Abstract
Louisiana and other Gulf South states comprise a “Stone Belt” where calcium oxalate stone formers (CaOx SFs) are found at a high rate of approximately 5%. In these patients, the agglomeration of small stone crystals, which are visible in nearly all morning urine collections, forms stones that can become trapped in the renal parenchyma and the renal pelvis. Without therapy, about half of CaOx SFs repeatedly form kidney stones, which can cause excruciating pain that can be relieved by passage, fragmentation (lithotripsy), or surgical removal. The absence of stones in “normal” patients suggests that there are stone inhibitors in “normal” urines.
At the Ochsner Renal Stone Clinic, 24-hour urine samples are collected by the patient and sent to the Ochsner Renal Stone Research Program where calcium oxalate stone agglomeration inhibition [tm] measurements are performed. Urine from healthy subjects and inactive stone formers has demonstrated strongly inhibited stone growth [tm] in contrast to urine from recurrent CaOx SFs. [tm] data from 1500 visits of 700 kidney stone patients have been used to evaluate the risk of recurrence in Ochsner's CaOx SF patients. These data have also been used to demonstrate the interactive roles of certain identified urinary stone-growth inhibitors, citrate and Tamm-Horsfall protein (THP), which can be manipulated with medication to diminish recurrent stone formation. Our goal is to offer patients both financial and pain relief by reducing their stones with optimized medication, using medical management to avoid costly treatments.
Abnormal urine composition is believed to cause renal stone formation. Although many people, healthy and otherwise, exhibit calcium oxalate crystalluria in their concentrated morning urines, only some form stones. This observation suggests that kidney stone patients have an “evil urine,” deficient in stone inhibition factors. Though renal stone patients tend to be hypercalciuric, hyperoxaluric, and hypocitraturic, these factors alone do not allow the treating physician to clearly predict which patients will form more stones.
While kidney stone disease is prevalent in all industrialized nations, Louisiana and the Gulf States comprise what is known as the “Stone Belt” where kidney stones occur at a rate of approximately 5% (1). Of patients who initially form kidney stones, about half recur without therapy. The recurrence rate without treatment is about 10% at 1 year, 33% at 5 years, and 50% at 10 years (2). The search for reliable, rapid, and accurate tools to enable the clinician to diagnose, predict, and prevent kidney stone recurrence continues.
A new tool developed at Ochsner was found to be predictive of stone formation rates (3, 4) by evaluating the effect of calcium oxalate stone former (CaOx SF) outpatient urine on in vitro calcium oxalate monohydrate (COM) crystal agglomeration inhibition [tm]. Using enzyme-linked immunoadsorbent assay (ELISA), we examined the consequences of the removal of Tamm-Horsfall protein (THP), the dominant protein in the urine, by ultrafiltration and determined the concentration of disaggregated THP in unfiltered and ultrafiltered urine (4). It was clear from our results that THP and citrate, as well as their interaction, increased [tm] (4). Moreover, we showed that THP and citrate could be regulated by alkali medication (5).
Using [tm], the effectiveness of alkali medication could be monitored and optimized to avoid more invasive and costly urological procedures. In stone-forming patients, long-term use of alkali medication reduced stone recurrence over five-fold and the necessity of stone treatment procedures by more than ten-fold (6). Outpatient measurements of [tm] are performed on CaOx SFs exclusively at Ochsner.
Materials & Methods
Patient Selection
Over a period of 7 years, more than 700 CaOx SFs were studied at the Ochsner Renal Stone Clinic (ORSC) in 1500, often consecutive, patient visits. All patients had urographic and/or other physical evidence of forming at least one stone, usually a calcium-containing oxalate stone. Quantitative oxalate, uric acid, or phosphate analyses were performed on most of these stones.
Agglomeration [tm], Citrate, and THP
For each published study, a smaller cohort of the 700 patients was selected (4–7).
Citrate and THP Interaction (4) (n = 53)
During an 18-month period, 53 CaOx SFs (14 women and 39 men) with a mean age of 47 (± 1.9) years were studied. For comparison, 22 healthy subjects (6 women and 16 men) with a mean age of 40 (± 1.5) years were selected from apparently healthy stone-free laboratory technicians and research fellows who were not on medication during the 48 hours preceding or during the study. Whereas the mean age of the control group was approximately 7 years less than that of the patient population, the ratios of men to women in the groups were 2.7 and 2.8, respectively. Age-based differences in COM parameters had not been observed in larger populations in our prior investigations (3).
Urinary THP Increased After Potassium Citrate Therapy (5) (n=33)
During a 4-year period, we evaluated 33 CaOx SFs (20 men and 13 women) between 20 and 72 years of age (mean age 46 years: 49 for men, 43 for women). Of the 33 study patients, 16 had a positive family history of renal stone formation, 15 had a negative family history, and in 2 the family history was unknown. All patients were active recurrent calcium stone formers, with a lifetime range of 2 to 105 stones formed (mean 12) and the previous 3-year range of 0 to 30 stones formed (mean 5). In 27 patients, crystallographic analysis confirmed stones containing COM. In six patients, crystallographic stone analysis was not performed, but these patients all had radiographic evidence of calcium-containing stones.
Agglomeration [tm] During Long-term Potassium Citrate Therapy (6) (n=80)
Clinical records and radiographic and ultrasound reports of 80 patients (55 men and 25 women) between the ages of 20 and 72 years (mean age of 48) were studied. Over a 4-year period, the patients were treated with potassium citrate therapy (K-Cit-Rx) for recurrent calcium oxalate urolithiasis at the ORSC. A history of procedures for urolithiasis, including extracorporeal shock wave lithotripsy, percutaneous nephrostolithotomy, open lithotomy, or ureteroscopic basket extraction, was also documented. To be included in the study, patients were required to have been medically evaluated on a regular basis in the ORSC (every 3 to 6 months during the first year and annually thereafter), and to have had radiographic follow-up with abdominal radiographs or ultrasound reports dictated and signed by a radiologist.
pH, Citrate, THP, and [tm] in CaOx SFs Before and During K-Cit-Rx (7) (n=151)
In our most recent study, citrate, oxalate, urate, THP, pH, [tm], and 24-hour urine volume were simultaneously determined for 151 patients in 464 multiple visits to the ORSC. Patient visits were separated and grouped into initial visit (IV, n=151) and subsequent visit (SV, n=313). A higher proportion of SV patients was presumed to be on K-Cit-Rx after their IV.
Urine Collection from CaOx SFs
Twenty-four hour urine samples were collected on an outpatient basis and maintained at room temperature. Thymol crystals were added as a preservative prior to collection.
Stone Formation Rate (6)
The frequency of stone formation was assessed as the number of new stones formed during the study. Stones that were excreted or apparently radiopaque on x-ray film within 6 months after lithotripsy were considered residual fragments and were not counted as new stone formation. In order to exclude residual fragments resulting from lithotripsy when assessing new stone formation, we allowed the possible residual fragments 6 months to increase in size. Any stone still visible after this 6-month lag time was considered a residual fragment. Any stone that became visible after this 6-month lag time was considered a new stone. Patients were labeled as inactive (0 stones/ 3 years), active (1 to 3 stones/ 3 years), and very active (>3 stones/ 3 years). The relationship between [tm] and the stone formation rate was analyzed in each group.
Crystallization Studies (3, 4)
Agglomeration of COM crystals was measured by the uptake of 45Ca tracer into standardized COM seed crystals in the system detailed previously (Figure 1), which measures the effect of diluted urine (20%) on COM kinetics. The initial calcium oxalate concentration product was varied to determine the lowest point at which sharp uptake of 45Ca tracer due to crystal growth began to occur. This point, by definition, was the equilibrium concentration product (expressed as the solubility in mmol/L) allowed the measurement of the time-dependent uptake of 45Ca tracer into the seed crystals at a known starting calcium oxalate supersaturation. These kinetics permitted the calculation of agglomeration inhibition values ([tm] in minutes).
Figure 1- Agglomeration inhibition [tm] measurements. (Reprinted with permission from W.B.Saunders Company from: Erwin DT, Kok DJ, Alam J, et al. Calcium oxalate stone agglomeration reflects stone-forming activity: citrate inhibition depends on macromolecules larger than 30 kilodalton. American Journal of Kidney Diseases 1994; 24: 893–900.)
CaOx SF Urinalyses
Urinary oxalate concentration was determined enzymatically with a diagnostics kit from Sigma (St. Louis, MO). Urinary citrate was measured by adapting a citrate lyase enzymic method (8). Calcium, phosphate, urate, and creatinine were determined by routine clinical procedures in Ochsner's Department of Pathology. The ELISA assays for determining the concentration of THP were performed exactly as previously described (9). The purified THP, isolated from the urine of an apparently healthy male volunteer, was used as a standard in each assay. Patient samples were diluted until parallelism with the standard curve was obtained. Three consecutive points from the section parallel to the standard were used to derive the THP concentration. The THP content of each purified THP sample was determined spectrophotometrically in sterile deionized water using the extinction coefficient of 10.8 of a 1% solution in a 1 cm light path at 277 nm (10). The prepared log-log standard curves (Figure 2) yielded mean values for urinary THP consistent with those reported for this assay.
Figure 2- Tamm-Horsfall glycoprotein (THP) ELISA curves. Serial dilutions of the THP standard (THP STD) from 5ug/100 ul-0.04 ug/100 ul and ultrafiltered stone-forming patient urine samples diluted from 1:20 to 1:160 were parallel at high dilution. The standard THP concentration was determined in a Beckman DU-64 spectrophotometer (Beckman Instruments, Inc, Fullerton, CA) using the extinction coefficient of 10.8 for a 1% deionized distilled water solution of THP in a 1-cm light path at a wavelength of 277 nm. THP-concentration values for patients were calculated from the average of triplicate determinations of three points parallel to the standard curve. (Reprinted with permission from W.B. Saunders Company from: Erwin DT, Kok DJ, Alam J, et al. Calcium oxalate stone agglomeration reflects stone-forming activity: citrate inhibition depends on macromolecules larger than 30 kilodalton. American Journal of Kidney Diseases 1994; 24: 893–900.)
Statistical Methods
Data were analyzed and figures and graphs were produced using Graphpad Software (1998: Graphpad Inc. San Diego, CA). All values are presented as mean values ± SEM or SD. Differences were considered as significant at p < 0.05. Parametric or nonparametric t-test comparisons and secondary analyses were used as indicated by the computer software and are specified in the figures or their legends. Correlation lines and Spearman's correlation coefficients (r) were computer generated.
Results
Table 1 compares the analyte values we determined in the pre-filtered 24-hour urine collections from stone-forming patients and healthy controls for various urine parameters. The patient group showed an increased urinary calcium excretion (p < 0.007) and a tendency toward increased oxalate excretion (p < 0.1), but we did not observe the statistically significant hypocitraturia reported in a larger study by Kok, et al (3). Hypercalciuria (> 7.5 mmole/24 h), hyperoxaluria (> 0.5 mmole/24 h), and hypocitraturia (< 2.0 mmole/24 h) were present in 38%, 26%, and 26% of our patient population, respectively.
Comparison (Mann-Whitney) of 24-hour urine composition from calcium oxalate stone-formers (n=53) and healthy controls (n=22)
The crystallization parameter, [tm], was not different for patients and healthy controls when compared as a whole. Differences in [tm] were apparent, however, when the data from stone-forming patients were subdivided into the three subgroups (inactive, active, and very active) according to the frequency of their new stone formation. While the mean [tm] of inactive stone-forming patients was indistinguishable from that of healthy controls, [tm] was significantly decreased in the active stone-forming patients (p < 0.05 vs controls) and even further in the very active stone-forming patients (p < 0.01 vs active) (Figure 3).
Figure 3- The relationship between calcium oxalate monohydrate crystal inhibition [tm] and the activity of calcium oxalate stone formation in stone-formers and healthy controls. (H) Healthy controls (n=22), (I) inactive = no stones (n=19), (A) active=1-3 stones (n=15), very active = more than 3 stones (n=13) over the last three years of observation. Differences in group mean [tm]-values were considered significant at p<0.05 following analysis of variance of the groups followed by a Mann-Whitney secondary test. (Reprinted with permission from W.B. Saunders Company from: Erwin DT, Kok DJ, Alam J, et al. Calcium oxalate stone agglomeration reflects stone forming activity: citrate inhibition depends on macromolecules larger than 30 kilodalton. American Journal of Kidney Diseases 1994; 24: 893–900.)
When the [tm] value was related to the urine constituents measured, a significant positive linear relationship was found with urinary citrate concentration (p < 0.0001, r = 0.44, n = 53) (Figure 4). In addition, [tm] showed a significant positive correlation with urinary THP concentration (p < 0.0001, r = 0.56, n = 53), as shown in Figure 5. There were no significant linear correlations between [tm] and the other analytes we measured in this patient cohort.
Figure 4- The computer generated linear regression relationship line (InStat, GraphPad Software, Inc., 1993 update) between calcium oxalate monohydrate agglomeration inhibition [tm] and urinary citrate concentration (mM) in calcium oxalate stone-former 24-hour urine collections. This correlation was significant (p<0.0001, n=53). The dashed lines indicate 95% confidence limits
Figure 5- The computer generated linear regression relationship line (GraphPad, 1993 update) between calcium oxalate monohydrate agglomeration inhibition [tm] and urinary Tamm-Horsfall glycoprotein concentration (mg/L) in the 24-hour urine collections of calcium oxalate stone-formers. This correlation was significant (p<0.0001, n=53). The dashed lines depict 95% confidence limits
When [tm] values were regressed against the products of the known urinary COM crystallization inhibitors THP and citrate (11), the linear regression coefficient increased (r = 0.67; Table 2) above that observed with either inhibitor alone (r = 0.56 and 0.44, respectively). When the THP-citrate concentration product was divided by urinary calcium concentration, the correlation remained the same (r = 0.64).
Linear Regression Values for Patient Urinary Citrate and Tamm-Horsfall Glycoprotein (THP) Concentrations and Their Mathematical Combinations as They Relate to Crystal Agglomeration Inhibition [tm]
The effect of increased [tm], urinary THP, and citrate excretion with alkali treatment was examined in a limited number of patients (5). After K-Cit-Rx, the urinary pH increased in 27 patients (82%). The mean pre-alkali treatment pH was 6.0 and the mean post-alkali treatment pH was 6.4, a highly significant (p < 0.0013) difference. Urinary citrate excretion rate increased in 26 patients (79%). Despite the seven patients with decreased citrate, the citrate in the entire group increased from a pre-alkali mean of 1.9 mmol/24 h to 2.6 mmol/24 h post-alkali (p < 0.0004).
THP increased in 24 patients (73%) (5). Of the nine patients whose THP value decreased, five (56%) also had a decreased urinary citrate excretion rate. Despite these nine patients, the mean THP of the group increased from 94.0 mg/24 h pre-alkali to 199.3 mg/24 hours post-alkali (p <0.0016). If data from the five patients who had a decrease in both citrate and THP were excluded, the mean THP increase would have been an even more dramatic 96.8 mg/24 h pre-alkali vs 225.0 mg/24 h post-alkali (p <0.0006).
The [tm] increased in the majority of patients in the study (19 of 33 patients [58%]). Although 14 patients showed a slight decrease in [tm], the group as a whole had an increased mean [tm] from 177.1 minutes pre-alkali to 221.0 minutes post-alkali (p < 0.024). In an earlier study (4), we observed a synergistic effect between urinary citrate and THP. Multiplying these values again, we found the product to be more significantly related to [tm] (p < 0.0003) than either urinary parameter alone (Table 2).
In a subsequent Ochsner study, data were organized in terms of age (years), duration of K-Cit-Rx (months), K-Cit dose (mEq/day), family history of stone disease, total number of stones passed, present radiographic stone status, average number of stones passed over 3 years, stone forming activity, number of follow-up urine studies, and the chemical composition of stones (Table 3). Mean 24-hour pretreatment urine values were compared with mean values during K-Cit-Rx.
Clinical History (n = 80)
Mean values ± SEM were calculated for all patients for pH, volume (L), calcium (mmol/d), citrate (mmol/d), oxalate (mmol/d), uric acid (mg/d), creatinine (mg/d), the in vivo effect of urine on the COM agglomeration inhibition [tm] (expressed in minutes), average number of stones passed per year, and average number of stone-related procedures (see patient selection) performed per year (Table 4).
Effect of Potassium Citrate Therapy (K-Cit-Rx) (n = 80)
A family history of stone disease could be documented in 44/80 patients (55%). Crystallographic analysis revealed a calcium phosphate component in the stones of three patients, while four had a uric acid component. The other stones were presumed to represent predominantly calcium oxalate due to their radiographic visibility. Chemical stone analysis was documented in 32 of the 80 patients.
Most importantly, clinically relevant relationships were observed after long-term K-Cit-Rx in calcium stone formers (Table 4). After treatment, we observed a decrease in the number of stones passed per year (∼5 fold), a decrease in the number of procedures (∼10 fold), and increased [tm] (∼25%). A dramatic increase in pH and a smaller decrease in calcium were observed between pretreatment and during treatment.
Though the citrate excretion rate tended to be higher (p=0.2039) during K-Cit-Rx, we did not observe a statistically significant increase (in these outpatients on free diets). The calculated difference between urinary citrate concentration after K-Cit-Rx (1.31±0.091 SEM) and before K-Cit-Rx (1.15±0.061) more closely approached statistical significance (p=0.087). These unexpected results might be partially accounted for by the fact that the pretreatment citrate excretion rate was below 2 mmol/d in only 37 patients; only four in this group had decreased citrate excretion after K-Cit-Rx (data not shown). In contrast, 13 of the 43 patients with baseline citrate excretion above 2 mmol/d either decreased or failed to show an increased citrate excretion after K-Cit-Rx.
Linear regression analyses of citrate excretion data from these patients indicated a significant linear correlation between [tm] and 24-hour citrate excretion rate before (p=0.0073) and during (p=0.0011) K-Cit-Rx. Linear correlations between [tm] and 24-hour urine citrate concentration were also observed before (p=0.019) and during (p=0.0001) K-Cit-Rx. By contrast, when [tm] was regressed against increased urinary pH during K-Cit-Rx, the correlation approached significance (p= 0.13); however, there was no suggestion of a correlation between [tm] and urinary pH before medication (p=0.77). Not surprisingly, no statistical correlation was noted when [tm] was regressed against urine volume before K-Cit-Rx (p=0.68) or during K-Cit-Rx (p=0.14).
No differences were observed for oxalate, uric acid, urine volume, or creatinine excretion rate before or during treatment (Table 4).
In our most recent and comprehensive study of CaOx SFs at Ochsner (7), SV pH and citrate excretion increased vs IV (5.97 to 6.23 mm/24 h [p < 0.0001] and 2.16 to 2.69 mm/24 h [p < 0.0001], respectively). This is consistent with this large group of 151 patients presumed to be on long-term K-Cit-Rx (7). The increased citrate excretion rate observed in this study (7), but not in our previous study (6) with smaller patient groups (n=80), substantiates that K-Cit-Rx increases citrate excretion when large groups of CaOx SFs are compared. The 24-hour excretion rate of uric acid also increased from 484.4 (IV) to 542.3 mg/24 h (SV) (p< 0.01) (Table 5).
Urine Composition from COSF Before (IV) and During (SV) K-Cit-Rx
Interestingly, linear regression analyses of 24-hour urine THP and urinary urate concentration showed a positive correlation in the SV (p<0.001) group (Figure 6). In both IV and SV groups, [tm] and THP were positively correlated (p<0.0001, p<0.027, respectively) (Figure 7), as in earlier studies (4), confirming THP's inhibitory role.
Figure 6- Linear regression relationship between urinary urate concentration [U] and Tamm-Horsfall protein concentration [THP] during potassium citrate therapy (K-Cit-Rx) at subsequent patient visits (SV, n=313, p<0.001). (Reprinted with permission from Editoriale Bios, Parma, Italy from: Cole FE, Romani W, Kok DJ, et al. Urine pH, citrate, urate (U), TammHorsfall protein (THP), and calcium oxalate (CO) monohydrate agglomeration inhibition [tm] in calcium oxalate stone formers (COSF) before and during potassium citrate therapy (KCITRx). In: Kidney Stones: Proceedings of the 8th European Symposium on Urolithiasis. Borghi L, Meschi T, Briganti A, et al, editors. Parma, Italy: Editorial Bios, 1999:527530.)
Figure 7- The relationship between calcium oxalate monohydrate inhibition [tm] by patient urine and Tamm-Horsfall protein concentration [THP] at initial patient visits (IV, n=151, p<0.001) and at subsequent visits (SV, n=313, p=0.027). (Reprinted with permission from Editoriale Bios, Parma, Italy from: Cole FE, Romani W, Kok DJ, et al. Urine pH, citrate, urate (U), TammHorsfall protein (THP), and calcium oxalate (CO) monohydrate agglomeration inhibition [tm] in calcium oxalate stone formers (COSF) before and during potassium citrate therapy (KCITRx). In: Kidney Stones: Proceedings of the 8th European Symposium on Urolithiasis. Borghi L, Meschi T, Briganti A, et al, editors. Parma, Italy: Editorial Bios, 1999:527530.)
Discussion
Because renal stone formation is a physiochemical process, it has been surprising that more of the 99% of subjects with CaOx crystalluria did not form renal stones. This observation has led to the hypothesis of an inhibitor(s) in normal urine preventing urinary crystal coalescence from forming stones large enough to be retained in the renal tree. At Ochsner, we have used the new tool of [tm] measurements to examine the effects of the urinary constituency of CaOx SFs, before and during K-Cit-Rx, on the inhibition of CaOx crystal clumping in artificial urine in vitro. These data represent a comprehensive assessment of the efficacy of [tm] in predicting the subsequent stone-forming activity of CaOx SFs.
Conservatively, we defined stone activity as new stone formation determined by x-ray, or spontaneous excretion of stones that were not previously present. Increased size of previously recognized stones was not considered active stone disease. Our results were nearly identical with those of prior reports from The Netherlands (3, 12, 13) on populations with comparable age and sex. We confirmed that urine from normal control subjects is capable of inhibiting both the growth (data not shown) and the agglomeration (4–7) of calcium oxalate crystals. The capacity to inhibit crystal agglomeration, [tm], was significantly diminished in the urine from CaOx SFs, a decrease directly related to the frequency of stone formation in the patients (Figure 3). While the urine from nonactive stone-formers (no new stones formed in the previous 3 years) was comparable to that of normal controls, the urine from active stone-formers (1 or 2 new stones formed) showed a significant decrease in the capability to inhibit crystal agglomeration. This capacity was even further reduced in the urine from very active stone-formers (three or more new stones formed in the previous 3 years). Taken together, these observations suggest that these noninvasive [tm] measurements may be clinically useful, reflecting stone recurrence in treating CaOx SFs.
We also confirmed the beneficial effects of alkali therapy (12) in CaOx SF patients and demonstrated that use of K-Cit-Rx in CaOx SFs results in an increased [tm] and stone recurrence (4–7). However, it is apparent from earlier studies (12) and the Ochsner studies summarized here (4–6) that the relationship between [tm] and citrate concentration is not absolute. Patient data from Ochsner confirmed the earlier findings (12) identifying a subset of patients who did not increase [tm] or reduce stone recurrence with alkali medication, in spite of normalizing their citrate excretion. These data supported the hypothesis that other urinary compounds were also involved in the inhibitory process. Studies of Ochsner's CaOx SFs have demonstrated that at least some of these compounds are high molecular weight (MW) compounds. Upon removal of molecules >30 kD from the urine, the ability to increase [tm] was completely lost (4). One of these compounds was likely to be 90 kD urinary Tamm-Horsfall glycoprotein (14) which, like citrate concentration, showed a correlation with increased [tm] (4). Other possibly inhibitory compounds, which were not measured in studies of Ochsner CaOx SFs, include nephrocalcin (15), uropontin (16), and crystal matrix protein (17).
Interestingly, while the citrate concentration remained unchanged before and after ultrafiltration in our study, the correlation between [tm] and citrate was absent in ultrafiltered urine (4). We hypothesized that citrate requires the presence of high MW compounds (such as THP, and possibly nephrocalcin, osteopontin, crystal matrix protein, or other macromolecules [18] that were removed by ultrafiltration) to effect [tm]. These data should be kept in mind when trying to optimize a protocol, using alkali therapy, which aims primarily at increasing citrate excretion. Our finding at Ochsner (4) of a synergistic action of THP and citrate on [tm] (Table 2) supports independent data from Hess (11, 19). In contrast to the data of Hess et al (11) from highly active recurrent stone-formers, we did not observe in our stone-forming patient population an effect of urine calcium concentration upon the apparent synergism between citrate and THP concentrations population (4, 5). Moreover, by analysis of particle sedimentation rate at saturated conditions, Hess et al (11) demonstrated that the addition of citrate to a solution of THP, at conditions where THP tends to self-aggregate (low pH and high ionic strength), greatly enhanced the inhibition of calcium oxalate aggregation by normal THP. When citrate was added to THP isolated from stone-formers, the action on crystal kinetics reversed from promotion of aggregation to inhibition. This dualistic ability of THP to act as both a promoter and as an inhibitor of crystal aggregation has been a controversial point in kidney stone research for some time.
In an early Ochsner study with a small patient cohort (5), pH increased an average of 0.4 in calcium stone-forming patients after K-Cit-Rx. The urinary citrate excretion also increased by a mean of 0.74 mmol/24 h. Surprisingly, THP also increased a significant amount, more than doubling from 94.0 mg/24 h to 199.3 mg/24 h. If the data for probable noncompliant patients were eliminated (e.g., patients whose urinary citrate did not increase on oral potassium citrate therapy), these values would be even more dramatic, with THP increasing from 96.8 mg/24 h pre-alkali to 225.0 mg/24 h post-alkali (p<0.0006). To our knowledge, this was the first report of increased THP excretion associated with oral alkalinization by K-Cit-Rx (5). The mechanism responsible for this effect, whether representing increased de novo synthesis or “washout” of pre-existing THP, or both, remains to be determined.
In this study with K-Cit-Rx (5), there was also a corresponding increase in mean [tm] from 177.9 minutes to 220.98 minutes. This suggests that the time it takes for crystals to agglomerate in urine of alkalinized patients might increase by as much as 24.8% over those without alkali treatment. Therefore, with alkali therapy, there would be less time for crystals to agglomerate and, theoretically, less chance of calcium stone formation (20). The main observation of our earlier study (5) was that recurrent calcium stone-forming patients, when treated over a short-term period with K-Cit-Rx (5), had increased urinary excretion of citrate and THP with a corresponding increase in [tm]. Secondly, since [tm] was inversely related to stone formation in two studies of different calcium stone-forming populations (3, 4) and since agglomeration of preformed calcium oxalate crystals is theoretically the rate-limiting factor in the formation of stones (3, 20), these observations could help explain, at least in part, the effectiveness of K-Cit-Rx in CaOx SFs.
Other important clinical data were observed in 80 Ochsner CaOx SFs during K-Cit-Rx over an average duration of 15 months. Improvement in control of their disease was documented by a five-fold decrease in the stone passage rate and a ten-fold decrease in the need for remedial operative procedures. As important, this radiologically verified clinical improvement in the passage of new stones was accompanied by a marked increase in the ability of the urine during K-Cit-Rx to increase [tm]. Thus, [tm], observed to be an indicator for the risk of stone recurrence before (3) and during (5, 6, 12) K-Cit-Rx, can be used as a guide for medical treatment of renal stones to help monitor the clinical effects during long-term treatment with K-Cit-Rx (6).
Although an optimum protocol for the use of alkaline citrate in the management of stone disease remains to be completely defined (21–23), only one of our observations in a single study (6) was unexpected: the overall citrate excretion did not rise (p=0.2039) on alkali treatment. Information from patient interviews and the fact that the pH increased (p<0.0001) support that there was good compliance with the medication regimen. There is one report that increased citrate excretion during citrate therapy was reversible with increased time (21). The most likely explanation in this Ochsner patient cohort was that a majority of patients had low normal baseline citrate excretion. Alternatively, the citrate data may suggest that the rise in pH alone can be beneficial, perhaps by making other macromolecular inhibitory substances (e.g. THP [4,5,11], nephrocalcin [15,24], CMP [17,25], uropontin [16,26], UAP [27]) more inhibitory or by preventing dissolution of hydroxyapatite particles that are involved in heterogeneous nucleation with calcium oxalate in the distal tubule/collecting duct (28, 29). Our use of urine at a 20% dilution in our in vitro crystallization assay (3, 4) may reflect exactly in vivo conditions at a halfway point inside the collecting ducts where crystal agglomeration is most likely to occur (20, 28).
In our most recent study (7), repeated observations of a large group of CaOx SFs during long term K-Cit-Rx found that urinary citrate, urate, and pH increased, whereas THP and urine volume did not. Noted earlier (4) was the positive correlation observed in this large group of 151 patients (7) between the increase of urinary [THP] and the ability of CaOx SF patient urines to inhibit [tm], confirming the inhibitory nature of THP during short and longer term K-Cit-Rx. In a prior independent study, it was observed that THP excretion increased with urine volume during the recovery period after exercise (30). A later study from another laboratory (31), however, found THP excretion to be volume independent, with the suggestion that the increase during recovery after exercise was due to “washing out pre-existing THP.” A “washout” of pre-existing THP could also be responsible for the THP excretion observed early in the course of alkali therapy (5). Increased THP excretion was not observed in this most recent investigation with a large group of patients studied on longer term K-Cit-Rx (7). Our most recent data support a THP “washout” phenomenon.
Urinary urate and THP concentrations were linearly related in our most recent study (7). Elsewhere, THP has been observed to increase after protein loading (31, 32). As suggested by others (31), the positive linear correlation we observed between urinary urate and THP concentrations could be non-specific and related to protein intake. A chronic high protein intake, associated both with high nucleic acid content (a precursor of urate [33])and with an increased protein synthesis due to high levels of circulating amino acids (31), would also support concurrent increased THP synthesis. Chronic high protein diets are also associated with increased nephrolithiasis and a decreased urinary ability to inhibit agglomeration (32). Short-term protein-loading results in a decreased ability to inhibit calcium oxalate agglomeration (32), which suggests a lower inhibitory THP concentration. It appears that short-term changes in THP excretion due to acid (protein) (32) or alkali loading (5, 12) differ from the long-term effects of acid and alkali loading. The reason for this needs to be explored further.
Conclusion
Urinary parameters such as 24-hour calcium, oxalate, sodium, or urine supersaturation have not provided physicians with the initially hoped for information needed to predict new stone formation. The best procedure, thus far, for improving [tm] has been alkalinization. Monitoring [tm], in addition to the routine radiologic follow-up of stone burden, helps in determining the efficacy of medication in preventing stone recurrence (5–7, 12). Our data support the utility of [tm] determinations on urine samples before and during therapy as a noninvasive tool to help support other monitors of clinical improvement of the disease. [tm] data might also be used to help identify patients requiring more aggressive medical therapy and help guide the physician in adjusting the dose of medication over long term therapy in order to decrease stone-forming rates and remedial procedures during K-Cit-Rx. Effective treatment can be assisted by evaluating the level of [tm] and increasing it to a normal range.
Acknowledgments
The authors would like to acknowledge the artistic work of Barbara Siede, Medical Illustrator. We would also like to acknowledge the following individuals:
Staff
Jawad Alam, PhD,
Scientific Research Staff
Fellows
David M. Ward, MD
John Allen, MD
Pedro A. Marcucci, MD
Kenneth Moore, MD
Research Support
Jean Vaughn, Laboratory Supervisor
Byrnes T, Carriere, Laboratory Technician
Obakeye Coker, Laboratory Technician
Carla James, Research Coordinator
Gwen Thezan, LPN
Medical Students
Dante Galliano, BS
David Whitehead, BS
Joey Turnipseed, BS
- Ochsner Clinic and Alton Ochsner Medical Foundation
References
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