The Characteristics Of Cystine Stone Patients And The Study Of Mechanism Of The Formation Of Cystine Stone

Chapter 1 IntroductionCystine stone is a rare disease,resulting from cystinuria,which is an autosomalrecessive or incompletely recessivedisease characterized by renal andintestinal transport defects of dibasic amino acids including cystine,ornithine,lysine,and arginine.Cystine stones recurrentfrequently and grow fast,resulting in the high risk of renal failure.Thus,this disease still is challenge in today’s Urology.However,the literatures focused on cystine stones are rare,especially in China.Therefore,we are ready to investigate the epidemic date,clinical and genetic characteristics of Chinese cystine stone patients,and study the mechanism of the formation of cystine stones.Chapter 2 The epidemical characteristicsof cystine stone Section 1 The gender and age related distribution of cystine stones according to the analysis of 5248 urinary stonesObjective:To investigate the gender and age related distribution of Chinese cystine stones.Methods:Between 1995 and 2008,5248 urinary stones were analyzed in our center by infrared spectroscopy.The demographic date of stone patients was also recorded.The special or rare composition was conformed by polarizing microscopic analysis and scanning electron microscopic analysis.Results:Among 5248 urinary stones,59 of them were composed of cystine,accounting for 1.12%.Among the 59 patients with cystine stones,39 of them were males and 20 were females,with a sex ratio of 1.91:1.No age trend was observed in these 59 patients,but when adjusted for the cases in each age group,the proportion of cystine stones in each age group decreased with age.Among 177 pediatric urinary stones,16 of them were cystine stones,accounting for 9.04%,which was significantly higher than that in adult stones.Conclusion:our results indicate that cystine stones are very rare in adult patients,but accounts a relatively high proportion in pediatric stone patients.Section 2 The retrospective analysis of the features of onset of cystine stone patientsObjective:To investigate thefeatures of onset of cystine stone patients.Method:The 30 cystine stone patients diagnosed in our center were included.Among these patients,19 were males and 11 were females,with a sex ratio of 1.72:1.The age of them ranged from 4 to 64 year old,with mean age of 26.3±15.4 year old.The demographic date of stone patients was also recorded.The date,including the age of first onset,the age of definite diagnosis,the information of stones,recurrence and surgical treatments,was recorded.Results:The median age of the first onset was 12 year old whereas the median age of definite diagnosis was 23 year old.Among 30 patients,28 of them was with upper urinary tract stones,of which most were bilateral and multiple.The mean diameter of the largest stone in each patient was 2.8 ± 1.3cm,and the most of these stones were quasi-circular.Staghorn stones were also frequently observed in our patients.The frequency of recurrence and surgical treatment in our patients were 0.6±0.3 and 0.47 ±0.32 per year,respectively.Themean intervalto recurrence2.4± 2.0 years.Furthermore,there was no association between age of the onset and the frequency of recurrence and surgical treatment.Conclusion:Our results indicate that the age of onset of cystine stones is relatively low but do not associated with the frequency of recurrence and surgical treatment.We also firstly found that a relatively long interval between the age of the onset and that of the definite diagnosis of cystine stones in China.Cystine stones in most of our patients were bilateral and multiple,with a large size.Furthermore,our patients suffered a high frequency of recurrence and surgical treatments.Chapter 3 The mechanism of the formation cystine stones Objective:To study the toxicity of cystine crystals on the renal proximal tubular cells.Methods:Humanrenalproximaltubularepithelial cell line(HK-2)was treated with different concentration of cystine crystals,and HK2 treated with calcium oxalate crystals was considered as the positive control.After 6 h cultures,the levels of MCP-1 mRNA in cells were quantified by qPCR.After 24 h culture,the cell proliferation rates,and the concentration of lactic dehydrogenase(LDH),malondialdehyde(MDA)and MCP-1 in culturesolution were evaluated.When HK2 cells were co-treated with cystine crystals and super oxygendehydrogenises(SOD)for 24h,the cellproliferation rates,and the concentration of were evaluated.Results:Cystine crystals significantly inhibited cell proliferation of HK2 cells.They also significantly increased the expression of MCP-1 mRNA in cells and the concentrations of LDH,MDA and MCP-1 in culturesolution.But the levels of LDH,MDA and MCP-1 after HK2 cells treated with cystine crystals were significantly lower than those treated with calcium oxalate crystals.When HK2 cells were co-treated with cystine crystals and SOD,the concentrations of LDH,MDA and MCP-1 in culturesolution were statistically decreased compared with the date after HK2 cells treated only with cystine crystals.Conclusion:Cystine crystals could induce the injury of renal proximal tubular cells,in which lipid oxidation may play an important role.Furthermore,antioxidase could protect cells against the toxicity of cystine crystals.Chapter 4 The clinical characteristics of cystine stone patientsSection 1 Quantitative analysis of cystine in urine by reverse phase high-performance liquid chromatography methodObjective:To establish the reverse phase high-performance liquid chromatography(RP-HPLC)method for quantitative analysis of cystine in urine in our center.Methods:Quantitative analysis of cystinewasperformed by aAgilent 1100HPLC system,a Agilent HC-C18(4.6x250mm,5μm)column,and a Agilent Zorbax Entend-C18(4.6x12.5mm,5μm)guard column.The mobile phase was 37:63(v/v)0.05 Msodium acetate-methanol adjusted topH 3.5 with 2.5 M citric acid;the flowrate was 1 ml/min.The assay used UV detectionat 286 nm.Use acrylonitrile to protect cysteine from oxidization to cystine,andderivatizethe cystine with dansyl chloride.Loratadine,used as internalstandard,Peak-area measurement wasused for quantification.All analysis was performedunder the same conditions at roomtemperature.Results:The retention times ofthe Loratadine and cystine adduct were 17.5 and 22.5 min,respectively.No chromatographic interferences from the urinewere observed.Theintra-day precision,calculatedfrom the peak-area ratios,was within therange 1.1-5.2%.The inter-day precision,calculated from the peak-arearatios,ranged from 1.9 to 6.2%.The correlation coefficient(r)for cystinewas 0.9999Conclusion:Our results indicated that this RP-HPLC method is simple,rapid and accurate quantification analysis of cystine in urine.Section 2 The clinical characteristics of cystine stone patients Objective:To compare the clinical characteristics between Chinese cystine stone patients and calcium stone patients,and investigate whether daily excretion of cystine associates with the clinical features of cystine stones.Method:The 30 cystine patients were included in this study,and 30 age and gender pared calcium stone patients and normal persons were also included as the calcium oxalate stone group and negative control group,respectively.Comprehensive metabolic evaluation was performed in these stone patients and normal persons included in this study.The individual therapy for each cystine stone patient,according to thedaily excretion of cystine,was performed,and subsequently a short follow up for these patients was performed.Results:Among 30 cystine stone patients,only 1 of them has mild increase in the level of serum creatinine and one has minor increase in serum uric acid.But when compared to calcium oxalate stone patients and normal persons,the cystine stone patients have much higher levels of serum urea nitrogen,creatinine and uric acid.We also found the association between the numbers of surgical procedures and renal function in cystine stone patients.Cystine stone patients had lower levels of urine oxalate and uric acid excretion than calcium oxalate stone patients,but had higher level of urine citric acid excretion.When compared to normal persons,cystine stone patients only had a lower level of urine creatinine excretion.In regard to the frequency of metabolic disorders,cystine stone patients had a lower frequency of hypocitraturia than calcium stone patients,but when compared to normal persons,cystine stone patients had higher frequency of hyperoxaluria and hypercalciuria.The AP(CaOx)index EQ of cystine stone patients was significantly lower than that of calcium stone patients,but equal to normal persons.We also found lower levels of serum creatinine and uric acid in mild cystinuria patients than moderate and severe cystine stone patients.Individual therapy according todaily excretion of cystine of each patient could prevent the recurrence of cystine stone successfully.Conclusion:Cystine stone patients have a high risk of the injury of renal function due to the high frequency of surgical treatments,but their renal function could be in normal range in the short term after the onset of stones,because of strong compensation of kidneys.Most of our cystine stone patients have metabolic disorders,but they do not increase the risk of the formation of calcium stones.Mild cystinuria patients and those patients taking individual therapy have better clinical prognosis.Chapter 5 Thegenetic characteristics of cystine stone patientsObjective:To evaluate the potential value of gene analysis in the diagnosis of cystinuria,and investigate the SLC3A1和SLC7A9 gene mutations in 23 Chinese cystinuria stone patients.Methods:Extract total DNA from 23 Chinese cystine stone patients,and then perform the polymerase chain reactionin amplification of code region and the sequence of exon/intron-splicing junction,and subsequently the amplified fragments were directly sequenced.Results:We found 29 different mutations in our 23 patients.Among these patients,12 of them were polymorphic sites.Except the 12 polymorphic sites,we found 12 out of 46 mutations in the SLC3A1 gene and 20 out of 46 mutations in the SLC7A9gene.The age of onset of cystine stones in homozygote patients was lower than that in heterzygote patients.Among the 29 different mutations,13 of them were firstly reported,of which 7 mutations were in SLC 3A1 gene:c.839A>G,c.850 G>T,C.1113C>A,c.1227C>G,c.1373G>T,c.1399A>G and c.1668 T>A;and the other 6 mutations in SLC3A9 gene:c.691G>A,c.711A>T,c.956G>A,c.1001A>G,c.1097T>A and c.1317C>A.Conclusion:Our findings suggest amarked genetic and allelic heterogeneity of cystinuria inour cystine stone patients.Gene analysis could be considered as one of techniques for the diagnosis of cystinuria.