A Renal-Clearable Macromolecular Reporter for Near-Infrared Fluorescence Imaging of Bladder Cancer.

Bladder cancer (BC) is a prevalent disease with high morbidity and mortality; however, in vivo optical imaging of BC remains challenging because of the lack of cancer-specific optical agents with high renal clearance. Herein, a macromolecular reporter (CyP1) was synthesized for real-time near-infrared fluorescence (NIRF) imaging and urinalysis of BC in living mice. Because of the high renal clearance (ca. 94 % of the injection dosage at 24 h post-injection) and its cancer biomarker (APN=aminopeptidase N) specificity, CyP1 can be efficiently transported to the bladder and specially turn on its NIRF signal to report the detection of BC in living mice. Moreover, CyP1 can be used for optical urinalysis, permitting the ex vivo tracking of tumor progression for therapeutic evaluation and easy translation of CyP2 as an in vitro diagnostic assay. This study not only provides new opportunities for non-invasive diagnosis of BC, but also reveals useful guidelines for the development of molecular reporters for the detection of bladder diseases.

The paradoxical role of urinary macromolecules in the aggregation of calcium oxalate: a further plea to increase diuresis in stone metaphylaxis.

This study was designed to get information on aggregation (AGN) of urinary calcium oxalate crystals (CaOx) which seems to occur in stone formation despite a protecting coat of urinary macromolecules (UMs). CaOx crystallization was directly produced in urine, control and albumin solution by Ox titration and was spectrophotometrically followed. A rapid decrease of optical density indicating AGN was absent in 14 of 15 freshly voided urines of 5 healthy controls. However, in the presence of UM-coated hydroxyapatite all urines with relative high sodium concentration, being an indicator of concentrated urine, showed a pronounced AGN which was abolished when these urines were diluted. Albumin relatively found to be an inhibitor of AGN showed after temporary adsorption on Ca Phosphate (CaP) massive self-AGN and changed to a promoter of CaOx AGN. Self-AGN after adsorption on surfaces especially of CaP, being an important compound of Randall's plaques, can thus explain this paradoxical behavior of UMs. Aggregated UMs probably bridge zones of electrostatic repulsion between UM-coated crystals with identical electrical surface charge. These zones extend by urine dilution which decreases ionic strength. Diminution of urinary concentration by increasing diuresis seems, therefore, to be important in stone metaphylaxis.

The Isolation and Quantitation of Fetuin-A-Containing Calciprotein Particles from Biological Fluids.

Multiple overlapping systemic and local inhibitory networks have evolved to prevent the unwanted deposition of mineral at ectopic sites. Fetuin-A is a liver-derived glycoprotein abundant in plasma that binds and stabilizes nascent mineral ion nuclei to form soluble colloidal high molecular weight complexes, called calciprotein particles (CPP). The binding of fetuin-A to mineral retards crystal ripening and precipitation from the aqueous phase, thereby facilitating the regulated clearance of mineral debris from the extracellular fluid. However, persistent disturbances in this humoral homeostatic system, as frequently seen in patients with Chronic Kidney Disease, may lead to the accumulation and aggregation of these nanoparticles in extraosseous tissues like the vasculature, driving inflammatory cascades, aberrant tissue remodeling, and functional impairment. Consistent with this conceptual framework, higher circulating CPP levels are associated with reduced renal function, increments in systemic inflammatory markers, derangements in bone morphogenetic cytokines, higher vascular calcification scores, aortic stiffening and an increased risk of death. This chapter describes optimized sample collection and preparative procedures for the isolation and enrichment of CPP from biological fluids. Methods for CPP quantitation are critically reviewed and detailed.

Inhibition of urinary macromolecule heparin on aggregation of nano-COM and nano-COD crystals.

PURPOSE: This research aims to study the influences of heparin (HP) on the aggregation of nano calcium oxalate monohydrate (COM) and nano calcium oxalate dihydrate (COD) with mean diameter of about 50 nm. METHOD: The influences of different concentrations of HP on the mean diameter and Zeta potential of nano COM and nano COD were investigated using a nanoparticle size Zeta potential analyzer. RESULTS: HP could be adsorbed on the surface of nano COM and nano COD crystals, leading to an increase in the absolute value of Zeta potential on the crystals and an increase in the electrostatic repulsion force between crystals. Consequently, the aggregation of the crystals is reduced and the stability of the system is improved. The strong adsorption ability of HP was closely related to the -OSO3- and -COO- groups contained in the HP molecules. X-ray photoelectron spectroscopy confirmed the coordination of HP with Ca2+ ions of COM and COD crystals. CONCLUSION: HP could inhibit the aggregation of nano COM and nano COD crystals and increase their stability in aqueous solution, which is conducive in inhibiting the formation of calcium oxalate stones.

Urine analysis in microfluidic devices.

Microfluidics has attracted considerable attention since its early development in the 1980s and has experienced rapid growth in the past three decades due to advantages associated with miniaturization, integration and automation. Urine analysis is a common, fast and inexpensive clinical diagnostic tool in health care. In this article, we will be reviewing recent works starting from 2005 to the present for urine analysis using microfluidic devices or systems and to provide in-depth commentary about these techniques. Moreover, commercial strips that are often treated as chips and their readers for urine analysis will also be briefly discussed. We start with an introduction to the physiological significance of various components or measurement standards in urine analysis, followed by a brief introduction to enabling microfluidic technologies. Then, microfluidic devices or systems for sample pretreatments and for sensing urinary macromolecules, micromolecules, as well as multiplexed analysis are reviewed, in this sequence. Moreover, a microfluidic chip for urinary proteome profiling is also discussed, followed by a section discussing commercial products. Finally, the authors' perspectives on microfluidic-based urine analysis are provided. These advancements in microfluidic techniques for urine analysis may improve current routine clinical practices, particularly for point-of-care (POC) applications.

Aggregation of freshly precipitated calcium oxalate crystals in urine of calcium stone patients and controls.

Aggregation (AGN) of freshly precipitated calcium oxalate crystals was photometrically studied in urine of 30 calcium stone patients and 30 controls, in solutions containing urinary macromolecules (UMS) and in an inhibitor free control solution (CS). Crystals were produced by oxalate titration and crystallization was monitored measuring optical density (OD). Tests were repeated adding hydroxyapatite (HAP) to urine and UMS and adding citrate and pyrophosphate (PPi) to UMS of the controls. AGN was recognized as a rapid OD decrease being at least three times faster than sedimentation of single crystals (p < 0.001) and used to calculate an extent of AGN (EA%). The time between the end of titration and the beginning of AGN was determined as suspension stability (SS). The main effect of urinary inhibitors was retardation of AGN without changing EA, SS being higher in urine than UMS (p < 0.001) and in UMS than CS (p < 0.001). In urine of 63% of controls but only in 33% of patients, no AGN was recorded (p < 0.05). The high inhibitory activity of urine could not be reproduced in UMS even in combination with 3.5 mM citrate or 0.05 mM PPi. 0.05 mg/mL HAP reduced SS in all urine samples to low values and increased the rate of rapid OD decrease, being a measure for the size of aggregates. Retarding AGN of crystals during their passage through the kidney seems to be an important mechanism to prevent stone formation during crystalluria. The promotion of AGN by HAP reveals a new role of Randall's plaques in nephrolithiasis.

Mechanism underlying the low prevalence of pediatric calcium oxalate urolithiasis.

PURPOSE: Urinary macromolecules in children show stronger inhibition of Ca oxalate crystal growth, aggregation and adhesion than in adults. To investigate the mechanism of Ca oxalate urolithiasis we evaluated the differences in inhibitory activity against oxalate induced renal cell injury between adults and children. MATERIALS AND METHODS: Urine samples were collected from healthy men and their sons. The protective effects of urinary macromolecules against oxalate induced injury to Madin-Darby canine kidney cells (ATCC) were examined by lactate dehydrogenase assay and immunostaining. Variations in the relative abundance of proteins involved in stone formation, such as osteopontin and calgranulin B, were analyzed. RESULTS: The urine of children had significantly higher urinary macromolecule and glycosaminoglycan concentrations than that of adults (p <0.01). Urinary macromolecules inhibited oxalate induced Madin-Darby canine kidney cell injury in a concentration dependent manner and stronger activity was observed in children (p <0.05). TUNEL staining and 8-OHdG immunostaining indicated stronger inhibition of apoptosis and oxidative stress in Madin-Darby canine kidney cells pretreated with pediatric urinary macromolecules (p <0.01). Osteopontin and calgranulin B expression correlated positively (p = 0.03). These proteins showed greater down-regulation in children (p <0.01). Osteopontin expression also correlated positively with lactate dehydrogenase release (p = 0.03). CONCLUSIONS: A reason for the low prevalence of pediatric urolithiasis is that pediatric urinary macromolecules have stronger inhibitory effects against oxalate induced renal cell injury and oxidative stress induced apoptosis. Furthermore, results suggest that osteopontin and calgranulin B expression is down-regulated in children due to this inhibitory effect and, thus, stone nidus formation is controlled.

Role of calcium in the aggregation of particles coated by urinary macromolecules.

OBJECTIVES: In urine, aggregation (AGN) of crystal occurs although they are coated by negatively charged urinary macromolecules (UM) and isolated at a distance from each other, where attraction forces become extremely weak. Calcium (Ca) bridges or viscous binding by UM could explain this AGN. METHODS: Suspensions of Ca oxalate monohydrate (COM) and carboxylated latex (CL) were prepared in buffered water and UM solutions which were obtained from the urine of 6 healthy men. Surface potential or zetapotential (ZP) of the particles was measured at various concentrations of Ca and UM. AGN of CL was monitored determining particle size and optical density. RESULTS: The ZP of COM was neutralized by Ca and stabilized around -15 mV by UM. In CL, a stable ZP of about -20 mV was achieved at >3 mM Ca. However, to induce AGN further addition of Ca was necessary. AGN then increased independently of ZP with increasing Ca and decreased with increasing UM concentration. CONCLUSIONS: In CL, two different Ca effects could be distinguished, one on ZP and another on AGN. The latter is probably based on the formation of Ca bridges between particles and is inhibited by a steric barrier of adsorbed UM. A similar mechanism is postulated for crystal aggregation.

Stabilization of calcium oxalate suspension by urinary macromolecules, probably an efficient protection from stone formation.

INTRODUCTION: Crystal aggregation (AGN) destabilizes crystal suspensions and during crystalluria probably favors crystal apposition to kidney calcifications and preexisting stones. We analyzed inhibition of AGN and stabilization of calcium oxalate suspensions by urinary macromolecules (UM), urine and solutions with urinary citrate concentration. MATERIALS AND METHODS: Solutions of UM (UMS) were obtained by a hemofiltration procedure from urine of 6 healthy men. Calcium oxalate suspensions were prepared in all solutions and urine by adjusting Ca2+ to 1.5 mM and by an oxalate titration to 1.0 mM. Crystallization was monitored measuring optical density (OD). Stability of suspensions (SS) was defined as the time without sedimentation and zeta potential (ZP) of crystals was measured. AGN was visualized by scanning electron microscopy and quantified by maximal OD. RESULTS: UMS inhibited AGN and increased ZP and SS. Most inhibitory activity of urine could be attributed to UM. 3.3-fold dilution of UM reduced SS only by 30%. CONCLUSIONS: During crystalluria, UM of healthy men are supposed to protect from stone formation by inhibiting AGN and stabilizing crystal suspensions. As a probably important aspect, this protection was found to be limited in time and may favorably be influenced by an increase of diuresis.

Urinary macromolecules and renal tubular cell protection from oxalate injury: comparison of normal subjects and recurrent stone formers.

AIM: To determine whether urinary macromolecules (UMM), which are the high molecular weight substances in urine, can provide protection against the oxalate-associated injury to the renal tubular cells. METHODS: UMM were extracted from 24-h urine of 12 healthy adult male volunteers and 13 recurrent-stone-former male patients. Urine parameters in relation to urolithiasis were measured, including the level of glycosaminoglycans (GAG) in the UMM. Madin-Darby canine kidney (MDCK) cells were used to evaluate the protective activity of UMM from oxalate-induced cytotoxicity by LDH release measurement and methyl-thiazolyl tertrazolium (MTT) assay. RESULTS: Considering urinary parameters, citrate was significantly higher in urine from normal subjects than stone-former subjects; the other parameters show no differences between the groups. Total UMM and the level of GAG in the UMM were also significantly higher in the normal subject group. Compared with normal subject and stone-former subject UMM, after cells were treated with the UMM and then exposed to oxalate solution, LDH release was significantly higher in stone-former group. In the MTT assay, we found that more viable cells were observed after treatment with UMM compared to control in both groups. Moreover, UMM from the normal subjects showed higher protective activity against oxalate-related cytotoxicity than UMM from the stone-former subjects. CONCLUSION: UMM protected renal epithelial cells from oxalate-related injury. This protective activity was found to be higher in normal subject UMM than stone-former UMM. Among other factors, a higher concentration of GAG and citrate in normal subject UMM might affect some parts in this finding.

Urinary macromolecular inhibition of crystal adhesion to renal epithelial cells is impaired in male stone formers.

BACKGROUND: Retention of microcrystals that form in tubular fluid could be a critical event in kidney stone formation. This study was performed to determine if urinary macromolecules from stone-forming (SF) individuals have reduced ability to inhibit crystal adhesion to renal cells. METHODS: A first morning whole urine (WU) sample was obtained from 24 SF subjects (17 males and 7 females) and 24 age-, race-, and sex-matched controls (C). An aliquot of urine was centrifuged and an ultrafiltrate (UF) free of macromolecules >10 kD and 10x concentrate (U(conc)) were prepared. RESULTS: Supplementing UF with increasing amounts of U(conc) to return the macromolecule concentration to 0.25x, 0.5x, or 1x of baseline progressively decreased crystal binding to cells. This effect was blunted in the male SF group compared to controls (P < 0.05, SF vs. C, for UF plus 0.25x macromolecules). No difference was apparent in the female groups. In order to identify responsible macromolecule(s), calcium oxalate monohydrate (COM) crystals were coated with U(conc) and adherent proteins then released and probed by Western blot. Coated COM crystals from male controls contained 3.5-fold more Tamm-Horsfall protein (THP) than SF subjects (P < 0.01). COM crystal coating with other proteins did not consistently differ between the groups. COM crystal coating by urinary prothrombin fragment 1 (UPTF1, P < 0.05) and crystal adhesion inhibitor (CAI) (P= 0.09) correlated with decreased crystal binding to cells, whereas coating with osteopontin (OPN) correlated with increased adhesion tendency (P < 0.05). CONCLUSION: Urinary macromolecules >10 kD coat COM crystals and block their adhesion to renal cells. This capacity appears to be blunted in male but not female SF individuals. Multiple urinary proteins may play a role in renal cell-urinary crystal interactions, and THP appears to be one of the more important ones.

Regulation by macromolecules of calcium oxalate crystal aggregation in stone formers.

Based on the structure of kidney stones, it is likely that they form as aggregations of preformed crystals, mostly calcium oxalate monohydrate (COM). In this study, we examined the ability of a macromolecular mixture isolated from the urine of normal individuals and stone formers to inhibit aggregation of preformed COM seed crystals in a simple ionic solution using measurements of changes in the particle size distribution (PSD) of preformed COM crystal aggregates. We also examined the effect in this assay of a number of synthetic homopolymers, naturally occurring urine macromolecules, and binary mixtures thereof. The macromolecular mixtures from urine of normals and most stone formers reduced the degree of aggregation of the seed crystals, whereas 22% of stone former urine macromolecules either did not disaggregate or actually promoted further aggregation. Stone formers within one family shared this property, but a non-stone forming sibling did not. Polyanions, either synthetic or naturally occurring, induced disaggregation to an extent similar to that exhibited by normal urine macromolecules, while polycations had no effect on the PSD. However, mixing a polyanion, either poly-aspartate or osteopontin, with the polycation poly-arginine, changed their behavior from disaggregation to aggregation promotion. The disaggregating behavior of normal urinary macromolecules provides a defense against aggregation, but a minority of stone forming individuals lacks this defense, which may contribute to stone formation.

Structural and functional modification of THP on nitration: comparison with stone formers THP.

OBJECTIVE: The crucial steps involved in the lithogenic process are governed by the macromolecular components of urine, of which proteins play a major role. Structurally abnormal proteins have been reported to be present in the urine of stone formers. Free radical injury has come a long way in explaining some of the pathophysiological events of renal lithiasis. Thus, our present work was designed to study the impact of the potent oxidant peroxynitrite on the biochemical components of the urinary Tamm-Horsfall glycoprotein (THP). MATERIALS AND METHODS: Nitration on THP was carried out using peroxynitrite (ONOO(-)). After nitration, biochemical components like thiols, S-nitrosothiol, hexose, hexosamine and sialic acid were determined and these factors were compared with those of stone formers and normal THP. Crystallization behavior of control, nitrated NS-THP and stone formers THP was studied. RESULTS: There was a significant decrease in thiol, hexose, hexosamine and sialic acid contents in stone formers and nitrated NS-THP, when compared to that of the control THP. In contrast to this, S-nitrosothiol content was significantly increased in stone formers and nitrated NS-THP (p < 0.001) when compared with the control THP. NO(x) metabolites were significantly elevated in stone formers THP when compared with that of control THP. When subjected to CaOx crystallization, stone formers THP and nitrated NS-THP promoted both CaOx nucleation and aggregation, while normal THP was found to be an inhibitor of the above processes. CONCLUSION: From our results we conclude that nitration of THP could represent one of the prime events in modifying kinetic behavior of THP, thus converting THP into a heterogeneous nucleator of renal calculi formation.

Hemodynamic factors in the genesis of diabetic microangiopathy.

There are many candidate mechanisms to explain the phenomenon of delayed microvascular disease in the diabetic. All may play some part in determining the genesis, the evolution or the ultimate degree and form of the angiopathy. General metabolic and humoral factors may provide the pathogenetic background against which special local conditions, e.g., in the retina or renal cortex, will determine the morphology of the angiopathy and its functional and structural consequences. Some of the processes occurring in the diabetic person may, however, be of major importance in initiating and maintaining conditions for the evolution of microvascular disease. The hemodynamic changes and the vascular responses to them that we have described are, we suggest, very likely to be an important component of this sort. Unlike the later structural changes, these hemodynamic phenomena are to be found very early in the diabetic state. Of most clinical importance, perhaps, is that they appear, with the achievement of adequate metabolic correction, to be reversible.

Urinary macromolecular substances as natural inhibitors of calcium oxalate crystal aggregation.

We studied the inhibitory effect of urinary macromolecular substances (UMM), more than 10,000 mol. wt., on aggregation of calcium oxalate crystals in a crystal-seed system and a non-crystal-seed system. In both assay systems, UMM from non-stone-farmers showed a marked inhibitory activity on crystal aggregation, whereas UMM from recurrent stone-formers showed a weak inhibitory activity. The distinction between the two types of UMM was greater in the non-crystal-seed system. The macromolecular inhibitors seemed to be composed of proteins or protein-complexes inasmuch as approximately 70 to 90 per cent of the macromolecular inhibitory activity was destroyed by protein digestion with pronase. Urinary acid glycosaminoglycans, which have been considered possible inhibitors, played a small role in the inhibition of crystal aggregation.

Uromucoids and urinary stone formation.

Crystal formation was studied in fresh urine samples after rapid concentration to standard osmolarity in a rotary evaporator at 37 degrees C. Uromucoids promoted calcium oxalate and calcium phosphate crystal formation and also induced clustering of calcium phosphate precipitates. It is postulated that uromucoid precipitation is the first stage in stone formation. Minerals then deposit on the uromucoid precipitate and the whole complex becomes attached to the renal tubules.