Alleviation of cadmium phytotoxicity through melatonin modulated physiological functions, antioxidants, and metabolites in tomato (Solanum lycopersicum L.).

The rising concentration of cadmium (Cd) builds a harmful effect on human and plant health associated with food chain contagion. Melatonin (MT) is an indole compound. Hence, the experiment was conducted to understand the physiological and biochemical mechanism of Cd detoxification by exogenous MT in tomato. Pots were filled with 30 ppm of Cd spiked soil and different concentration of exogenous MT was given to the plants through seed treatment (250 ppm), foliar spray viz., 25, 50, and 100 ppm, and both, whereas the foliar spray was given at 30 days after transplanting (DAT) and 46 DAT. When the plants are exposed to Cd stress, it reduces the gas exchange characters. The results revealed that foliar spray of 25 ppm of exogenous MT recorded the highest photosynthetic rate, stomatal conductance, and osmotic potential. MT had a direct interaction with reactive oxygen species scavenging by elevating endogenous antioxidant enzymes as well as the metabolites in plants. The contribution of MT foliar spray of 25 ppm at 30 and 46 DAT can mitigate Cd stress and it has potential implications for ensuring food safety and food security in marginal agriculture.

Preparation and Characterization of Optimized Hydrochar from Paper Board Mill Sludge.

The amount of Paper Board Mill Effluent Treatment Plant Sludge (PBM-ETPS) dumped from paper mills are huge and its conversion into hydrochar for the purpose of energy has broad prospects. This study investigated the optimum conditions for the production of PBM-ETPS derived hydrochar (PBM-ETPSH) through Reponse Surface Methodology (RSM) for more surface area and pore volume with minimal hydrogen to carbon (H/C) and oxygen to carbon (O/C) ratios. The PBM-ETPSH had higher heating value (HHV) of 18.39 MJ kg-1 with fixed carbon percentage of 15.6. Our results showed a reduction in H/C (35.05%) and O/C (43.7%) ratios confirming the coalification of optimized PBM-ETPSH. Thermogravimetric investigations of blending PBM-ETPSH with coal in 1:1 ratio increased its HHV to 22.25 MJ kg-1 making it suitable as an energy alternative for paper mills.

Solid state parameters, structure elucidation, High Resolution X-Ray Diffraction (HRXRD), phase matching, thermal and impedance analysis on L-Proline trichloroacetate (L-PTCA) NLO single crystals.

Nonlinear optical single crystal of L-Proline trichloroacetate (L-PTCA) was successfully grown by Slow Evaporation Solution Technique (SEST). The grown crystals were subjected to single crystal X-ray diffraction analysis to confirm the structure. From the single crystal XRD data, solid state parameters were determined for the grown crystal. The crystalline perfection has been evaluated using high resolution X-ray diffractometer. The frequencies of various functional groups were identified from FTIR spectral analysis. The percentage of transmittance was obtained from UV Visible spectral analysis. TGA-DSC measurements indicate the thermal stability of the crystal. The dielectric constant, dielectric loss and ac conductivity were measured by the impedance analyzer. The DC conductivity was calculated by the cole-cole plot method.

Electrolytic recovery of dilute copper from a mixed industrial effluent of high strength COD.

In this study, the electrochemical treatment has been investigated in the real acidic effluent of copper-phthalocyanine dye manufacturing plant. Galvanostatic batch electrolyses have been carried out in an undivided cell using stainless steel as cathode, dimensionally stable anode (DSA) and graphite as anodes at different current densities and temperatures. The influence of these variables on current efficiency, cell voltage, energy consumption and deposit quality was reported. Under optimized conditions, the maximum copper recovery of 98% and COD removal efficiency of 87.3% with the energy consumption of about 11.23 kWh/kg of Cu and 6.08 kWh/kg of COD, respectively at 30 degrees C were achieved in the acidic raw effluent using 2D parallel-plate cathode. While in 3D stainless steel turning cathode reactor, 99.5% of copper can efficiently be recovered from dilute solution with an acceptable current efficiency of about 56.8% with minimum energy consumption of 2.37 kWh/kg of Cu. The experimental results suggested that the efficiency of copper removal is hindered by the presence of organic species in the mixed industrial effluent.

Immunological detection of nitrosative stress-mediated modified Tamm-Horsfall glycoprotein (THP) in calcium oxalate stone formers.

The generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in hyperoxaluric condition has been proved experimentally. This may result in the formation of the cytotoxic metabolite peroxynitrite, which is capable of causing lipid peroxidation and protein modification. The presence of nitrotyrosine in proteins has been associated with several pathological conditions. The present study investigated the presence of nitrotyrosine in the stone formers Tamm-Horsfall glycoprotein (THP). In vitro nitration of control THP was carried out using peroxynitrite. New Zealand white rabbits were immunized with peroxynitrated THP at 15-day intervals. Antisera collected following the third immunization were assayed for antibody titres using solid-phase ELISA. Antibodies were purified by affinity chromatography. The carbonyl content of control, stone formers and nitrated THP were determined. Western blotting was carried with control, stone formers and nitrated THPs. Immunodiffusion studies demonstrated cross-reaction with nitrated bovine serum albumin. Significant amounts (p < 0.001) of carbonyl content were present in both stone formers and nitrated THPs. Western blot analysis confirmed the presence of nitrated amino acid 3-nitrotyrosine in stone formers, which could bring about structural and functional modifications of THP in hyperoxaluric patients. A cross-reaction with nitrated bovine serum albumin confirms that the raised antibody has certain paratopes similar to the epitope of nitrated protein molecules. Detection of 3-nitrotyrosine in stone formers THP indicates that it is one of the key factors influencing the conversion of THP to a structurally and immunologically altered form during calcium oxalate stone formation.

Characterization of histone (H1B) oxalate binding protein in experimental urolithiasis and bioinformatics approach to study its oxalate interaction.

The rat kidney H1 oxalate binding protein was isolated and purified. Oxalate binds exclusively with H1B fraction of H1 histone. Oxalate binding activity is inhibited by lysine group modifiers such as 4',4'-diisothiostilbene-2,2-disulfonic acid (DIDS) and pyridoxal phosphate and reduced in presence of ATP and ADP. RNA has no effect on oxalate binding activity of H1B whereas DNA inhibits oxalate binding activity. Equilibrium dialysis method showed that H1B oxalate binding protein has two binding sites for oxalate, one with high affinity, other with low affinity. Histone H1B was modeled in silico using Modeller8v1 software tool since experimental structure is not available. In silico interaction studies predict that histone H1B-oxalate interaction take place through lysine121, lysine139, and leucine68. H1B oxalate binding protein is found to be a promoter of calcium oxalate crystal (CaOx) growth. A 10% increase in the promoting activity is observed in hyperoxaluric rat kidney H1B. Interaction of H1B oxalate binding protein with CaOx crystals favors the formation of intertwined calcium oxalate dehydrate (COD) crystals as studied by light microscopy. Intertwined COD crystals and aggregates of COD crystals were more pronounced in the presence of hyperoxalauric H1B.

Epitaxial deposition of calcium oxalate on uric acid rich stone matrix is induced by a 29 kDa protein.

BACKGROUND: Association of macromolecules particularly the role of proteins in urolithiasis has been studied for last few centuries, but still a complete profile of stone matrix proteins that mediate co-precipitation of uric acid and calcium oxalate has not been characterized. We isolated and characterize proteins from uric acid rich stone matrix, which have oxalate binding activity. METHODS: Matrix proteins were isolated from uric acid rich stone matrix using EDTA as a demineralizing agent. The radiolabelled solubilized proteins were fractionated with increasing ionic concentration by DEAE cellulose column chromatography to identify the oxalate binding protein. It was purified using Sephadex G-200 column chromatography. Amino acid composition was determined and monoclonal antibody was produced against the oxalate binding uric acid rich stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers urine, their oxalate binding activity assayed and cross reactivity with the produced monoclonal antibody were checked using ELISA and Western blotting. RESULTS: Matrix on DEAE column chromatography elution yielded 3 protein peaks and they were named as fraction I, II and III among which fraction I had higher oxalate binding activity which was further purified with Sephadex G-200 column which yielded 2 protein peaks designated as Ia and Ib. Fraction Ib with molecular weight 29 kDa exhibited the maximum oxalate binding activity. Forty percent of this 29 kDa protein is comprised of basic amino acids. Monoclonal antibody (IgG1) was produced against the 29 kDa stone matrix protein. Urinary uric acid binding proteins were isolated from stone formers, 4 protein peaks were obtained named as fraction I to IV. Among them, fraction IV having molecular weight of approximately 29 kDa cross reacted up to 85.6% with 29 kDa stone matrix protein. Moreover, urinary 29 kDa protein exhibited oxalate binding activity of 94.16 +/- 6.08 pmol/mg protein at pH 5.5. CONCLUSION: The 29 kDa protein isolated from uric acid rich stone matrix and urine are one and the same, thereby insinuating that 29 kDa protein might play a major role in epitaxial deposition of calcium oxalate over uric acid core, consequently favoring the lithogenic events like uric acid and calcium oxalate nucleation, aggregation and retention.

Uric acid: an abettor or protector in calcium oxalate urolithiasis? Biochemical study in stone formers.

BACKGROUND: Free radical induced renal damage leads to crystal retention and formation of large stones. However, the scenario behind uric acid (UA) stone formation is still a mystery, as uric acid, a risk factor of stone formation, seems to be a potent antioxidant that can protect cells from damage by reactive oxygen species. This study was intended to evaluate the role of uric acid in stone formers by assessing the oxidative stress status of the stone patients. METHODS: Determination of urinary stone forming risk factors and oxidative stress factors like plasma lipid peroxidation, protein carbonyls of stone formers and histopathological changes and uric acid deposition in stone patients kidney biopsy were studied. RESULTS: Increased concentrations of urinary uric acid and oxalate in both uric acid as well as calcium oxalate stone formers were observed, whereas calcium is increased in calcium stone formers and not in the uric acid stone patients. Inhibitors such as citrate and glycosaminoglycans (GAGs) were found to be significantly decreased in all the stone patients. Histopathological studies confirmed the deposition of crystals in the damaged tubules and De Galantha staining authenticates that the damage is caused due to uric acid crystals. Increased oxidative stress is dictated by the concentrations of lipid peroxidation and protein carbonyls in stone formers. Moreover, increased activities of urinary marker enzymes substantiate the tubular damage. CONCLUSION: We speculated that uric acid acts as a calculi forming salt rather than an antioxidant and it has no role in preventing oxidative stress pertaining to urolithiasis.

Oxidative stress in urogenital tuberculosis patients: a predisposing factor for renal stone formation--amelioration by vitamin E supplementation.

BACKGROUND: Previous studies have shown that urogenital tuberculosis (GuTb) patients treated or untreated with regular anti-Tb regimen excrete comparatively high levels of urinary stone forming constituents than normal subjects. Enhanced oxidative stress is also considered as a prime factor that accelerates urolithiasis. The present study was aimed to determine antioxidant status and lipid peroxidation of these individuals in order to assess their risk for kidney stone formation. METHODS: GuTb patients and age-matched normal subjects were divided into four groups: I: normal subjects (n=60), II: GuTb patients a day before treatment (n=72), III: GuTb patients after treatment with isoniazid (300 mg), rifampicin (450 mg) and pyrazinamide (1.5 g) per day for 60 days (n=42), and IV: GuTb patients supplemented with vitamin E (200 mg/day) along with regular chemotherapy for 60 days (n=30). Blood was collected and tested for various markers of oxidative stress. RESULTS: Increased levels of lipid peroxidation, protein carbonyls (PCO), advanced oxidative protein products (AOPP) and reduced antioxidant defenses by impairment in enzyme activities like superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione and decreased plasma concentrations of non enzymatic antioxidants like vitamins C and E were observed in the treated and untreated GuTb patients. CONCLUSIONS: These biochemical disparities may lead to membrane disintegrity, which is favorable for retention of mirolithis. Advocation of vitamin E enhanced the antioxidant status of the plasma, thereby preventing membrane injury, consequently reducing the risk of stone formation in urogenital tuberculosis patients, who were treated with their routine anti-tuberculosis drug regimen.

Calcium oxalate monohydrate binding protein: a diagnostic biomarker for calcium oxalate kidney stone formers.

Urinary oxalate is a biomarker for calcium oxalate kidney stone disease; however, its assay is insensitive and nonspecific. Calcium oxalate monohydrate (COM) binding protein (45 kDa) is a promoter of calcium oxalate kidney disease, which is markedly upregulated by oxalate induced oxidative stress. The current study was carried out to evaluate whether COM binding protein can serve as a diagnostic marker for calcium oxalate kidney stone formers. COM binding protein was isolated, purified and antibody was raised against it in rabbits. Urine samples (24 h) were collected from patients suffering from various kidney diseases such as acute nephritis, chronic nephritis, nephrotic syndrome, calcium oxalate (CaOx) stone formers, uric acid stone formers, struvite stone formers and calcium phosphate stone formers. This COM binding protein was quantified by an in house ELISA method and the excretion was found to lie between 2 and 3 mg in control samples, while in CaOx stone formers it was detected between 11 and 19 mg. Urinary risk factors were assayed. We conclude that COM binding protein can serve as a diagnostic marker for CaOx stone formers.

Nuclear pore complex oxalate binding protein p62: expression in different kidney disorders.

BACKGROUND: Urolithiasis is a multifactorial process that starts with the formation of microcrystals in the urine and terminates as mature renal calculi. The oxalate binding protein plays a vital role in the transport of oxalate. The physiological significance of the presence of oxalate binding protein in the nuclear pore complex is not well understood. METHODS: The nuclear envelope was extracted from human cadaver kidneys. 14C oxalate was labeled, nuclear pore complex proteins were extracted and loaded onto Sephadex G-200, and further purified in DEAE-Sephadex A-50 column. The radioactive protein peak was pooled, concentrated and checked for purity in SDS-PAGE. The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and was homogeneous. Urine samples of healthy individuals with no history of kidney disease served as control. Blood and urine samples were collected from kidney and autoimmune disorder patients and checked for the expression of p62 protein by ELISA. RESULTS: Extracted and purified nuclear pore complex oxalate binding protein had a molecular weight of 62 kDa. A threefold increase in oxalate excretion was observed in hyperoxaluric patients compared to control subjects. The protein expression was found to be higher in hyperoxaluric patients vs. controls, chronic renal failure (CRF) and acute renal failure (ARF), whereas decreased expression was observed in nephrotic syndrome (NS) patients. p62 autoantibodies was observed in hyperoxaluria (HO), systemic lupus erythematosus (SLE) and primary biliary cirrhosis (PBC), whereas it was absent in controls. CONCLUSION: Increased expression of p62 may be due to membrane damage induced by oxalate stress, and may be used as a diagnostic marker. This study also confirms the presence of p62 autoantibodies in HO patients.

Salubrious effect of vitamin E supplementation on renal stone forming risk factors in urogenital tuberculosis patients.

The incidence of renal calculi has been evaluated to be 25% in urogenital tuberculosis patients. The stone could be caused due to the host, the pathogenic organism, or possibly by the treatment. Studies were carried out to find out the efficacy of vitamin E supplementation in reducing the risk of stone formation in renal tuberculosis patients. The study constituted four groups, Group I with 30 normal volunteers, the second group comprised of 36 renal tuberculosis patients (GuTb) a day before treatment. Third group comprised of 24 patients with regular anti tuberculosis drug regimen for sixty days. In the fourth group, 12 patients were treated with anti tuberculosis drug regimen along with supplementation of antioxidant vitamin E (200 mg/day) for sixty days. Hyperuricosuria and hypercalciuria were observed in group II and group III patients, along with increased excretion of oxalate and creatinine, accompanied by decreased excretion of inhibitors such as citrate and glycosaminoglycans (GAGs). Renal damage was evident with increased leakage of Lactate dehydrogenase (LDH), Alkaline phosphatase (ALP) and gamma-Glutamyl transferase (gamma-GT) in renal tuberculosis patients. From the results of the above study, it is obvious that increased urinary oxalate levels leads to cellular damage in GuTb patients, which is a prerequisite for crystal retention as revealed by the elevated urinary marker enzymes. Antioxidant therapy prevents membrane injury thereby reducing the risk of stone formation. Hence vitamin E supplementation has a salubrious effect in preventing stone forming tendency with routine anti tuberculosis drug regimen.

Expression of nuclear pore complex oxalate binding protein p62 in experimental hyperoxaluria.

Proteins are thought to play a major role in stone formation. Oxalate binding protein plays a vital role in the transport of oxalate. This study was aimed at determining whether hyperoxaluria induces the expression of nuclear pore complex oxalate binding protein p62 which has the transport function. Hyperoxaluria was induced in male Wistar rats by feeding 0.75% ethylene glycol in water. The oxalate binding activity of the nuclear pore complex protein increased markedly during experimental hyperoxaluria, whereas nuclear lamina had no binding at all. There was an alteration in the elution profile of the nuclear pore complex oxalate binding protein during the hyperoxaluric condition. The protein was purified and had a molecular weight of 62 kDa (data not shown). The purified protein showed cross-reactivity with the monoclonal antibody (MAb 414) and it showed homogeneity. The expression of this protein (p62) during the hyperoxaluric condition was determined by ELISA and a 3-fold increase was observed when compared to control rats. The increased expression is further confirmed by Western blotting and immunohistochemistry. The increase in p62 protein expression may be either due to increased expression of certain genes or degradation of the cell membrane by oxalate-induced cell injury. Thus, the present study suggests that the increased expression of this protein (p62) may be due to the oxalate induction.

Modulatory effect of the 23-kD calcium oxalate monohydrate binding protein on calcium oxalate stone formation during oxalate stress.

AIMS: To isolate, characterize, and quantify the 23-kD calcium oxalate monohydrate (COM) binding protein in the urine of controls and calcium oxalate stone formers and to study its role in kidney stone formation. METHODS: Calcium oxalate crystals were prepared and allowed to interact with human control kidney homogenate as well as urine of controls and calcium oxalate stone formers. EDTA extract was used for the separation of the 23-kD COM-binding protein (partially purified). This partially purified 23-kD COM-binding protein was further separated by DEAE-cellulose column chromatography. SDS-PAGE confirmed the molecular weight. An antibody was raised against the renal 23-kD COM-binding protein in rabbits. The 23-kD COM-binding protein was quantified in the urine from controls and stone formers by ELISA. Thiol group quantification, oxalate-binding assay, and calcium oxalate crystal nucleation and aggregation were performed. Morphological changes of the calcium oxalate crystals induced by the urinary 23-kDa protein were determined using scanning electron microscopy. The expression of this protein using different concentrations of oxalate was also determined in an in vitro model. RESULTS: The urinary excretion of the 23-kD COM-binding protein varies between 0.5 and 1.5 mg/24 h in controls, while in stone former its excretion was found to range from 5 to 7 mg/24 h. The protein isolated from urine was found to inhibit crystal nucleation and aggregation in controls, while the protein isolated from stone formers exhibited less inhibitory activity with reduced thiol groups. The 23-kD COM-binding protein derived from control urine formed COM crystals and intertwined calcium oxalate dihydrate crystals in a crystal growth system, while protein isolated from stone formers' urine induced aggregation of COM crystals. This protein expression was found to be increased with increasing concentration of oxalate in renal epithelial cells of the African green monkey kidney (VERO) cell line. CONCLUSIONS: Increased expression and excretion of the 23-kD protein was observed in oxalate stress conditions, and in stone formers this protein exhibited a promoting activity. The increased excretion of this protein with promoting activity favors the lithogenic process in stone formers.

Combined effects of ammonia and manganese on astrocytes in culture.

Ammonia has been strongly implicated in the pathogenesis of hepatic encephalopathy (HE), and astrocytes appear to be the primary target of ammonia neurotoxicity. Recent work has shown that manganese also plays a role in the pathogenesis of HE and causes astrocyte morphologic and functional changes similar to ammonia. We therefore investigated whether a combination of these compounds could produce additive/synergistic effects. Cultured astrocytes treated with 5 mM ammonia (NH4Cl) along with 100 microM manganese acetate (MnAc) for 3 h showed a 55-65% increase in free radical production over ammonia or manganese alone (P < 0.05). There was also a 50% decrease in the mitochondrial membrane potential (beta psi(m)) at 24 h following treatment with NH4Cl (5 mM) plus MnAc (50 microM) (P < 0.05), as compared to ammonia or manganese alone. Astrocytes treated with ammonia or manganese alone for 24 h showed no cell death, as determined by LDH release and light microscopic examination. However, cultures treated with ammonia plus manganese showed 80-90% necrotic cell death as estimated by light microscopy and 59% cell death as determined by LDH release. LDH release by ammonia plus manganese was blocked by the antioxidant superoxide dismutase (25 units/ml) as well as by the nitric oxide synthase inhibitor N(omega)-nitro-L-argininemethyl ester (500 microM). In conclusion, ammonia plus manganese exert additive/synergetic effects on the induction free radicals, mitochondrial inner membrane depolarization and cellular integrity, which may contribute to the tissue injury associated with chronic forms of HE.

Studies on calcium oxalate binding proteins: effect of lipid peroxidation.

OBJECTIVE: Urolithiasis and free radicals have long been associated. In this study, we have isolated calcium oxalate monohydrate (COM) binding proteins from rat kidney before and after lipid peroxidation (LPO) and studied its properties on calcium oxalate crystal growth. MATERIALS AND METHODS: LPO was carried out using t-butyl hydroperoxide, cumene hydroperoxide and an ascorbate system. The COM binding proteins from control and peroxidised tissues were isolated using a modified procedure. Protein was extracted using 25 mM EDTA, and the extract was loaded onto a DEAE cellulose column and eluted with Tris-HCl buffer (pH 6.5), 0.05 M NaCl in the above buffer and 0.3 M NaCl in the same buffer. Three major protein fractions were obtained, and they were designated as fractions I, II and III according to their order of elution. The proteins were subjected to calcium oxalate crystal nucleation and aggregation. RESULTS: A positive correlation was observed between LPO and COM adsorption, while a negative correlation was observed between reduced glutathione and COM adsorption. Peroxidised protein did not show any alteration in the elution profile on the DEAE cellulose column. The -SH content of the peroxidised fractions were lower than that of the control fractions, but their oxalate binding activities were increased. Peroxidised fraction I promoted crystal growth to a greater extent than the control fraction I. Peroxidised fractions II and III were less inhibitory in nature compared to their control fractions. Light-microscopic examination of the crystals formed in the presence of the peroxidised fractions showed the formation of large aggregates of COM. CONCLUSION: Peroxidation of the renal proteins favoured their adsorption to COM crystals. -SH depletion increased the oxalate binding activity and also their affinity to the COM crystals. The peroxidised fraction I was found to favour the formation of large aggregates, suggesting that peroxidation may be one of the mechanisms altering the crystal inhibitory activity of the proteins in hyperoxaluria.

Effect of A. lanata leaf extract and Vediuppu chunnam on the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria.

Urolithiasis is one of the third most common afflictions found in humans. The efficacy of the two Siddha drugs, Aerva lanata and Vediuppu chunnam as antilithic agents using a urolithic rat model were tested in this study. Hyperoxaluria was induced in rats using 0.75% ethylene glycol in drinking water. Aerva lanata(3.0 mg kg(-1)body weight) and Vediuppu chunnam (3.5 mg kg(-1)body weight) were given orally for 28 days. Urinary risk factors of urolithiasis were monitored at the end of 7th, 14th, 21st and 28th days. Urinary volume was increased in hyperoxaluric as well as drug-treated rats. Increased urinary excretion of calcium, oxalate, uric acid, phosphorus and protein in hyperoxaluric rats was brought down significantly by the administration of A. lanata or Vediuppu chunnam. Decreased magnesium excretion in hyperoxaluric rats was normalized by drug treatment. The drug increases the urine volume, thereby reducing the solubility product with respect to calcium oxalate and other crystallizing salts such as uric acid, which may induce epitaxial deposition of calcium oxalate. Drug alone treated rats did not show any adverse effects. Combination therapy was found to be more effective and this indigenous medicine can be used successfully as an antilithic agent.

Effect of experimental hyperoxaluria on renal calcium oxalate monohydrate binding proteins in the rat.

OBJECTIVE: To determine the functional role of calcium oxalate binding proteins in the nucleation, aggregation and retention of calcium oxalate crystals under physiological and hyperoxaluric conditions. Materials and methods Hyperoxaluria was induced in rats using 0.75% of ethylene glycol in drinking water. Calcium oxalate binding proteins were isolated and fractionated by cellulose column chromatography. Three major protein peak fractions were obtained (73 kDa in Tris-HCl buffer, 20 kDa in 0.05 mol/L NaCl buffer and 23 kDa in 0.3 mol/L buffer). Oxalate binding and the inhibition of crystal nucleation and aggregation by these fractions were determined. RESULTS: The adsorption of calcium oxalate monohydrate (COM) was ubiquitous in rat tissues and subcellular organelles, but the percentage adsorption varied; maximum absorption occurred in kidneys and pancreas, with microsomes showing maximal adsorption in the kidney. Hyperoxaluric rat tissues showed a greater percentage adsorption. Microsomes were enriched with the 20 kDa protein, while nuclei contained the 23 kDa protein in higher concentrations. COM-binding proteins derived from hyperoxaluric rat kidney had a greater content of 74 kDa and 23 kDa proteins with increased oxalate-binding activities. In the crystal-growth studies, the 74 kDa protein was a promoter, while the other protein fractions inhibited crystallization. In hyperoxaluria, the crystal-growth promoting activity of the 74 kDa protein was further increased, while the inhibition by the 20 and 23 kDa proteins was decreased. The 74 kDa protein derived from control rats formed single COM crystals in a crystal growth system, while the hyperoxaluric rat fraction induced the aggregation of COM crystals. CONCLUSION: COM-binding proteins (the 74 and 23 kDa fractions) were expressed more in hyperoxaluric rats. In hyperoxaluria the 74 kDa protein tended to promote crystal nucleation and aggregation, and the 20 and 23 kDa proteins were less inhibitory, which increases the risk of stone formation.

A novel basic protein from human kidney which inhibits calcium oxalate crystal growth.

OBJECTIVES: To isolate calcium oxalate-binding proteins from human kidney and characterize the functional properties. MATERIALS AND METHODS: Calcium oxalate crystals were prepared and allowed to interact at two different pH values with Triton-extracted human kidney homogenate. The proteins in the homogenate were isolated and fractionated on a cellulose column, and purified by high-performance liquid chromatography. The protein with the greatest oxalate binding activity at pH 4.5 was analysed for its amino-acid composition and characterized by Scatchard plot analysis, crystal growth, nucleation and aggregation studies. RESULTS: Three major protein fractions were eluted when calcium oxalate monohydrate was adsorbed at both pH values (designated as fractions I-III, according to their order of elution). The yield of fraction I and III was increased when adsorbed at an acidic pH. However, only fraction III had maximum oxalate binding activity at pH 4.5. When purified, this protein had maximum oxalate binding activity of approximately 270 pmol/mg protein and a molecular weight of approximately 23 kDa. Amino acid analysis showed that 18% of the total molar proportion was of basic amino acids, e.g. lysine and arginine, while acidic amino acids accounted for only 11%. Both alanine and glycine constituted approximately 41% of the total molar proportion. Modifications to the lysine group abolished oxalate-binding activity of the protein. The protein inhibited crystal growth by 82% at 0.8 micromol/L, while it inhibited the nucleation and aggregation of the crystals by 6% and 28%, respectively, at 49 nmol/L. The inhibition of both nucleation and aggregation was higher at pH 5.7 than at pH 7.4. Significantly, the protein induced the formation of intertwined calcium oxalate dihydrate crystals in a medium known to induce the formation of individual dihydrate crystals. CONCLUSION: The protein described here is the first reported basic inhibitor of calcium oxalate crystal growth with oxalate-binding activity at pH 4.5 that modulates calcium oxalate crystallization. It is suggested that this protein may play a physiologically significant role in inhibiting stone formation in acidic urine.

Uric acid-binding proteins in calcium oxalate stone formers and their effect on calcium oxalate crystallization.

OBJECTIVES: To study the effect of urinary uric acid-binding proteins of controls and stone formers on calcium oxalate crystal nucleation and aggregation. MATERIALS AND METHODS: Urine samples were collected over 24 h from 20 stone formers and from 20 age-matched normal controls. Uric acid crystallization was induced by adding equal volumes of 2.5 mmol/L uric acid. The bound proteins were separated on a cellulose column, and by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The effect of the separated fractions on calcium oxalate crystal nucleation and aggregation was assessed. RESULTS: The protein bound to unit mass of uric acid crystals was higher in hyperoxaluric urine than in control urine. On cellulose-column separation, the uric acid-crystal binding proteins produced three major protein peaks, i.e. fraction I (buffer), fraction II (0.05 mol/L sodium chloride in Tris-HCl buffer) and fraction III (0.3 mol/L sodium chloride in buffer), with a minor peak obtained on elution with increasing concentrations of sodium chloride in Tris-HCl buffer (pH 7.0). Fraction I derived from either stone formers or controls promoted calcium oxalate crystallization. Fraction II from the control samples was a strong inhibitor, whereas hyperoxaluric fraction II was less inhibitory. CONCLUSION: Uric acid-binding proteins isolated either from the urine of stone formers or controls modulated calcium oxalate crystal growth. Proteins isolated from stone formers were less inhibitory of crystal nucleation and aggregation. These proteins may act as a bridge, leading to the epitaxial deposition of calcium oxalate over a urate core.