A Multi-Site Study Confirms Abnormal Glycosylation in the Tamm-Horsfall Protein of Patients With Interstitial Cystitis

Parsons, C. Lowell; Proctor, Jeffrey; Teichman, Joel; Nickel, J. Curtis; Davis, Edward L.; Evans, Robert J.; Zupkas, Paul; Phillips, Cody; Shaw, Timothy I.; Naidu, Natasha; Argade, Sulabha

doi:10.1016/j.juro.2011.02.2699

Cited by 18 publications

(16 citation statements)

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“…Urinary toxic factors bind to and are neutralized by THP . Patients with IC compared with normal subjects have a defective THP with reduced sialic acid content, which is critical to its protective activity . Overall, we believe that the balance between all of the urinary cations and anions is critical to prevent damage to the vital bladder mucus.…”

Section: Discussionmentioning

confidence: 89%

Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis

et al. 2014

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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.

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Section: Discussionmentioning

confidence: 89%

Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis

et al. 2014

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“…To counteract potentially toxic amino containing compounds, evolution has provided a remarkable, well conserved urinary protein in vertebrates produced in the renal tubules that will electrostatically attract and sequester these cations, THP. 15,20,25,26 THP is richly sialylated, making it strongly anionic, and will bind cations, rendering them biologically inactive, analogous to the role of albumin in serum. [15][16][17]20,21,26 A healthy bladder depends on the presence of THP and a level of toxic amino metabolites that is not excessive enough to overcome the protective effect of THP and damage the GAG layer.…”

Section: Discussionmentioning

confidence: 99%

Role of urinary cations in the etiology of interstitial cystitis: A multisite study

Parsons

Argade

Evans³

et al. 2020

Int J of Urology

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Objective To determine whether patients with interstitial cystitis have elevated levels of toxic urinary cations, to identify and quantify these cationic metabolites, and to assess their cytotoxicity. Methods Isolation of cationic fraction was achieved by solid phase extraction using an Oasis MCX cartridge on urine specimens from interstitial cystitis patients and controls. C18 reverse phase high‐performance liquid chromatography was used to profile cationic metabolites, and they were quantified by the area under the peaks and normalized to creatinine. Major cationic fraction peaks were identified by reverse phase high‐performance liquid chromatography and liquid chromatography–mass spectrometry. HTB‐4 urothelial cells were used to determine the cytotoxicity of cationic fraction and of individual metabolites. Results The reverse phase high‐performance liquid chromatography analysis was carried out on cationic fraction metabolites isolated from urine samples of 70 interstitial cystitis patients and 34 controls. The mean for controls versus patients was 3.84 (standard error of the mean 0.20) versus 6.71 (0.37) mAU*min/µg creatinine, respectively (P = 0.0001). The cationic fraction cytotoxicity normalized to creatinine for controls versus patients in mean percentage was −7.79% (standard error of the mean 3.32%) versus 20.03% (standard error of the mean 2.75%; P < 0.0005). The major toxic cations were 1‐methyladenosine, 1‐methylguanine, N2,N2‐dimethylguanosine and L‐tryptophan. Conclusions These data confirm significant elevation of toxic cations in the urine of interstitial cystitis patients. These toxic cations likely represent a primary cause of interstitial cystitis, as they can injure the bladder mucus and initiate an epithelial leak.

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“…THP was isolated from urine via the salt precipitation method of Tamm and Horsfall as described previously . The purity of the resulting THP was varified by SDS‐PAGE with silver stain.…”

Section: Patients Subjects and Methodsmentioning

confidence: 99%

“…2HCl (DMB)‐HPLC . The protein was quantified by Superdex‐200 size‐exclusion chromatography (Superdex‐200 SEC) with ultraviolet (UV) detection at 277 nm . THP was dissolved at 1 mg/mL in 0.02 m sodium phosphate buffer (pH 6.8) containing 4 m urea, and a 100 μL (100 μg) sample was injected onto the Superdex‐200 column.…”

Section: Patients Subjects and Methodsmentioning

confidence: 99%

“…The biological activity of THP to function as a protective molecule was found to reside in the SA content on the N‐terminal glycans . In several studies, including a ‘blinded’ multisite study , the SA content of THP in patients with IC has been shown to be reduced compared with asymptomatic control subjects. In both studies the THP concentration in urine was equal in both control subjects and patients with IC.…”

Section: Introductionmentioning

confidence: 99%

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Tamm‐Horsfall protein‐associated nucleotides in patients with interstitial cystitis

Argade

Shaw

et al. 2013

BJU International

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What's known on the subject? and What does the study add?• The nucleotides associated with Tamm-Horsfall protein (THP) identified in this study are 2′(3′) isomers, which are uncommon and very little is known about their biochemical pathway and role in interstitial cystitis (IC). The current study provides additional evidence of our earlier finding that THP is abnormal in IC patients. This can adversely affect THP's protective function, and suggests that THP plays a role in the pathogenesis of the disease. Objective• To identify and characterise Tamm-Horsfall protein (THP)-associated metabolites present in patients with interstitial cystitis (IC). Identification of these metabolites would give us insight into the complex interaction of THP with urinary metabolites and its effect on the protective function of THP. Patients, Subjects and Methods• THP was isolated from the urine specimens of 146 patients with IC and 87 control subjects, and was analysed for total sialic acid (SA) content by 1,2-diamino-4,5-methylenedioxybenzene. 2HCl (DMB)-high performance liquid chromatography (HPLC).• THP-associated metabolites were isolated by Superdex-200 size-exclusion chromatography (SEC) as a second peak (SP2) and the SP2 was further fractionated into six major components by C18 reverse phase-HPLC.• Ion-pair HPLC analysis was performed to identify and quantify THP-associated metabolites.• The metabolite structures were confirmed by reversed-phase HPLC combined with electrospray ionization (ESI), liquid chromatography-tandem mass spectrometry (LC-MS and LC-MS/MS) and by high-resolution ESI-time of flight mass spectrometry (HR-ESI-TOFMS). Results• The THP-associated metabolites (SP2) were more prevalent in patients with IC than in the controls (40.4% vs 12.6%, P < 0.001) as determined by Superdex-200 SEC.• Superdex-200 SEC showed higher SP2 content in patients with IC than in controls, as determined by area under the peak/100 mg of THP. The mean (SEM), for patients was 84.3 (8.1) vs 18.0 (2.4) milli-absorption unit*min, for controls (P < 0.001).• Total SA content of THP was much lower in SP2-positive patients with IC than those who were SP2-negative. The mean (SEM) was 41.6 (3.2) vs 92.6 (2.2) nmol/mg THP, respectively (P < 0.001).• Ion-pair HPLC identified SP2 metabolites as nucleotides, namely 5′-CMP, 3′-UMP, 3′-AMP, 3′-GMP, 2′-AMP and 2′-GMP. The total nucleotide content of SP2 was significantly higher in patients with IC than in controls. The mean (SEM) was 25.3 (2.9) vs 2.2 (0.2) mg/mg THP, respectively (P < 0.001).• LC-MS and LC-MS/MS confirmed the nucleotides based on retention time on HPLC, and mass to charge ratio (m/z) of the parent ion and the daughter ions. HR-ESI-TOFMS gave further confimation of the nucleotide sturctures with high mass accuracy. Conclusions• In the THP of patients with IC, there is a direct correlation between reduced SA levels and high prevalence of nucleotides associated with it.• The THP of patients with IC has a much higher content of these nucleotides than control, and these unique nucleotide...

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A Multi-Site Study Confirms Abnormal Glycosylation in the Tamm-Horsfall Protein of Patients With Interstitial Cystitis

Abstract: These multisite data validate that abnormal glycosylation of Tamm-Horsfall protein occurs in patients with interstitial cystitis and may have a role in interstitial cystitis causation.

Cited by 18 publications

References 20 publications

Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis

Role of urinary cations in the aetiology of bladder symptoms and interstitial cystitis

Role of urinary cations in the etiology of interstitial cystitis: A multisite study

Tamm‐Horsfall protein‐associated nucleotides in patients with interstitial cystitis

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