Multicolor imaging of calcium-binding proteins in human kidney stones for elucidating the effects of proteins on crystal growth.
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ABSTRACT:
SUBMITTER: Tanaka Y
PROVIDER: S-EPMC8390759 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Calcium nephrolithiasis may be considered as a complex disease having multiple pathogenetic mechanisms and characterized by various clinical manifestations. Both genetic and environmental factors may increase susceptibility to calcium stones; therefore, it is crucial to characterize the patient phenotype to distinguish homogeneous groups of stone formers. Family and twin studies have shown that the stone transmission pattern is not mendelian, but complex and polygenic. In these studies, heritability of calcium stones was calculated around 50%Calcium-sensing receptor (CaSR) is mostly expressed in the parathyroid glands and in renal tubules. It regulates the PTH secretion according to the serum calcium concentration. In the kidney, it modulates electrolyte and water excretion regulating the function of different tubular segments. In particular, CaSR reduces passive and active calcium reabsorption in distal tubules, increases phosphate reabsorption in proximal tubules and stimulates proton and water excretion in collecting ducts. Therefore, it is a candidate gene for calcium nephrolithiasis.In a case-control study we found an association between the normocitraturic stone formers and two SNPs of CaSR, located near the promoters region (rs7652589 and rs1501899). This result was replicated in patients with primary hyperparathyroidism, comparing patients with or without kidney stones. Bioinformatic analysis suggested that the minor alleles at these polymorphisms were able to modify the binding sites of specific transcription factors and, consequently, CaSR expression.Our studies suggest that CaSR is one of the candidate genes explaining individual predisposition to calcium nephrolithiasis. Stone formation may be favored by an altered CaSR expression in kidney medulla involving the normal balance among calcium, phosphate, protons and water excretion.
| S-EPMC3339356 | biostudies-literature
Project description:Crystallization of L-cystine is a critical step in the pathogenesis of cystine kidney stones. Treatments for this disease are somewhat effective but often lead to adverse side effects. Real-time in situ atomic force microscopy (AFM) reveals that L-cystine dimethylester (L-CDME) and L-cystine methylester (L-CME) dramatically reduce the growth velocity of the six symmetry-equivalent {100} steps because of specific binding at the crystal surface, which frustrates the attachment of L-cystine molecules. L-CDME and L-CME produce l-cystine crystals with different habits that reveal distinct binding modes at the crystal surfaces. The AFM observations are mirrored by reduced crystal yield and crystal size in the presence of L-CDME and L-CME, collectively suggesting a new pathway to the prevention of L-cystine stones by rational design of crystal growth inhibitors.
| S-EPMC5166609 | biostudies-literature
Project description:Understanding the mechanisms of kidney stone formation, development patterns and associated pathological features are gaining importance due to an increase in the prevalence of the disease and diversity in the presentation of the stone composition. Based on the microstructural characteristics of kidney stones, it may be possible to explain the differences in the pathogenesis of pure and mixed types of stones. In this study, the microstructure and distribution of mineral components of kidney stones of different mineralogy (pure and mixed types) were analyzed. The intact stones removed from patients were investigated using synchrotron radiation X-ray computed microtomography (SR-μCT) and the tomography slice images were reconstructed representing the density and structure distribution at various elevation planes. Infrared (IR) spectroscopes, X-ray diffraction (XRD) and scanning electron microscopy (SEM) were used to confirm the bulk mineral composition in the thin section stones. Observations revealed differences in the micro-morphology of the kidney stones with similar composition in the internal 3-D structure. Calcium oxalate monohydrate stones showed well-organised layering patterns, while uric acid stones showed lower absorption signals with homogenous inner structure. Distinct mineral phases in the mixed types were identified based on the differential absorption rates. The 3-D quantitative analysis of internal porosity and spatial variation between nine different types of stones were compared. The diversity among the microstructure of similar and different types of stones shows that the stone formation is complex and may be governed by both physiological and micro-environmental factors. These factors may predispose a few towards crystal aggregation and stone growth, while, in others the crystals may not establish stable attachment and/or growth.
| S-EPMC6430423 | biostudies-literature
Project description:Calcium oxalate kidney stones contain low amounts of proteins, some of which have been implicated in progression or prevention of kidney stone formation. To gain insights into the pathophysiology of urolithiasis, we have characterized protein components of calcium oxalate kidney stones by proteomic approaches. Proteins extracted from kidney stones showed highly heterogeneous migration patterns in gel electrophoresis as reported. This was likely to be mainly due to proteolytic degradation and protein-protein crosslinking of Tamm-Horsfall protein and prothrombin. Protein profiles of calcium oxalate kidney stones were obtained by in-solution protease digestion followed by nanoLC-MALDI-tandem mass spectrometry, which resulted in identification of a total of 92 proteins in stones from 9 urolithiasis patients. Further analysis showed that protein species and their relative amounts were highly variable among individual stones. Although proteins such as prothrombin, osteopontin, calgranulin A and calgranulin B were found in most stones tested, some samples had high contents of prothrombin and osteopontin, while others had high contents of calgranulins. In addition, calgranulin-rich stones had various neutrophil-enriched proteins such as myeloperoxidase and lactotransferrin. These proteomic profiles of individual kidney stones suggest that multiple systems composed of different groups of proteins including leucocyte-derived ones are differently involved in pathogenesis of individual kidney stones depending on situations.
| S-EPMC3706363 | biostudies-literature
Project description:BACKGROUND:The dogma that urine is sterile in healthy individuals has been overturned by recent studies applying molecular-based methods. Mounting evidences indicate that dysbiosis of the urinary microbiota is associated with several urological diseases. In this study, we aimed to investigate the urinary microbiome of male patients with calcium-based kidney stones and compare it with those of healthy individuals. RESULTS:The diversity of the urinary microbiota in kidney stone patients was significantly lower than that of healthy controls based on the Shannon and Simpson index (P?<?0.001 for both indices). The urinary microbiota structure also significantly differed between kidney stone patients and healthy controls (ANOSIM, R?=?0.11, P?<?0.001). Differential representation of inflammation associated bacteria (e.g., Acinetobacter) and several enriched functional pathways were identified in the urine of kidney stones patients. Meanwhile, we found the species diversity, overall composition of microbiota and predicted functional pathways were similar between bladder urine and renal pelvis urine in kidney stone patients. CONCLUSIONS:A marked dysbiosis of urinary microbiota in male patients with calcium-based kidney stones was observed, which may be helpful to interpret the association between bacteria and calcium-based kidney stones.
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Project description:Idiopathic hypercalciuria is a frequent defect in calcium kidney stone formers that is associated with high intestinal calcium absorption and osteopenia. Characteristics distinguishing hypercalciuric stone formers from hypercalciuric patients without kidney stone history (HNSFs) are unknown and were explored in our study.We compared 172 hypercalciuric stone formers with 36 HNSFs retrospectively selected from patients referred to outpatient clinics of the San Raffaele Hospital in Milan from 1998 to 2003. Calcium metabolism and lumbar bone mineral density were analyzed in these patients. A strontium oral load test was performed: strontium was measured in 240-minute urine and serum 30, 60, and 240 minutes after strontium ingestion; serum strontium concentration-time curve and renal strontium clearance were evaluated to estimate absorption and excretion of divalent cations.Serum strontium concentration-time curve (P<0.001) and strontium clearance (4.9±1.3 versus 3.5±2.7 ml/min; P<0.001) were higher in hypercalciuric stone formers than HNSFs, respectively. The serum strontium-time curve was also higher in hypercalciuric stone formers with low bone mineral density (n=42) than in hypercalciuric stone formers with normal bone mineral density (n=130; P=0.03) and HNSFs with low (n=22; P=0.01) or normal bone mineral density (n=14; P=0.02). Strontium clearance was greater in hypercalciuric stone formers with normal bone mineral density (5.3±3.4 ml/min) than in hypercalciuric stone formers and HNSFs with low bone mineral density (3.6±2.5 and 3.1±2.5 ml/min, respectively; P=0.03). Multivariate regression analyses displayed that strontium absorption at 30 minutes was positively associated calcium excretion (P=0.03) and negatively associated with lumbar bone mineral density z score (P=0.001) in hypercalciuric stone formers; furthermore, hypercalciuric patients in the highest quartile of strontium absorption had increased stone production risk (odds ratio, 5.06; 95% confidence interval, 1.2 to 20.9; P=0.03).High calcium absorption in duodenum and jejunum may expose hypercalciuric patients to the risk of stones because of increased postprandial calcium concentrations in urine and tubular fluid. High calcium absorption may identify patients at risk of bone loss among stone formers.
| S-EPMC4974885 | biostudies-other
Project description:Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs--iRFP670, iRFP682, iRFP702 and iRFP720--from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.
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Project description:Calcium oxalate raphide crystals are found in bundles in intravacuolar membrane chambers of specialized idioblasts cells of most plant families including many crop plants and are found in most tissues. Raphides are needle-shaped, often with barbed ends, and their morphology and biomineralization pattern have been a focus of much research. In the family Araceae, raphides have been proposed to cause acridity in crops such as taro (Colocasia esculenta (L.) Schott). Acridity is irritation that causes swelling of lips, mouth and throat, as well as itchiness and pain when raw or insufficiently cooked tissues are eaten; this response varies among taro consumers. Since raphides in some other foods do not cause acridity, and since acridity can be inactivated by cooking and/or protease treatment, it is possible that a toxin or allergen-like compound is associated with the crystals. Using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) peptide sequencing of selected peptides from purified raphides and the results from taro apex transcriptome sequencing, we have shown that profilins, known allergens, are present on raphides. One of the five raphide profilins genes was highly expressed in the apex and had a 17-amino-acid insert that significantly increased that profilin’s epitope peak. This insert was predicted to be coiled and exposed on the surface of the folded peptide. A second profilin had a 2-amino-acid insert that also had a greater B-cell epitope prediction. The taro profilins showed 83 to 92% similarity to known characterized profilins while other raphide peptides showed greater than 90% similarity to other higher plants. The presence on the raphides of peptides normally associated with mitochrondria (ATP synthase), chloroplasts (chaperonin cpn 60 kDa), cytoplasm (actin, profilin) and vacuole (V-type ATPase) indicates a multistage biocrystallation process ending with possible invagination of the tonoplast and addition of mucilage that may be derived from the Golgi. Actin might play a crucial role in the generation of the needle-like raphides. We also showed that commercial allergen test strips for hazelnuts, where profilin is a secondary allergen, have potential for screening in a plant breeding program to reduce acridity and during food processing to avoid over-cooking.
2022-02-19 | GSE191235 | GEO
Project description:Long-term nephrocalcinosis leads to kidney injury, fibrosis, and even chronic kidney disease (CKD). Reports have proved macrophages can transition into myofibroblasts(MMT), leading to collagen deposition and fibrosis in CKD. However, the effect of MMT in calcium oxalate (CaOx) crystal-induced kidney fibrosis remains unclear. This study demonstrated that histone methyltransferase EZH2-mediated MMT contributes to CaOx crystal-induced fibrosis. We identified abundant MMT cells by immunofluorescence and flow cytometry in kidney tissues of patients with CaOx-related CKD, CaOx nephrocalcinosis mouse model, and CaOx-treated RAW264.7 macrophage cells. A high level of MMT cell infiltration was associated with a decline in the glomerular filtration rate in CaOx nephrocalcinosis patients. Clodronate Liposomes-mediated macrophage depletion attenuates calcium oxalate crystal-induced fibrosis in mice. Subsequently, transcriptomic and single-cell sequencing revealed that EZH2 was highly expressed in kidneys with CaOx deposition, especially in macrophages. Further study demonstrated that EZH2 inducible knock-out or pharmacological inhibition by GSK-126 attenuated MMT and renal fibrosis in vivo and in vitro. Mechanistically, ChIP and transcriptomic sequencing showed that EZH2 inhibition reduced the enrichment of H3K27me3 on the DUSP23 gene promoter and elevated DUSP23 expression. The Co-IP and molecular docking analysis showed that DUSP23 mediated the dephosphorylation of pSMAD3 (S423/425), the key regulator of MMT. In addition, DUSP23 over-expression could alleviate SMAD3-mediated MMT. Thus, our study found that EZH2 promotes kidney fibrosis by meditating MMT via the DUSP23/SMAD3 pathway in nephrocalcinosis.
2024-10-26 | GSE280007 | GEO
Project description:Calcium oxalate kidney stones, the most prevalent type of kidney stones, undergo a multi-step process of crystal nucleation, growth, aggregation, and secondary transition. The secondary transition has been rather overlooked, and thus, the effects on the disease and the underlying mechanism remain unclear. Here, we show, by periodic micro-CT images of human kidney stones in an ex vivo incubation experiment, that the growth of porous aggregates of calcium oxalate dihydrate (COD) crystals triggers the hardening of the kidney stones that causes difficulty in lithotripsy of kidney stone disease in the secondary transition. This hardening was caused by the internal nucleation and growth of precise calcium oxalate monohydrate (COM) crystals from isolated urine in which the calcium oxalate concentrations decreased by the growth of COD in closed grain boundaries of COD aggregate kidney stones. Reducing the calcium oxalate concentrations in urine is regarded as a typical approach for avoiding the recurrence. However, our results revealed that the decrease of the concentrations in closed microenvironments conversely promotes the transition of the COD aggregates into hard COM aggregates. We anticipate that the suppression of the secondary transition has the potential to manage the deterioration of kidney stone disease.
| S-EPMC10987347 | biostudies-literature