1 Section of General Internal Medicine and 2 Department of Urology, Nephrology and Rheumatology, University Hospital, Berne, Switzerland
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Abstract |
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Methods. By means of a commercially available ELISA (Pharmacia and Upjohn/Elias, Germany), we measured THP in 24-h urines of 104 SF (74 males/30 females, age 1674 years) who had formed 8.7±2.4 stones (range 1240), and of 71 C (41 males/30 females, age 2262 years). Types of stones formed by SF were 88 calcium, eight uric acid, six infection, and two cystine. All values are means±SE.
Results. The normal range (5th to 95th percentile) of UTHPxV was 9.335.0 mg/day in males and 9.036.3 mg/day in females respectively. Mean UTHPxV was 21.3±1.2 mg/day (range 3.451.6) in male and 15.2±1.6 mg/day (range 1.832.3) in female SF (P=0.008 vs male SF). Since UTHPxV was positively correlated with CCrea (r=0.312, P=0.001) in SF as well as with UCreaxV (r=0.346, P=0.0001) and with body surface (r=0.271, P=0.0003) in all study subjects, mean THP/Crea (mg/mmol) was used for all further calculations. Overall, THP/Crea was lower in SF (1.42±0.07 vs 1.68±0.08, P=0.015), mainly due to increased THP/Crea in female C (2.08±0.11, P=0.0036 vs female SF, P=0.0001 vs male C and vs male calcium SF), which also explains decreased THP/Crea values in calcium SF (1.46±0.08, P=0.041 vs C). In addition, THP/Crea was reduced in uric acid SF (1.11±0.21, P=0.049 vs C). Whereas THP/Crea was not related to age, urine volume, intake of dairy calcium, or urinary markers of protein intake, either in C or in SF, it correlated significantly with urinary Citrate/Crea, both in C (r=0.523, P=0.0001) and in SF (r=0.221, P=0.025). In C only, but not in SF, THP/Crea was correlated with urinary Calcium/Crea (r=0.572, P=0.0001) and with Oxalate/Crea (r=0.274, P=0.022).
Conclusions. Both in C and SF, urinary THP excretion is related to body size, renal function and urinary citrate excretion, whereas dietary habits apparently do not affect THP excretion. Uric acid and calcium stone formation predict reduced THP excretion in comparison with C, whereas female gender goes along with increased urinary THP excretion in C. Possibly most relevant to kidney stone formation is the fact that THP excretion rises only in C in response to increasing urinary calcium and oxalate concentrations, whereas this self-protective mechanism appears to be missing in SF.
Keywords: hypercalciuria; hyperoxaluria; inhibitors of crystallization; modulators of crystallization; nephrolithiasis; TammHorsfall protein (THP)
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Introduction |
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Because THP has been detected in highly variable amounts in kidney stones [5], it has been proposed as playing a role in nephrolithiasis. In principle, macromolecules with very high negative charge densities and strong affinities for urinary crystals become irreversibly incorporated into these crystals and therefore are part of the stone matrix [6]. However, THP, cannot be found within urinary calcium oxalate crystals [7], which indicates that its binding to crystal surfaces is not irreversible. By reversibly staying at crystal surfaces, THP mainly affects the aggregation of preformed crystals [6,8,9]. Much controversy, however, exists about whether THP is an inhibitor or a promoter of crystal aggregation [4]. Previous studies [8,9] have demonstrated that THP at urine-like concentrations is a very powerful inhibitor of calcium oxalate crystal aggregation in vitro. With rising concentrations of calcium, sodium, and hydrogen ions as well as of THP itself, inhibitory activity is progressively lost, and some abnormal THPs from recurrent stone formers even become promoters of aggregation [9].
Because THP at higher concentrations possibly starts to promote crystal aggregation, increases in urinary THP excretion rates might be of pathophysiological relevance in nephrolithiasis. Of particular interest may be that a high-protein diet has been shown to increase urinary THP excretion in rats significantly [10], since high intake of meat protein is a frequently diagnosed risk factor for kidney stone formation [11]. No data, however, are available on effects of increased protein intake on THP excretion in humans.
Using quantitative electroimmunodiffusion, Bichler et al. [12] as well as Samuell [13] found that average urinary THP excretion rates in humans were 4050 mg/day, without differences between normal subjects and calcium renal stone formers. In patients with uric acid stones as well as in those with stag horn calculi or renal tubular acidosis, however, Bichler et al. [12] described significantly lower THP excretion rates. The same was found by Wikström and Wieslander [14] in calcium kidney stone formers in comparison with healthy controls. Moreover, as part of tubular dysfunction, these authors described a particularly low THP excretion rate in patients with renal tubular acidosis [14]. More recent studies using radioimmunoassay or ELISA revealed no difference in THP excretion rates between healthy controls and calcium stone formers in two studies [15,16], whereas Romero et al. [17] showed a significant decrease of THP excretion in recurrent calcium kidney stone formers. The results of their study, however, were probably hampered by the fact that THP was measured in urine samples previously stored at -20°C. This has been shown to produce wide variations in results, whereas measurements in samples stored at -70°C or in fresh urines produce results with constant reproducibility [18].
The aim of the present study was to measure urinary THP excretion rates prospectively in non-selected kidney stone formers as well as in healthy subjects and to identify possible determinants of THP excretion rates, such as age, gender, renal function, type of stones, urine volume and intakes of protein and other nutrients.
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Subjects and methods |
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All SF underwent ambulatory metabolic evaluation while adhering to their free-choice diet [19]. They were never studied until at least 2 months had elapsed after renal colic or urological intervention. Medications known to interfere with calcium, citrate, or uric acid metabolism were discontinued at least 2 weeks before evaluation. Besides the blood and urine samples, which were obtained in the fasting state, two (in five SF) or three (in 99 SF) 24-h urine collections were performed by every patient. For comparison, 71 healthy subjects (C) without family history of kidney stones, 41 men and 30 women, aged 37.0±1.3 years (range 2262), collected one 24-h urine while on self-selected free-choice diet, whereas no fasting blood and urine samples were obtained.
Twenty-four hour urines were collected in 3-l plastic bottles containing 10 g of boric acid as preservative agent [19]; an internal study in collaboration with the Laboratory of Clinical Chemistry at the University of Berne had revealed that concentration measurements of urinary constituents as well as of pH did not differ from those obtained with thymol, a widely used preservative agent [20]. Urine samples were analysed for calcium (Ca), phosphate (P), magnesium (Mg), sodium (Na), potassium (K), chloride (Cl), uric acid (UA), urea, and creatinine (Crea) by autoanalyser techniques. Urine pH was measured by a Metrohm 654 pH meter (Metrohm, Herisau, Switzerland). Oxalate (Ox) was measured after oxidation by oxalate oxidase, citrate (Cit) using the citrate lyase method, and sulphate (Sulph) by high pressure liquid chromatography, as previously described [19].
In SF only, creatinine clearance (CCrea) was calculated from 24-h urine creatinine excretion and normalized for 1.73 m2 body surface. Body surface was determined from body length and weight, based on nomograms [21]. In all study subjects, net gastrointestinal absorption of alkali (GI-Alkali, expressed in mEq/day) was derived from excretion rates of non-combustible cations and anions according to the formula
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Urinary THP was measured by a commercially available indirect non-competitive enzyme linked immunosorbent assay [24] (Synelisa TammHorsfall Protein, Pharmacia & Upjohn/Elias Diagnostics, Freiburg, Germany), whereby monoclonal mouse anti-human-THP antibodies, immobilized on pins, bind THP antigen from standards (03.582050120 mg/l in phosphate-buffered saline) and urine samples. These antigen-antibody complexes associate with an enzyme-labelled polyclonal sheep anti-human-THP antibody, which subsequently converts added substrate to form a coloured solution. The colour formation is monitored at 492 nm. All incubations were carried out at room temperature, and measurements were performed in duplicate. Coefficients of variation were 5.05.2% for intra-assay variability and 7.89.2% for inter-assay variability.
All freshly collected 24-h urine specimens were carefully shaken for 2 min at room temperature in order to avoid losing large THP polymers for analysis due to settling. Immediately thereafter, 10-µl aliquots were aspired and diluted 1 : 100 with the denaturing sample buffer supplied with the ELISA kit. These samples were stored in airtight containers at room temperature until ELISA measurements were performed after 2050 days. Internal pilot studies in collaboration with the manufacturer (unpublished) had revealed that this procedure yielded highly stable and reproducible results even after 200 days of storage.
All values are presented as means±SE. Urinary measurements of individual SF are expressed as means of two or three urine collections. For comparisons between groups, the non-parametric MannWhitney U-test was used, whereas Wilcoxon signed-rank test was applied for within-group comparisons. Simple and multiple linear regression analyses were performed for correlation studies.
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Results |
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Whereas UTHPxV was not related to the subjects' age, either in C or in SF, it correlated positively with CCrea, which had been determined in SF only (r=0.312, P=0.001). Moreover, in the whole study population (SF and C), UTHPxV was positively correlated with UCreaxV (r=0.346, P=0.0001) as well as with body surface (r=0.271, P=0.0003). Altogether, these findings suggest that UTHPxV increases with body size and decreases when renal function becomes progressively impaired. Therefore, THP/Crea (mg/mmol) was used for all further calculations, and every urinary parameter was normalized for urinary creatinine (as a combined marker of renal function and body size).
Overall, THP/Crea was lower in SF (1.42±0.07) than in C (1.68±0.08, P=0.015). As apparent from Tables 1 and 2
, where 24-h urine data of all male or female SF as well as of male or female calcium SF are compared with respective controls, this difference is primarily due to an increase in THP/Crea among female C, whose value of 2.08±0.11 was significantly higher than in female SF (1.53±0.15, P=0.0036) as well as in male C (1.37±0.08, P=0.0001) and in male SF (1.38±0.09, P=0.0001). This increase in THP/Crea among healthy women is also responsible for the significantly lower THP/Crea values in the subgroup of calcium SF (Ca-SF, 1.46±0.08 vs 1.68±0.08 in C, P=0.041). In comparison with C, THP/Crea was also reduced in the group of seven male and one female uric acid SF (1.11±0.21 vs 1.68±0.08, P=0.049); however, with a value of 1.77, THP/Crea was not diminished in the one particularly active uric acid SF who had passed 240 gravel-like stones.
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Table 3 summarizes clinical and urinary data of male SF in comparison with female SF. When considering the whole group of SF, activity of stone disease, dairy calcium intake and urinary THP/Crea were not different between male and female SF, whereas urinary stone-forming compounds (Ca, Ox, UA) and chelating agents (Cit, Mg) as well as urinary markers of intakes of protein (Urea, P, Sulph), salt (Na, Cl), and alkali (K, GI-Alkali) were significantly increased in female SF in comparison with their male counterparts. Urine volume was equal in female and male SF; however, when urine volumes were normalized for body size and renal function, i.e. urine creatinine, they were higher in female than in male SF. Identical differences were observed when comparing male with female Ca-SF.
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Discussion |
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The amounts of THP that are being excreted daily by humans in the present as well as in previous studies [1216] vary between about 5 and 600 mg (!), depending on the assay system used for THP concentration measurements. Although differences in experimental techniques and THP antibodies may explain such huge variations, the state of disaggregation of THP molecules achieved during sample preparation is a main determinant of the number of exposed antibody binding sites [15]. In the present study, measurements in highly diluted urine samples at alkaline pH most probably guaranteed a very high degree of disaggregation of excreted THP molecules, and thus maximum interaction of antibody binding sites with the THP antibody. However, the reduced amount of excreted urinary THP that we and others [12] have measured in uric-acid SF may be, at least partly, due to these patients lower urinary pH, which may have induced pronounced self-aggregation of THP molecules in vivo. This could induce flocculation [8,9] and increased adhesion of THP molecules to urothelial cells, thereby lowering the amount of THP that can be measured in urine.
In general, available studies do not suggest that urinary THP excretion is different between kidney stone formers and non-stone formers [12,13,15,16], except for specific subgroups published in older studies, where urinary THP excretion was found to be reduced in patients with renal tubular acidosis [12,14], uric acid [12], or stag horn stones [12]. In the present comparison of 104 unselected common kidney stone patients with 71 healthy controls, urinary THP excretion was reduced in the main sub group of calcium SF (85% of all SF), as recently also found by others [17]. This was, however, solely because female stone formers excreted significantly less THP than female controls, whereas such a difference was completely absent in men. In addition, urinary THP excretion was reduced in uric acid SF, as previously described by others [12], and possibly due to increased precipitation of THP molecules in vivo at low urine pH (see previous paragraph). Urinary THP excretion, however, did not differ between subgroups of SF with calcium, uric acid, or infection nephrolithiasis.
We find increased urinary THP/Crea ratios in healthy women in comparison with stone-forming women as well as non-stone-forming and stone-forming men. This is in accordance with Samuell [13], who also demonstrated increased urinary THP/Crea ratios in healthy women in comparison with men. The difference, however, disappeared when values were no more corrected for urinary creatinine [13], as it does in our study, where urinary creatinine is also significantly lower in women than in men. The correction for creatinine, however, is justified, since we clearly demonstrate that daily urinary THP excretion is related to body-size and renal function. In other words, relative to body-size and renal function, healthy women excrete significantly more THP than healthy men and kidney stone-formers of either sex. The pathophysiological relevance of this finding remains to be elucidated.
It has been inferred from the work of Schoel and Pfleiderer [25] that the total amount of THP excreted in human urine must be synthesized de novo by the kidneys each day. Because rats fed a high-protein diet excrete more THP [10] and humans with idiopathic calcium nephrolithiasis have increased renal mass when their meat-protein intake is exaggerated [19], enlarged kidneys on high meat-protein intake might produce more THP. Therefore, an important question of the present study was whether THP excretion would be related to urinary markers of protein intake, such as urea, phosphate, and sulphate. Indeed, a rise in urinary excretion of abnormal THPs in certain stone formers with subsequent promotion of crystal aggregation [9] might provide an additional explanation for the well-known link between high protein intake and nephrolithiasis [11]. Unlike in rats fed a high-protein diet [10], however, THP excretion in humans on free-choice diet in the present study did not correlate with protein intake and therefore does not provide this additional link. On the other hand, as already described by Thornley et al. [15] and confirmed by the present study, urinary THP excretion is positively related to renal function, i.e. CCrea.
Another remarkable finding of the present study is the positive correlation of urinary THP excretion with urinary citrate, although of lesser significance in stone formers. This is in keeping with previous work by Fuselier et al. [26], who demonstrated for the first time that the increase in urinary citrate following oral potassium citrate therapy in calcium stone formers was correlated with an increase in urinary THP excretion. Since urinary citrate is a well-known marker of alkali consumption and of intracellular acidbase changes in tubular cells [27], increases in intracellular pH values of tubular cells may have additionally increased the state of disaggregation of urinary THP molecules already in vivo, i.e. before sample preparation in the laboratory. By such a mechanism, an additional number of antibody-binding sites may have been exposed to the THP antibody [15] and thus have produced higher THP concentration measurements. Alternatively, increases in intracellular pH values may directly have stimulated de novo synthesis of THP [26].
A new and possibly most relevant finding of the present study is that THP excretion is positively related to urinary excretions of the two most important stone-forming ions, calcium and oxalate, in healthy subjects; previous studies did not find such a correlation [15,28]. On the other hand, this correlation cannot be found in the whole group of kidney stone formers as well as in the large subgroup of calcium SF. Inasmuch as this type of correlation indeed suggests a causal relationship, the exact mechanism whereby THP excretion rises in response to increasing urinary calcium and oxalate, as well as the pathophysiological relevance of the fact that this correlation is missing in stone formers are not known at this stage. However, as demonstrated in animal models of nephrolithiasis, the production of crystallization inhibitors such as uropontin and THP is stimulated after induction of moderate hyperoxaluria, which most probably reflects a self-protective response against stone formation [29]. Inasmuch as THP is an important inhibitor of calcium oxalate crystal aggregation [4,6,8,9], the positive correlation of urinary THP excretion with excretions of calcium and oxalate in healthy subjects might indicate that humans are normally protected from exaggerated crystal aggregation with subsequent stone formation during periods of hypercalciuria and hyperoxaluria. Moreover, the fact that this correlation does not exist in patients with nephrolithiasis would suggest that such a self-protective mechanism could be defective in kidney stone formers.
In conclusion, the present study lists determinants of urinary THP excretion in humans (Table 6): both in healthy subjects as well as in kidney stone formers, urinary THP excretion is related to body size, renal function, and urinary citrate excretion (as a marker of renal intracellular acidbase status), whereas dietary habits (intakes of fluids, dairy calcium, and protein) apparently do not affect THP excretion. Uric acid stone formation as well as calcium stones in women predict reduced THP excretion in comparison with healthy subjects, in whom female gender goes along with increased urinary THP excretion. Finallyand possibly most relevant to kidney stone formationa rise in THP excretion correlates with increasing urinary calcium and oxalate excretions only in healthy subjects, whereas such a potentially self-protective correlation is missing in kidney stone-formers.
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Acknowledgments |
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Notes |
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References |
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