Urinary Tamm-Horsfall Protein Research Articles

Diagnostic value of urinary Tamm-Horsfall protein and 24 h urine osmolality for recurrent calcium oxalate stones of the upper urinary tract: Cross-sectional study.

We aimed to investigate the diagnostic value of urinary Tamm-Horsfall protein (THP) and 24 h urine osmolality for recurrent calcium oxalate (CaOx) stones. Clinical data of 120 patients with upper urinary tract stones admitted to our hospital between January 2020 and January 2022 were retrospectively reviewed. Patients were divided into recurrence (53 patients) and non-recurrence (67 patients) groups based on postoperative stone recurrence. Meanwhile, 50 healthy patients were selected as the control group. Urinary THP levels, 24 h urine osmolality, and biochemical indices were compared between the three groups; their diagnostic values for stone recurrence were evaluated using receiver operating characteristic (ROC) curves. Urinary THP, 24 h urine osmolality, and biochemical indices were significantly higher in the recurrence group than in the non-recurrence and control groups (P < 0.05). The 24 h urine osmolality was positively correlated with urinary oxalic acid and calcium excretion. ROC curve analysis showed that optimal cutoff values of urinary THP and 24 h urine osmolality for the diagnosis of stone development were ≥27.01 mg/L and ≥577.69 mOsm/(kg H2O), respectively. Furthermore, these indices combined significantly improved the accuracy of diagnosis. Urinary THP and 24 h urine osmolality were higher in patients with recurrent CaOx stones. Detection of both parameters combined can accurately diagnose stone recurrence.

Tamm–Horsfall protein in humane urine: sex-dependent differences in the excretion and N-glycosylation pattern

Tamm–Horsfall protein (THP) is a highly N-glycosylated protein from epithelial cells of the ascending limb of Henle loop. It is secreted into the urine as part of the innate immune response against uropathogenic pathogens. As women are more likely to suffer from urinary tract infections, biomedical studies were conducted to investigate sex-differences in THP excretion, as well as differences in the THP N-glycosylation pattern. A total of 238 volunteers (92 men, 146 women, 69 with hormonal contraceptives) participated in this study, providing urine samples. Women showed a clear tendency to have higher THP concentration and excretion rates than men (p < 0.16). Regular intake of hormonal contraceptives had no significant influence on urinary THP concentration compared to no regular intake. The individual N-glycosylation pattern of THP in urine samples from randomly selected individuals (10 female, 10 male) was investigated after enzymatic release and MS analysis of the oligosaccharides. Female subjects tended to have an increased proportion of oligomannose type N-glycans and non-fucosylated glycans, whereas men had an increased proportion of fucosylated complex-type glycans. The higher level of oligomannose-type glycans in THP from women might be explained by a self-defence mechanism to overcome the higher infections pressure by the female anatomical properties.

Tamm-Horsfall Protein Expression in Urine of Buffaloes at the Estrus and Diestrus Stages of Estrous Cycle

Background: Identifying buffaloes in estrus is crucial for enhancing the efficiency of reproduction as silent heat is a major concern in this species. Review of literature indicated the role of Tamm-Horsfall protein (THP) is associated with the physiological events around the estrus stage. Hence, the present study was aimed to quantify the expression level of THP in urine of buffaloes to ascertain its suitability as a marker for identifying buffaloes in estrus. Methods: The grouping of animals as mid-diestrus (Group-I/G-I/Control group), regular estrus (Group-II/G-II) and silent estrus (Group-III/G-III) buffaloes was done using a combinatorial approach. The expression of THP in the urine of three groups of animals was quantified by western blot. Result: The results of the present study revealed higher levels of urinary THP in G-II animals when compared to G-III and G-I animals, although it was statistically non-significant (p greater than 0.05). The present study revealing for increased urinary expression of THP in G-II and G-III animals when compared to G-I animals probably construes the antimicrobial role of THP in the female reproductive tract and its role during estrus to prevent microbiota population beyond the physiological levels. For THP to be considered as biomarker for detecting buffaloes in silent heat there is need for further studies involving large number of animals. Nonetheless, an easy method has been developed that facilitated THP detection and quantification in urine of buffaloes by western blot.

Systematic analysis of modulating activities of native human urinary Tamm-Horsfall protein on calcium oxalate crystallization, growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix

Functions of Tamm-Horsfall protein (THP), the most abundant human urinary protein, have been studied for decades. However, its precise roles in kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in calcium oxalate monohydrate (COM) kidney stone formation. THP was purified from the human urine by adsorption method using diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4–40 μg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 μg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large calcium-binding domains (residues 65–107 and 108–149) and three small oxalate-binding domains (residues 199–207, 361–368 and 601–609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for calcium-affinity and/or oxalate-affinity demonstrated that THP exerted a high affinity with only calcium, not oxalate. Functional validation revealed that saturation of THP with calcium, not with oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with calcium ions.

The kidney releases a nonpolymerizing form of uromodulin in the urine and circulation that retains the external hydrophobic patch domain.

Uromodulin [Tamm-Horsfall protein (THP)] is a glycoprotein uniquely produced in the kidney. It is released by cells of the thick ascending limbs apically in the urine and basolaterally in the renal interstitium and systemic circulation. Processing of mature urinary THP, which polymerizes into supramolecular filaments, requires cleavage of an external hydrophobic patch (EHP) at the COOH-terminus. However, THP in the circulation is not polymerized, and it remains unclear if nonaggregated forms of THP exist natively in the urine. We propose that an alternative processing path, which retains the EHP domain, can lead to a nonpolymerizing form of THP. We generated an antibody that specifically recognizes THP with retained EHP (THP + EHP) and established its presence in the urine in a nonpolymerized native state. Proteomic characterization of urinary THP + EHP revealed its COOH-terminus ending at F617. In the human kidney, THP + EHP was detected in thick ascending limb cells and less strongly in the renal parenchyma. Using immunoprecipitation followed by proteomic sequencing and immunoblot analysis, we then demonstrated that serum THP has also retained EHP. In a small cohort of patients at risk for acute kidney injury, admission urinary THP + EHP was significantly lower in patients who subsequently developed acute kidney injury during hospitalization. Our findings uncover novel insights into uromodulin biology by establishing the presence of an alternative path for cellular processing, which could explain the release of nonpolymerizing THP in the circulation. Larger studies are needed to establish the utility of urinary THP + EHP as a sensitive biomarker of kidney health and susceptibility to injury.NEW & NOTEWORTHY In this work, we discovered and characterized a novel form of uromodulin that does not polymerize because it retains an external hydrophobic patch at the COOH-terminus. These findings establish an alternative form of cellular processing of this protein and elucidate new aspects of its biology. We also provide evidence suggesting that measuring urinary nonpolymerizing uromodulin could be a promising assay to assess the risk of acute kidney injury.

Mechanism of Huayu Jianpi Fangshi decoction in urolithiasis prevention: a randomized trial.

This study aims to explore the mechanism of the Huayu Jianpi Fangshi decoction in urolithiasis prevention. The present study was designed as a randomized, double-blinded, placebo-controlled clinical trial. Sixty patients with the qi stagnation and blood stasis, spleen deficiency, and dampness obstruction types of urolithiasis were randomly divided into two groups: the treatment group and the control group (n=30 in both groups). Patients in the treatment group were treated with the Huayu Jianpi Fangshi decoction, while patients in the control group were treated with the Huayu Fangshi placebo decoction. Both treatments were taken orally two times per day. All patients received treatment over the course of four weeks. The main outcome indicators included the Tamm-Horsfall protein (THP) expression levels, osteopontin, and inter-α-trypsin inhibitor heavy chain 3 (ITIH3) in the patients' urine as well as changes in 24-h urinary citric acid, urinary magnesium levels, and Traditional Chinese Medicine (TCM) syndrome scores. The results of the present study revealed a significant increase in the total citric acid excretion level (244.75±59.62 vs. 297.48±57.91 mmol/L, P<0.01), significant decrease in the total urinary THP level (10.83±7.73 vs. 6.37±6.10 mg/L, P<0.05), significant decrease in the total ITIH3 level (9.51±6.32 vs. 6.14±4.46 mg/L, P<0.05) in the patients' 24-h urine, and a significant elevation of the total TCM syndrome score (5% vs. 23%, P<0.01) in the treatment group when compared with the control group. The Huayu Jianpi Fangshi decoction can increase the excretion of urinary citric acid, decrease the expression levels of urinary THP and ITIH3 in patients with cured urolithiasis, and reduce the TCM syndrome score.

The association between thyroid dysfunction (TD) and diabetic kidney disease (DKD) in type 2 diabetes mellitus (T2DM).

Epidemiological studies have identified an association between thyroid dysfunction (TD) and various kidney diseases. In this study, the prevalence of TD in type 2 diabetic mellitus (T2DM) patients with diabetic kidney disease (DKD) was evaluated to analyse the potential association between TD and DKD in T2DM patients. A total of 2108 T2DM patients from Anhui Provincial Hospital were recruited in this study. Demographic and clinical characteristics were collected from 834 T2DM patients with DKD and 1274 T2DM patients without DKD (non-DKD). All patients were stratified into a number of groups based on UACR (urine albumin-to-creatinine ratio) or eGFR (estimated glomerular filtration rate): (a) A1: normoalbuminuria (<30), A2: microalbuminuria (30-300) and A3: macroalbuminuria (>300); (b) F1: normal filtration (60-139), F2: hyper filtration (≥140) and F3: low filtration (<60). Significant differences were observed between the non-DKD and DKD groups (P<.05) in age, sex ratio, duration, systolic blood pressure, total cholesterol, low density lipoprotein cholesterol, serum creatinine, blood urea nitrogen, free triiodothyronine (FT3), free thyroxine (FT4) and sensitive thyrotropin hormone (sTSH). The macroalbuminuira and low filtration groups had the lowest levels of FT3 and FT4 and the highest level of sTSH, compared with all other groups (P<.0167). The prevalence of subclinical hypothyroidism in the DKD group was significantly higher than that in the non-DKD group (χ2 =13.92, P<.01). Logistic regression analysis showed that hypothyroidism was associated with increased UACR or reduced eGFR in T2DM patients. Compared with controls, T2DM patients with hypothyroidism exhibited a higher UACR and urinary excretion of transferrin, as well as a lower excretion of urinary Tamm-Horsfall protein (THP) (P<.0167). Subclinical hypothyroidism is more prevalent in T2DM patients with DKD than in T2DM patients without DKD. Hypothyroidism is associated with albuminuria and decreased eGFR in T2DM patients.

Management of Tamm-Horsfall Protein for Reliable Urinary Analytics.

PurposeUrinary extracellular vesicles (uEVs) are a novel source of biomarkers. However, urinary Tamm–Horsfall Protein (THP; uromodulin) interferes with all vesicle isolation attempts, precipitates with normal urinary proteins, thus, representing an unwanted “contaminant” in urinary assays. Thus, the aim is to develop a simple method to manage THP efficiently.Experimental designThe uEVs are isolated by hydrostatic filtration dialysis (HFD) and treated with a defined solution of urea to optimize release of uEVs from sample. Presence of uEVs is confirmed by transmission electron microscopy, Western blotting, and proteomic profiling in MS.ResultsUsing HFD with urea treatment for uEV isolation reduces sample complexity to a great extent. The novel simplified uEV isolation protocol allows comprehensive vesicle proteomics analysis and should be part of any urine analytics to release all sample constituents from THP trap.Conclusions and clinical relevanceThe method brings a quick and easy protocol for THP management during uEV isolation, providing major benefits for comprehensive sample analytics.

MP03-05 HYPERPHOSPHATURIA OR HYPEROXALURIA EXACERBATES INTRA-RENAL CALCIFICATION BUT NOT STONE FORMATION IN TAMM-HORSFALL PROTEIN KNOCKOUT MICE

INTRODUCTION AND OBJECTIVES:Mice lacking one or both alleles of Tamm-Horsfall protein (THP) gene, thus excreting low or no urinary THP, are prone to developing spontaneous calcium phosphate (apatit...

A study of urinary Tamm-Horsfall protein excretion in adult type 2 diabetes mellitus .

To study the excretion of urinary Tamm-Horsfall protein (THP) with a wide range of urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) in a population with type 2 diabetes mellitus (T2DM). A retrospective investigation was performed in 3,599 adult inpatients with T2DM in Anhui Provincial Hospital of China. Demographic information, urinary THP data, and other lab reports were obtained from the Electronic Patient Record (EPR). (1) In T2DM, average HbA1c was 8.6%, and 55.2% of the patients had an HbA1c > 9%. The percentages with normal excretion, decreased excretion, and increased excretion of urinary THP were 49.8%, 41.7%, and 8.5%, respectively. Serum creatinine (Scr), blood urea nitrogen (BUN), and UACR levels were highest, and eGFR was lowest, in the decreased urinary THP group. (2) The proportion of decreased urinary THP excretion rose, and the level of urinary THP declined when UACR increased or eGFR decreased. (3) Urinary THP showed a negative correlation to Scr, BUN, triglyceride (TG), and UACR (rs = -0.173, -0.228, -0.060 and -0.237, respectively), but a positive correlation with eGFR and high-density lipoprotein cholesterol (HDL-C) (rs =0.128 and 0.040, respectively). (4) Logistical regression analysis showed that Scr, BUN, TG, UACR, and eGFR were independent risk factors for decreased urinary THP (OR=3.477, 1.461, 1.160, 2.124, and 2.087, respectively). Increased albuminuria and decreased GFR are predictors of decreased urinary THP excretion. Distal convoluted tubule lesion may precede glomerular damage in a portion of T2DM patients. .

Tamm-Horsfall Protein is a Potent Immunomodulatory Molecule and a Disease Biomarker in the Urinary System.

Tamm–Horsfall protein (THP), or uromodulin (UMOD), is an 80–90-kDa phosphatidylinositol-anchored glycoprotein produced exclusively by the renal tubular cells in the thick ascending limb of the loop of Henle. Physiologically, THP is implicated in renal countercurrent gradient formation, sodium homeostasis, blood pressure regulation, and a defense molecule against infections in the urinary system. Investigations have also revealed that THP is an effective binding ligand for serum albumin, immunoglobulin G light chains, complement components C1 and C1q, interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, and interferon-γ through its carbohydrate side chains for maintaining circulatory and renal immune homeostasis. Thus, THP can be regarded as part of the innate immune system. UMOD mutations play crucial roles in congenital urolithiasis, hereditary hyperuricemia/gout, and medullary cystic kidney diseases. Recent investigations have focused on the immunomodulatory effects of THP on immune cells and on THP as a disease biomarker of acute and chronic kidney diseases. Our studies have suggested that normal urinary THP, through its epidermal growth factor (EGF)-like domains, binds to the surface-expressed EGF-like receptors, cathepsin G, or lactoferrin to enhance polymorphonuclear leukocyte phagocytosis, proinflammatory cytokine production by monocytes/macrophages, and lymphocyte proliferation by activating the Rho family and mitogen-activated protein kinase signaling pathways. Furthermore, our data support both an intact protein core structure and carbohydrate side chains are important for the different protein-binding capacities of THP. Prospectively, parts of the whole THP molecule may be used for anti-TNF-α therapy in inflammatory diseases, autoantibody-depleting therapy in autoimmune disorders, and immune intensification in immunocompromised hosts.

Urinary Tamm-Horsfall protein, albumin, vitamin D-binding protein, and retinol-binding protein as early biomarkers of chronic kidney disease in dogs.

Proteinuria is a marker and mediator of chronic kidney disease (CKD). In clinical practice, the urinary protein‐to‐creatinine ratio (UP/C) is of limited usefulness, because it indicates only the magnitude of proteinuria and not the origin of the loss (glomerular or tubular). The complete assessment of proteinuria includes quantitative and qualitative evaluations, both of which are required in order to optimize the therapy. In addition to measuring the UP/C, we performed SDS‐PAGE and western blotting to determine the expression of albumin, vitamin D‐binding protein (VDBP), retinol‐binding protein (RBP), and Tamm‐Horsfall protein (THP) in urine samples of 49 dogs: healthy (control) dogs (n = 9); and dogs with CKD (n = 40), stratified by stage. In the dogs with stage 3 or 4 CKD, there was a predominance of tubular proteins. Neither VDBP nor RBP was observed in the urine of the control dogs. Among the dogs with stage 1 or 2 CKD, VDBP and RBP were detected in those without proteinuria or with borderline proteinuria. The expression of urinary albumin was significantly higher in the stage 4 group than in any other group (P ≤ 0.01). In the stage 4 group, urinary THP was either undetectable or lower than in the control group (P ≤ 0.01). In conclusion, urinary VDBP and RBP might act as early markers of kidney injury, and a decrease in urinary THP could be an indicator of CKD progression.

Protein Complexes in Urine Interfere with Extracellular Vesicle Biomarker Studies.

Urine exosomes (extracellular vesicles; EVs) contain (micro)RNA (miRNA) and protein biomarkers that are useful for the non-invasive diagnosis of various urological diseases. However, the urinary Tamm-Horsfall protein (THP) complex, which forms at reduced temperatures, may affect EV isolation and may also lead to contamination by other molecules including microRNAs (miRNAs). Therefore, we compared the levels of three miRNAs within the purified EV fraction and THP- protein-network. Urine was collected from healthy donors and EVs were isolated by ultracentrifugation (UC), two commercial kits or sepharose size-exclusion chromatography (SEC). SEC enables the separation of EVs from protein-complexes in urine. After UC, the isolation of EV-miRNA was compared with two commercial kits. The EV isolation efficiency was evaluated by measuring the EV protein markers, Alix and TSG101, CD63 by Western blotting, or miR-375, miR-204 and miR-21 by RT-qPCR. By using commercial kits, EV isolation resulted in either low yields or dissimilar miRNA levels. Via SEC, the EVs were separated from the protein-complex fraction. Importantly, a different ratio was observed between the three miRNAs in the protein fraction compared to the EV fraction. Thus, protein-complexes within urine may influence EV-biomarker studies. Therefore, the characterization of the isolated EV fraction is important to obtain reproducible results.

Hints to the diagnosis of uromodulin kidney disease.

BackgroundUromodulin kidney disease (UKD) is an inherited kidney disease caused by a uromodulin (UMOD) gene mutation. The UMOD gene encodes the Tamm–Horsfall protein (THP), which is the most abundant protein in healthy human urine. Because of its rarity, the incidence of UKD has not been fully elucidated. The purpose of the present study is to clarify the frequency of UKD among patients who underwent renal biopsy.MethodsImmunostaining for THP was performed for patients <50 years of age with renal insufficiency and hyperuricemia without overt urinalysis abnormality from renal biopsy databases. Serum and urinary THP concentrations were evaluated in available individuals.ResultsFifteen patients were selected for immunostaining from a total of 3787 patients. In three independent patients, abnormal THP accumulation in renal tubular cells was observed. A novel missense A247P UMOD mutation was detected in two of the three patients, including one having a typical family history of familial juvenile hyperuricemic nephropathy. Serum and urinary THP concentrations of all available patients with UMOD A247P mutation were significantly lower than those of controls.ConclusionsIn the present study, UKD was detected in <1 in 1000 subjects who underwent renal biopsies. However, in subjects meeting all of the above criteria, abnormal THP accumulation was detected in 20% (3/15), suggesting that renal biopsy with immunostaining for THP is a good tool for diagnosing UKD. Also, low serum THP concentration detected in the present subjects might be a good diagnostic marker or important in understanding the pathogenesis of UKD.

対角線電気泳動法を用いた健常人尿中Tamm-Horsfall protein解析

対角線電気泳動法を用いた健常人尿中Tamm-Horsfall protein解析

Relationship between urinary Tamm–Horsfall protein excretion and renal function in dogs with naturally occurring renal disease

Tamm-Horsfall protein (THP) is physiologically excreted in urine, but little is known about the role of THP in the diagnosis of renal disease in dogs. The aim of this study was to evaluate to which extent naturally occurring renal disease affects the urinary excretion of THP. Dogs were divided into 5 groups according to plasma creatinine concentration, urinary protein-to-creatinine ratio (UP/UC), and exogenous plasma creatinine clearance (P-ClCr ) rates: Group A (healthy control dogs; n = 8), nonazotemic and nonproteinuric dogs, with P-ClCr rates > 90 mL/min/m(2) ; group B (n = 25), nonazotemic and nonproteinuric dogs with reduced P-ClCr rates (51-89 mL/min/m(2) ); group C (n = 7), nonazotemic but proteinuric dogs with P-ClCr rates 53-98 mL/min/m(2) ; group D (n = 8), azotemic and borderline proteinuric dogs (P-ClCr rates: 22-45 mL/min/m(2) ); and group E (n = 15), azotemic and proteinuric dogs (not tested for P-ClCr ). THP was measured by quantitative Western blot analysis, and the ratio of THP-to-urinary creatinine (THP/UC) was calculated. The THP/UC concentrations were not different among dogs of groups A-D, but were reduced in dogs of group E (P < .001). THP/UC correlated negatively with serum creatinine (P < .01) and UP/UC (P < .01), but was not significantly associated with P-ClCr . Decreased levels of THP/UC were present in moderately to severely azotemic and proteinuric dogs. This suggests tubular injury in these dogs and that THP might be useful as urinary marker to study the pathogenesis of renal disease.

Effect of urinary Tamm-horsfall protein concentration changes under centrifugation and its association with urolithiasis formation in rats

To explore the concentration changes of Tamm-Horsefall protein (THP) under centrifugation in rat urine and discuss its association with urolithiasis formation. A total of 40 Wistar rats were divided randomly into 4 groups of flying with stone (A), flying without stone (B), stone without flying (C) and control (D). After centrifugation, the THP concentrations of each group were measured by enzyme-linked immunosorbent assay (ELISA). Then urinary system was dissected, stained with hematoxylin & eosin and observed under electron microscopy to examine the distribution and number of each section. The SPSS 13.0 software was used for data analyses. Group A showed significant difference in THP concentrations with groups C and D ( (11 ± 4) vs (15 ± 6), (17 ± 4) ng/ml, P = 0.037 and 0.005).No statistically significant difference existed between groups A and B ((11 ± 5) ng/ml, P = 0.998) or groups C and D (P = 0.422). Group B had significant difference in THP concentrations with groups D (P = 0.036). Regarding the number of stones in ureter, Group A had statistically significant difference with B (P = 0.029).However, there was no difference in the number of bladder stones.In kidney stones, there was significant difference (P = 0.029) on "+ +" rating. Centrifugation may reduce the urinary concentration of THP so as cause urolithiasis formation in rats.

Comparison of urinary exosomal protein solubilization methods for two-dimensional gel electrophoresis

Background: Urinary exosomal proteins have recently emerged as important candidates for elucidating the mechanisms underlying physiological events and disease-related metabolism in the kidney. Here, we evaluated standard sample preparation methods for two-dimensional gel electrophoresis (2DE) to determine which one yielded the maximum protein recovery from urinary exosomes for protein identification.Materials and Methods: Urinary exosomes were purified from a healthy subject by using ultracentrifugation. The final pellets were dissolved with PBS or RIPA buffer. After being desalted, these exosomal protein solutions were each treated with 1 of 4 rehydration buffers (Rbs) containing detergents in the following formulations: CHAPS (Rb1), CHAPS and Triton X-100 (Rb2), dodecyl maltoside (Rb3), and ASB-14 (Rb4).Results: For all Rbs, a much greater number of protein spots was detected in the samples isolated with RIPA than with PBS. Only minor differences were observed in the number of protein spots for Rb1-3. The largest protein spots were detected using the combination of RIPA buffer and Rb4; however, the background on the 2DE gel was high in the region of >66 kD and at the lower pH values. For all combinations, the co-precipitation of the urinary Tamm-Horsfall protein masked the protein spots in the 66-100 kD region.Conclusion: For extracting a large number of proteins with a relatively clear background on silver-stained 2DE gels, the optimal exosomal protein-dissolving buffer is RIPA buffer. All of the evaluated Rbs, except for the one containing ASB-14 as a detergent, is suitable for solubilizing exosomal proteins on 2DE.

Tamm–Horsfall protein gene is associated with distal tubular dysfunction in patients with type 1 diabetes

The first changes in the diabetic kidney are glycogen deposits in the epithelial cells of the thick ascending limb of Henle, which leads to decreased production of Tamm-Horsfall protein (THP). The production of THP is also influenced by nitric oxide (NO). The aims of this study were to investigate whether low excretion of THP, a sign of distal tubular dysfunction, in patients with type 1 diabetes was associated with polymorphisms in the THP gene and the endothelial NO synthase (eNOS) gene. Urine was collected from 301 patients with type 1 diabetes, 164 with normoalbuminuria, 91 with microalbuminuria and 46 with macroalbuminuria. Urinary THP concentration below median (3.12 mg/l) was defined as tubular dysfunction. Representative polymorphisms were analysed in the THP and eNOS genes. Patients with tubular dysfunction had longer diabetes duration and higher blood pressure than patients without tubular dysfunction. Tubular dysfunction was common in patients with macroalbuminuria (70% of patients) and it was associated with the AA+AT genotypes of rs12444268 in the THP gene [odds ratio (OR) 1.8, 95% confidence interval (CI) 1.1-2.8], and the GG genotype of rs1799983 in the eNOS gene (OR 1.6, 95% CI 1.03-2.6). When adjusting for other associated factors, diabetes duration, glycosylated haemoglobin (HbA(1c)), mean arterial pressure and albuminuria, the THP rs12444268 and macroalbuminuria were independently associated with tubular dysfunction. Distal tubular dysfunction was associated with the THP gene and macroalbuminuria in patients with type 1 diabetes.

Qualification and application of an ELISA for the determination of Tamm Horsfall Protein (THP) in human urine and its use for screening of Kidney Stone Disease

Kidney stone disease affects 1 - 20% of the general population. At present, the diagnosis of a stone is done using radiography method when noticeable symptoms appeared. We developed a non-invasive quantitative assay for urinary THP, namely ELISA; whereby our previous study and other reports had shown the usefulness of THP as biomarker for kidney stone disease. Since urine is biological fluid that is easily obtainable, this method could be used as a screening assay for kidney stone prior to confirmation with radiography. The ELISA gave assay linearity r(2) > 0.999 within the range of 109 ng/mL to 945 ng/mL THP. Assay precisions were < 4% (C.V.) for repeatability and < 5% (C.V.) for reproducibility. Assay accuracy range from 97.7% to 101.2% at the various THP concentrations tested. Assay specificity and sensitivity were 80% and 86%, respectively. The cut-off points at P < 0.05 were 37.0 and 41.2 mug/mL for male and female, respectively. The assay is cost effective and rapid whereby the cost for assaying each urine sample in duplicate is approximately USD0.35 and within 5 hours, 37 samples can be assayed alongside full range of standards and 3 QC samples in each plate. Furthermore, sample preparation is relatively easy where urine sample was diluted 10 times in TEA buffer. The usability of the ELISA method for diagnosis of kidney stone disease is evaluated with 117 healthy subjects and 58 stone formers.