorated after eight weeks of treatment with either DBT or the angiotensin-converting enzyme inhibitor, benazepril. DBT, but not benazepril, partially attenuated the increases in blood glucose, triglycerides and cholesterol in STZ-diabetic rats. Additionally, the increased expression of transforming growth factor-beta (1) mRNA in the renal cortex due to streptozotocin-induced diabetes was modestly attenuated by these treatments. However, eight weeks of treatment with DBT failed to modify the concentration of angiotensin II in plasma or kidney, indicating that the ability of the preparation to retard the progression of kidney disease was not attributable to inhibition of the renin-angiotensin system. We propose that DBT alleviates renal alterations in diabetes and slows the progression of diabetic nephropathy by suppressing transforming growth factor-beta (1) mRNA expression. The preparation may therefore be useful as an adjuvant therapy for controlling diabetes and its complications.     

Excretion of urinary cadmium, copper, and zinc in cadmium-exposed and nonexposed subjects, with special reference to urinary excretion of β2-microglobulin and metallothioneinand metallothionein

The objectives of this study were to examine the association between urinary excretion of cadmium (U-Cd), copper (U-Cu), and zinc (U-Zn) and the severity of two different indicators of renal toxicity (urinary excretion of beta2-microglobulin [U-beta2-MG] and metallothionein [U-MT]) in Cd-exposed subjects compared to controls, and to assess the physiologic mechanisms by which the exposure to environmental Cd affects U-Cd, U-Cu, and U-Zn. The target population included 3508 Cd-exposed and 294 nonexposed participants who received a health survey conducted among the population of the Kakehashi River basin. Increases of U-Cd, U-beta2-MG, and U-MT in the Cd-exposed population were observed relative to excretion of these substances in controls. Regression analysis using a general linear model revealed that the correlations between U-Cd or U-Cu, and U-beta2-MG and between U-Cd, U-Cu or U-Zn, and U-MT were statistically significant in both sexes, but the correlation between U-Zn and U-beta2-MG excretion was significant only in men. These results suggest U-Cd and U-Cu is affected by dysfunction in renal tubular absorption (indicated by U-beta2-MG), whereas not only U-Cd and U-Cu but also U-Zn appear to be a function of renal cellular desquamation (indicated by U-MT).     

Inhibitory effects of beta 2-microglobulin on in vitro calcification of osteoblastic cells

Elevated levels of aluminum and beta 2-microglobulin have been demonstrated in chronic dialysis patients. The role of aluminum in the pathogenesis of renal osteodystrophy has also been shown. We report on the effects of beta 2-microglobulin on calcification in vitro using osteoblastic cells, clone MC3T3-El. At concentrations comparable to those in plasma of chronic dialysis patients, both beta 2-microglobulin and aluminum suppressed calcification while collagen synthesis and alkaline phosphatase activity were maintained. These observations may be related to the impaired bone mineralization frequently observed in chronic dialysis patients.     

Evaluation of tubulointerstitial lesions' severity in patients with glomerulonephritides: an NMR-based metabonomic study

An 1H NMR-based metabonomic approach was used to investigate the correlation of histopathologically assessed tubulointerstitial lesions with the urinary metabolite profile in 77 patients with glomerulonephritides submitted to renal biopsy. The presence of renal damage was predicted with a sensitivity of 96% and a specificity of 99%. Patients with mild, moderate, and severe tubulointerstitial lesions were progressively differentiated from the healthy individuals in the Orthogonal Signal Correction Partial Least-Squares-Discriminant Analysis (OSC/PLS-DA) models with a statistically significant separation between those with mild and with severe lesions. The onset of the tubulointerstitial lesions is characterized by decreased excretion of citrate, hippurate, glycine, and creatinine, whereas further deterioration is followed by glycosuria, selective aminoaciduria, total depletion of citrate and hippurate, and gradual increase in the excretion of lactate, acetate, and trimethylamine-N-oxide. NMR-based metabonomic urinalysis could contribute to the early evaluation of the severity of the renal damage and possibly to the monitoring of kidney function.     

Some biological properties of beta2-microglobulin and its antibody.

beta2-Microglobulin on the surface of lymphocytes exists in two molecular forms, first as part of the HL-A antigen complex, and second as a free molecule unbound to other membrane macromolecules. Quantitative determinations of the numbers of molecules of beta2-microglobulin per lymphocyte when compared to published values for the numbers of HL-A molecules per cell are consistent with an excess of beta2-microglobulin compared to HL-A antigens on the cell surface. Turnover studies of beta2-microglobulin from the lymphocyte surface indicated that both beta2-microglobulin and HL-A components are metabolized at similar rates. beta2-Microglobulin appears to be released in the free form. HL-A antigens, if released from the cell surface, appear to be released unbound to beta2-microglobulin. The effects of anti-beta2-microglobulin antibody on lymphocyte activation, namely on the mixed lymphocyte reaction and on antigen induced proliferative response, were studied. Anti-beta2-microglobulin completely inhibited the mixed lymphocyte reaction and the antigen induced proliferative response.     

Use of ultrasound for evaluating structural changes of the renal parenchyma in chronic primary glomerulopathies

Ultrasound was used to measure echogenesis, renal cortex thickness and kidney length in 59 patients with various types of the primary glomerulonephritis. The obtained results have shown that the measured parameters correlated with the progress in renal failure. An increase in the intensity of echo from the cortex, thinning of the renal cortex, and the decrease in renal length are typical signs of the advancement of renal failure. As renal function deteriorates in the consequence of destruction of its parenchyma, ultrasound examination might be an illustration of the pathological changes in the kidney. Considering the fact that ultrasound is perfectly safe, it may be used in monitoring of the disease progress and the results of treatment.     

Renal synthesis of leukaemia inhibitory factor (LIF), under normal and inflammatory conditions

Leukaemia inhibitory factor (LIF) is a pleiotropic cytokine that is particularly involved in nephrogenesis and repair of the extracellular matrix. Transgenic mice overexpressing LIF have mesangial proliferative glomerulonephritis. Also, during local inflammatory reactions, such as kidney graft rejection or urinary tract infections, urinary LIF excretion is enhanced. The aim of the study therefore was to study LIF production by normal and inflammatory diseased kidneys (glomerulonephritis or graft rejection), maintained in short cultures. To determine the responsibility of the kidney itself in LIF synthesis, we measured LIF secretion into the culture supernatants of human mesangial or renal tubular epithelial cells. Fragments from diseased kidneys, whether grafts or not, released more LIF than normal human kidney fragments, mesangial or renal tubular epithelial cells. However, LIF production was delayed in renal transplants compared to glomerulonephritic samples taken from untreated patients. In every case, LIF production was enhanced by interleukin 1beta (IL-1beta) and inhibited by IL-4 or dexamethasone, except in two severe rejection episodes. So, LIF appeared to respond to pro- and anti-inflammatory stimuli, in vitro and in vivo. Considering its biological effects, LIF could play a role in inflammatory renal diseases.     

Deficiency in beta(2)-microglobulin, but not CD1, accelerates spontaneous lupus skin disease while inhibiting nephritis in MRL-Fas(lpr) nice: an example of disease regulation at the organ level

When mutations that inactivate molecules that function in the immune system have been crossed to murine lupus strains, the result has generally been a uniform up-regulation or down-regulation of autoimmune disease in the end organs. In the current work we report an interesting dissociation of target organ disease in beta(2)-microglobulin (beta(2)m)-deficient MRL-Fas(lpr) (MRL/lpr) mice: lupus skin lesions are accelerated, whereas nephritis is ameliorated. beta(2)m deficiency affects the expression of classical and nonclassical MHC molecules and thus prevents the normal development of CD8- as well as CD1-dependent NK1(+) T cells. To further define the mechanism by which beta(2)m deficiency accelerates skin disease, we studied CD1-deficient MRL/lpr mice. These mice do not have accelerated skin disease, excluding a CD1 or NK1(+) T cell-dependent mechanism of beta(2)m deficiency. The data indicate that the regulation of systemic disease is not solely governed by regulation of initial activation of autoreactive lymphocytes in secondary lymphoid tissue, as this is equally relevant to renal and skin diseases. Rather, regulation of autoimmunity can also occur at the target organ level, explaining the divergence of disease in skin and kidney in beta(2)m-deficient mice.     

Limited proteolysis of beta 2-microglobulin at Lys-58 by complement component C1s

We have now demonstrated that activated complement component C1s cleaves beta 2-microglobulin at the position identical to that at which beta 2-microglobulin is cleaved in serum of patients suffering from lung cancer. The main cleavage is in the disulphide loop C-terminal to Lys-58, generating a modified form of beta 2-microglobulin with a two-chain structure. The C-terminal Lys-58 in the A chain is highly susceptible to removal by a carboxypeptidase-B-like activity causing the formation of des-Lys58-beta 2-microglobulin. This is the first demonstration of a noncomplement protein substrate for the proteolytic activity of C1s. The C1s-induced cleavage of beta 2-microglobulin can be inhibited in the presence of C1 esterase inhibitor, demonstrating a regulatory function of C1 esterase inhibitor in the C1s-induced cleavage of beta 2-microglobulin.