Naoya Amasaki

Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin

We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin (bone sialoprotein) (nucleotides 265-886 and 1183-1424). Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that osteopontin is presumably involved in stone formation as stone matrix.

Osteopontin mRNA in the Kidney on an Experimental Rat Model of Renal Stone Formation without Renal Failure

We had sequenced a cDNA of calcium oxalate urinary stone protein extracted with 0.1 M EDTA. cDNA sequences showed complete homology between urinary stone protein and human osteopontin (OPN, bone sialoprotein I). In the present study, we investigated the expression of OPN mRNA in the rat kidney in an experimental model of renal stone formation using glyoxylic acid and 1,25-dihydroxyvitamin D3 (D3). In the renal stone formation model with and without renal failure, OPN mRNA was shown to be localized by in situ hybridization using an OPN cRNA probe mainly in the distal convoluted tubule of the kidney, and was enhanced compared with the normal control which was sporadically positive. By Northern blot analysis, the expression of OPN mRNA was shown to be increased by about 5.2-fold in the renal stone formation model and 2.3-fold in D3-administered rats compared with controls. However, no change in the expression of OPN mRNA was observed in an acute renal failure model induced by gentamicin or in rats administered glycoxylic acid alone. Therefore, the promotion of OPN mRNA expression was intrinsic to this stone formation model, and not secondary to acute renal failure because of obstruction by microcrystals in the renal tubules or gentamicin.

Analysis of osteopontin DNA in patients with urolithiasis

Abstract We previously reported the importance of osteopontin (OPN) in the formation of urinary calculus. Since OPN protein is present in normal kidneys, we investigated the difference in OPN at the DNA level between normal subjects and urolithiasis patients. There has not been any genetic investigation of OPN in familial urolithiasis previously reported worldwide. To confirm hereditary predisposing factors for urolithiasis, changes in OPN DNA within a family were investigated in relation to the presence or absence of urinary calculus. Leukocyte OPN DNA from two normal subjects and five patients with urinary calculus was investigated by SSCP analysis: OPN DNA nucleotide sequence was determined, based on the result of SSCP analysis. As a result, a mutation of GCC to GCT, encoding amino acid position 250 (Ala-250) was found. To confirm the frequency of mutation at this site, OPN DNA was extracted from peripheral blood in 36 normal subjects (Con group), 25 patients with familial urolithiasis (FSF), and 40 patients with recurrent urinary calculus and who had had two or more previous episodes (RSF). The degree of mutation at Ala-250 was then examined by restriction fragment length polymorphism (RFLP) method. As described above, the nucleotide codon encoding the amino acid sequence position 250, Ala-250, was GCC in two normal subjects. This is the original codon. In five patients with urolithiasis it was GCT, showing a substitution of C with T. On examining the frequency of this mutation, the ratio of normal homozygous GCC was 11/36 in the Con group, 1/25 in FSF and 1/40 in RSF. The ratio of heterozygous GCC/GCT was 16/36 in the Con group, 15/25 in FSF and 26/40 in RSF, and the ratio of homozygous GCT was 9/36 in the Con group, 9/25 in FSF and 13/40 in RSF. Furthermore, the gene frequency of the normal codon GCC was 0.528 in the Con group, 0.3 in FSF and 0.35 in RSF, showing a significantly higher incidence in the Con group (P < 0.05). The gene frequency of mutated GCT was 0.472 in Con group, 0.7 in FSF and 0.65 in RSF, showing a significantly higher incidence in urolithiasis patients (P < 0.05). On investigating the inheritance of Ala-250 in five families in which both parent and offspring demonstrated urolithiasis, the nucleotide substitution in Ala-250 in parents with urolithiasis was inherited by their offspring. In all five families the offspring developed urinary calculus. This study showed that there is no difference in OPN structure between the Con group and urolithiasis patients. However, it was predicted that due to the frequency of normally coded GCC being high in the Con group a difference in the amount of OPN might be caused by a difference in transcription velocity between the two groups. Furthermore, it was suggested that examining the inheritance of Ala-250 within a family is a diagnostic method for identifying the predisposing hereditary factors for urolithiasis patients.

Characteristics and Usage of Different Ureteral Stent Catheters

Presuming that complications associated with ureteral stenting vary in type and occurrence depending on the material and cross-section of the stent, six types of stents immersed in 48 different preparations of artificial urine for 1 month to observe surface changes with a scanning electron microscope. As a result, there was less encrustation on the silicone material compared with other material types, probably due to the smoothness of the surface. This may be related with higher frequency of spontaneous removal or migration to the bladder of this catheter type. Because silicone catheters have softer and thicker walls with a narrow lumen, they may be appropriate for long-term stenting, but not for urinary drainage. In alkaline bacteriuria, struvite encrustation was observed on all stents. This reaction was especially intense with Towers peripheral stents, which had most irregular and uneven surfaces. In aseptic alkaline urine, calcium phosphate crystals partly covered with proteinaceous debris were noted on catheter surfaces. Although in some patients encrustation of uric acid occurred in the bladder portion of the stents, there was no uric acid encrustation in this experimental study.

Renal Damages after Extracorporeal Shock Wave Lithotripsy Evaluated by Gd-DTPA-Enhanced Dynamic Magnetic Resonance Imaging

Renal damages after extracorporeal shock wave lithotripsy (ESWL) were evaluated by magnetic resonance imaging (MRI) including Gd-DTPA-enhanced dynamic MRI in 37 patients with renal stone by spin echo methods (T1 and T2-weighted scan) and small tip angle gradient echo method (T2-weighted scan). Sixty-eight percent of the patients had changes in the MRI findings after ESWL. The frequently observed findings were perirenal fluid collection (38%), loss of corticomedullary junction (35%), and increased signal intensity of muscle and other adjacent tissue (34%). Preoperative Gd-DTPA-enhanced dynamic MRI showed low intensity band which suggests Gd-DTPA secretion from the glomerulus into the renal tubulus. In all cases the low intensity band became unclear after ESWL because of renal contusion due to ESWL. MRI, including Gd-DTPA-enhanced dynamic MRI, is considered to be a good procedure for evaluation of renal damages due to ESWL.

Influence of urinary sialic acid on calcium oxalate crystal formation.

Using seed crystal method, whole-urine method, and scanning electron microscopy, the inhibitory effects of sialic acid and osteopontin (OPN) on aggregation/growth of CaOx crystals were investigated. Using the seed crystal method, sialic acid showed an inhibitory effect on CaOx crystal aggregation/growth in a concentration-dependent manner, but almost no effect was observed using the whole-urine method. OPN showed an inhibitory effect on aggregation/growth in both experimental systems. The inhibitory effect of asialo-OPN on aggregation/growth was approximately 20% lower than that of OPN in the experiment using the seed crystal method and approximately 15% lower in the experiment using the whole-urine method. Scanning electron microscopy showed that OPN and sialic acid inhibit the aggregation of CaOx crystals. The above findings show that sialic acid accounts for about 15–20% of the involvement of OPN in CaOx crystallization.

Continuous Evaluation for Retroperitoneal Hematoma following Extracorporeal Shock Wave Lithotripsy

In this paper we report on a patient with extensive retroperitoneal hematoma due to extracorporeal shock wave lithotripsy (ESWL) for a renal stone. The patient had a negative history of previous primary renal disease or risk factors for retroperitoneal hemorrhage after ESWL. The hematoma was treated conservatively and it was completely absorbed 9 months after ESWL as evaluated by computed tomography. Gd-DTPA-enhanced dynamic magnetic resonance imaging (dynamic MRI) was performed immediately after formation of the hematoma. The decrease in renal blood flow in the affected kidney could be observed in this patient by dynamic MRI and its time intensity curve.

Localization and inhibitory activity of alpha(2)HS-glycoprotein in the kidney.

Abstractα2HS-Glycoprotein (HS), a crystal surface binding substance extracted from human urine, is considered to be one of the urinary macromolecular inhibitors in urolithiasis. In the present study, reverse transcription–polymerase chain reaction was used to examine HS mRNA expression, and immunohistochemical staining was used to reveal its localization in the human kidney. The inhibitory effects of recombinant human HS and native human HS on calcium oxalate crystal growth were examined in a seed crystal system. HS mRNA was found to be expressed in the human kidney, and it was located in the epithelial cells of distal and proximal renal tubular cells. However, neither recombinant HS nor native HS had an inhibitory effect on crystals in the protein concentration of urine of healthy humans. HS in the urine, therefore, does not seem to be a potent inhibitor in stone formation.

Regulation of the expression of OPN mRNA in the rat as an experimental model of renal stone disease.

We have sequenced a cDNA of calcium oxalate urinary stone protein extracted with EDTA. cDNA sequences showed complete homology between urinary stone protein and human osteopotine (OPN, bone sialoprotein 1). In this study, we investigated the expression of OPN mRNA in rat kidney serving as experimental models for several conditions that are considered to be risk factors in human renal stone formation. In the renal stone formation model, the expression of OPN mRNA in the distal convoluted tubule of the kidney was enhanced compared with the control which was found sporadically positive by in situ hybridization. By Northern blot analysis, the expression of OPN mRNA was increased in pyelonephritis and hydronephrosis models compared with the control, but no changes were observed in dietary–acid or base–loading models. The expression of OPN mRNA was markedly inhibited in the renal stone formation model by concomitant administration of estradiol and/or progesterone.

Clinical Efficiency of Type-IV Collagen in Extracorporeal Shock Wave Lithotripsy

We previously reported a patient who developed anti-glomerular basement membrane antibody-induced glomerulonephritis (GBM-GN) after extracorporeal shock wave lithotripsy (ESWL). To determine whether ESWL causes anti-GBM antibody production thereby inducing GBM-GN, we measured serum levels of type-IV collagen (the most major component of GBM) before and after ESWL. Serum samples were obtained from 13 patients with renal stones and 15 with ureteral stones. The serum type-IV collagen level was determined by a radioimmunoassay method. No significant changes were observed in the type-IV collagen level after ESWL in each patient group. Type-IV collagen was abundant in the GBM but was not released into the blood after ESWL. Therefore, in patients with urinary tract stones, it is not likely that type-IV collagen, a hidden antigen in the renal glomeruli, is exposed or released into the blood after ESWL, resulting in autoantibody production and GBM-GN.