Tamm-Horsfall protein (THP) is theorized to play a critical role in preventing kidney stone formation. There is conflicting literature on THP analysis in kidney stone patients; therefore, this study was conducted using sensitive and specific bio-analytical techniques to better understand differences in THP, which play a potential role in nephrolithiasis pathogenesis. THP was isolated from urine samples of 34 male and 19 female kidney stone patients and 30 male and 24 female control subjects using diatomaceous earth. Protein was quantified by Superdex-200 size-exclusion chromatography. Sialic acid was determined by 1,2-diamino-4,5-methylenedioxybenzene high-performance liquid chromatography. Neutral and amino sugars were determined by high pH anion-exchange chromatography (HPAEC) with pulsed amperometric detection. THP N-glycans were derivatized with 2-aminobenzamide (2-AB) and profiled by HPAEC with fluorescence detection. N-glycan structures were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Results indicate that kidney stone patients had 32% lower protein content compared to controls, while sialic acid content was lower by 29 and 24% in male and female kidney stone patients, respectively, compared to controls. The neutral and amino sugars were also lower by 18 and 20% for male and female kidney stone patients, respectively, compared to controls. All results were statistically significant (p<0.001). These results are supported by 2-AB profiling of THP N-glycans and by MALDI-TOF MS of highly sialylated N-glycans in the range of m/z 3000-6000. This study demonstrates quantitative and qualitative differences in THP, which can be crucial contributing factors for nephrolithiasis.
ObjectivesTo identify and characterise urinary cationic metabolites, defined as toxic factors, in patients with interstitial cystitis (IC) and in control subjects. To evaluate the cytotoxicity of the urinary cationic metabolite fraction of patients with IC vs control subjects and of individual metabolites in cultured urothelial cells. Subjects and MethodsCationic fractions (CFs) were isolated from the urine specimens of 62 patients with IC and 33 control subjects by solid-phase extraction. CF metabolites were profiled using C18 reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection, quantified by area-under-thepeaks using known standards, and normalized to creatinine. RP-HPLC and liquid chromatography (LC)-mass spectrometry (MS)/tandem MS (MS/MS) were used to identify major CF peaks. HTB-4 urothelial cells were used to determine the cytotoxicity of CFs and of individual metabolites with and without Tamm-Horsfall protein (THP). ResultsRP-HPLC analysis showed that metabolite quantity was twofold higher in patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was 3.1 (0.2) vs 6.3 (0.5) mAU*min/μg creatinine (P < 0.001). LC-MS identified 20 metabolites. Patients with IC had higher levels of modified nucleosides, amino acids and tryptophan derivatives compared with control subjects. The CF cytotoxicity was higher for patients with IC compared with control subjects. The mean (SEM) for control subjects vs patients was −2.3 (2.0)% vs 36.7 (2.7)% (P < 0.001). A total of 17 individual metabolites were tested for their cytotoxicity. Cytotoxicity data for major metabolites were all significant (P < 0.001): 1-methyladenosine (51%), 5-methylcytidine (36%), 1-methyl guanine (31%), N 4 -acetylcytidine (24%), N 7 -methylguanosine (20%) and L-Tryptophan (16%). These metabolites were responsible for higher toxicity in patients with IC. The toxicity of all metabolites was significantly lower in the presence of control THP (P < 0.001). ConclusionsMajor urinary cationic metabolites were characterised and found to be present in higher amounts in patients with IC compared with control subjects. The cytotoxicity of cationic metabolites in patients with IC was significantly higher than in control subjects, and control THP effectively lowered the cytotoxicity of these metabolites. These data provide new insights into toxic factor composition as well as a framework in which to develop new therapeutic strategies to sequester their harmful activity, which may help relieve the bladder symptoms associated with IC.
Patients with IC had significantly higher levels of cationic metabolites with higher cytotoxicity compared to controls. THP of these patients had reduced ability to sequester cytotoxicity of cationic metabolites. Patients who significantly improved on therapy had the same levels and toxicity of cationic metabolites as symptomatic males, suggesting that these cations may be the cause of epithelial dysfunction in IC.
INTRODUCTION AND OBJECTIVES: Lower urinary tract symptoms (LUTS) such as frequency and nocturia are overlapped between patients of interstitial cystitis/bladder pain syndrome (IC/BPS) with or without Hunner's lesion (HL and NHL, respectively) and those with overactive bladder (OAB). To detect IC/BPS in patients with LUTS, we measured several urine markers from patients with HL, NHL, OAB and healthy controls. For each maker, we determined whether the urine level was significantly different among them, and whether the marker level was correlated to symptom scores.METHODS: Urine was collected from 19 patients with HL ( 9) and NHL (10) before hydrodistention, 19 patients with OAB controlled by medication and 15 normal volunteers. Multiplex analyses of 40 cytokines, chemokines and growth factors were performed with a multiple antigen assay (Milliplex MAP Kit) on urine specimens. All participants completed the O'Leary-Sant score including symptom indexes (OSSI) and problem indexes (OSPI), and visual analog scale (VAS) pain score.RESULTS: Several markers were significantly increased in IC/ BPS patients compared with OAB and control patients, including vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), CD40 ligand (CD40L) and chemokines such as CCL3, CCL5, CCL11 and CXCL10 although there was no significant difference in these markers between HL and NHL patients. As the results of univariate and multivariate regression analysis, OSSI and OSPI were significantly correlated positively with CXCL10, and FGF-2 and CXCL10. VAS score was significantly correlated positively with FGF-2, interleukin-6 (IL-6), CCL3, CCL7, CCL11 and CXCL10 (Table ).CONCLUSIONS: Patients with HL and NHL had increased urine markers associated with immune and inflammatory responses including T-helper type 1 related chemokines such as CCL3, CCL5, CCL11 and CXCL10, which may be useful to detect IC/BPS in patients with LUTS. In addition, changes in CXCL10 may be positively correlated with symptom scores of IC/BPS patients.
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