CN1965858A - Medicament for preventing and treating renal calculus through regulating concentration of citric acid in urine - Google Patents

Abstract

The invention discloses that lithium carbonate is used to prepare a medicine for regulating and controlling the concentration of urinary citric acid and preventing and treating kidney stones.

Description

Drugs that regulate urinary citric acid concentration to prevent and treat kidney stones

technical field

The invention relates to the use of a salt, specifically refers to the use of lithium carbonate to prepare a medicine for regulating and controlling the concentration of urinary citric acid and preventing and treating kidney stones.

Background technique

According to reports, there is no significant difference in the absorption of citric acid from the gastrointestinal tract of hypocitrauria patients and normal people. Hypocitraturia is not the result of gastrointestinal malabsorption but is the result of excessive reabsorptive transport by the kidneys. Citric acid is freely filtered from the glomerulus, and the concentration of citrate in the excreted urine depends on the plasma citrate concentration and its reabsorption by the renal proximal tubules. Its excessive reabsorption will result in decreased excretion of citric acid from urine. Through the in vitro study of renal proximal tubule tissue, it was confirmed that the first step of kidney reabsorption of citrate is its transport by dicarboxylic acid transporters (NaDC1 and NaDC3) located in the brush border and basement membrane of renal proximal tubules . Other similar studies have confirmed that the absorption of blood citrate is mainly mediated by the Na ⁺ -dependent citrate transporter (NaCT) distributed in tissues such as the liver. Thus, the functional status of these transporters plays a decisive role in the regulation of urinary citrate.

NaDC-1 is a sodium ion-dependent dicarboxylic acid transporter. Its substrate affinity is low, and its main physiological function is to absorb citric acid cycle intermediates, such as citric acid and succinate, but mainly succinic acid. NaDC-3 is a sodium-dependent, high-affinity substrate dicarboxylate transporter that is present in the kidney, brain, liver, and placenta. The function of NaDC-3 in the kidney is mainly to generate the driving force of the organic anion transporter OAT1 to promote the entry of organic anions into the renal tubular wall cells through the basement membrane. NaCT is one of the newly discovered members of this transporter family. It is expressed in the liver, testis and brain. It has a high affinity with citrate and is the main transporter of citrate dicarboxylate. It also transports other dicarboxylic and tricarboxylic acids but with very low affinity.

Excessive reabsorption of citric acid in urine by the kidneys is one of the important causes of hypocitraturia. Hypocitraturia occurs more frequently in patients with kidney stones, most of which are congenital. The mechanism causing congenital hypocitraturia is still unclear, but in its pathogenesis, Na ⁺ -dependent dicarboxylic acid transporters (NaDC1 and NaDC3) and Na ⁺ -dependent citrate transporter (NaCT) may play a role Important role, see He, YN, Chen, XC, Yu, ZH: Sodium dicarboxylate cotransporter-1expression in renal tissues and its role in rat experimental nephrolithiasis.J Nephrol, 17:34, 2004; HE, YN, CHEN, XM, YU, ZH, et al: The change of human Na+/dicarboxylate co-transporter 1 expression in the kidney and its relationship with pathogenesis of nephrolithiasis Natl Med J China, 81: 1066, 2001; Pajor, AM: Molecular cloning and functional expression of a sodium-dicarboxylate co- trans-porter from human kidney. Am J Physiol, 270: F6422648, 1996.

Studies have also found that citric acid can effectively prevent the formation of kidney stones by complexing with calcium ions, inhibiting calcium coagulation in urine and the formation of calcium oxalate crystals, see Ryall, R.: Urinary inhibitors of calcium oxalate crystallization and their potential role in stone formation .. World J Urol, 15:155, 1997. At this stage, it is known that oral citrate (such as potassium citrate, magnesium potassium citrate, etc.) can increase the concentration of urinary citric acid, thereby effectively inhibiting the formation of kidney stones, see Hamm, L., Hering-Smith, K.: Pathophysiology of hypocitraturicnephrolithiasis . Endocrinol Metab Clin North Am, 31: 885, 2002, and, Hojgaard, I., Tiselius, H.: The effects of citrate and urinary macromolecules on the aggregation of hydroxyapatite crystals solutions with a composition dissimilar to that ol sub t in the , 26:89, 1998. However, these drugs have poor mouthfeel and severe gastrointestinal reactions, thus limiting their clinical application. In addition, local perfusion of citric acid solution can also effectively dissolve calcium-containing stones, see Zhang, X.B., Wang, Z.P., Duan, J.M.: Urothelial injury to the rabbitbladder caused by calcium dissolving agents including two new citrate solutions. Urol Res, 33:309 , 2005, and, Zhang, X.B., Wang, Z.P., Duan, J.M.: New chemistry for urological calcium phosphate calculi-a study in vitro. BMC Urol 5:9 2005. However, its unbearable side effects, such as damage to the urinary system mucosa and damage to the body such as local catheterization required for perfusion, make it difficult for these therapies to be widely used in clinic. Therefore, it is necessary to continue to explore new therapeutic approaches, find out new drugs to control the concentration of citrate in urine and new drugs to treat kidney stones.

Contents of the invention

Through a series of experimental studies, the present invention provides a new medicine for preparing and controlling the content of citric acid in urine and treating kidney stones.

The invention uses lithium carbonate to prepare medicines for controlling the concentration of citric acid in urine and medicines for treating kidney stones.

At this stage, lithium carbonate is a commonly used drug in psychiatry for the treatment of mania and so on. Lithium carbonate can effectively reduce symptoms, and its safety has been confirmed. But so far, no report has been found to treat other diseases with lithium carbonate.

The research of the present invention shows that the use of lithium carbonate treatment can significantly increase the urinary citric acid concentration of humans and rats, thereby effectively inhibiting the development of kidney stones; it can make crystal deposition caused by ammonium oxalate, dilation of renal tubules and infiltration of peritubular inflammatory cells, etc. Damage was significantly reduced. It shows that lithium carbonate has a significant preventive effect on calcium oxalate stones. According to a large number of clinical reports that lithium carbonate is used to treat human manic psychosis, oral administration of lithium carbonate is safe, and no obvious toxic and side effects have been found on humans in therapeutic doses of lithium carbonate, and the incidence of adverse reactions may be lower than that of other oral administration. Citrates, such as potassium citrate and magnesium potassium citrate, etc.; and the efficiency of this drug in increasing urinary citric acid levels is significantly higher than that of the latter. However, lithium accumulation poisoning may occur. Therefore, during the period of taking this medicine, the blood lithium concentration should be checked regularly, so as to adjust the dosage accordingly.

Description of drawings

Figure 1 Changes in urinary citrate concentration after lithium carbonate treatment. In the figure, x±s, ^b P<0.05 compared with other groups at the same time point.

Fig. 2 Changes of plasma citrate concentration in rats after treatment with lithium carbonate. In the figure, x±s, ^b P>0.05 compared with other groups at the same time point.

Figure 3 Changes in plasma citrate concentration after lithium carbonate treatment. In the figure: x±s, ^c P<0.01 compared with lithium carbonate treatment group. ^b P<0.05 compared with rats in 36.9mg and 52.4mg lithium carbonate treatment groups. ^e P<0.05 compared with rats in the 52.4mg/d lithium carbonate treatment group.

Fig. 4 to Fig. 7 show the pathological changes of the renal pathology in the calculus model rats. The specimens were taken from calculus model rats, and the cortical tissue sections were fixed in formalin and routinely stained for observation and comparison. in:

Figure 4 is the kidney tissue of the control group (×200).

Fig. 5 is the kidney tissue (×200) after modeling with 5% ammonium oxalate for 14 days. It can be seen from the figure that the renal tubules are obviously dilated, and inflammatory cells leak into the interstitium.

Fig. 6 is the kidney tissue (×200) of the calculus model rat treated with lithium carbonate for 7 days. It can be seen from the picture that the renal tubules are mildly dilated and a small amount of inflammatory cells exudate.

Fig. 7 shows that the stone model rats were treated with lithium carbonate for 14 days. It can be seen from the figure that the pathological damage was further alleviated compared with the above lithium carbonate treatment for 7 days.

Figure 8 Changes in urinary citric acid concentration in patients after lithium carbonate treatment. In the figure ^b P<0.05 compared with the placebo group at the same time point.

Detailed ways

The present invention provides relevant experiment and result below:

Animal experiments The experimental animals used were Waster rats.

Take 120 Waster rats and randomly divide them into 4 groups according to body weight:

(1) Control group. Normal feed, free drinking tap water.

(2) 5% ammonium oxalate group. Feeding feed containing 5% ammonium oxalate, free drinking tap water (the same below) + tap water gavage, 2 times/day.

(3) Lithium carbonate group. Normal feed, free drinking tap water + 18.2mg/ml lithium carbonate gavage, 2 times/day.

(4) 5% ammonium oxalate + lithium carbonate group. After feeding with 5% ammonium oxalate feed for 7 days, 18.2 mg/ml lithium carbonate was added to the stomach, 2 times/day.

In the experiment, (3) group, i.e. lithium carbonate group, was given lithium carbonate 18.2mg/ml.1ml gavage from the beginning, twice a day; After feeding with 5% ammonium oxalate feed for 7 days, add lithium carbonate 18.2mg/ml.1ml orally, twice a day. Simultaneously observe the dose-effect relationship on rats when two doses of lithium carbonate 26.4mg/day and 52.4mg/day are used for treatment. Before the start of the experiment, all rats were fed for 7 days to adapt to the metabolic cage environment. On the 3rd, 7th and 14th days after lithium carbonate treatment, the rats were placed in metabolic cages to collect urine samples for 24 hours, and thymol and 0.5ml 12N hydrochloric acid was used to prevent citric acid from being decomposed by urinary bacteria, and then blood samples were collected via the right ventricular approach. The right kidney was placed in 10% formalin for histological examination, and the left kidney was used for examination of renal crystals.

Enzymatic detection of serum citrate and oxalate. Other biochemical indicators such as blood and urine creatinine, calcium, phosphorus, potassium, magnesium and uric acid etc. were detected by a fully automatic biochemical analyzer (SYNCHRON CX7, manufactured by Beckman Coulter). Urine pH was detected with a pH meter (Mettler Toledo 320-S, sensitivity 0.01), and crystalluria was detected with a polarizing microscope.

Kidney Crystal Deposition Detection

Grading of renal crystal deposition: 0 = no crystal deposition; 1 = a small amount of crystals dispersed in the renal medulla; 2 = a moderate amount of crystals dispersed in the renal medulla and papilla; 3 = a large amount of crystals dispersed in the renal medulla, renal papilla and cortex; 4 = A large number of calculi are distributed in the renal papilla accompanied by hydronephrosis or renal calculus.

Kidney Pathological Examination

Routine paraffin sections of renal cortex tissue, HE staining. According to renal tissue damage from mild to severe, the grades are 0, 1, 2 and 3: (0 = no obvious damage; 1 = mild dilation of part of the renal tubules, inflammatory cell infiltration damage in peri-tubular tissues < 20%; 2 = many dilated renal tubules, inflammatory cell infiltrating damage of peritubular tissue < 40%; 3 = most dilated renal tubules severely, inflammatory cell infiltrating damage of peritubular tissue > 40%).

In addition to the animal experiments, the present invention has also carried out corresponding experiments on the treatment effect of human lithium carbonate.

Randomly select 55 patients (39 males, 17 females, average ages 35.8 and 30.6 years) who are receiving lithium carbonate treatment from the psychiatric department, collect blood and urine samples, and observe the changes of relevant biochemical indicators. Exclude obstructive urinary tract disease, chronic urinary abscess, renal failure (serum creatinine greater than 168 μmol/L, normal not greater than 132 μmol/L), renal tubular acidosis and patients who had undergone lithotripsy within 6 months. All 55 patients signed informed consent. Among them, 35 patients were given 0.5g lithium carbonate bid, oral treatment; the remaining 17 patients received placebo treatment.

All the above experiments were processed statistically, and SPSS13.0 statistical software was used for data processing, and LSD-t test or Dunnett t-test was used for statistical analysis. P<0.05 is considered significant.

The corresponding experimental results are as follows:

1. Effect of lithium carbonate on biochemical indicators in rats

After 3 days of treatment with lithium carbonate, the concentration of urinary citric acid in rats was significantly increased. Compared with the control group, the concentration of urinary citric acid in the lithium carbonate group increased by 4 times (P<0.05). However, with the prolongation of treatment time and the increase of dose, there was no significant further increase. The level of blood citrate in the lithium carbonate treatment group increased slightly, but there was no significant difference compared with the control group (P>0.05, Fig 2). Blood Li ⁺ concentrations increased with treatment dose, see Figure 3. Under the treatment of 36.9mg/d lithium carbonate, the average Li ⁺ concentration was 0.82mmol/L. In rats in groups (2), (3) and (4), urinary uric acid slightly increased from day 14, but there was no significant difference compared with the control group. All rats treated with lithium carbonate had significantly elevated urine pH. Group (4), that is, 5% ammonium oxalate + lithium carbonate group, its urine creatinine level increased to some extent from the 7th day, while the urinary creatinine level of the group (3) using only lithium carbonate increased from the 14th day increased. Serum creatinine concentrations in groups (2), (3) and (4) all increased to varying degrees, but only group (4) had significant significance compared with the control group on day 21, see Table 1. Other serum biochemical indicators of the tested mice, such as Na ⁺ , K ⁺ , Mg ⁺ and P, had no significant changes.

Ammonium oxalate-induced kidney stone model

After feeding ammonium oxalate for 3 days, the concentrations of oxalic acid and calcium in the urine of the mice increased significantly, and there was no significant fluctuation over time, and the serum oxalic acid concentration did not increase significantly, see Table 2. Crystalluria occurred in 87% of the rats tested, but no rat kidney crystal deposition was found. On the 7th day, the proportion of crystalluria in rats increased to 100%, and moderate to large crystal deposition (87%) appeared in the renal medulla and papillae. On day 14, all ammonium oxalate-treated rats showed massive crystal deposition, see Table 2. Pathological examination showed that there was only slight tubular dilatation on day 3, see Table 3, and obvious tubular dilation, crystal deposition and obvious peritubular inflammatory cell infiltration were seen on day 7, see Figure 5. Except for the lithium carbonate treatment group, the kidney damage of all the above-mentioned rats fed with ammonium oxalate was obviously aggravated, as shown in Tables 2 and 3. However, no obvious kidney damage occurred in the rats treated with lithium carbonate, and hyperoxaluria and crystalluria persisted in the stone+lithium carbonate group. However, after the addition of lithium carbonate treatment, the kidney damage was gradually alleviated, and basically returned to normal on the 21st day, see Figure 6 and Figure 7.

2. Changes in biochemical indicators of patients

After lithium carbonate treatment, the patient's urinary citrate concentration increased significantly and did not increase with the increase of lithium carbonate dose (Figure 6). Serum citrate concentration did not change significantly. Serum Li ⁺ concentration increased with lithium carbonate dose. The mean serum lithium concentrations of patients treated with 1.0g/d. lithium carbonate and 1.5g/d. lithium carbonate were 0.62(0.62±0.12)mmol/L and 0.80(0.80±0.21)mmol/L, respectively. The patient's body weight did not decrease significantly, and there were no obvious changes in other biochemical indicators.

The above experiments showed that after 3 days of treatment with lithium carbonate, the concentration of urinary citrate was significantly increased and there was no significant further increase with the prolongation of treatment time and dose increase. The mechanism of this change in urine is: Li ⁺ can replace Na ⁺ to bind to one of the binding sites of these dicarboxylic acid transporters, and this combination can significantly affect its transport function, thereby reducing the increase in urine and blood citrate concentrations, Indirectly increase the concentration of citric acid in excreted urine. Due to the limitation of the ion replacement site and the limitation of the citrate content in the filtered urine, increasing the dose or prolonging the treatment time had no further significant effect on the urinary citrate concentration. On the other hand, this shows that the lower therapeutic dose has reached the maximum regulatory effect, so there is no need to try to increase the therapeutic dose in order to further improve the efficacy.

Fig. 8 shows the change of urinary citrate concentration after applying placebo and lithium carbonate to treat patients respectively. Placebo is the placebo group and LiC is the lithium carbonate treatment group. It can be seen that the concentration of urinary citric acid in patients increased significantly after treatment with lithium carbonate.

In order to evaluate the therapeutic effect of lithium carbonate on kidney stones, we fed rats with 5% ammonium oxalate feed to establish a rat kidney stone model. Ammonium oxalate is an effective inhibitor of kidney stone formation, which can effectively increase the formation of urinary stones in rats, see Khan, S.: Animal models of kidney stone formation: analysis. World J Urol, 15: 236, 1997. In this experiment, rats fed with 5% ammonium oxalate feed for 3 days showed obvious hypocitricuria, hyperoxaluria and crystalluria, and a large amount of crystal deposition was seen in the renal medulla and papilla of rats on 7 days. On the 14th day, in addition to the above pathological changes, a large number of stones and a large number of inflammatory cell infiltration around the renal tubules could still be seen. After the application of lithium carbonate, the crystal deposition and inflammatory cell infiltration around the renal tubules were gradually reduced, and basically disappeared at 21 days, indicating that this therapy has a good effect on experimental calculi in rats.

The above experiments show that lithium carbonate can be used to prepare a medicine for regulating the concentration of citric acid in urine and treating kidney stones. The use of lithium carbonate can significantly increase the concentration of citric acid in human and rat urine, thereby effectively inhibiting the development of kidney stones; it can significantly reduce the crystal deposition caused by ammonium oxalate, dilation of renal tubules, and infiltration of peritubular inflammatory cells. Table 1 Changes of other biochemical indicators in rat urine (mmol/L) The changes of urinary Cr, pH, Oxalate and Ca ²⁺ from day 5 are listed in the table. x±s, ^b P<0.05 compared with other groups at the same time point. After ammonium oxalate modeling, Ca2 ⁺ and oxalate increased significantly, and urinary pH increased significantly after lithium carbonate and lithium citrate treatment. project control group 5% ammonium oxalate group Lithium carbonate group 5% ammonium oxalate + lithium carbonate group Cr10d14d21dpH10d14d21dOxalate10d14d21dCa ²⁺ 10d14d21d 2.32 ^± 0.30b ^{23.1 ±} 0.29b 2.33± ^0.26b 5.90±0.45b ^6.02 ±0.45b 6.06± ^0.49b 0.19±0.080.23±0.110.22±0.112.60±0.472.52±0.352.56±0.30 ^2.42 ±0.32 ^b 2.49±0.33 ^b 3.04±0.55 ^b 5.89±0.61 ^{b 5.93±0.74 b 6.10} ±0.63 ^b 0.96±0.35 ^b 0.95±0.37 b 1.02±0.38 ^b 3.87±0.56 ^b 3.79±0.66 ^{b 3.634} 3.95±0.754.25±0.864.97±0.817.10±0.357.15±0.307.05±0.450.20±0.100.19±0.110.18±0.122.58±0.462.60±0.472.58±0.45 3.16±0.624.37±0.794.84±0.897.12±0.447.10 ^± 0.497.06±0.460.95 ^± 0.37b 0.97±0.41b ^0.99 ±0.41b 3.95±0.53b ^{3.84 ±} 0.55b 3.88± ^0.62b Table 2 The ratio (%) of crystalluria and kidney crystal deposition in rats. Collect the data of 3, 7, 10, 14 and 21d of 4 groups of rats respectively. x±s, ^c P＜0.01vs group A, B and E; ^b P<0.05vs group C and group D; ^b P<0.05vs group C and D; ^f P<0.05vs group A, B and E group no Crystalluria Crystal deposition 3 7 10 14 twenty one 3 7 10 14 twenty one Control group 5% ammonium oxalate group Lithium carbonate group 5% ammonium oxalate + lithium carbonate group 30303030 ^{075c 075c 0100c 0100c 0100c 0100c 0100c 0100c 0100c 0100c 012.5c 012.5b} 050 ^c 050 ^{f 050c 025b 060c 012.5b} 070 ^c 00 ^b Table 3 Pathological changes of rat kidneys on day 1. The pathological sections were taken from the renal cortex of rats with kidney stones, fixed in formalin, routinely sectioned, observed and counted after HE staining ( ^b P<0.05 VS group C and D). group no renal tubule dilatation Inflammatory cell infiltration 0+ 1+ 2+ 3+ 0+ 1+ 2+ 3+ Control group 5% ammonium oxalate group Lithium carbonate group 5% ammonium oxalate + lithium carbonate group 30303030 3003018 00012 0900b ^02100b 3003021 0606 ^{01203b 01200b}

Claims (2)

1. Lithium carbonate is used to prepare the medicine for controlling the concentration of citric acid in urine. 2. Lithium carbonate is used in the preparation of medicines for the treatment of kidney stones.

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