Glycosaminoglycans as inhibitors of calcium oxalate crystal growth and aggregation.

Evidence is presented that the glycosaminoglycans, chondroitin 6-sulphate and chondroitin 4-sulphate, are the major inhibitors of calcium oxalate crystal growth and aggregation in dilute normal urine.     

Inhibitors of the growth and aggregation of calcium oxalate crystals in vitro

A sensitive method has been developed for measuring the effect of inhibitors of crystallisation on the growth and aggregation of calcium oxalate crystals in vitro. Of the known inhibitors tested the highly charged anions pyrophosphate and ethane-1-hydroxy-1,1-diphosphonate (EHDP) were effective down to concentrations of 0.1–1 μmole/l and the polyanions heparin and chondroitin sulphate A down to concentrations of 1 nmole/l. Within the normal range of urea concentrations found in urine the neutral molecule urea caused a small but significant degree of inhibition. At higher concentrations the degree of inhibition increased sharply. In the concentrations tested the cations magnesium and methylene blue had no effect on the growth and aggregation of crystals.These findings suggest that highly charged anions and polyanions may have some clinical value as a possible treatment for recurrent calcium oxalate stone-formation in man.     

Enzymatic determination of urinary chondroitin sulphate: applications in renal stone disease and acromegaly

A method was developed for the determination of urinary chondroitin sulphate (CS), including dermatan sulphate and chondroitin 4 and 6-sulphates, using an enzymatic degradation with chondroitinase-ABC followed by precipitation with Alcian blue, whereby CS was determined as the difference between undigested and chondroitinase digested material. The method was linear in the range 0-100 mg l-1 with a detection limit of 1 mg l-1 and allowed determinations on small urine volumes without pretreatment of the urine. It could be demonstrated that males excreted more CS than females, and growing children had the highest urinary content of CS. Renal calcium stone formers did not differ from healthy controls in urinary CS. Patients with acromegaly had a higher excretion of CS compared with controls. There was also, in these patients, a positive correlation between the serum growth hormone levels and the urinary CS, indicating that CS-excretion may be an estimate of the activity of the pituitary disorder.     

Influence of urine on "in vitro" crystallization rate of calcium oxalate: determination of inhibitory activity by a [14C]oxalate technique.

A simple radiochemical method is proposed for the in vitro assay of the inhibitory activity of urine with respect to calcium oxalate crystal growth using [14C]oxalate as a tracer. The method shows an improved sensitivity over existing methods and indicates that citrate, pyrophosphate and chondroitin sulphate are active inhibitors of calcium oxalate crystal growth down to concentrations of 10(-5), 10(-7) and 10(-10) mol/l respectively. The inhibitory activity in the urines of 12 recurrent calcium stone-formers was significantly lower than in the urines of matched control subjects (P less than 0.01), confirming the clinical usefulness of the test.     

The influence of serum and serum proteins on calcium oxalate crystal growth and aggregation

The effects of serum, albumin, alpha-globulin, and a mixture of alpha- and beta-globulin on the growth and aggregation of calcium oxalate crystals were measured in a standard seeded crystallisation system. At concentrations below 0.01% blood, which corresponded to microscopic haematuria, all were potent inhibitors of crystal aggregation whilst only having a minor effect on crystal growth. It was found that albumin, alpha-globulin and beta-globulin can account for the total inhibitory effect of serum on crystal growth and crystal aggregation.     

A micropuncture and renal clearance study in the rat of the urinary excretion of heparin, chondroitin sulphate and metabolic breakdown products of connective tissue proteoglycans

Commercial heparin and acid glycosaminoglycans (AGAG) prepared from normal female human urine were tritiated catalytically in aqueous solution. Nasal septal chondroitin sulphate was tritiated by NaB3H4 reduction of the aldehydes produced by very limited periodate oxidation. The refined products were characterized by electrophoresis and biochemical analysis. The tritiated products were infused into Munich-Wistar rats, and their kidney clearances were measured and compared with that of inulin. The passage of heparin and chondroitin sulphate at the glomerulus was restricted, as shown by the ratios of their concentrations in Bowman's space fluid and plasma. Comparison with whole kidney urine/plasma ratios indicated that some tubular reabsorption also occurred. The low whole kidney clearance of AGAG from normal female human urine probably also resulted from restricted glomerular passage and tubular reabsorption. The low whole kidney clearances, primarily caused by restricted transglomerular movement, are discussed on the bases of (a) the molecular sizes and shapes of the AGAG, (b) an electrostatic barrier at the basement membrane and (c) binding of AGAG to plasma colloids, thus reducing plasma levels of free AGAG. The implications of these findings in (a) heparin therapy and (b) urinalysis of disorders of connective tissue metabolism are discussed.     

Binding of glycosaminoglycan inhibitors to calcium oxalate crystals in relation to ionic strength

Several inhibitors of calcium oxalate crystallization have been identified and shown to exhibit quantitative and qualitative differences in efficacy. Glycosaminoglycans are potent inhibitors of crystal growth and aggregation, and the efficiency seems to increase with an increasing charge density. In order to investigate the mechanism of inhibition, we performed binding experiments of radioactivity labelled heparin, chondroitin sulphate and the low-molecular mass heparin analogue pentosan polysulphate to calcium oxalate crystals and subsequent displacements by increasing the amounts of non-radioactive ligands or increasing ionic strength. Ligands with a high charge density bound more readily and with a seemingly higher affinity than ligands with a low charge density, but were also more susceptible to displacement when the ionic strength was increased. It is concluded that a higher affinity to the crystals may be the reason why highly charged glycosaminoglycans are more efficient inhibitors of calcium oxalate crystal growth.     

血清及其成分对铜绿假单胞菌生物膜形成的影响

目的研究血清及其主要成分对铜绿假单胞菌生物膜形成的影响。方法在96孔板中用结晶紫染色法检测血清、清蛋白和转铁蛋白对铜绿假单胞菌生物膜形成的影响;激光共聚焦显微镜观察血清处理后铜绿假单胞菌生物膜的形态。结果马血清能显著促进PAO1生物膜的形成,使生物膜的形成量从2.26±0.42增加到3.42±0.08(t=4.71,P0.01);而人血清能抑制其生物膜的形成,使生物膜形成量减少至0.81±0.10(t=4.71,P0.01)。马血清可显著增加生物膜的总量并使生物膜呈片状分布;而人血清可显著抑制生物膜的形成,并使其呈散在点状分布。马血清可促进部分临床菌株生物膜的形成,而人血清能显著抑制所有临床菌株生物膜的形成。2.5 g/L清蛋白可使PAO1生物膜形成量从1.96±0.22增加到2.54±0.18(t=3.55,P0.05),而5 g/L转铁蛋白可使PAO1生物膜形成量从1.85±0.36减少到0.84±0.24(t=4.03,P0.05)。结论马血清和清蛋白可显著促进铜绿假单胞菌生物膜的形成,而人血清和转铁蛋白可抑制生物膜的形成。     

Crystal inhibition: the effects of polyanions on calcium oxalate crystal growth

The inhibition of calcium oxalate crystal growth by the glycosaminoglycans, chondroitin sulphates and heparin, by the low-molecular-weight heparin analogue pentosan polysulphate and by Tamm-Horsfall glycoprotein extracted from human urine, was measured by using a seeded crystal procedure and compared with the inhibition by pyrophosphate. It was found that the most pronounced inhibition was obtained with the polyanions with the highest charge density, i.e., heparin and pentosan polysulphate. Tamm-Horsfall glycoprotein caused an inhibition of a similar magnitude as urinary chondroitin sulphates. Urinary polyanions with a high affinity to Sepharose 4B were more efficient inhibitors than those with a low or no affinity to the gel. It is concluded that urinary polyanions are important inhibitors of calcium oxalate crystal growth and that the potency of inhibition increases with the charge density.     

The relationship between the dermal content and the 24-hour excretion of analytically identical glycosaminoglycans in humans

Using a material consisting of related dermal specimens and 24-h urine samples from 17 psoriatics and 20 non-psoriatics it has been shown for the first time that the excretion of dermatan sulphate and a fraction of chondroitinase ABC resistant GAG ('heparan sulphate') are positively associated with the tissue content of the analytically identical glycosaminoglycans in dermis. The excretion of chondroitin 4/6 sulphate, total hydroxyproline and a peptide-bound fraction of this does not, however, mirror the tissue content of the corresponding constituents in dermis. There was no difference in the type of association between tissue and urine measurements in psoriatics and non-psoriatics. The results were analysed using multiple regression to avoid the unwanted effect of diverse concomitant variables (6 variables).     

Human parathyroid cell proliferation in response to calcium, NPS R-467, calcitriol and phosphate

It remains uncertain how calcium, phosphate and calcitriol regulate parathyroid cell growth. The present study was aimed at examining possible direct effects of these modulators and of the calcimimetic NPS R-467 on parathyroid cell growth in vitro. Cell proliferation was determined by [3H]thymidine incorporation and cell cycle antigen Ki 67 expression in a parathyroid cell culture model derived from uraemic patients. The effect of NPS R-467 on parathyroid hormone (PTH) secretion and intracellular [Ca2+]i response was also examined. Increasing the [Ca2+] in the medium from 0.5 to 1.7 mM increased DNA synthesis (P < 0.005) and the number of Ki 67-positive cells (P < 0.005). However, NPS R-467 (0.01-1 microM) inhibited 3[H]thymidine incorporation by 35% in the presence of 0.5 mM [Ca2+]e. Exposure of cells to Ca2+ or NPS R-467 led to a rapid increase of intracellular Ca2+, although the pattern of increase differed. Addition of calcitriol (10-10-10-7 M) to the culture medium suppressed [3H]thymidine incorporation dose-dependently. Finally, high levels of phosphate (3.5 mM) in the medium led to a significant (P < 0.05) increase in [3H]thymidine incorporation. The observed stimulatory effect of Ca2+ in the medium in vitro appears to be at variance with the inhibitory effect of calcimimetic NPS R-467 in vitro. In an attempt to solve these apparent discrepancies, and based on the notion of a reduced calcium-sensing receptor (CaR) expression in parathyroid tissues of uraemic patients, we hypothesize that Ca2+ may regulate parathyroid cell proliferation via two different pathways, with predominant growth inhibition in cases of high CaR expression or activation, but prevailing stimulation of proliferation in cases of low CaR expression.     

Binding of glycosaminoglycans to sodium urate and uric acid crystals

The binding of heparin, chondroitin sulphate and the low molecular weight heparin analogue pentosan polysulphate to sodium urate and uric acid crystals was studied by the use of radioactively labelled glycosaminoglycans were used in the binding step, and varying amounts of unlabelled glycosaminoglycans for the competition experiments. The experiments were carried out in 140 mmol/l NaCl at pH 6, with or without 5 mmol/l CaCl2 in the solution. A reversible and almost complete binding of heparin and chondroitin sulphate to sodium urate crystals did occur in the presence of calcium, whereas pentosan polysulphate bound incompletely. The binding was much less pronounced in the calcium-free conditions. Uric acid crystals did not bind any of the three inhibitors, not even with calcium present. The clinical relevance depends on whether sodium urate microcrystals are present in the urine of calcium stone patients, to cause a binding and thereby a masking or inactivation of these inhibitors in the urine, which seems to be possible in the presence of calcium. However, the potential of pentosan polysulphate for the treatment of calcium stone patients does not seem to be at risk from this effect.     

Urinary glycosaminoglycans in patients with hypothyroidism and in healthy subjects

Although the changes in urinary glycosaminoglycans have been investigated in several endocrinopathies, no information was hitherto available on the content and composition of urinary glycosaminoglycans in hypothyroidism. Urinary glycosaminoglycans were therefore investigated in patients with hypothyroidism and in healthy subjects. The total daily excretion of urinary glycosaminoglycans was found to be significantly increased (by 41%) in hypothyroidism. Two electrophoretic bands were always detected in both examined groups: a major band of chondroitin sulphate and a minor band of heparan sulphate. Heparan sulphate and chondroitin sulphate levels were respectively 114% and 42% higher in patients with hypothyroidism than in controls. The respective increases in chondroitin-4-sulphate and chondroitin-6-sulphate were 31% and 41%. The relative quantities of chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate and non-sulphated chondroitin sulphate were unchanged in the two examined groups. The changes observed in the levels of the excreted glycosaminoglycans may reflect the altered metabolism of connective tissue in hypothyroidism.     

Effect of Bovine Parathyroid Hormone 1–34 Fragment on Renal Production and Excretion of Adenosine 3', 5' Monophosphate in Man

Abstract. Biologically active bovine parathyroid hormone (b PTH) 1–34 fragment infused over one hour in normal subjects produced an immediate and sharp increase in the excreted fractions of filtered bicarbonate, sodium and potassium, followed by the return to pre-infusion levels as soon as the administration of b PTH was stopped. There was a gradual but steady increase in the excreted fraction of filtered phosphate but a decrease in the excreted fraction of filtered calcium and magnesium. The excreted fractions of these ions were still abnormal 150 minutes after the completion of PTH infusion. The urinary excretion of 3'5′-cyclic AMP increased immediately about one hundred-fold but returned rapidly to pre-infusion levels. Urinary clearance of cyclic AMP approximated glomerular filtration rate in control periods and was twenty to thirty times greater during b PTH infusion. In subjects overloaded with bicarbonate, b PTH brought about a decrease in bicarbonate T_m and the same effects on the urinary excretion of other electrolytes. 3'5′-cyclic AMP excretion was clearly higher in control periods and reached higher levels during b PTH infusion When compared to subjects without an alkaline load. 3'5′-cyclic AMP excretion and fractional clearance were also clearly higher in subjects not given b PTH when control periods were compared to periods with bicarbonate infusion or after acetazolamide administration. During distal blockade obtained by simultaneous administration of chlorothiazide and ethacrynic acid, there was a delay in the rise of 3'5′-cyclio AMP excretion after b PTH administration. It can be concluded from these studies that the pattern of excretion of 3'5′-cyclic AMP is similar to that of bicarbonate, sodium and potassium. The increase of 3'5′-cyclic AMP excretion when urinary pH is above 7 suggests a diffusion trapping mechanism for the secretion into the lumen of this nucleotide. Distal diuretics used in distal blockade did not inhibit 3'5′-cyclic AMP production but delayed its secretion into urine.     

Comparison of sulphated glycosaminoglycan and hyaluronate synthesis and secretion in cultured hepatocytes, fat storing cells, and Kupffer cells

The extracellular matrix of normal liver contains several types of proteoglycans including heparan sulphate, chondroitin sulphate isomers, dermatan sulphate, and the glycosaminoglycan, hyaluronic acid. In the present study both the synthesis and secretion as well as the pattern of radioactively labeled proteoglycans and hyaluronic acid of hepatocytes, fat-storing cells (Ito cells), and Kupffer cells maintained in monolayer cultures under mostly identical conditions were compared to assess their relative contribution to hepatic proteoglycan synthesis. Fat-storing cells were identified as the main type of cell producing and secreting proteoglycan and hyaluronic acid. More than 70% of labeled proteoglycan and hyaluronic acid were secreted into the medium. Heparan sulphate is the main type of proteoglycan in hepatocytes, whereas in the medium of fat-storing cells, chondroitin sulphate and dermatan sulphate comprise the major fractions. Hyaluronic acid was not detectable in hepatocyte cultures and found only in low amounts in the medium of Kupffer cells. The results point to a stringent quantitative and qualitative cellular compartmentation of proteoglycan synthesis in liver with fat-storing cells as the most important cell type for matrix proteoglycan and hyaluronic acid production.     

Macromolecules inhibit calcium oxalate crystal growth and aggregation in whole human urine

Removal of macromolecules with Mr greater than 10,000 had no discernible effect on the detectable nucleation of calcium oxalate crystals from undiluted human urine, but promoted the deposition of crystalline material and markedly increased the degree of aggregation of the precipitated crystals. Calcium oxalate crystals and crystal aggregates precipitated from ultrafiltered urine were, on average, 68% larger than those deposited from whole urine. These findings suggest that urinary macromolecules play a key role in preventing calcium oxalate kidney stone disease by inhibiting the formation of large crystal aggregates and thereby reducing the probability of particle retention in the kidney tubules.     

Sera from patients with collapsing focal segmental glomerulosclerosis increase albumin permeability of isolated glomeruli

The collapsing variant of focal segment glomerulosclerosis (FSGS) is characterized by heavy proteinuria and rapid progression to renal failure. Its cause is not known. We have characterized a substance in the circulation of patients with classic FSGS that increases in vitro permeability of glomeruli to albumin (P(alb)) and causes proteinuria when injected into rats. Inclusion of normal serum prevents the increase in P(alb) caused by this FSGS factor. We investigated the effect of sera from patients with collapsing FSGS on P(alb), as well as the effect of inclusion of normal serum. Isolated glomeruli were incubated with serum from each of 11 patients with collapsing FSGS (1:50 dilution) or with patient serum and an equal volume of pooled normal serum. P(alb) was determined on the basis of changes in glomerular volume in response to an oncotic gradient. Sera from 10 of the 11 patients with collapsing FSGS increased P(alb) of isolated glomeruli to a value of 0.5 or greater. In each of the 5 cases tested, inclusion of normal serum abolished the increase in P(alb). Sera of patients with collapsing FSGS increased glomerular P(alb). Our finding that the increase in P(alb) is abolished by normal serum suggests that the substance and its mechanism of action are similar or identical to the FSGS factor we have isolated from the plasma of patients with recurrent FSGS. The presence of a circulating factor in collapsing FSGS has implications for prognosis and treatment in primary and recurrent collapsing FSGS.     

Determination of hyaluronic acid,dermatan sulphate,heparan sulphate and chondroitin 4/6 sulphate in human dermis,and a material of reference

Using a new method based on electrophoresis and enzymatic digestion with chondroitinase AC small skin biopsies of 15 healthy individuals were assayed for qualitative and quantitative content of glycosaminoglycans. The content of collagen was measured as hydroxyproline. The following results were obtained: (1) The mean substance content and standard deviation of hyaluronic acid, dermatan sulphate, heparan sulphate chondroitin 4 or 6 sulphate and hydroxyproline in dried defatted tissue is presented. (2) There is a negative correlation with age for hyaluronic acid and no correlation with age for any of the other glycosaminoglycans or the hydroxyproline. (3) The sex has no major influence on any of the quantities measured. (4) The observed substance content of water in wet tissue is presented.     

Determination of hyaluronic acid, dermatan sulphate, heparan sulphate and chondroitin 4/6 sulphate in human dermis, and a material of reference

Using a new method based on electrophoresis and enzymatic digestion with chondroitinase AC small skin biopsies of 15 healthy individuals were assayed for qualitative and quantitative content of glycosaminoglycans. The content of collagen was measured as hydroxyproline. The following results were obtained: (1) The mean substance content and standard deviation of hyaluronic acid, dermatan sulphate, heparan sulphate chondroitin 4 or 6 sulphate and hydroxyproline in dried defatted tissue is presented. (2) There is a negative correlation with age for hyaluronic acid and no correlation with age for any of the other glycosaminoglycans or the hydroxyproline. (3) The sex has no major influence on any of the quantities measured. (4) The observed substance content of water in wet tissue is presented.