An evaluation of Tamm–Horsfall protein glycans in kidney stone formers using novel techniques

Abstract

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.

Isolation of Extracellular Vesicles: General Methodologies and Latest Trends

“…The gel-like structure formed in the body by THP catches bacteria and interferes with the spread of infection [ 146 ]. In addition, THP is capable of binding calcium ions [ 147 ]. The THP forms complexes with negatively charged membranes via the bridges of calcium ions and the formed gel-like network retains EVs; this eventually decreases the EV yield [ 94 ].…”

Interstitial Cystitis/Bladder Pain Syndrome

“…38 Additionally, mast cells, which have been found in greater number in the urothelium of patients with IC, have been found to have estrogen receptors; increase in estrogen may lead to degranulation of histamine from mast cells, and subsequent release of substance P. [39][40][41] Other postulated etiologies include deficiency in Tamm-Horsfall protein (an anionic protein which binds to and thereby neutralizes urinary toxins) and antiproliferative factor (APF) which inhibits the normal proliferation of bladder epithelial cells. 42,43 Characterization of the latter factor, which initially received significant attention, has not been proven to be easily replicable of note, and research efforts no longer focus on APF at present.…”

Tamm–Horsfall glycoprotein engages human Siglec‐9 to modulate neutrophil activation in the urinary tract

“…Although total levels of THP are unchanged, patients with interstitial cystitis have only half of the THP sialic acid content of healthy controls 25 . Similarly, mass spectrometry of urine THP from kidney stone patients revealed >20% less sialic acid content than healthy controls 26 . Patients with type I diabetes are more susceptible to UTI, have more glucose and less sialic acid in their THP glycans than healthy controls 27 , and may exhibit reduced THP urinary excretion rates 28 .…”

“…25 Similarly, mass spectrometry of urine THP from kidney stone patients revealed 420% less sialic acid content than healthy controls. 26 Patients with type I diabetes are more susceptible to UTI, have more glucose and less sialic acid in their THP glycans than healthy controls 27 and may exhibit reduced THP urinary excretion rates. 28 Although reduced THP N-acetylgalactosamine (GalNAc), another component of the Sda antigen, has also been reported in patients with UTI, 29 terminal GalNAcβ1-4 did not contribute to THP/Siglec-9 interactions (Supplementary Figure 1C).…”