Paleerath Peerapen

p38 MAPK mediates calcium oxalate crystal-induced tight junction disruption in distal renal tubular epithelial cells

We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 μM SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 μg/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na+/K+-ATPase-α1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.

Effects of calcium oxalate monohydrate crystals on expression and function of tight junction of renal tubular epithelial cells

Tight junction has a crucial role in regulating paracellular transports (as a barrier) and in separating apical from basolateral compartments to maintain cell polarity (as a fence). Tight junction can be disrupted by various stimuli, including oxidative stress, pathogens and proinflammatory cytokines. However, association of defective tight junction with kidney stone pathogenesis remains unknown. We therefore examined whether calcium oxalate monohydrate (COM) crystals, which are the major crystalline composition in kidney stones, have any effects on expression and function of tight junction of polarized renal tubular epithelial cells. Western blot analysis revealed marked decrease in levels of occludin and zonula occludens-1 (ZO-1) in COM-treated polarized Madin-Darby canine kidney (MDCK) cells. Immunofluorescence staining revealed not only the decline of these tight junction proteins but also their redistribution and dissociation in COM-treated cells. Additionally, transepithelial resistance was significantly decreased, indicating impaired tight junction barrier and increased paracellular permeability in COM-treated cells. Subcellular fractionation followed by western blot analysis of Na+/K+-ATPase-α1 revealed that this basolateral membrane marker was also detectable in apical membrane fraction of COM-treated cells, but not in apical membrane fraction of control cells. Immunofluorescence study confirmed the translocation of Na+/K+-ATPase-α1 (from basolateral to apical membranes) in COM-treated cells, indicating impaired fence function of the tight junction. Moreover, dihydrorhodamine assay using flow cytometry revealed the significantly higher level of hydrogen peroxide in the COM-treated cells. These data provide the first evidence to demonstrate decreased expression and defective barrier and fence functions of the tight junction of renal tubular epithelial cells exposed to COM crystals that may be fundamental for subsequent renal tubulointerstitial injury, which in turn enhances the stone pathogenesis.

High calcium enhances calcium oxalate crystal binding capacity of renal tubular cells via increased surface annexin A1 but impairs their proliferation and healing.

Hypercalciuria is associated with kidney stone formation and impaired renal function. However, responses of renal tubular cells upon exposure to high-calcium environment remain largely unknown. We thus performed a proteomic analysis of altered proteins in renal tubular cells induced by high-calcium and evaluated functional significance of these changes. MDCK cells were maintained with or without 20 mM CaCl(2) for 72 h. Cellular proteins were then analyzed by two-dimensional electrophoresis (2-DE) (n = 5 gels derived from 5 independent culture flasks per group). Spot matching and quantitative intensity analysis revealed 20 protein spots (from a total of 700) that were differentially expressed between the two groups. These altered proteins were then identified by Q-TOF-MS and MS/MS analyses, including those involved in calcium binding, protein synthesis, carbohydrate metabolism, lipid metabolism, cell proliferation, mitosis regulation, apoptosis, cell migration, oxidative stress, and ion transport. Protein network analysis and functional validation revealed that high-calcium-exposed cells had 36.5% increase in calcium oxalate monohydrate (COM) crystal-binding capacity. This functional change was consistent to the expression data in which annexin A1 (ANXA1), a membrane-associated calcium-binding protein, was markedly increased on the apical surface of high-calcium-exposed cells. Pretreatment with anti-ANXA1 antibody could neutralize this increasing crystal-binding capacity. Moreover, high-calcium exposure caused defects in cell proliferation and wound healing. These expression and functional data demonstrate the enhanced crystal-binding capacity but impaired cell proliferation and wound healing in renal tubular cells induced by high-calcium. Taken together, these phenomena may contribute, at least in part, to the pathogenic mechanisms of hypercalciuria-induced nephrolithiasis and impaired renal function. Our in vitro study offers several candidates for further targeted functional studies to confirm their relevance in hypercalciuria and kidney stone disease in vivo.

Systematic evaluation for effects of urine pH on calcium oxalate crystallization, crystal-cell adhesion and internalization into renal tubular cells

Urine pH has been thought to be an important factor that can modulate kidney stone formation. Nevertheless, there was no systematic evaluation of such pH effect. Our present study thus addressed effects of differential urine pH (4.0–8.0) on calcium oxalate (CaOx) crystallization, crystal-cell adhesion, crystal internalization into renal tubular cells, and binding of apical membrane proteins to the crystals. Microscopic examination revealed that CaOx monohydrate (COM), the pathogenic form, was crystallized with greatest size, number and total mass at pH 4.0 and least crystallized at pH 8.0, whereas COD was crystallized with the vice versa order. Fourier-transform infrared (FT-IR) spectroscopy confirmed such morphological study. Crystal-cell adhesion assay showed the greatest degree of crystal-cell adhesion at the most acidic pH and least at the most basic pH. Crystal internalization assay using fluorescein isothiocyanate (FITC)-labelled crystals and flow cytometry demonstrated that crystal internalization into renal tubular cells was maximal at the neutral pH (7.0). Finally, there were no significant differences in binding capacity of the crystals to apical membrane proteins at different pH. We concluded that the acidic urine pH may promote CaOx kidney stone formation, whereas the basic urine pH (i.e. by alkalinization) may help to prevent CaOx kidney stone disease.

In vitro evidence of the promoting effect of testosterone in kidney stone disease: A proteomics approach and functional validation

UNLABELLED Incidence of kidney stone disease in males is 2- to 4-fold greater than in females. This study aimed to determine effects of testosterone on kidney stone disease using a proteomics approach. MDCK renal tubular cells were treated with or without 20nM testosterone for 7days. Cellular proteins were extracted, resolved by 2-DE, and stained with Deep Purple fluorescence dye (n=5 gels derived from 5 independent samples/group). Spot matching, quantitative intensity analysis, and statistics revealed significant changes in levels of nine protein spots after testosterone treatment. These proteins were then identified by nanoLC-ESI-Qq-TOF MS/MS. Global protein network analysis using STRING software revealed α-enolase as the central node of protein-protein interactions. The increased level of α-enolase was then confirmed by Western blotting analysis, whereas immunofluorescence study revealed the increased α-enolase on cell surface and intracellularly. Functional analysis confirmed the potential role of the increased α-enolase in enhanced calcium oxalate monohydrate (COM) crystal-cell adhesion induced by testosterone. Finally, neutralization of surface α-enolase using anti-α-enolase antibody successfully reduced the enhanced COM crystal-cell adhesion to the basal level. Our data provided in vitro evidence of promoting effect of testosterone on kidney stone disease via enhanced COM crystal-cell adhesion by the increased surface α-enolase. BIOLOGICAL SIGNIFICANCE The incidence of kidney stone disease in male is 2- to 4-fold greater than in female. One of the possible factors of the male preference is the higher testosterone hormone level. However, precise molecular mechanisms that testosterone plays in kidney stone disease remained unclear. Our present study is the first exploratory investigation on such aspect using a proteomics approach. Our data also provide a novel mechanistic aspect of how testosterone can impact the risk of kidney stone formation (i.e. the discovery that testosterone increases alpha-enolase expression on the surface of renal tubular cells that is responsible, at least in part, for crystal-cell adhesion).

Caffeine prevents kidney stone formation by translocation of apical surface annexin A1 crystal-binding protein into cytoplasm: In vitro evidence

Recent large 3 cohorts have shown that caffeinated beverage consumption was associated with lower risk of kidney stone disease. However, its protective mechanisms remained unknown and had not been previously investigated. We thus evaluated protective effects of caffeine (1 μM–10 mM) on calcium oxalate monohydrate (COM) kidney stone formation, using crystallization, crystal growth, cell-crystal adhesion, Western blotting, and immunofluorescence assays. The results showed that caffeine reduced crystal number but, on the other hand, increased crystal size, resulting in unchanged crystal mass, consistent with crystal growth that was not affected by caffeine. However, caffeine significantly decreased crystal-binding capacity of MDCK renal tubular cells in a dose-dependent manner. Western blotting and immunofluorescence study of COM crystal-binding proteins revealed significantly decreased level of annexin A1 on apical surface and its translocation into cytoplasm of the caffeine-treated cells, but no significant changes in other COM crystal-binding proteins (annexin A2, α-enolase, HSP70, and HSP90) were observed. Moreover, caffeine decreased intracellular [Ca2+] but increased [Ca2+] secretory index. Taken together, our findings showed an in vitro evidence of the protective mechanism of caffeine against kidney stone formation via translocation of annexin A1 from apical surface into cytoplasm to reduce the crystal-binding capacity of renal tubular epithelial cells.

Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12

Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP).

Protein Network Analysis and Functional Studies of Calcium Oxalate Crystal-Induced Cytotoxicity in Renal Tubular Epithelial Cells

Our previous expression study has reported a set of proteins with altered levels in renal tubular cells after exposure to calcium oxalate monohydrate (COM) crystals, which are the main composition of kidney stones. However, their functional significance remained largely unknown. In this study, protein network analysis revealed that the significantly altered proteins induced by COM crystals were involved mainly in three main functional networks, including i) cell proliferation and wound healing; ii) oxidative stress and mitochondrial function; and iii) cellular junction complex and integrity. Cell proliferation and wound healing assays showed that the COM‐treated cells had defective proliferation and tissue healing capability, respectively. Oxyblot analysis demonstrated accumulation of the oxidized proteins, whereas intracellular ATP level was significantly increased in the COM‐treated cells. Additionally, level of zonula occludens‐1 (ZO‐1), a tight junction protein, was significantly decreased, consistent with the significant declines in transepithelial resistance (TER) and level of RhoA signaling molecule in the COM‐treated cells. These findings indicate significant perturbations in mitochondrial and oxidative stress axis that cause defective cell proliferation, tissue healing capability, junctional protein complex, and cellular integrity of renal tubular epithelial cells exposed to COM crystals that may play important roles in kidney stone pathogenesis.

Alterations of proteins in MDCK cells during acute potassium deficiency.

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury.

Physiologic changes of urinary proteome by caffeine and excessive water intake.

Abstract Background: Diurnal variations and physiologic changes of urinary proteome have been suggested in the urinary proteomics field. However, no clear evidence has been demonstrated. The present study thus aimed to define changes in urinary proteome by physiological stimuli, i.e. caffeine intake and excessive water drinking, both of which cause physiologic diuresis. Methods: Urine samples were collected from 30 healthy individuals under three different conditions: (i) morning void as the control; (ii) after drinking a cup of coffee; and (iii) after drinking 1 L of water within 20 min. Thereafter, differentially excreted proteins were analyzed by 2-DE proteomics approach and validated by Western blotting and ELISA. Results: Spot matching, quantitative intensity analysis, and ANOVA followed by Tukey’s post-hoc multiple comparisons and the Bonferroni correction revealed significant differences in levels of five protein spots among three different conditions. These proteins were identified by quadrupole time-of-flight mass spectrometry (Q-TOF MS) and/or MS/MS analyses as kininogen 1 isoform 3, β-actin, prostaglandin D synthase (PGDS), fibrinogen α-chain and immunoglobulin light chain. Among these, the decreased level of immunoglobulin was successfully validated by Western blotting and ELISA. Conclusions: These data indicated that caffeine intake and excessive water drinking could affect urinary excretion of some proteins and may affect urinary proteome analysis.