Evidence for size and charge permselectivity of rat ascending colon. Effects of ricinoleate and bile salts on oxalic acid and neutral sugar transport.

We have measured unidirectional transmural fluxes of oxalate and neutral sugars across rat ascending colon in vitro, under short-circuit conditions, to characterize permeability barriers selective for size and charge. Ionic oxalate appears to be transported preferentially to sodium oxalate. Mucosal addition of taurocholate (1 mM), deoxycholate (1 mM), or ricinoleate (1 mM) increased bidirectional oxalate fluxes, and the ricinoleate effects were independent of medium calcium. Bidirectional fluxes of uncharged sugar molecules fell sharply at molecular weights above 76 (molecular radius above 3 A), and oxalate transport was retarded relative to that of uncharged molecules of similar size, suggesting that there is both size and charge permselectivity. Ricinoleate increased fluxes of all neutral molecules tested but changed neither the exclusion limits nor the cation selectivity of the epithelium. Bile salts and ricinoleate increase oxalate transport, probably by making more channels available, but do not alter size and charge selectivity.

Evidence that serum calcium oxalate supersaturation is a consequence of oxalate retention in patients with chronic renal failure.

Serum oxalate rises in uremia because of decreased renal clearance, and crystals of calcium oxalate occur in the tissues of uremic patients. Crystal formation suggests that either uremic serum is supersaturated with calcium oxalate, or local oxalate production or accumulation causes regional supersaturation. To test the first alternative, we ultrafiltered uremic serum and measured supersaturation with two different methods previously used to study supersaturation in urine. First, the relative saturation ratio (RSR), the ratio of the dissolved calcium oxalate complex to the thermodynamic calcium oxalate solubility product, was estimated for 11 uremic (before and after dialysis) and 4 normal serum samples using a computer program. Mean ultrafiltrate oxalate predialysis was 89 +/- 8 microM/liter (+/- SEM), 31 +/- 4 postdialysis, and 10 +/- 3 in normals. Mean RSR was 1.7 +/- 0.1 (predialysis), 0.7 +/- 0.1 (postdialysis), and 0.2 +/- 0.1 (normal), where values greater than 1 denote supersaturation, less than 1, undersaturation. Second, the concentration product ratio (CPR), the ratio of the measured calcium oxalate concentration product before to that after incubation of the sample with calcium oxalate monohydrate crystal, was measured in seven uremic and seven normal serum ultrafiltrates. Mean oxalate was 91 +/- 11 (uremic) and 8 +/- 3 (normal). Mean CPR was 1.4 +/- 0.2 (uremic) and 0.2 +/- 0.1 (normal). Predialysis, 17 of 18 uremic ultrafiltrates were supersaturated with respect to calcium oxalate. The degree of supersaturation was correlated with ultrafiltrate oxalate (RSR, r = 0.99, r = 29, P less than 0.001; CPR, r = 0.75, n = 11, P less than 0.001). A value of ultrafiltrate oxalate of 50 microM/liter separated undersaturated from supersaturated samples and occurred at a creatinine of approximately 9.0 mg/dl.

Evidence for secondary hyperparathyroidism in idiopathic hypercalciuria.

Circulating levels of immunoreactive parathyroid hormone (PTH) were measured in 40 patients with idiopathic hypercalciuria (IH) before and during reversal of hypercalciuria with thiazide, and in four normal subjects before and during induction of hypercalciuria with furosemide. 26 patients with IH had elevated serum PTH levels. The remaining patients had normal levels. Although the correlation was not complete, high PTH levels were generally found in patients who had more severe average urinary calcium losses. When initially elevated. PTH levels fell to normal or nearly normal values during periods of thiazide administration lasting up to 22 months. When initially normal, PTH levels were not altered by thiazide. Reversal of hyperparathyroidism by thiazide could not be ascribed to the induction of hypercalcemia, since serum calcium concentration failed to rise in a majority of patients. Renal hypercalciuria produced by furosemide administration elevated serum PTH to levels equivalent to those observed in patients with IH. The findings in this study help to distinguish between several current alternative views of IH and its relationship to hyperparathyroidism. Alimentary calcium hyperabsorption cannot be the major cause of IH with high PTH levels, because this mechanism could not elevate PTH. Idiopathic hypercalciuria cannot be a variety of primary hyperparathyroidism, as this disease is usually defined, because PTH levels are not elevated in all patients and, when high, are lowered by reversal of hypercalciuria. Primary renal loss of calcium could explain the variable occurrence of reversible hyperparathyroidism in IH, since renal hypercalciuria from furosemide elevates serum PTH in normal subjects. Consequently, a reasonable working hypothesis is that IH is often due to a primary renal defect of calcium handling that leads, by unknown pathways, to secondary hyperparathyroidism.

Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid.

We have determined that the organic matrix of calcium oxalate kidney stones contains a glycoprotein inhibitor of calcium oxalate crystal growth (nephrocalcin) that resembles nephrocalcin present in the urine of patients with calcium oxalate stones and differs from nephrocalcin from the urine of normal people. Pulverized calcium oxalate renal stones were extracted with 0.05 M EDTA, pH 8.0; nephrocalcin eluted in five peaks using DEAE-cellulose column chromatography, and each peak was further resolved by Sephacryl S-200 column chromatography. Four of the five DEAE peaks corresponded to those usually found in nephrocalcin from urine; the fifth eluted at a lower ionic strength than any found in urine. Amino acid compositions and surface properties of nephrocalcins isolated from kidney stones closely resembled those of nephrocalcins isolated from urine of stone-forming patients: they differed from normal in lacking gamma-carboxyglutamic acid residues, and in forming air-water interfacial films that were less stable than those formed by nephrocalcin from normal urine.

Evidence that blood ionized calcium can regulate serum 1,25(OH)2D3 independently of parathyroid hormone and phosphorus in the rat.

This study asks whether arterial blood ionized calcium concentration (Ca++) can regulate the serum level of 1,25-dihydroxy-vitamin D3 [1,25(OH)2D3] independently of serum phosphorus and parathyroid hormone (PTH). We infused either PTH (bovine 1-34, 10 U/kg body wt/h) or saline into awake and unrestrained rats for 24 h, through a chronic indwelling catheter. PTH raised total serum calcium and arterial blood ionized calcium, yet serum 1,25(OH)2D3 fell from 35 +/- 6 (mean +/- SEM, n = 10) with saline to 12 +/- 3 pg/ml (n = 11, P less than 0.005 vs. saline). To determine if the decrease in serum 1,25(OH)2D3 was due to the elevated Ca++, we infused PTH into other rats for 24 h, along with varying amounts of EGTA. Infusion of PTH + 0.67 micron/min EGTA reduced Ca++, and 1,25(OH)2D3 rose to 90 +/- 33 (P less than 0.02 vs. PTH alone). PTH + 1.00 micron/min EGTA lowered Ca++ more, and 1,25(OH)2D3 increased to 148 +/- 29 (P less than 0.01 vs. saline or PTH alone). PTH + 1.33 micron/min EGTA lowered Ca++ below values seen with saline or PTH alone, and 1,25(OH)2D3 rose to 267 +/- 46 (P less than 0.003 vs. all other groups). Thus, during PTH infusion lowering Ca++ with EGTA raised 1,25(OH)2D3 progressively. There were no differences in serum phosphorus concentration or in arterial blood pH in any group infused with PTH. The log of serum 1,25(OH)2D3 was correlated inversely with Ca++ in all four groups infused with PTH (r = -0.737, n = 31, P less than 0.001), and also when the saline group was included (r = -0.677, n = 41, P less than 0.001). The results of this study indicate that serum 1,25(OH)2D3 may be regulated by Ca++ independent of PTH and serum phosphorus levels in the rat. Since 1,25(OH)2D3 regulates gastrointestinal calcium absorption, there may be direct feedback control of 1,25(OH)2D3, by its regulated ion, Ca++.

Urine glycoprotein crystal growth inhibitors. Evidence for a molecular abnormality in calcium oxalate nephrolithiasis.

One reason that some people are prone to calcium oxalate nephrolithiasis is that they produce urine that is subnormal in its ability to inhibit the growth of calcium oxalate crystals. We have identified in human urine a glycoprotein (GCI) that inhibits calcium oxalate crystal growth strongly, and at low concentrations (10(-7) M); in this study, we have isolated GCI molecules from the urine of normal people and patients with calcium oxalate stones. GCI from stone formers is abnormal in three ways: it contains no detectable gamma-carboxyglutamic acid (Gla), whereas normal GCI contains 2-3 residues of Gla per mole; about half of the GCI in urine of patients inhibits crystal growth 4-20 times less than normal GCI as judged by its performance in a kinetic growth assay, in vitro; at the air-water interface, patient GCI has a film collapse pressure approximately half of normal. GCI molecules from the urine of patients with calcium oxalate nephrolithiasis are intrinsically abnormal, and these abnormalities could play a role in the genesis of stones.

Nephrocalcin: biosynthesis by human renal carcinoma cells in vitro and in vivo.

Renal carcinoma cells removed surgically from two patients (one primary tumor and one bone metastasis) were maintained in short-term culture. Media conditioned by these cells contained calcium oxalate monohydrate crystal growth inhibitor, a glycoprotein named nephrocalcin (NC). NC was also detected in both cell lines by an enzyme-linked immunosorbent assay using anti-NC antibody raised in rabbits. The glycoprotein was purified from the culture medium and found to have an amino acid composition similar to that of normal human urinary NC. However, NC from the renal carcinoma cells, isolated in multiple forms by DEAE-cellulose column chromatography, contained larger amounts of carbohydrate residues than normal NC. Purified NCs showed a dissociation constant of 10(-6) to 10(-8) M toward calcium oxalate monohydrate crystal. Three renal carcinoma cell lines maintained in long-term culture failed to produce NC. Our study demonstrates that NC is produced by renal cell carcinoma cells (in vitro) from primary and metastatic tumors. Preliminary data suggest that urinary levels of NC corresponded with disease progression in patients with metastatic disease, suggesting that NC may be useful clinically as a tumor marker.

Calcium-uric acid nephrolithiasis.

A small fraction of patients with nephrolithiasis form mixed stones containing calcium and uric acid or pass both calcium and uric acid stones; 23 of 539 patients we have studied fall in this category. These mixed stone formers tend to have unusually frequent stone recurrences. Although the patients are often considered to have a variant of uric acid nephrolithiasis, a high proportion harbor calcium as well as uric acid disorders. The usual treatment for uric acid lithiasis may fail to prevent calcium stone recurrence, unless concomitant calcium disorders are simultaneously corrected. Dual treatment may be very effective in preventing continued stone disease.

Evidence for mild reversible hyperparathyroidism in distal renal tubular acidosis.

Circulating levels of immunoreactive parathyroid hormone were measured in six patients with distal renal tubular acidosis before and during two years of long-term alkali therapy. Parathyroid hormone level was elevated modestly in five patients before treatment and fell, gradually during treatment to normal or near normal levels. Urine calcium level fell, serum calcium level rose, and renal phosphorus reabsorption rose during treatment. Stopping treatment briefly caused reversion of serum parathyroid hormone and calcium levels and renal phosphorus reabsorption to pretreatment values within eight weeks. Mild hyperparathyroidism is present in renal tubular acidosis and reverses with alkali treatment.

Formation of a single calcium stone of renal origin. Clinical and laboratory characteristics of patients.

Of 859 consecutive patients with calcium renal stones, 182 had formed only one stone before entering our program. Ninety-three (51.1%) had idiopathic hypercalciuria or hyperuricosuria; 36 (19.8%) others had a systemic disorder producing stones. The remainder, 53 (29.1%) patients, had no metabolic disorder. The frequency of hypercalciuria was lower than among patients with multiple stones. Compared with recurrent stone formers, patients with single stones were older when they passed their stones, suffered higher rates of complication such as surgery, cystoscopy, and urinary tract infection, and had a lower frequency of idiopathic hypercalciuria. During treatment, 11% formed a new stone in an average follow-up period of three years, a relapse rate similar to that of multiple stone formers; however, those patients with single stones who did relapse formed new stones at lower rates than multiple stone formers. Compared with recurrent stone formers, patients with solitary stones were more often treated with diet alone. Since there were no substantial differences between solitary and recurrent stone disease, patients who have formed a single stone should be evaluated and they should be treated no differently from other patients with stone disease.

Elucidation of multiple forms of nephrocalcin by 31P-NMR spectrometer.

Four forms of nephrocalcin have been routinely isolated from mammalian kidney tissues and urine using DEAE-cellulose column chromatography with a linear NaCl gradient. We have demonstrated that these four forms of nephrocalcin, isolated from bovine kidneys, contain different amounts of phosphate residues, and that alkaline phosphatase digestion converts these to only one form of nephrocalcin. The changes in the nephrocalcin before and after removal of phosphate residues were measured by 31P-NMR spectrometer. Loss of phosphate residues decreased the dissociation constant of nephrocalcin 10-fold toward calcium oxalate monohydrate crystals, suggesting the phosphate residues appear to be important in the inhibitory effects of calcium oxalate monohydrate crystal growth.

Factors that predict relapse of calcium nephrolithiasis during treatment: a prospective study.

We evaluated 522 patients with idiopathic, recurrent calcium nephrolithiasis using a comprehensive clinical and laboratory protocol, and obtained additional laboratory measurements during their subsequent years of treatment in our program. In 57 patients, a new calcium stone ultimately formed during treatment (relapse), whereas 189 others have been free of recurrence during at least two years (average 4.3 +/- 2.2 [SD] years) of follow-up. Compared with the patients who remained stone-free, the patients with relapse (1) had a shorter interval between the time they entered our program and the time their last recurrent, pretreatment stone formed; (2) excreted more calcium *in mg/kg of body weight pr 24 hours) in their urine during treatment (2.79 +/- 1.08 versus 2.39 +/- 0.98 [SD] for relapse and stone-free); and (3) increased their urine volume less during treatment compared with pretreatment values (delta in liters per 24 hours was -0.02 +/- 0.48 versus 0.23 +/- 0.54 for relapse and stone-free). The two groups were otherwise the same. All comparisons used only data obtained prior to relapse. A discriminant function using only these three characteristics correctly identified 72 percent of patients with relapse and 67 percent of those who remained stone-free.

Effects of low-calcium diet on urine calcium excretion, parathyroid function and serum 1,25(OH)2D3 levels in patients with idiopathic hypercalciuria and in normal subjects.

We have used a low-calcium diet providing only 2 mg/kg (body weight) per 24 hours of calcium to distinguish between "renal" and "absorptive" idiopathic hypercalciuria. Sixteen of 27 hypercalciuric subjects excreted calcium in excess of intake during days seven, eight and nine of he diet, suggesting some element of renal hypercalciuria; however, all patients had low or normal serum PTH and urine cAMP levels. In general, fasting urine calcium was elevated in these 16 subjects and normal in the remaining 11, who conserved calcium more normally. SErum 1,25(OH)2D3 levels were the same in patients and normal subjects, even though PTH levels of the patients were below those of he normal subjects. Urine magnesium excretion and phosphorus excretion were both increased in the patients who excreted calcium in excess of intake. Our findings suggest that renal and absorptive hypercalciuria may not be distinct entities but rather the two extremes of a continuum of behavior. A uniform elevation of intestinal calcium absorption and a variable defect of renal calcium reabsorption could explain our results far better than the hypothesis of distinct absorptive and renal forms of hypercalciuria.

Pathogenesis and treatment of calcium stones.

Calcium stones arise from imbalances between urinary excretions of insoluble salts and water. Idiopathic hypercalciuria and hyperparathyroidism are the calcium disorders usually associated with elevated levels of calcium in the urine. Renal tubular acidosis is associated with a disordered acid-base status that results in low urine citrate. Hypocitraturia itself is a cause of calcium stones because it leaves urine calcium free to complex with either oxalate or phosphate. Elevated urine oxalate is commonly associated with dietary excesses, bowel disease, and, rarely, primary hyperoxaluria. Hyperuricosuria, usually of dietary origin, when reversed can cause a fall in new calcium stones.

Preliminary report of a new treatment strategy for advanced pelvic malignancy: surgical resection and radiation therapy using afterloading catheters plus an inflatable displacement prosthesis in the treatment of advanced primary and recurrent rectal cancer.

An unsolved problem in colon and rectal surgery involves the treatment of locally invasive primary and recurrent rectal cancer. An approach is described that uses intracavitary iridium-192 sources in combination with a pelvic displacement prosthesis to augment external beam radiation doses to sites of residual disease identified at surgery. This approach should permit administration of tumoricidal doses of radiation to positive surgical margins minimizing radiation toxicity to the small bowel. The radiation source and all prosthetic materials are removed at the bedside within 2 weeks of surgery, ensuring accurate radiation dosimetry, minimizing infectious complications, and sparing the patient the need for full high-dose pelvic irradiation.

A modified cyanide-nitroprusside method for quantifying urinary cystine concentration that corrects for creatinine interference.

Cystinuria, an inherited disease, is clinically diagnosed by detecting cystine in urine. A colorimetric method using sodium cyanide and sodium nitroprusside is a simple qualitative test used to detect cystinuria. Several colorimetric methods have been proposed for quantitative analysis of cystine; however, we found that none of them were satisfactory because the results were not reproducible. The causes of non-reproducible results were: (1) insufficient reduction time for conversion of cystine to cysteine, and (2) the interference of creatinine. In this report, we present a method to quantitate cystine in urine. We also found that ascorbic acid and ferric chloride, but not zinc chloride, interfered with the color reaction. Using this method, 15 normal urine samples (10 males and 5 females) and 12 cystine stone forming patients' (5 males and 7 females) urine were analyzed. The method was compared to commercially available urine controls. Only captopril showed a dose dependent response and color intensity at 521 nm. Thiola and D-penicillamine showed little effect on cystine determination.

Renal stone disease in older adults.

The pathophysiology of stone disorder in older adults, as compared to their younger counterparts, has not been thoroughly investigated. This article examines the differences in serum and urine chemistries between groups that are younger and older than 60 years of age. The principal finding is that stone formation occurs at lower urinary supersaturations in older patients, suggesting that other unexplored factors are significant contributors. The authors then review the possible effect of age on the morbidity of stone disease and the implications of stone disease for the development and management of osteoporosis.

Clinical characteristics and pathogenetic mechanisms in hyperuricosuric calcium oxalate renal stone disease.

Studies of patients with hyperuricosuria have provided four kinds of evidence linking their hyperuricosuria to calcium stone formation and identifying them as a distinct entity apart from other calcium stone formers. The evidence includes a more virulent natural history of stone disease, in vitro evidence for heterogeneous nucleation of calcium oxalate crystallization by seed crystals for uric acid or sodium hydrogen urate, and a reduction by uric acid or urate of the inhibitory activity which urine normally has upon calcium oxalate crystal growth. The fourth bit of evidence, the dramatic reduction in stone formation rates with allopurinol treatment, will be discussed in detail in a following paper.

The effects of allopurinol treatment on stone formation on hyperuricosuric calcium oxalate stone-formers.

The syndrome of hyperuricosuric calcium oxalate nephrolithiasis, consisting of calcium oxalate stones, hyperuricosuria and the absence of known causes of calcium stones, follows a course of particularly severe stone disease. This course can be dramatically altered by the reduction in new stone formation by the chronic administration of allopurinol. The mechanisms proposed in linking hyperuricosuria to calcium oxalate stones are both dependent upon the presence of oversaturation of the urine with one or both species of uric acid. Allopurinol reduces the saturation of the urine with respect to sodium hydrogen urate and reduces urine concentration of undissociated uric acid, thus directly altering the pathogenetic mechanisms proposed for this disorder.