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Trigonelline (TRIG) is a natural compound in an alkaloid family found in diverse plants. This compound exerts anti-inflammatory, anti-allergic, anti-oxidative and anti-fibrotic activities in several disease models. However, its beneficial role in endothelial injury, especially induced by diabetes, is unclear. We, therefore, evaluated the effects of TRIG on the cellular proteome of human endothelial (EA.hy926) cells followed by functional validation in high-glucose (HG)-induced endothelial deteriorations. Label-free quantification using nanoLC-ESI-Qq-TOF MS/MS revealed 40 downregulated and 29 upregulated proteins induced by TRIG. Functional enrichment analysis using DAVID and REVIGO tools suggested the involvement of these altered proteins in several biological processes and molecular functions, particularly cell-cell adhesion, ATP metabolic process, cell redox homeostasis, cadherin binding, and ATP hydrolysis activity. Experimental validation showed that HG triggered endothelial-to-mesenchymal transition (EndMT) (as demonstrated by increased spindle index and mesenchymal markers, i.e., fibronectin and vimentin, and decreased endothelial markers, i.e., PECAM-1 and VE-cadherin), increased oxidized proteins, and reduced intracellular ATP, active mitochondria, endothelial tube/mesh formation and VEGF secretion. However, TRIG successfully abolished all these defects induced by HG. These data indicate that TRIG prevents HG-induced EndMT, oxidative stress, mitochondrial dysfunction, and impaired angiogenic activity in human endothelial cells.

Resveratrol is a natural phenolic compound that belongs to stilbenoid group found in diverse plants. Health benefits and therapeutic potentials of resveratrol have been widely recognized in various diseases. In kidney stone disease, it can alleviate oxalate-induced hyperproduction of free radicals in renal epithelial cells. Nevertheless, its direct effects on calcium oxalate (CaOx) crystal, which is the major stone component, remained unclear. This study therefore addressed the direct effects of resveratrol (at 1, 10 or 100 μM) on each step of CaOx kidney stone formation. The results revealed that resveratrol had no significant effects on CaOx crystallization. However, resveratrol significantly decreased CaOx crystal growth and adhesion to renal epithelial cells at all concentrations, and induced crystal internalization into the cells (a process related to crystal degradation by endolysosomes) in a concentration-dependent manner. On the other hand, resveratrol promoted crystal aggregation. These data indicate that resveratrol serves as a dual modulator on CaOx stone formation. While it inhibits CaOx stone development by reducing crystal growth and adhesion to renal cells and by inducing crystal internalization into the cells, resveratrol promotes crystal aggregation, which is one of the mechanisms leading to kidney stone formation.

Trigonelline is a phytoalkaloid commonly found in green and roasted coffee beans. It is also found in decaffeinated coffee. Previous report has shown that extract from trigonelline-rich plant exhibits anti-lithiatic effects in a nephrolithiatic rat model. Nevertheless, cellular mechanisms underlying the anti-lithiatic properties of trigonelline remain hazy. Herein, we used nanoLC-ESI-Qq-TOF MS/MS and MaxQuant-based quantitative proteomics to identify trigonelline-induced changes in protein expression in MDCK renal cells. From a total of 1006 and 1011 proteins identified from control and trigonelline-treated cells, respectively, levels of 62 (23 upregulated and 39 downregulated) proteins were significantly changed by trigonelline. Functional enrichment and reactome pathway analyses suggested that these 62 altered proteins were related to stress response, cell cycle and cell polarity. Functional validation by corresponding experimental assays revealed that trigonelline prevented calcium oxalate monohydrate crystal-induced renal cell deteriorations by inhibiting crystal-induced overproduction of intracellular reactive oxygen species, G0/G1 to G2/M cell cycle shift, tight junction disruption, and epithelial-mesenchymal transition. These findings provide cellular mechanisms and convincing evidence for the renoprotective effects of trigonelline, particularly in kidney stone prevention.

Annexin A1 (ANXA1) is a multifunctional calcium-binding protein that can bind to membrane phospholipids. Under high-calcium condition, ANXA1 expression increases on renal epithelial cell surface, leading to enhanced adhesion of calcium oxalate (CaOx) crystal (stone material) onto the cells. To regulate various cellular processes, ANXA1 interacts with many other intracellular protein partners. However, components of the ANXA1-interacting protein complex remain unclear. Herein, we characterized the interacting complexes of apical membrane (ApANXA1) and cytosolic (cyANXA1) forms of ANXA1 in apical membrane and cytosolic compartments, respectively, of renal epithelial cells under high-calcium condition using proteomic and bioinformatic approaches. After fractionation, the ApANXA1- and CyANXA1-interacting partners were identified by immunoprecipitation followed by nanoLC‑ESI‑Qq-TOF tandem mass spectrometry (IP-MS/MS). The ANXA1-interacting partners that were common in both apical membrane and cytosolic compartments and those unique in each compartment were then analyzed for their physico-chemical properties (molecular weight, isoelectric point, amino acid contents, instability index, aliphatic index, and grand average of hydropathicity), secondary structure (α-helix, β-turn, random coil, and extended strand), molecular functions, biological processes, reactome pathways and KEGG pathways. The data demonstrated that each set of these interacting proteins exhibited common and unique characteristics and properties. The knowledge from this study may lead to better understanding of the ApANXA1 and CyAXNA1 biochemistry and functions as well as the pathophysiology of CaOx kidney stone formation induced by high-calcium condition.

Abstract Increasing evidence of association between kidney stone disease (KSD) and renal cell carcinoma (RCC) has been reported. Nevertheless, mechanism underlying such association remained unknown. Herein, we investigated the effects of calcium oxalate monohydrate (COM), a major crystalline component causing KSD, on induction of carcinogenic features in non-cancerous renal cells. COM crystals induced morphological changes from epithelial to fibroblast-like spindle shape. Additionally, COM increased spindle index and mesenchymal markers (fibronectin and vimentin) but declined epithelial markers (E-cadherin and zonula occludens-1). Moreover, COM down-regulated ARID1A, a tumor suppressor gene recently reported to be reversely associated with RCC, at both mRNA and protein levels. COM also down-regulated other RCC-related tumor suppressor genes, PTEN and VHL, but up-regulated oncogene TPX2. Finally, COM enhanced invading capability, cell-aggregate formation, chemoresistance to cisplatin, and secretion of an angiogenic factor (VEGF). These data indicate that COM crystals trigger epithelial-mesenchymal transition (EMT) and several carcinogenic features in the non-cancerous renal cells. These mechanisms may explain and strengthen the association between KSD and RCC.

Trigonelline is the second most abundant bioactive alkaloid found in coffee. It is classified as a phytoestrogen with similar structure as of estradiol and exhibits an estrogenic effect. A previous study has reported that fenugreek seed extract rich with trigonelline can reduce renal crystal deposition in ethylene glycol-induced nephrolithiatic rats. However, direct evidence of such anti-lithogenic effects of trigonelline and underlying mechanisms have not previously been reported. Our study therefore addressed the protective effects and mechanisms of trigonelline against kidney stone-forming processes using crystallization, crystal growth, aggregation and crystal-cell adhesion assays. Also, proteomics was applied to identify changes in receptors for calcium oxalate monohydrate (COM), the most common stone-forming crystal, on apical membranes of trigonelline-treated renal tubular cells. The analyses revealed that trigonelline significantly reduced COM crystal size, number and mass during crystallization. Additionally, trigonelline dose-dependently inhibited crystal growth and crystal-cell adhesion, but did not affect crystal aggregation. Mass spectrometric protein identification showed the smaller number of COM crystal receptors on apical membranes of the trigonelline-treated cells. Western blotting confirmed the decreased levels of some of these crystal receptors by trigonelline. These data highlight the protective mechanisms of trigonelline against kidney stone development by inhibiting COM crystallization, crystal growth and crystal-cell adhesion via downregulation of the crystal receptors on apical membranes of renal tubular cells.

Objective: The present study was conducted to evaluate the toxicity and teratogenic effects of MSG in chick embryo development. Methods: Various concentrations of MSG solution, 1 mg, 1.5 mg, 2.5 mg and 3 mg of MSG/gm egg weight were injected into the fertilized hen’s egg at 21 hrs of incubation and further incubated until day 3, 6 and 10. The day 3 and day 6 were processed for serial section, while the day 3 was processed for total mount and day 10 were observed totally. Results: The effects of MSG were growth retardation, embryonic death and congenital malformations of several organs such as brain (rachischisis and anencephaly), eye (microphthalmia and anophthalmia), ectopiacordis, ectopia viscera, opening of posterior neuropore (spinal bifida) and under development of heart and viscerae. Conclusion: MSG produced congenital deformities and growth retardation in chick embryos which may predict to cause the same results to human embryos. Pregnant women should be advised not to consume MSG to avoid potential harmful effects to the unborn child.