The effect of preincubation of seed crystals of uric acid and monosodium urate with undiluted human urine to induce precipitation of calcium oxalate in vitro : implications for urinary stone formation

BACKGROUND: Previous studies demonstrated that crystals of uric acid (UA) and sodium urate (NaU) can induce the precipitation of calcium oxalate (CaOx) from its inorganic metastable solutions, but similar effects have not been unequivocally shown to occur in urine. The aim of this investigation was to determine whether preincubation of these seeds with urine alter their ability to induce deposition of CaOx from solution and thus provide a possible explanation for discrepancy of results obtained from aqueous inorganic solutions and undiluted urine. MATERIALS AND METHODS: The effects of commercial seed crystals of UA, NaU and CaOx (6 mg/100 ml) on CaOx crystallization were compared in a solution with the same crystals that had been preincubated for 3 hours with healthy male urine. A Coulter Counter was used to follow the crystallization and decrease in soluble (14) C-oxalate was measured to determine the deposition of CaOx. The precipitated particles were examined by scanning electron microscopy (SEM). The preincubated seeds were demineralized and proteins released were analyzed by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Analysis of (14) C-oxalate data revealed that while treated UA seeds did not affect CaOx deposition, those of NaU and CaOx inhibited the process by 51.9 (p<0.05) and 8.5% (p<0.05) relative to their respective untreated counterparts. Particle size analysis showed that the average modal sizes of particles precipitated in the presence of treated seed crystals of UA, NaU, and CaOx were very similar to those deposited in the presence of their respective untreated controls. These findings were confirmed by SEM which also showed that seed crystals of UA and NaU, untreated and treated, were attached like barnacles upon the surfaces of CaOx crystals which themselves were bigger than those precipitated in the presence of CaOx seeds. SDS-PAGE analysis of the demineralized treated seeds showed that they all selectively adsorbed urinary proteins, and perhaps other urinary macromolecules and low molecular weight components, on their surface. CONCLUSIONS: It was concluded that preincubation with urine, such as occurs in vivo, only slightly reduces the ability of seed crystals of CaOx, but not of UA, to cause deposition of CaOx. The most striking effect was on NaU seeds where the preincubation quite dramatically attenuated their promotory effect on the mineral deposition. This may explain the discrepancy between findings of studies carried out in inorganic solutions and undiluted human urine. This stresses the invalidity of directly extrapolating results obtained in inorganic solutions to likely effects in urine and more importantly, on stone formation.     

Urinary glycosaminoglycans and glycoproteins in a calcium oxalate crystallization system

This study measures the effects of total urinary glycosaminoglycans (GAGs), glycoproteins (GPs) and individual GAGs on the nucleation rates (Bo), growth rates (G) and suspension densities (Mт) of calcium oxalate (CaOx) crystallization by the mixed suspension mixed product removal (MSMPR) system. Total urinary GAGs, glycoproteins and individual GAGs including heparan sulfate (HS), chondroitin sulfate (CS) and Hyaluronic acid (HA) were added into the artificial urine (AU) and then introduced into the MSMPR test chamber and the crystal sizes and numbers were analyzed by a particle counter. The effects of added GAGs and GPs on CaOx crystallization were reflected by the changes on the crystallization indexes including the Bo, G and Mт of CaOx that were calculated based on the crystal size and numbers. Total urinary GAGs showed no statistical significance on CaOx crystallization. However, individual GAGs such as HA, CS and HS enhanced Bo and suppressed the G when measured individually. CS and HS enhanced the Mт while HA shown no significant change in the Mт of CaOx. Total urinary GPs showed an increase in the G and Mт of crystals. Although total urinary GAGs showed no statistically significant effect on CaOx crystallization, individual GAGs (CS, HS) promoted the CaOx crystallization by increasing the suspension density of smaller crystals, indicative of reduced risk of stones while HA showed no significance in the M(T) of CaOx formed. Urinary GPs indicated increased sizes and M(T) suggesting larger crystals and/or aggregates.     

Nucleation of calcium oxalate crystals on an imprinted polymer surface from pure aqueous solution and urine

Calcium oxalate (CaOx) is the most common component of human kidney stones. Heterogeneous nucleation is regarded as the key mechanism in this process. In this study, we have used an imprinted 6-methacrylamidohexanoic acid/divinylbenzene co-polymer as a biomimetic surface to nucleate CaOx crystal formation. The polymer was imprinted with either calcium oxalate monohydrate (COM) or dihydrate (COD) template crystals. These were washed out of the polymer, which was then immersed in various test solutions. The test solutions were an aqueous solution of calcium chloride and sodium oxalate, artificial urine and a sample of real urine. Crystals that formed on the polymer surface were characterized by X-ray powder diffraction, Fourier transform infrared spectroscopy, atomic absorption spectroscopy and scanning electron microscopy. Results showed that in the aqueous solution the COM-imprinted polymer induced the nucleation of COM. The COD-imprinted polymer induced only trace amounts of COD crystallization, together with larger quantities of COM. In artificial and real urines, COM also specifically precipitated on the COM-imprinted surface. The results show that, at least to some extent, the imprinted polymers direct formation of their morphologically matched crystals. In the case of COD, however, it appears that either rapid hydrate transformation of COD to COM occurs, or the more stable COM polymorph is directly co-precipitated by the polymer. Our results support the hypothesis that heterogeneous nucleation plays a key role in CaOx stone formation and that the imprinted polymer model could provide an additional and superior diagnostic tool for stone researchers to assess stone-risk in urine.Abbreviations COD calcium oxalate dihydrate - COM calcium oxalate monohydrate - COT calcium oxalate trihydrate - dvb divinylbenzene - 6-maaha 6-methylacrylamidohexanoic acid     

Effect of Aerva lanata on calcium oxalate urolithiasis in rats

Calcium oxalate (CaOx) stone was induced in rats using 0.75% of ethylene glycol in drinking water for 28 days. Ethylene glycol treated rats showed significant increase in the activities of oxalate synthesizing enzymes such as glycolic acid oxidase (GAO) in liver and lactate dehydrogenase (LDH) in liver and kidney. CaOx crystal deposition, as indicated by increased excretion of stone-forming constituents in urine, such as calcium, oxalate, uric acid, phosphorus and protein and decreased concentration of inhibitors, such as citrate and magnesium was observed in ethylene glycol induced urolithic rats. Histopathological studies also confirmed the deposition of CaOx crystals. Administration of Aerva lanata aqueous suspension (2g/kg body wt/dose/day for 28 days) to CaOx urolithic rats had reduced the oxalate synthesizing enzymes, diminished the markers of crystal deposition in the kidney. The results of the present study confirmed that A. lanata can be used as an curative agent for urolithiasis.     

Intracrystalline proteins and the hidden ultrastructure of calcium oxalate urinary crystals: implications for kidney stone formation

The external appearance of urinary calcium oxalate (CaOx) crystals suggests that they are solid, homogeneous structures, despite their known association with proteins. Our aim was to determine whether proteins comprising the organic matrix of CaOx crystals are superficial or intracrystalline in order to clarify the role of urinary proteins in the formation of kidney stones. CaOx crystals were precipitated from centrifuged and filtered, or ultrafiltered, healthy human urine. They were then treated with dilute NaOH to remove bound proteins, partially demineralized with EDTA, or fractured and subjected to limited proteolysis before examination by low-resolution scanning electron microscopy or field emission scanning electron microscopy. Crystals precipitated from centrifuged and filtered urine had a complex interior network of protein distributed throughout the mineral phase, which appeared to comprise closely packed subcrystalline particles stacked in an orderly array among an amorphous organic matrix. This ultrastructure was not evident in crystals deposited in the absence of macromolecules, which were completely solid. This is the first direct evidence that crystals generated from cell-free systems contain significant amounts of protein distributed throughout a complex internal cribriform ultrastructure. Combined with mineral erosion in the acidic lysosomal environment, proteins inside CaOx crystals would render them susceptible to attack by urinary and intracellular renal proteases and facilitate their further dissolution or disruption into small particles and ions for removal by exocytosis. The findings also have broader ramifications for industry and the materials sciences, as well as the development and resorption of crystals in biomineralization systems throughout nature.     

Calcium Oxalate Crystals in Developing Seeds of Soybean

Young developing soybean seeds contain relatively large amountsof calcium oxalate (CaOx) monohydrate crystals. A test for Caand CaOx indicated that Ca deposits and crystals initially occurredin the funiculus, where a single vascular bundle enters theseed. Crystals formed in the integuments until the embryo enlargedenough to crush the inner portion of the inner integument. Crystalsthen appeared in the developing cotyledon tissues and embryoaxis. All crystals formed in cell vacuoles. Dense bodies andmembrane complexes were evident in the funiculus. In the innerintegument, cell vacuoles assumed the shape of the future crystals.This presumed predetermined crystal mould is reported here forthe first time for soybean seeds. As crystals in each tissuenear maturity, a wall forms around each crystal. This intracellularcrystal wall becomes contiguous with the cell wall. Integumentcrystals remain visible until the enlarging embryo crushes theinteguments; the crystals then disappear. A related study revealedthat the highest percent of oxalate by dry mass was reachedin the developing +16 d (post-fertilization) seeds, and thendecreased during late seed maturation. At +60 d, CaOx formationand disappearance are an integral part of developing soybeanseeds. Our results suggest that Ca deposits and crystals functionallyserve as Ca storage for the rapidly enlarging embryos. The oxalate,derived from one or more possible metabolic pathways, couldbe involved in seed storage protein synthesis. Copyright 2001Annals of Botany Company Calcium, crystals, development, Glycine max, ovule, oxalate, seed, soybean     

Cholesterol crystal formation and growth in model bile solutions

Cholesterol monohydrate crystal formation was studied in supersaturated model bile solutions, containing unlabeled cholesterol, sodium cholate and soybean phosphatidylcholine, and tracer amounts of [3H]cholesterol. Solutions were either seeded with cholesterol crystals to initiate growth, or not seeded to allow self-nucleation and subsequent crystal growth to occur. Crystal growth at 37 degrees C was measured by two methods. First, radioactive cholesterol crystals were isolated by filtration, and the mass of cholesterol that had precipitated was calculated. In unseeded solutions, there was a long lag period before crystal growth was detected. This lag time was decreased by increases in the cholesterol concentration, temperature, and lipid concentration. In seeded solutions, crystal growth also was dependent on the cholesterol concentration, temperature, and lipid concentration. The second method used to measure crystal growth involved the Coulter Counter. At 37 degrees C, reproducible results were not obtained using unseeded solutions due to blocking of the counter aperture with large crystals. In seeded solutions, crystal growth could be measured as an increase in total particle volume. However, comparison of growth rate estimates from the Coulter Counter with those obtained radiochemically revealed poor agreement between the two methods. It is probable that the Coulter Counter is inaccurate in measuring the volume of cholesterol monohydrate crystals due to their anisometric shape.     

Ferroptosis in calcium oxalate kidney stone formation and the possible regulatory mechanism of ANKRD1

The objective of this study was to explore the role of ferroptosis in the formation of calcium oxalate (CaOx) kidney stones and the regulatory mechanism of the ankyrin repeat domain 1 (ANKRD1) gene. The study found that the Nrf2/HO-1 and p53/SLC7A11 signaling pathways were activated in the kidney stone model group, and the expression of the ferroptosis marker proteins SLC7A11 and GPX4 was significantly reduced, while the expression of ACSL4 was significantly increased. The expression of the iron transport-related proteins CP and TF increased significantly, and Fe²⁺ accumulated in the cell. The expression of HMGB1 increased significantly. In addition, the level of intracellular oxidative stress was increased. The gene with the most significant difference caused by CaOx crystals in HK-2 cells was ANKRD1. Silencing or overexpression of ANKRD1 by lentiviral infection technology regulated the expression of the p53/SLC7A11 signaling pathway, which regulated the ferroptosis induced by CaOx crystals. In conclusion, CaOx crystals can mediate ferroptosis through the Nrf2/HO-1 and p53/SLC7A11 pathways, thereby weakening the resistance of HK-2 cells to oxidative stress and other unfavorable factors, enhancing cell damage, and increasing crystal adhesion and CaOx crystal deposition in the kidney. ANKRD1 participates in the formation and development of CaOx kidney stones by activating ferroptosis mediated by the p53/SLC7A11 pathway.     

Heavy metal stress reduces the deposition of calcium oxalate crystals in leaves of Phaseolus vulgaris

Calcium oxalate (CaOx) crystals in plants may serve as a sink for the absorption of excess calcium, and they could play an important role in heavy metal detoxification. In this study, the effect of heavy metals and different calcium concentrations on the growth of calcium oxalate crystals in leaves of Phaseolus vulgaris was investigated. Different analytical techniques were used to determine the influence of exogenous lead and zinc on CaOx deposition and to detect a presence of these metals in CaOx crystals. We found a positive correlation between the calcium concentration in the nutrient medium and the production of calcium oxalate crystals in leaves of hydroponically grown plants. On the other hand, addition of the heavy metals to the nutrient medium decreased the number of crystals. Energy dispersive X-ray spectrometry did not detect the inclusion of heavy metals inside the CaOx crystals. Our investigation suggests that CaOx crystals do not play a major role in heavy metal detoxification in P. vulgaris but do play an important role in bulk calcium regulation.     

Allicin attenuates calcium oxalate crystal deposition in the rat kidney by regulating gap junction function

Renal calculus is a global common urological disease that is closely related to crystal adhesion and renal tubular epithelial cell impairment. Gap junctions (GJs) and their components (connexins and Cxs) are involved in various pathophysiology processes, but their roles in renal calculi progression are not well defined. Our previous RNA microarray analysis suggests that GJs are one of the key predicted pathways involved in the renal calcium oxalate (CaOx) crystal rat model. In the current study, we found that the Cx43 and Cx32 expression and the GJ function decreased significantly after stimulation with CaOx or sodium oxalate (NaOx) in NRK-52E, MDCK, and HK-2 cells, and Cx43 expression also decreased in renal tissues in renal CaOx crystal model rats. Inhibition of Cx43 in NRK-52E cells by small interference RNA significantly increased the CD44 and androgen receptor expression, and the adhesion between CaOx crystals and cells, which were consistent with the function of GJ inhibitors. On the other hand, after GJ function and Cx43 expression were increased by allicin, diallyl disulfide, or diallyl trisulfide, the impairment of NRK-52E cells by NaOx or other GJ inhibitors and the adhesion between CaOx crystals and renal cells decreased significantly. Furthermore, allicin also increased Cx43 expression and inhibited crystal deposition in rat kidneys. Taken together, our results provide a basis that GJs and Cx43 may participate in renal CaOx stone progression and that allicin, together with its analogues, could be potential drugs for renal calculus precaution.     

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.     

Peeping into Human Renal Calcium Oxalate Stone Matrix: Characterization of Novel Proteins Involved in the Intricate Mechanism of Urolithiasis

Background

The increasing number of patients suffering from urolithiasis represents one of the major challenges which nephrologists face worldwide today. For enhancing therapeutic outcomes of this disease, the pathogenic basis for the formation of renal stones is the need of hour. Proteins are found as major component in human renal stone matrix and are considered to have a potential role in crystal–membrane interaction, crystal growth and stone formation but their role in urolithiasis still remains obscure.

Methods

Proteins were isolated from the matrix of human CaOx containing kidney stones. Proteins having MW>3 kDa were subjected to anion exchange chromatography followed by molecular-sieve chromatography. The effect of these purified proteins was tested against CaOx nucleation and growth and on oxalate injured Madin–Darby Canine Kidney (MDCK) renal epithelial cells for their activity. Proteins were identified by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF MS) followed by database search with MASCOT server. In silico molecular interaction studies with CaOx crystals were also investigated.

Results

Five proteins were identified from the matrix of calcium oxalate kidney stones by MALDI-TOF MS followed by database search with MASCOT server with the competence to control the stone formation process. Out of which two proteins were promoters, two were inhibitors and one protein had a dual activity of both inhibition and promotion towards CaOx nucleation and growth. Further molecular modelling calculations revealed the mode of interaction of these proteins with CaOx at the molecular level.

Conclusions

We identified and characterized Ethanolamine-phosphate cytidylyltransferase, Ras GTPase-activating-like protein, UDP-glucose:glycoprotein glucosyltransferase 2, RIMS-binding protein 3A, Macrophage-capping protein as novel proteins from the matrix of human calcium oxalate stone which play a critical role in kidney stone formation. Thus, these proteins having potential to modulate calcium oxalate crystallization will throw light on understanding and controlling urolithiasis in humans.     

Theaflavin protects against oxalate calcium-induced kidney oxidative stress injury via upregulation of SIRT1

Renal tubular cell injury induced by calcium oxalate (CaOx) is a critical initial stage of kidney stone formation. Theaflavin (TF) has been known for its strong antioxidative capacity; however, the effect and molecular mechanism of TF against oxidative stress and injury caused by CaOx crystal exposure in kidneys remains unknown. To explore the potential function of TF on renal crystal deposition and its underlying mechanisms, experiments were conducted using a CaOx nephrocalcinosis mouse model established by glyoxylate intraperitoneal injection, and HK-2 cells were subjected to calcium oxalate monohydrate (COM) crystals, with or without the treatment of TF. We discovered that TF treatment remarkably protected against CaOx-induced kidney oxidative stress injury and reduced crystal deposition. Additionally, miR-128-3p expression was decreased and negatively correlated with SIRT1 level in mouse CaOx nephrocalcinosis model following TF treatment. Moreover, TF suppressed miR-128-3p expression and further abolished its inhibition on SIRT1 to attenuate oxidative stress in vitro. Mechanistically, TF interacted with miR-128-3p and suppressed its expression. In addition, miR-128-3p inhibited SIRT1 expression by directly binding its 3''-untranslated region (UTR). Furthermore, miR-128-3p activation partially reversed the acceerative effect of TF on SIRT1 expression. Taken together, TF exhibits a strong nephroprotective ability to suppress CaOx-induced kidney damage through the recovery of the antioxidant defense system regulated by miR-128-3p/SIRT1 axis. These findings provide novel insights for the prevention and treatment of renal calculus.     

Attenuation of oxalate-induced nephrotoxicity by eicosapentaenoate-lipoate (EPA-LA) derivative in experimental rat model

Hyperoxaluria is one of the major risk factors for the formation of urinary calcium oxalate stones. Calcium oxalate crystals and their deposition have been implicated in inducing renal tubular damage. Lipoic acid (LA) and eicosapentaenoic acid (EPA) have been shown to ameliorate the changes associated with hyperoxaluria. This prompted us to investigate the nephroprotectant role of EPA-LA, a new derivative, in vivo in hyperoxaluric rats. Elevation in the levels of calcium, oxalate and phosphorus, the stone-forming constituents, were observed in calculogenic rats as a manifestation of crystal deposition.Tubular damage to the renal tissue was assessed byassaying the excretion of marker enzymes in the urine. Damage to the tubules was indicated by increased excretion of alkaline phosphatase (ALP), lactate dehydrogenase (LDH), gamma-glutamyl transferase (gamma-GT), beta-Glucuronidase (beta-GLU) and N-Acetyl beta-D glucosaminidase (NAG). Fibrinolytic activity was found to be reduced. Administration of EPA, LA and EPA-LA reduced the tubular damage and decreased the markers of crystal deposition markedly, which was substantiated by the reduction in weight of bladder stone formed. Our results highlight that EPA-LA is the most effective drug in inhibiting stone formation and mitigating renal damage caused by oxalate toxicity, thus confirming it as a nephroprotectant. Further work in this direction is warranted to establish the therapeutic effectiveness of this new derivative.     

Macropinocytosis is the Major Mechanism for Endocytosis of Calcium Oxalate Crystals into Renal Tubular Cells

During an initial phase of kidney stone formation, the internalization of calcium oxalate (CaOx) crystals by renal tubular cells has been thought to occur via endocytosis. However, the precise mechanism of CaOx crystal endocytosis remained unclear. In the present study, MDCK renal tubular cells were pretreated with inhibitors specific to individual endocytic pathways, including nystatin (lipid raft/caveolae-mediated), cytochalasin D (actin-dependent or macropinocytosis), and chlorpromazine (CPZ; clathrin-mediated) before exposure to plain (non-labeled), or fluorescence-labeled CaOx monohydrate (COM) crystals. Quantitative analysis by flow cytometry revealed that pretreatment with nystatin and CPZ slightly decreased the crystal internalization, whereas the cytochalasin D pretreatment caused a marked decrease in crystal uptake. Immunofluorescence study and laser-scanning confocal microscopic examination confirmed that the cytochalasin D-pretreated cells had dramatic decrease of the internalized crystals, whereas the total number of crystals interacted with the cells was unchanged (crystals could adhere but were not internalized). These data have demonstrated for the first time that renal tubular cells endocytose COM crystals mainly via macropinocytosis. These novel findings will be useful for further tracking the endocytosed crystals inside the cells during the course of kidney stone formation.     

Experimental calcium-oxalate crystal production and dissolution by selected wood-rot fungi

Twenty-six species of white-rotting Agaricomycotina fungi (Basidiomycota) were screened for their ability to produce calcium-oxalate (CaOx) crystals in vitro. Most were able to produce CaOx crystals in malt agar medium in the absence of additional calcium. In the same medium enriched with Ca²⁺, all the species produced CaOx crystals (weddellite or whewellite). Hyphae of four species (Ganoderma lucidum, Polyporus ciliatus, Pycnoporus cinnabarinus, and Trametes versicolor) were found coated with crystals (weddellite/whewellite). The production of CaOx crystals during the growth phase was confirmed by an investigation of the production kinetics for six of the species considered in the initial screening (Pleurotus citrinopileatus, Pleurotus eryngii, Pleurotus ostreatus, P. cinnabarinus, Trametes suaveolens, and T. versicolor). However, the crystals produced during the growth phase disappeared from the medium over time in four of the six species (P. citrinopileatus, P. eryngii, P. cinnabarinus, and T. suaveolens). For P. cinnabarinus, the disappearance of the crystals was correlated with a decrease in the total oxalate concentration measured in the medium from 0.65 ??g mm⁻² (at the maximum accumulation rate) to 0.30 ??g mm⁻². The decrease in the CaOx concentration was correlated with a change in mycelia morphology. The oxalate dissolution capability of all the species was also tested in a medium containing calcium oxalate as the sole source of carbon (modified Schlegel medium). Three species (Agaricus blazei, Pleurotus tuberregium, and P. ciliatus) presented a dissolution halo around the growth zone. This study shows that CaOx crystal production is a widespread phenomenon in white-rot fungi, and that an excess of Ca²⁺ can enhance CaOx crystal production. In addition, it shows that some white-rot fungal species are capable of dissolving CaOx crystals after growth has ceased. These results highlight a diversity of responses around the production or dissolution of calcium oxalate in white-rot fungi and reveal an unexpected potential importance of fungi on the oxalate cycle in the environment.     

A study on calcium oxalate crystals in <Emphasis Type="Italic">Tinantia anomala</Emphasis> (Commelinaceae) with special reference to ultrastructural changes during anther development

Calcium oxalate (CaOx) crystals in higher plants occur in five forms: raphides, styloids, prisms, druses, and crystal sand. CaOx crystals are formed in almost all tissues in intravacuolar crystal chambers. However, the mechanism of crystallization and the role of CaOx crystals have not been clearly explained. The aim of this study was to explore the occurrence and location of CaOx crystals in organs of Tinantia anomala (Torr.) C.B. Clarke (Commelinaceae) with special attention to ultrastructural changes in the quantity of tapetal raphides during microsporogenesis. We observed various parts of the plant, that is, stems, leaves, sepals, petals, anthers, staminal trichomes and stigmatic papillae and identified CaOx crystals in all parts except staminal trichomes and stigmatic papillae in Tinantia anomala. Three morphological forms: styloids, raphides and prisms were found in different amounts in different parts of the plant. Furthermore, in this species, we identified tapetal raphides in anthers. The number of tapetal raphides changed during microsporogenesis. At the beginning of meiosis, the biosynthesis of raphides proceeded intensively in the provacuoles. These organelles were formed from the endoplasmic reticulum system. In the tetrad stage, we observed vacuoles with needle-shaped raphides (type I) always localised in the centre of the organelle. When the amoeboid tapetum was degenerating, vacuoles also began to fade. We observed a small number of raphides in the stage of mature pollen grains.     

Metabolic evaluation of urolithiasis patients from eastern Croatia

Metabolic parameters were determined in fasting blood serum, fasting first morning urine, and 24-hour urine of male patients with recurrent calcium oxalate stones (N = 26, age 39.1 +/- 6.2 years) as well as in male healthy controls (N = 18, age 35.0 +/- 7.1 years), recruited from the eastern part of Croatia. The 24-hour urinary calcium excretion was significantly higher (p < 0.01) for patients (5.6 +/- 2.5 mmol) than for controls (3.7 +/- 1.9 mmol), but potassium excretion was higher (p < 0.01) for controls (74.5 +/- 33.8 mmol) than for patients (49.2 +/- 15.7 mmol). The mean ionic activity product of calcium and oxalate ions, IAP(CaOx), calculated from the fasting first morning urine parameters, was 25% higher for patients than for controls, but the difference was not statistically significant (p > 0.05). Very strong correlation (r = 0.97) was obtained between IAP(CaOx) values and calculated Ogawa indices that were recommended for estimating the potential risk for calcium oxalate stone formation.     

Exosomes from miR‐20b‐3p‐overexpressing stromal cells ameliorate calcium oxalate deposition in rat kidney

Hyperoxaluria‐induced calcium oxalate (CaOx) deposition is the key factor in kidney stone formation, for which adipose‐derived stromal cells (ADSCs) have been used as a therapeutic treatment. Studies revealed that miR‐20b‐3p is down‐regulated in hypercalciuric stone‐forming rat kidney. To investigate whether ADSC‐derived miR‐20b‐3p‐enriched exosomes protect against kidney stones, an ethylene glycol (EG)‐induced hyperoxaluria rat model and an in vitro model of oxalate‐induced NRK‐52E cells were established to explore the protective mechanism of miR‐20b‐3p. The results showed that miR‐20b‐3p levels were decreased following hyperoxaluria in the urine of patients and in kidney tissues from animal models. Furthermore, treatment with miR‐20b‐3p‐enriched exosomes from ADSCs protected EG‐induced hyperoxaluria rats, and cell experiments confirmed that co‐culture with miR‐20b‐3p‐enriched exosomes alleviated oxalate‐induced cell autophagy and the inflammatory response by inhibiting ATG7 and TLR4. In conclusion, ADSC‐derived miR‐20b‐3p‐enriched exosomes protected against kidney stones by suppressing autophagy and inflammatory responses.