Involvement of urinary proteins in the rat strain difference in sensitivity to ethylene glycol-induced renal toxicity.

Ethylene glycol (EG) exposure is a common model for kidney stones, because animals accumulate calcium oxalate monohydrate (COM) in kidneys. Wistar rats are more sensitive to EG than Fischer 344 (F344) rats, with greater COM deposition in kidneys. The mechanisms by which COM accumulates differently among strains are poorly understood. Urinary proteins inhibit COM adhesion to renal cells, which could alter COM deposition in kidneys. We hypothesize that COM accumulates more in Wistar rat kidneys because of lower levels of inhibitory proteins in urine. Wistar and F344 rats were treated with 0.75% EG in drinking water for 8 wk. Twenty-four-hour urine was collected every 2 wk for analysis of urinary proteins. Similar studies were conducted for 2 wk using 2% hydroxyproline (HP) as an alternative oxalate source. Total urinary protein was higher in F344 than Wistar rats at all times. Tamm-Horsfall protein was not different between strains. Osteopontin (OPN) levels in Wistar urine and kidney tissue were higher and were further increased by EG treatment. This increase in OPN occurred before renal COM accumulation. Untreated F344 rats showed greater CD45 and ED-1 staining in kidneys than untreated Wistars; in contrast, EG treatment increased CD45 and ED-1 staining in Wistars more than in F344 rats, indicating macrophage infiltration. This increase occurred in parallel with the increase in OPN and before COM accumulation. Like EG, HP induced markedly greater oxalate concentrations in the plasma and urine of Wistar rats compared with F344 rats. These results suggest that OPN upregulation and macrophage infiltration do not completely protect against COM accumulation and may be a response to crystal retention. Because the two oxalate precursors, EG and HP, produced similar elevations of oxalate, the strain difference in COM accumulation may result more so from metabolic differences between strains than from differences in urinary proteins or inflammatory responses.

Identification of a uromodulin fragment for diagnosis of IgA nephropathy.

IgA nephropathy (IgAN) is one of the most common types of glomerulonephritis worldwide and is diagnosed only with a renal biopsy. The purpose of the present studies was to identify the potential biomarkers for the non-invasive diagnosis of IgAN. The combination of a magnetic bead separation system with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to analyze urinary peptides of IgAN patients, other glomerulopathy patients, and healthy controls. ClinProTools v.2.0 software was also applied to establish a diagnostic model for IgAN. Our results demonstrated that 11 features had optimal discriminatory performance (p <0.00001). Among these features, the peptide with m/z 1913.14 was identified as a fragment of uromodulin. Receiver operating characteristic (ROC) analysis for m/z 1913.14 showed that the area under the curve (AUC) was 0.998 for distinguishing IgAN versus healthy controls, and 0.815 for distinguishing IgAN versus other glomerulopathy. Analysis of urine peptides patterns by the magnetic bead separation system and MALDI-TOF-MS was a non-invasive diagnostic tool. We conclude that the urinary peptide with m/z 1913.14, which was identified as a uromodulin fragment, may be used as a biomarker for the non-invasive diagnosis of IgAN clinically.

Evidence for a role of uromodulin in chronic kidney disease progression.

BACKGROUND: Uromodulin (also known as Tamm-Horsfall protein) is the most abundant urinary protein in healthy individuals and exhibits diverse functions including prevention of ascending urinary tract infections by binding type I-fimbriated Escherichia coli. Although uromodulin is targeted to the apical membrane of thick ascending limb (TAL) cells and secreted into the lumen, detectable levels are also found in venous blood. Uromodulin has been shown to interact with and activate specific components of the immune system, and thus, may act as a signalling molecule for renal tubular damage. METHODS: In order to investigate the potential involvement of uromodulin in chronic kidney disease (CKD), we quantified uromodulin in paired urine and serum from 14 healthy volunteers and 77 CKD patients. Clinical parameters such as estimated GFR (eGFR), proteinuria and urinary N-acetyl-beta-D-glucosaminidase (NAG) were measured. Mean infiltration and atrophy score were assessed in patient biopsies. Additionally, tumour necrosis factor-alpha, interleukin-6 (IL-6), IL-8 and IL-1 beta were measured in serum samples. RESULTS: eGFR correlated positively with urinary uromodulin and negatively with serum uromodulin. Patients with abnormally low urinary uromodulin showed a broader range of serum uromodulin. Patients with both very low urinary and serum uromodulin had the highest tubular atrophy scores. There was a positive correlation of serum uromodulin with all cytokines measured. Additionally, in in vitro experiments, uromodulin caused a dose-dependent increase in pro-inflammatory cytokine release from whole blood. CONCLUSIONS: Our data suggest that TAL damage, or damage distal to the TAL, results in an elevated interstitial uromodulin, which stimulates an inflammatory response. Persistent chronic TAL damage reduces TAL cell numbers and attenuates urinary and serum uromodulin concentrations. The combined analysis of serum and urinary uromodulin provides new insights into the role of uromodulin in CKD and suggest that uromodulin may be an active player in CKD progression.

Uromodulin levels associate with a common UMOD variant and risk for incident CKD.

Common variants in the region of the UMOD gene, which encodes uromodulin (Tamm-Horsfall protein), associate with chronic kidney disease (CKD) and estimated GFR (eGFR). Whether uromodulin levels associate with UMOD variants or with the risk for developing CKD is unknown. We conducted an age- and gender-matched case-control study (n = 200) of incident CKD (eGFR <60 ml/min per 1.73 m(2)) within the Framingham Heart Study (FHS). Baseline urinary uromodulin concentrations were related to case-control status 9.9 yr later and to genotype at rs4293393. As a replication set, we tested the genotype association with uromodulin concentration in the Atherosclerosis Risk in Communities (ARIC) Study (n = 42). Geometric means of uromodulin concentrations were 51% higher in case than in control subjects (P = 0.016). The adjusted odds ratio of CKD per 1-SD higher concentration of uromodulin was 1.72 (95% confidence interval 1.07 to 2.77; P = 0.03) after accounting for CKD risk factors and baseline eGFR. We observed lower urinary uromodulin concentrations per each copy of the C allele at rs4293393 in both cohorts. In summary, elevated uromodulin concentrations precede the onset of CKD and associate with a common polymorphism in the UMOD region.

A further investigation on a MALDI-based method for evaluation of markers of renal damage.

The validity of the urinary protein profile to characterize the pathological states of diabetic, nephropathic and diabetic-nephropathic patients was considered on the basis of previously obtained results by MALDI/MS, showing a different abundance ratio of the collagen alpha1 and alpha5 chain precursor fragments at m/z 1219 and 2049 and of the uromodulin precursor fragment at m/z 1912 observed in healthy subjects and patients; a larger number of subjects was examined and the obtained results were statistically evaluated. The p values related to the observed differences indicate that they are statistically significant when comparing all patients versus healthy controls, diabetic with normo or microalbuminuria versus nephropathic with advanced renal disease patients and diabetic with normo or microalbuminuria versus diabetic with advanced nephropathy patients. The scatter plot matrix gives evidence of the strict inverse relationship between the abundances of ions at m/z 1912 and 1219, the correlation coefficient being particularly high (r = 0.921, p < 0.001). The relationship between the true positive rate (sensitivity) and false positive rate (1-specificity) for every possible cutoff value in abundance of the considered ionic species was investigated through the receiver-operating characteristic (ROC) curve. The obtained data indicate that a good differentiation of nephropathic patients with advanced renal disease and diabetic patients with advanced nephropathy versus healthy subjects can be easily obtained by this approach.

Surface aggregation of urinary proteins and aspartic Acid-rich peptides on the faces of calcium oxalate monohydrate investigated by in situ force microscopy.

The growth of calcium oxalate monohydrate in the presence of Tamm-Horsfall protein (THP), osteopontin, and the 27-residue synthetic peptides (DDDS)(6)DDD and (DDDG)(6)DDD (D = aspartic acid, S = serine, and G = glycine) was investigated via in situ atomic force microscopy. The results show that these four growth modulators create extensive deposits on the crystal faces. Depending on the modulator and crystal face, these deposits can occur as discrete aggregates, filamentary structures, or uniform coatings. These proteinaceous films can lead to either the inhibition of or an increase in the step speeds (with respect to the impurity-free system), depending on a range of factors that include peptide or protein concentration, supersaturation, and ionic strength. While THP and the linear peptides act, respectively, to exclusively increase and inhibit growth on the (101) face, both exhibit dual functionality on the (010) face, inhibiting growth at low supersaturation or high modulator concentration and accelerating growth at high supersaturation or low modulator concentration. Based on analyses of growth morphologies and dependencies of step speeds on supersaturation and protein or peptide concentration, we propose a picture of growth modulation that accounts for the observations in terms of the strength of binding to the surfaces and steps and the interplay of electrostatic and solvent-induced forces at the crystal surface.

Proteomics and detection of uromodulin in first-time renal calculi patients and recurrent renal calculi patients.

Renal calculi disease or known as kidney stone disease is the most common urological disorder in both men and women, although it is more prevalent in men. The lifetime chance for an individual to develop renal calculi is approximately 10% whereas the risk of recurrence in a 10-year period is 74%. Therefore, a diagnostic tool for screening or detecting renal calculi is greatly needed. In this study, we analyze urinary protein profiles from patients with renal calculi for the first time (RC), healthy subjects (HS), and patients with recurrent renal calculi (RRC) to identify a biomarker for detecting the disease. Urinary proteins were isolated by salt precipitation and the proteins resolved by SDS-PAGE. Target proteins were analyzed with LC/MS/MS. Thirty-two proteins were identified from healthy subjects and patients. Uromodulin was the most abundant urinary protein in HS but was a very faint band if detected at all from those that formed renal calculi for the first time (p < 0.05). Yet the excreted levels of urinary uromodulin in RRC were similar to those of the HS suggesting that uromodulin is a reliable biomarker for only RC. In addition, a few immunoglobulins that were commonly found in the urine of both RC and RRC, which include Ig alpha heavy chain 1, Ig gamma-2 c region, Ig gamma-3 heavy chain disease protein, Ig heavy chain variable region, Ig heavy constant region gamma 4, and Ig heavy chain. Ig heavy chain Fab frag and antibody a5b7 chain B may serve as potential biomarkers for renal calculi disease.

[Construction of urinary Tamm-Horsfall protein assay by enzyme-linked immunosorbent assay and establishment of reference range of healthy subject].

Tamm-Horsfall protein (THP) is the most abundant protein in the urine. THP is expressed in the renal tubule as a precursor protein having 640 amino acid residues and anchored by GPI anchor in the cell membrane. Thereafter, THP is secreted as a glycoprotein is a molecular weight of about 95-100 kD. Despite several investigations from the perspective of renal failure, the physiological role of this protein is not yet clear. It has been reported that THP is also related to certain conditions, such as kidney stone formation and urinary tract infection. To examine the excretion of THP into urine, we constructed an enzyme linked immunosorbent assay(ELISA) for the measurement of THP in the urine. THP was purified from healthy human urine using Diatomaceous Earth. Then we obtained an antibody to purified THP and constructed a sandwich ELISA assay system to test urine samples. The sensitivity of measurement was 0.78 ng/ml. In this assay, the concentration of THP in spot urine can be linearly measured from 0.78 ng/ml to 50 ng/ml. The CV of assay was 2.4 to 4.1%. The measurement was not disrupted by urinary albumin (approximately 15 mg/ml), hemoglobin (approximately 15 microg/ml), glucose (approximately 30 mg/ml) and ascorbic acid (approximately 10 mg/ml). Pretreatment by centrifugation or filtration of urine affected the concentration of THP because of the agglutinated form of THP. We showed that the urinary excretion rate remained almost constant in our test population, 1.00-1.65 mg/hr (average +/- 1SD 1.30 +/- 0.25) for healthy men and 0.61-1.51 mg/hr (0.90 +/- 0.30) for healthy women and 1.10 +/- 0.34 mg/hr overall.

What's new in the diagnosis and management of painful bladder syndrome/interstitial cystitis?

Painful bladder syndrome/interstitial cystitis (PBS/IC) is a controversial subject. Despite its many controversies, recent data on diagnostics show that cystoscopy and hydrodistension findings may not be sensitive or specific. Diagnosis is suggested primarily on the basis of history. Antiproliferative factor and Tamm-Horsfall protein are novel tests that may prove to be worthwhile pending future studies. Currently, there is no single diagnostic gold standard. Recent data on therapeutics show that, among oral therapies, amitriptyline and pentosan are efficacious. For best response, pentosan should be initiated early and used for a minimum of 6 months. Immune-modulating agents show promise but are limited by side effects. Intravesical alkalinized lidocaine with heparin may be effective for rapid symptom relief, pending results of prospective randomized trials. Intravesical botulinum toxin A, bacille Calmette-Guérin, and sacral neuromodulation may have a role in select patients.

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.

Abnormal expression and processing of uromodulin in Fabry disease reflects tubular cell storage alteration and is reversible by enzyme replacement therapy.

Uromodulin (UMOD) malfunction has been found in a range of autosomal dominant tubulointerstitial nephropathies associated with hyperuricaemia, gouty arthritis, medullary cysts and renal failure-labelled as familial juvenile hyperuricaemic nephropathy, medullary cystic disease type 2 and glomerulocystic kidney disease. To gain knowledge of the spectrum of UMOD changes in various genetic diseases with renal involvement we examined urinary UMOD excretion and found significant quantitative and qualitative changes in 15 male patients at various clinical stages of Fabry disease. In untreated patients, the changes ranged from normal to a marked decrease, or even absence of urinary UMOD. This was accompanied frequently by the presence of aberrantly processed UMOD lacking the C-terminal part following the K432 residue. The abnormal patterns normalized in all patients on enzyme replacement therapy and in some patients on substrate reduction therapy. Immunohistochemical analysis of the affected kidney revealed abnormal UMOD localization in the thick ascending limb of Henle's loop and the distal convoluted tubule, with UMOD expression inversely proportional to the degree of storage. Our observations warrant evaluation of tubular functions in Fabry disease and suggest UMOD as a potential biochemical marker of therapeutic response of the kidney to therapy. Extended comparative studies of UMOD expression in kidney specimens obtained during individual types of therapies are therefore of great interest.

Production and characterization of transgenic mice harboring mutant human UMOD gene.

Familial juvenile hyperuricemic nephropathy is caused by mutations in the UMOD gene encoding uromodulin. A transgenic mouse model was developed by introducing a human mutant UMOD (C148W) cDNA under control of the mouse umod promoter. Uromodulin accumulation was observed in the thick ascending limb cells in the kidney of transgenic mice. However, the urinary excretion of uromodulin in transgenic mice did not decrease and LC-MS/MS analysis indicated it was of mouse origin. Moreover, the creatinine clearance was not different between wildtype and transgenic animals. Consequently, the onset of the disease was not observed in transgenic mice until 24 weeks of age.

Proteomic techniques identify urine proteins that differentiate patients with interstitial cystitis from asymptomatic control subjects.

OBJECTIVE: The purpose of this study was to identify differences in urine proteins between patients with interstitial cystitis (IC) and asymptomatic control (AC) subjects with the use of proteomic techniques. STUDY DESIGN: Nine patients with IC and their age-, race-, and sex-matched AC subjects volunteered a urine specimen. Urine proteins were separated with the use of 2-dimensional polyacrylamide gels. Differing proteins underwent digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Computer-assisted data analysis was used to identify the corresponding protein. Differences in urine protein responses between patients with IC and AC subjects were evaluated by the Mann-Whitney U test to account for the nonnormal frequency distribution of the parameter estimate or chi-square when data were bimodal. RESULTS: Four proteins differed significantly between patients with IC and AC subjects. The AC subjects had a greater concentration of a uromodulin (P = .019) and two kininogens (P = .023, .046). The patients with IC had a greater concentration of inter-alpha-trypsin inhibitor heavy chain H4 (P = .019). CONCLUSION: These urine protein isoforms may be biomarkers for IC.

Urinary uromodulin carries an intact ZP domain generated by a conserved C-terminal proteolytic cleavage.

Uromodulin (or Tamm-Horsfall protein) is the most abundant protein in human urine under physiological conditions. Little is known about the molecular mechanism of uromodulin secretion. By extensive Mass Spectrometry analyses we mapped the C-termini of human and murine urinary proteins demonstrating that urinary uromodulin is generated by a conserved C-terminal proteolytic cleavage and retains its entire ZP domain.

Tubular function in diabetic children assessed by Tamm-Horsfall protein and glutathione S-transferase.

In a previous study, we found urinary excretion of Tamm-Horsfall protein (THP) to be persistently decreased in 25% of patients during the first year after diagnosis of diabetes mellitus. We thus wanted to study another marker for distal tubular function, pi glutathione S-transferase (pi-GST) and compare this and THP with proximal tubular function evaluated with alpha-GST and alpha-1-microglobulin (HC) in patients with longer duration of diabetes. One hundred and eighty-four diabetic and 16 control children were studied with timed overnight urine collections. Median age was 14 years, and median age at diagnosis was 8 years. The urinary excretion of alpha- and pi-GST was significant lower in diabetic than control children. There were no differences in the excretion of HC and THP. Diabetic children with decreased alpha-GST had higher albumin excretion, HbA 1c levels, and longer diabetes duration but decreased THP excretion and cystatin-C clearance compared with those with normal excretion. In contrast, a decreased pi-GST or THP excretion was not associated with such differences. Diabetic children with increased HC excretion had increased HbA 1c levels. Diabetic children, before the stage of microalbuminuria, may have signs of both proximal and distal tubular dysfunction, which is related to diabetes duration and poor metabolic control. Alpha-GST and pi-GST seem to be more sensitive than other parameters studied.

Intact protein core structure is essential for protein-binding, mononuclear cell proliferating, and neutrophil phagocytosis-enhancing activities of normal human urinary Tamm-Horsfall glycoprotein.

Tamm-Horsfall glycoprotein (THP) is synthesized in the particular sites of renal tubules acting as a defense molecule in the urinary system. In the present study, we found that THP contained high amount of Siaalpha(2,3)Gal/GalNAc, moderate amount of beta(1,4)GlcNAc oligomers and GlcNAc/branched mannose, and low amount of mannose residues, but no Siaalpha(2,6)Gal/GalNAc, in the side-chains of the molecule. THP exhibited high binding affinity with human TNF-alpha, IgG, C1q and BSA, moderate binding affinity with IL-8, and low binding affinity with IL-6 and IFN-gamma. For exploring the role of carbohydrate side-chains and protein core in the protein-binding and cell-stimulating activities, THP was enzyme-digested with carbohydrate-specific [neuraminidase (Nase), beta-galactosidase (Gase)], protein-specific [V8 protease (V8), proteinase K (PaseK)] and glycoconjugate-specific [carboxypeptidase Y (Case), O-sialoglycoprotein endopeptidase (Oase)] degrading enzymes. We found that THP digested with V8, Oase, and PaseK, significantly reduced its protein-binding, mononuclear cell proliferating, and neutrophil phagocytosis-enhancing activities. These results suggest that the intact protein core structure, but not carbohydrate side-chains, is essential for pleotropic functions of THP molecule.

Decreased urinary concentration of Tamm-Horsfall protein is associated with development of renal failure and cardiovascular death within 20 years in type 1 but not in type 2 diabetic patients.

OBJECTIVE: The first changes in the diabetic kidney are glycogen deposits in the epithelial cells of the thick ascending limb of Henle. These cells produce Tamm-Horsfall protein (THP). Is low excretion of THP associated with the development of renal insufficiency or cardiovascular disease? MATERIAL AND METHODS: Urine samples were collected at baseline in patients with type 1 (n = 131) and type 2 (n = 108) diabetes who were followed for a mean of 14 years (range 1-20 years) and 4.5 years (range 1-15 years), respectively. RESULTS: Twenty percent of type 1 and 54% of type 2 diabetic patients died and 24% and 29%, respectively developed uraemia. A decreased urinary concentration of THP (u-THP) was associated with an eight-fold increased risk of renal failure and cardiovascular death in type 1 but not in type 2 diabetic patients, irrespective of the degree of albuminuria and glycosylated haemoglobin and blood pressure levels. There were no differences in the degrees of albuminuria, serum creatinine or u-THP between the two types of diabetic patients at baseline. Low u-THP occurred in 8% and 9% of normoalbuminuric type 1 and type 2 diabetic patients, respectively. CONCLUSION: A decreased u-THP was associated with an eight-fold increased risk of cardiovascular death and uraemia in type 1 but not in type 2 diabetic patients.

Defective Tamm-Horsfall protein in patients with interstitial cystitis.

PURPOSE: Normal urinary Tamm-Horsfall protein shows a urothelial cytoprotective effect against potentially toxic compounds in urine that may injure the urothelium and cause bladder disease. One such disease is interstitial cystitis. In patients with interstitial cystitis this protective effect is decreased. We hypothesized that a difference in Tamm-Horsfall protein in patients with interstitial cystitis exists that may be involved in disease pathogenesis. MATERIALS AND METHODS: Using enzyme-linked immunosorbent assay the urinary Tamm-Horsfall protein concentration was determined in patients with interstitial cystitis and control subjects. Sialic acid content was measured by high performance liquid chromatography based assay. The structure of the protein glycosylation chains was analyzed using matrix assisted laser desorption/ionization-time of flight mass spectrometry. RESULTS: The mean Tamm-Horsfall protein concentration was not significantly different in patients with interstitial cystitis and controls (28.8 vs 28.2 mg/l urine and 36.8 vs 36.7 microg/mg creatinine, respectively, p = 0.6). The total mean sialic acid content of Tamm-Horsfall protein was almost 2-fold lower in 22 patients with interstitial cystitis compared with that in 20 controls (46.3 +/- 4.3 vs 75.3 +/- 4.1 nmol sialic acid per mg Tamm-Horsfall protein, respectively, p <0.0001). On matrix assisted laser desorption/ionization-time of flight mass spectrometry N-glycans released from Tamm-Horsfall protein revealed lower molecular weight di-antennary N-glycan structures and a resulting decrease in the number of terminal sialic acid residues in 10 patients with interstitial cystitis relative to those in 10 controls. CONCLUSIONS: Tamm-Horsfall protein is qualitatively different in patients with interstitial cystitis compared to controls. These data suggest that altered Tamm-Horsfall protein may be involved in interstitial cystitis pathogenesis and it may be useful for clinical diagnosis.

Inhibitors of calcification in blood and urine.

In bone and teeth formation, coordinated calcification is a highly desirable biological process. However, heterotopic calcification at unwanted tissue sites leads to dysfunction, disease and, potentially, to death and therefore requires prevention and treatment. With the recent discovery of calcification inhibitors we now know that biological calcification is not passive but a complex, active and highly regulated process. Calcification at vascular sites is the most threatening localization and manifests as part of atherosclerosis or arteriosclerosis. Atherosclerosis is often accompanied by intimal plaque calcification, whereas arteriosclerosis is characterized by calcification of the media. The severity of calcification of cerebral or coronary atherosclerotic plaques is associated with an increased incidence of events such as stroke or myocardial infarction. Medial calcification is the major cause of arterial stiffness, which contributes to left ventricular dysfunction and heart failure. Patients with chronic kidney disease are at especially increased risk for both intimal and medial calcification. In this context, it is currently thought that calcium-regulatory factors including fetuin-A, matrix Gla protein, osteoprotegerin, and pyrophosphates act in a local or systemic manner to prevent calcifications of the vasculature, and that dys-regulations of such calcification inhibitors may contribute to progressive calcifications. Nephrolithiasis represents another process of unwanted calcification responsible for significant morbidity. More than 80% of renal stones contain calcium. Urinary factors inhibiting calcification are citrate, glycosaminoglycans, Tamm-Horsfall protein, and osteopontin. This review summarizes current experimental and clinical data underlining the biological importance of these calcification inhibitors.