The changes in urinary crystal properties in patients with calcium oxalate (CaOx) calculi after oral administration of potassium citrate (K3cit) were investigated via atomic force microscopy (AFM), scanning electron microscopy (SEM), X-ray powder diffractometry (XRD), and zeta potential analyzer. The AFM and SEM results showed that the surface of urinary crystals became concave, the edges and corners of crystals became blunt, the average size of urinary crystallines decreased significantly, and aggregation of urinary crystals was reduced. These changes were attributed to the significant increase in concentration of excreted citrate to
Nephrolithiasis, characterized by renal calculi, is a disorder that has recently shown a trend of rising incidence around the world. Renal calculi mainly contain calcium oxalate (CaOx) crystals [
Although CaOx is supersaturated in urine, the formation of renal calculi in healthy people is difficult because of all kinds of inhibitors in urine, including citrate, magnesium, osteopontin, and tyrosine hydroxylase [
Potassium citrate (K3cit) is one of the main drugs for treatment and prevention of renal calculi. In July 1985, the US Food and Drug Administration approved K3cit as a single drug to treat CaOx calculi with low urinary citrate and uric acid calculi. As a clinical drug, K3cit is widely used for its advantages such as nontoxicity, low price, few side effects, and potential for long-term use. Studies have shown that after K3cit intake in patients with chronic renal calculi, the occurrence rate of new renal calculi was one-fifth of that without K3cit intake [
However, little is known about the change in urinary crystal properties in patients with CaOx calculi after K3cit administration. After ten years of urinary crystal study, we found that after K3cit intake, crystal depressions emerge on the surfaces of some urinary crystals in patients with CaOx calculi, which is direct evidence that citrate dissolves CaOx calculi
Sodium azide (NaN3) and all other reagents used were all analytically pure. All the glassware was washed clean with secondary distilled water.
Urine crystals were observed using a Philips XL-30 environmental scanning electron microscope (SEM) at 20 kV after being covered by an ultrathin layer of gold. Atomic force microscopy (AFM) was performed in contact mode with air by using commercial AFM (AutoProbe CP, ThermoMicroscopes, USA). Microfabricated silicon nitrite cantilevers (Park Scientific Instruments) were used. The 512 × 512 pixel AFM images were obtained using the software (ThermoMicroscopes Proscan Image Processing Software Version 2.1) provided with the instrument, which can eliminate low-frequency background noise in the scanning direction. X-ray diffraction (XRD) results were obtained using a D/max-
The participants in the study included 30 randomly selected lithogenic patients (18 men and 12 women; mean age = 53.1 years; range = 21~73 years; all of them were from the Lithotripsy Center of the First Affiliated Hospital of Jinan University) and 30 randomly selected healthy humans with no prior history of urinary stones (16 men and 14 women; mean age = 37.5 years; range = 22~56 years; all of them were from the graduates and teachers of Jinan University). Urinary stones were collected after surgery, disinfected with 75% alcohol (A.R. grade), rinsed with distilled water, and placed in a dust-free incubator at 40°C to dry. The urinary stones were then ground into powder by an agate mortar for X-ray diffraction (XRD) characterization, which showed that the quality fraction of CaOx in stones was between 80% and 100% and that the stones contained small amounts of calcium phosphate and uric acid.
The changes in urinary crystal property in 30 patients (from the same 30 patients above) before and after K3cit intake were studied for a week, and the dose of K3cit (in tablet) was set at 2.538 g/d. None of the patients had gastric intolerance.
Urine treatment and urinary crystallite collection were carried out according to the methods reported in the literature [
Ammonium metavanadate-assisted catalytic-kinetic spectrophotometry was performed to determine urinary citrate content [
Figure
SEM images of representative urinary crystals from patients with CaOx renal calculi after oral administration of K3cit for one week. Scale bar: 20
Aside from crystal depressions that emerged on the surface of crystals, the morphology of urinary crystals showed other changes, such as the rounding and smoothing of the corners and edges (Figure
To further study these concave crystals, the morphology was observed via AFM. Figure
AFM images of urinary crystal of one patient with CaOx calculi before (a) and after (b) K3cit intake. Image size: 3
Figure
XRD patterns of urinary crystals of two patients with CaOx calculi before ((a), (c)) and after ((b), (d)) K3cit intake. ★: COM;
Among the 30 stone patients, 21 patients (70%) had crystalluria, in which 10 patients had COM as main composition, 1 patient had COD as main composition, and 2 patients had both COM and COD. The other compositions were uric acid (UA, seven cases) and calcium phosphate (CaP, one case). However, after K3cit intake, only seven patients had still crystalluria, in which 1 patient had COM, 2 patients had COD, and 4 patients had both COM and COD. The other composition was urate, uric acid, and CaP.
Before K3cit intake, the citrate content in urine of patients with CaOx calculi was
Comparison between the properties of urine and urinary crystals from healthy control individuals and patients with CaOx calculi before and after K3cit intake. (a) Excretion amount of citrate; (b) excretion amount of GAGs; (c) urinary pH; (d) zeta potential (
The obtained values
Before K3cit intake, the zeta potential of urinary crystals in patients with renal calculi was −
Whether it was the group which had hypocitraturia or the group with normocitraturia, the citrate content in urine of patients increased after K3cit intake. But there was no linear relationship between the increment of citrate excretion and the amount of urine citrate before K3cit intake. The concave urinary crystals appeared in the urine with a high citrate excretion amount after K3cit intake.
Citrate is one of the most abundant anions in urine and an important inhibitor of calcium calculi [
Figure
When citrate excretion increases, the formation of CaOx calculi is possibly inhibited through the following mechanisms. Citric acid is a tricarboxylic acid with acid dissociation constants (pKa) of 2.9, 4.3, and 5.6 [
Nine patients of the 30 stone patients studied had urinary crystallites with concave surface after K3cit intake. Their citrate excretion amounts were in the region from 510 to 725 mg/L with an average of 631 mg/L, which was higher than the average value of citrate excretion amounts after K3cit intake (492 mg/L).
(1) K3cit makes the tips of the crystals round and smooth.
Citrate can maintain a complexation-dissociation equilibrium with the Ca2+ ions on the surface of CaOx crystals in urine, especially those surrounding and on the tips of CaOx crystals. These crystals are continuously dissolved because of the complexation with citrate. At the same time, the dissolved Ca2+ ions are continuously deposited on the surface of CaOx crystals. This continuous dissolution-deposition caused the morphology of crystals to be more rounded and blunt after K3cit intake (Figure
(2) K3cit inhibits aggregation of urinary crystals.
After the tips and edges of crystals became round and smooth, the tendency of crystal aggregation was reduced. In addition, the increase in zeta potential absolute value on the surface of urinary crystals after K3cit intake (Figure
(3) K3cit can decrease the size of urinary crystals.
The excretion amounts of citrate (Figure
(4) In addition, citrate is active in the nucleation phase of calcium oxalate and has a profound effect on the morphology of the crystals formed [
(5) K3cit inhibits the formation of UA crystals and reduces the heterogeneous nucleation process of COM crystals. A lot of UA crystals can be found among urinary crystals. The XRD spectra (Figure
(6) K3cit converts COM crystals to COD crystals. The SEM data (Figure
Urinary crystal depressions were first observed in patients with CaOx calculi after K3cit intake, and these depressions are direct proof of the dissolution of CaOx crystals by citrate, thereby preventing CaOx calculi formation. The cause of crystal depression formation was explained. After K3cit intake for one week, the aggregation of urinary crystals in patients with CaOx calculi significantly decreased, the species and amount of the crystals decreased, COD content increased, and COM content decreased. In addition, the zeta potential absolute value on the crystal surface increased because of the increase in urinary citrate and GAGs excretion amount as well as urine alkalization. These results inhibit CaOx calculi formation. The results in this work will help shed light on the prevention of kidney stones formation and the delay of its recurrence.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This research work was supported by the Natural Science Foundation of China (81170649 and 30672103).