heparitin-sulfate and Kidney-Calculi

Urinary polyanions recovered from the urine samples of kidney stone-formers and normal controls were subjected to preparative agarose gel electrophoresis, which yielded fractions 1-5 in a decreasing order of mobility. In both groups, chondroitin sulfates were identified in the fast-moving fractions and heparan sulfates in the slow-moving fractions. Furthermore, two types of heparan sulfates were identified based on their electrophoretic mobility: slow-moving and fast-moving. The fractionated urinary polyanions were then tested in an in vitro calcium oxalate crystallization assay and compared at the same uronic acid concentration, whereby, the chondroitin sulfates of stone-formers and heparan sulfates of normals enhanced crystal nucleation. Fraction 5 of the normals, containing glycoproteins (14-97 kDa) and associated glycosaminoglycans, were found to effectively inhibit crystallization. Papainization of this fraction in stone-formers revealed crystal-suppressive effects of glycoproteins, which was not seen in similar fractions of normals. It was concluded that glycoproteins could modulate the crystal-enhancing glycosaminoglycans such as chondroitin sulfates of stone-formers but not in normals. The differing crystallization activities of electrophoretic fraction 1 of normals and stone-formers revealed the presence of another class of glycosaminoglycan-hyaluronan. Hence, in the natural milieu, different macromolecules combine to have an overall outcome in the crystallization of calcium oxalate.

Topics: Adult; Calcium Oxalate; Chondroitin Sulfates; Crystallization; Electrophoresis, Agar Gel; Electrophoresis, Cellulose Acetate; Glycoproteins; Heparitin Sulfate; Humans; Kidney Calculi; Middle Aged; Urinary Calculi; Urolithiasis

Alterations in intracellular Ca2+ ([Ca2+]i) are generally associated with cellular distress. Oxalate-induced cell injury of the renal epithelium plays an important role in promoting CaOx nephrolithiasis. However, the degree of change in intracellular free calcium ions in renal epithelial cells during oxalate exposure remains unclear. The aim of this study is to determine whether acute short-term exposure to oxalate produces morphological changes in the cells, induces a change in cytosolic Ca2+ levels in renal tubular epithelial cells and whether the application of extracellular glycosaminoglycans (GAGs) prevents these changes. Cultured Mardin-Darby canine kidney cells were exposed to oxalate, and changes in cytosolic Ca2+ were determined under various conditions. The effect of heparin and heparan sulfate (HS) during oxalate exposure was examined. The change in the GAG contents of the culture medium was also determined. Transmission electron microscopy (TEM) was performed for morphological analysis. The degree of change in cytosolic Ca2+ strongly correlated with oxalate concentration. Cytosolic Ca2+ levels decreased in parallel with an increase in the concentration of oxalate. However, this decrease was strongly inhibited by pretreatment with heparin or HS. TEM revealed cytoplasmic vacuolization, the appearance of flocculent material and mitochondrial damage after oxalate exposure. On the other hand, pretreatment with heparin or HS completely blocked these morphological changes. The present data suggest that acute exposure to a high concentration of oxalate challenges the renal cells, diminishes their viability and induces changes in cytosolic Ca2+ levels. Heparin and HS, which are known as potent inhibitors of CaOx crystallization, may also prevent oxalate-induced cell changes by stabilizing the cytosolic Ca2+ level.

Topics: Animals; Calcium; Calcium Oxalate; Cell Line; Cell Survival; Cytoprotection; Dogs; Extracellular Fluid; Heparin; Heparitin Sulfate; Intracellular Membranes; Kidney; Kidney Calculi; Microscopy, Electron

We reported that expression of both HSPG and of bikunin are increased in calcium oxalate (CaOx) nephrolithic rat kidneys (lida et al., J. Am. Soc. Nephrol. 1999, Urol. Res. 1997). However, these findings were obtained from separate experiments. The present study evaluates whether levels of HSPG and bikunin expression differ in the rat kidney during calcium oxalate nephrolithiasis. Twenty-four male Wistar rats weighing 200-250 g were assigned to one of four groups (n = 6 each group) and administered with 0.5% ethylene glycol daily and 0.5 microgram of 1 alpha-OH-D3 every other day to induce CaOx nephrolithiasis. Animals were sacrificed 1 or 2 weeks later and both kidneys were excised. The cortex was separated from the medulla and papillary tips in the right kidney, then stored in liquid nitrogen for quantitative competitive-reverse transcription-polymerase chain reaction (QC-RT-PCR). The left kidney was fixed in 10% buffered formalin for histochemical studies. We assessed the variable gene expression of both HSPG and bikunin by QC-RT-PCR. Immunohistochemical analyses of left kidney tissue samples determined the localization of HSPG and bikunin. Normal rats serving as controls (n = 6 each) were also sacrificed and processed in the same manner as the experimental groups. QC-RT-PCR confirmed that HSPG and bikunin mRNA expression is significantly increased in nephrolithic kidneys (p < 0.05; Mann-Whitney test), and that medulla and papillary tips tended to express more mRNA of both. Immunohistochemical studies revealed that the production of HS and bikunin was increased in both the distal and proximal tubules of nephrolithic kidneys. These findings suggest that the increased expression of both HSPG and bikunin play an important role during calcium oxalate stone formation. In addition, this phenomenon might be associated with the progression of urothelial damage.

Topics: Animals; Calcium Oxalate; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Kidney; Kidney Calculi; Male; Membrane Glycoproteins; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Trypsin Inhibitor, Kunitz Soybean; Up-Regulation

The effect of glycosaminoglycans (GAGs) in urinary crystal inhibition has been shown in vitro, but their inhibitor role in vivo has not been precisely determined in stone-forming patients. The aim of this study was to compare the levels of total GAGs and their components in primary stone-forming patients and a healthy control group and to investigate the impact of shockwave lithotripsy (SWL).. Thirty-eight patients with primary kidney stones and 31 healthy controls were included in this prospective study. Total urinary GAG concentrations were determined by the dimethylene blue assay (DMB), and GAG fractions (chondroitin sulfate, heparan sulfate, and dermatan sulfate) were studied by cellulose acetate electrophoresis. Analysis was repeated after SWL in the stone patients.. Chondroitin sulfate was the major component secreted in the urine of the control subjects. Heparan sulfate was the major component in the urine of the stone patients with less chondroitin sulfate and dermatan sulfate (48%, 35%, 16.5%, respectively). Our study showed a significant increase in total urinary GAGs (4.75 v. 7.43 microg/mg of creatinine; P<0.0001) after SWL. Dermatan sulfate was the main component in this group (P<0.0001). The total urinary GAG concentrations remained high for at least 2 days after SWL.. The elevation in total GAGs after SWL indicates the presence of tissue injury, which also renders dermatan sulfate the principal excreted component. Studies with longer follow-up periods are needed to determine whether these changes in the excretion of GAG components persist.

Topics: Adult; Biomarkers; Chondroitin Sulfates; Creatinine; Dermatan Sulfate; Electrophoresis, Cellulose Acetate; Female; Heparitin Sulfate; Humans; Kidney Calculi; Lithotripsy; Male; Prognosis; Prospective Studies; Severity of Illness Index

While the pathogenic mechanisms responsible for calcium nephrolithiasis remain unknown, the influence of heparan sulphate proteoglycan (HSPG) on disease progression of other diseases, such as polycystic kidneys and diabetic glomerulosclerosis, makes it an important candidate for the study of stone formation. Using the indirect immunofluorescence assay and image analysis, we were able to quantify and visualize the loss of HSPG localized in the basement membrane of the glomerulus and the mucosa of ureter or renal pelvis in patients with recurrent calcium nephrolithiasis as compared to normal subjects. However, no significant change in HSPG was observed in the basement membrane of the tubular epithelium. The decreased HSPG in the glomerulus may reflect the potentially disrupted anion/neutral barrier for glomerular filtration, which would encourage the accumulation of stone solutes. The drop in HSPG staining intensity in the basement membrane of the mucosa of ureter/renal pelvis may suggest the tendency of adhesion of crystal to urothelial surfaces. Based on these immunological data, it appears that HSPG plays a modulatory role in the pathogenesis of this disease.

Topics: Adult; Basement Membrane; Calcium; Fluorescent Antibody Technique, Indirect; Heparitin Sulfate; Humans; Kidney; Kidney Calculi; Kidney Glomerulus; Kidney Pelvis; Kidney Tubules; Middle Aged; Tissue Distribution; Ureter