Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus.

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called 'butyrolactone autoregulators' which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R, 6R)- and (2S,3S,6S)-enantiomers] were also produced from (+/-)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S, 6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.

Identification and characterization of 6-dehydroVB-A reductase from Streptomyces antibioticus.

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyces hormone, virginiae butanolide A (VB-A), that induces virginiamycin production of S. virginiae at nanomolar concentrations. To characterize the biosynthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydro VB-A reductase that is responsible for the final reduction step in the biosynthesis. Assay protocols and stabilization conditions were established. The 6-dehydro VB-A reductase was found to require NADPH, not NADH, as a coenzyme. The K(m) values of the enzyme for NADPH and (+/-)-6-dehydro VB-A were determined to be 50 +/- 2 microM and 100 +/- 5 microM, respectively. Ultracentrifugation experiments revealed that 6-dehydro VB-A reductase was present almost exclusively in the 100,000 x g supernatant fraction, indicating that the enzyme is a cytoplasmic-soluble protein. The M(r) of the native 6-dehydro VB-A reductase was estimated to be 82,000 +/- 3000 by molecular sieve HPLC. The optimal pH was found to be 6.7 +/- 0.2.

Papillary renal cell carcinoma with numeric changes of chromosomes in a long-term hemodialysis patient: a karyotype analysis.

A patient with renal neoplasm was nephrectomized 5 months after successful renal transplantation. The tumor was clearly diagnosed after renal transplantation, although a computed tomography scan before renal transplantation had suggested its presence. The patient had been on hemodialysis for 11 years, with extensive acquired renal cystic disease before renal transplantation. Pathology revealed a renal cell tumor with granular cells and tubulopapillary structure, consistent with papillary renal cell carcinoma. Karyotype analysis revealed that this tumor, which was 1.7 cm in diameter, showed trisomy of chromosomes 5, 16, and 20 and deletion of chromosome Y. This preliminary study suggests that cytogenetic changes of papillary renal cell tumor associated with acquired cystic disease are similar to those in the general population.

[Glycerol-loading test in experimental acute renal failure using 31P magnetic resonance spectroscopy of the kidney].

We observed that administration of exogenous glycerol reduces beta-ATP level and increases glycerophosphate level using magnetic resonance spectroscopy (MRS) of the kidney. A new "glycerol-loading test" for use with MRS in rats was developed. Sprague-Dawley rats were anesthetized and the left kidney was exposed and placed on surface coil. 31P-MRS (109.25MHz) of the kidney was performed using 10 microseconds, 90' pulse width and 2.308 sec repetition time with an accumulation of 200 times. MRS was performed 24 hours after induction of acute renal failure in each model of acute renal failure. Glycerol-loading test was performed by the intravenous infusion of 10% glycerol through jugular vein over a 60 min period. MRS was recorded every 10 min during glycerol infusion and for 60 min after cessation of infusion. In normal rats, beta-ATP level in the kidney was decreased to 52.0% of the pre-loading value at 55 min glycerol infusion and recovered to 66.7% 55 min after glycerol infusion was stopped. In the cisplatinum model, the reduction of beta-ATP and the increase of glycerophosphate levels following glycerol-loading was similar to that in normal rats; however, the recovery of beta-ATP level after infusion was stopped was weaker. Rats treated with glycerol and HgCl2 showed rather severe acute renal failure, but the beta-ATP level at 55 min glycerol infusion was 87.3% and 92.4%, respectively, showing difference from that in normal rats. No uptake of glycerol was observed 3 hours after 45 min pedicle clamping.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of adenosine levels in the carotid artery, renal vein and inferior vena cava after glycerol or mercury injection in the rat.

The adenosine A1 receptor antagonist (FR113453) prevents glycerol- but not mercury-induced acute renal failure. To clarify this mechanism, adenosine concentration in the renal vein was measured serially. Plasma adenosine in the renal vein increased from the preinjection value of 120.6 +/- 15.4 (mean +/- SEM) pmol/ml to 426.9 +/- 107.5, 407.0 +/- 70.1 and 283.9 +/- 22.9 pmol/ml at 1, 5 and 60 min after intramuscular injection of 10 mg/kg of 50% glycerol into Sprague-Dawley rats. On the other hand, intramuscular vehicle (0.9% NaCl) injection and subcutaneous administration of 10 mg/kg of HgCl2 did not change or caused mild elevation of adenosine concentration in the renal vein. Furthermore, simultaneous blood collection from the carotid artery, renal vein and inferior vena cava revealed a greater increase in adenosine concentration in the inferior vena cava than in the artery or renal vein. These findings were not affected by the administration of FR113453 or vehicle (methylcellulose). The increase in adenosine in the inferior vena cava was derived from the release from the acutely damaged muscles due to glycerol injection. These findings suggest that the effect of adenosine A1 antagonist to prevent glycerol-induced acute renal failure is due to the inhibition of adenosine A1 receptor in the kidneys during the release of adenosine through the inferior vena cava. Therefore, the release of adenosine from the muscle and hemolysis plays an important role to induce acute renal failure in the glycerol-injected rat.

Amelioration of glycerol-induced acute renal failure in rats by an adenosine A1 receptor antagonist (FR-113453).

A potent adenosine A1 receptor antagonist, FR-113453, was tested for its preventive effect on glycerol-induced acute renal failure in rats. First, the optimum timing of FR-113453 administration was studied. Oral FR-113453 (100 mg/kg) given 1 h before or 5-10 min after glycerol injection produced a significant reduction of the serum creatinine at 24 h (4.3 +/- 0.8 mg/dL [vehicle] vs. 1.4 +/- 0.4 mg/dL [FR-113453], and 4.7 +/- 1.1 mg/dL vs. 1.3 +/- 0.6 mg/dL, respectively, p < 0.001). However, when FR-113453 was given 2 h after glycerol injection, the serum creatinine did not improve. Creatinine clearance at 24 h after the induction of acute renal failure was significantly better in rats given FR-113453 (100 mg/kg) 1 h before glycerol than in rats given vehicle alone (0.08 +/- 0.08 mL/min vs. 0.01 +/- 0.02 mL/min), (p < 0.01). The kidney weight was lower and less severe histologic changes were observed at 24 h in the FR-113453-treated group. Renal blood flow (measured using 85Sr microspheres) did not change at 24 h after glycerol injection (3.0 +/- 0.9 mL/min/g [vehicle] vs. 3.6 +/- 0.9 mL/min/g [FR-113453]), but renal vascular resistance was significantly reduced by FR-113453 (47.9 +/- 37.9 vs. 26.4 +/- 5.2 mm Hg/mL/min/g, p < 0.05). Beta-ATP levels (measured by 31P-magnetic resonance spectroscopy) were reduced in glycerol-induced acute renal failure, with no difference between the vehicle and FR-113453-treated groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of severity of acquired renal cysts between CAPD and hemodialysis.

The prevalence and severity of renal cystic changes in 15 continuous ambulatory peritoneal dialysis (CAPD) patients were compared with those in 15 hemodialysis patients matched by age, sex and duration of dialysis. Cyst grade and kidney volume were prospectively evaluated using CT scan. In 15 age- and sex-matched patients on long-term dialysis, follow-up for a mean of 32 months, beginning 73 months after the start of dialysis, revealed that, in both treatment modalities, a significant increase in cyst grade, kidney volume and the rate of increase in kidney volume as a reflection of cystic change (3.08 +/- 3.87 [mean +/- SD] ml/month in CAPD, 1.43 +/- 1.17 ml/month in hemodialysis) had occurred. However, differences in these parameters between CAPD and hemodialysis groups were not significant. In 7 patients in the CAPD group who were also treated by hemodialysis, it could not be statistically proven that the rate of increase in kidney volume during initial hemodialysis was different from that observed in subsequent CAPD (2.60 +/- 2.44 ml/month during hemodialysis, 5.13 +/- 4.94 ml/month during CAPD). This longitudinal prospective study suggests that the prevalence and severity of acquired renal cystic disease are the same in long-term CAPD and hemodialysis patients.

Cystic transformation in native kidneys in renal allograft recipients with long-standing good function.

Acquired renal cystic disease develops in the native kidneys of patients with renal allografts with long-standing poor function. However, there has been no long-term prospective study of the regression or development of cysts in native kidneys of renal allograft recipients with good long-term function (serum creatinine: 114 +/- 24 mumol/l). We followed 61 renal allograft recipients for 63.5 +/- 29.0 months (mean +/- SD) using computed tomography (CT scan) every 1 or 2 years after transplantation. The cyst grade at pretransplantation was significantly reduced at the first study after transplantation (0.98 +/- 1.39 vs. 0.57 +/- 0.96; p less than 0.01). Baseline study after transplantation revealed that the incidence of cysts in native kidneys was 22/61 (36.1%) and 26/61 (42.6%) in the follow-up study. In 32 patients (52.5%), there were no visible cysts in the native kidneys. In 9 patients (14.8%), there was no change in the number of cysts during follow-up, while in another 9 patients the number of cysts decreased. On the other hand, the number of cysts increased in 11 patients (18.0%) during follow-up. The mean follow-up duration was longer in the group with an increased number of cysts than in the groups with no visible cysts or a decrease in number. Biochemical analysis of cyst fluid from newly developed cysts in 2 patients showed differences from the pattern in acquired renal cystic disease of hemodialysis patients. These results indicate that after regression of acquired cysts in most allograft recipients, no or only a few cysts persist in patients with good graft function. The remaining cysts continue to regress further in some patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Ten-year prospective study on the development of renal cell carcinoma in dialysis patients.

A prospective study was undertaken to investigate the development of renal cell carcinoma in dialysis patients. Three renal cell carcinomas were detected among 96 hemodialysis patients in 1979, and screening by computed tomographic (CT) scan was continued yearly until 1989. During this 10-year period, one renal cell carcinoma was found in the second year and another in the ninth year. Autopsy performed on seven of 19 patients who died showed one case of small clear cell carcinoma accompanying acquired cystic disease. In 33 males, kidneys were found to have enlarged 2.7 +/- 1.7 times over the 10-year follow-up due to acquired cysts, while no change in kidney volume was noted in 24 females. Native kidneys in nine of 12 patients who maintained functioning grafts were reduced in size. The patient with the largest kidney enlargement (11.5 times) died from retroperitoneal bleeding in 1989. These prospective study results suggest that both the incidence and prevalence of renal cell carcinoma in dialysis patients is high. Furthermore, major complications of acquired renal cystic disease seem to occur predominantly in males.

Exercise-induced acute renal failure in 3 patients with renal hypouricemia.

Three cases of exercise-induced non-oliguric acute renal failure in patients with renal hypouricemia, an isolated defect of the renal urate transport system, are described. During acute renal failure, the serum uric acid levels were 5.6, 2.7 and 5.8 mg/dl, respectively, and were within normal limits. The values representing the fractional excretion of uric acid (FEUA) were 28.7, 60.0 and 12.7%, with accompanying serum creatinine levels of 8.1, 3.9 and 3.3 mg/dl, respectively. After recovery, the serum uric acid fell to 0.6, 0.7 and 1.0 mg/dl and the FEUA increased to 79.3, 52.8 and 43.2%, respectively. Two of the patients examined exhibited decreased reabsorption of filtered urate. These 3 examples of renal hypouricemia represented 23% of 13 cases of mild exercise-induced acute renal failure encountered within our experience.

Compensatory hypertrophy of renal grafts with triple drug therapy.

The kidney volume after transplantation was compared in two groups, one treated by conventional immunosuppression and the other receiving small amounts of ciclosporin (CsA) together with azathioprine and steroid (the so-called triple therapy). Fourteen pairs of donors and recipients were investigated in each group. The kidney volume was measured, using computed tomography (CT scan), before and after transplantation in the donors and after transplantation in the recipients during the allograft rejection-free period. The graft volume at 2-3 months after transplantation was smaller (215.9 +/- 30.9 ml, mean +/- SD) in patients who received small amounts of ciclosporin A (CsA) together with azathioprine and steroid than that (270.9 +/- 75.0 ml) in those treated by conventional immunosuppression. The remaining kidney in the donor after transplantation underwent a similar increase in volume in the conventional and triple therapy groups. It is suggested that even a small amount of CsA can significantly limit the compensatory renal growth.

Severity of acquired renal cysts in native kidneys and renal allograft with long-standing poor function.

To identify factors related to the development of uremic acquired cystic disease of the kidney, native and grafted kidneys were examined in four men and three women after kidney transplantation. The incidence and severity of cystic transformation of native kidneys and grafts were compared by plain computed tomographic scans. In a uremic environment (serum creatinine level of greater than or equal to 265 mumol/L [3 mg/dL] for an average of 5.0 years; range, 2.8 to 8.2 years), acquired renal cysts were formed in both the native kidneys and the graft in three of the male and one of the female patients. Cysts were extensive in the native kidneys but relatively infrequent in the grafts in three of the men. One male subject was found to have acquired cysts only in the native kidney. Acquired renal cysts developed even in grafts undergoing chronic rejection, and increased numbers were found in native kidneys that were in uremic conditions for long periods, both before and after renal transplantation. These results suggest that the duration of uremia is the most important factor in the development of acquired renal cysts.

Lectin peroxidase conjugate reactivity in acquired cystic disease of the kidney.

The origins of acquired cysts, hyperplastic epithelia of cyst walls, and renal cell carcinomas were investigated by evaluating their lectin conjugate reactivity. Paraffin-embedded blocks from 9 patients with acquired cystic disease were examined by the high-sensitivity lectin-antilectin immune peroxidase method. 11-176 lesions in each patient, 690 lesions in total, were stained both with Tetragonolobus lotus lectin (T) and peanut lectin (P); the former is specific for proximal tubules and the latter for distal tubules and collecting ducts. Out of 606 acquired cysts with single-layered epithelia, 559 (92.2%) were positive for T and negative for P, and 66 out of 75 (88.0%) cysts with hyperplastic multilayered epithelia were positive for T and negative for P. Three out of 4 solid adenomas and, to varying degrees, 5 renal carcinomas revealed the same reaction. These results suggest that almost all cysts accompanying acquired cystic disease of the kidney, including those with single-layered and multilayered epithelia, as well as solid adenomas and renal cell carcinomas, are derived from proximal tubules.