Repeated dose multi-drug testing using a microfluidic chip-based coculture of human liver and kidney proximal tubules equivalents.

A microfluidic multi-organ chip emulates the tissue culture microenvironment, enables interconnection of organ equivalents and overcomes interspecies differences, making this technology a promising and powerful tool for preclinical drug screening. In this study, we established a microfluidic chip-based model that enabled non-contact cocultivation of liver spheroids and renal proximal tubule barriers in a connecting media circuit over 16 days. Meanwhile, a 14-day repeated-dose systemic administration of cyclosporine A (CsA) alone or in combination with rifampicin was performed. Toxicity profiles of the two different doses of CsA on different target organs could be discriminated and that concomitant treatment with rifampicin from day6 onwards decreased the CsA concentration and attenuated the toxicity compared with that after treatment with CsA for 14 consecutive days. The latter is manifested with the changes in cytotoxicity, cell viability and apoptosis, gene expression of metabolic enzymes and transporters, and noninvasive toxicity biomarkers. The on chip coculture of the liver and the proximal tubulus equivalents showed its potential as an effective and translational tool for repeated dose multi-drug toxicity screening in the preclinical stage of drug development.

Melamine promotes calcium crystal formation in three-dimensional microfluidic device.

Melamine, which induces proximal tubular (PT) cell damage has a greater nephrotoxic effect when combined with cyanuric and uric acids; however, it is unknown whether such effect can stimulate calcium phosphate (CaP)/calcium oxalate (CaOx) stone formation. Here, we show that melamine acts as an inducer of CaP, CaOx and CaP + CaOx (mixed) crystal formations in a time and concentration-dependent manner by stabilizing those crystals and further co-aggregating with melamine. To explore the physiological relevance of such melamine-augmented calcium crystal formation, we used 2-dimensional (2D) and 3D microfluidic (MF) device, embedded with PT cells, which also resembled the effect of melamine-stimulated CaP, CaOx and mixed crystal formation. Significantly, addition of preformed CaP and/or CaOx crystal in the presence of melamine, further potentiated those crystal formations in 3D MFs, which helped the growth and aggregation of mixed crystals. Our data show that the mechanism of such predisposition of stone formation could be largely due to co-crystallization between melamine and CaP/CaOx and pronounced effect on induction of stone-forming pathway activation in 3D MF. Taken together, melamine-induced CaP and/or CaOx crystal formation ex-vivo will help us in understanding the larger role of melamine as an environmental toxicant in producing the pathology in similar cellular microenvironments.

Site-specific expression of IQGAP1 in human nephrons.

IQGAP1 is a multifunctional, 190-kDa scaffolding protein that plays an important role in the regulation of cell adhesion, migration, proliferation, differentiation, polarization and cytoskeletal remodeling. IQGAP1 is ubiquitously expressed in human organs and is highly expressed in the kidney. Currently, the site-specific expression of IQGAP1 in the human nephrons is unclear. We performed Western blotting analysis, immunohistochemistry and double-immunolabeling confocal microscopic analysis of IQGAP1 with specific biomarkers of each nephron segment to study the expression and distribution of IQGAP1 in human nephrons. We found that IQGAP1 was strongly expressed in human podocytes and glomerular endothelial cells, but weakly expressed in glomerular mesangial cells. In human renal tubules, IQGAP1 was strongly expressed in the collecting duct, moderately expressed in the proximal tubule, medullary loop, distal convoluted tubule and connecting tubule. IQGAP1 staining was much stronger in the apical membrane in the proximal tubule, thick descending limb and thick ascending limb of medullary loop and collecting duct. However, the expression of IQGAP1 was mainly in the basolateral membrane of the connecting tubule, and diffusely in the thin limb of medullary loop and distal convoluted tubule. The interaction between IQGAP1 and F-actin suggested that cytoskeleton regulation may be the underlying mechanism mediating the effect of IQGAP1 in human nephrons. To the best of our knowledge, this is the first report of specific expression and differential subcellular location of IQGAP1 in human nephrons. The site-specific expression pattern of IQGAP1 suggests that IQGAP1 may play diverse roles in various human nephron segments.

Cannabinoid-1 receptor regulates mitochondrial dynamics and function in renal proximal tubular cells.

AIMS: To evaluate the specific role of the endocannabinoid/cannabinoid type-1 (CB1 R) system in modulating mitochondrial dynamics in the metabolically active renal proximal tubular cells (RPTCs). MATERIALS AND METHODS: We utilized mitochondrially-targeted GFP in live cells (wild-type and null for the CB1 R) and electron microscopy in kidney sections of RPTC-CB1 R-/- mice and their littermate controls. In both in vitro and in vivo conditions, we assessed the ability of CB1 R agonism or fatty acid flux to modulate mitochondrial architecture and function. RESULTS: Direct stimulation of CB1 R resulted in mitochondrial fragmentation in RPTCs. This process was mediated, at least in part, by modulating the phosphorylation levels of the canonical fission protein dynamin-related protein 1 on both S637 and S616 residues. CB1 R-induced mitochondrial fission was associated with mitochondrial dysfunction, as documented by reduced oxygen consumption and ATP production, increased reactive oxygen species and cellular lactate levels, as well as a decline in mitochondrial biogenesis. Likewise, we documented that exposure of RPTCs to a fatty acid flux induced CB1 R-dependent mitochondrial fission, lipotoxicity and cellular dysfunction. CONCLUSIONS: CB1 R plays a key role in inducing mitochondrial fragmentation in RPTCs, leading to a decline in the organelle's function and contributing to the renal tubular injury associated with lipotoxicity and other metabolic diseases.

Exogenous H2S Inhibits Autophagy in Unilateral Ureteral Obstruction Mouse Renal Tubule Cells by Regulating the ROS-AMPK Signaling Pathway.

BACKGROUND/AIMS: The induction of excessive autophagy by increased levels of oxidative stress is one of the main mechanisms underlying unilateral ureteral obstruction (UUO)-induced vascular endothelial cell dysfunction. Hydrogen sulfide (H2S) has been shown to have an anti-oxidative effect, but its mode of action on excessive autophagy in vascular endothelial cells is unclear. METHODS: Surgery was used to induce UUO in male C57BL/6 mice as an in vivo model. Human renal epithelial cells (HK-2) were treated with H2O2 as an in vitro model. NaHS was used as an exogenous H2S donor. Transmission electron microscopy was applied to observe the structure of renal autophagosomes. The expression of proteins related to autophagy and apoptosis was detected by western blot analysis in vivo and in vitro. Flow cytometry (DCFH-DA) was used to examine the levels of intracellular reactive oxygen species (ROS). The terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to detect cell apoptosis. Compound C was used to analyze the association of AMPK with autophagy. RESULTS: Compared with the sham group, in which the ureter was exposed but not ligated, the cell apoptosis index, number of autophagosomes, protein expression of microtubule-associated protein 1 light-chain 3 (LC3)-II/I, beclin-1, and p-AMPK/AMPK were significantly increased in the UUO group. On the other hand, p62, cystathionine ß-synthase, and cystathionine γ-lyase protein expression levels and H2S concentration were significantly decreased (p < 0.05). These alterations were ameliorated by the addition of NaHS (p < 0.05). Similar results were observed in vitro. By using the AMPK inhibitor compound C, it was indicated that AMPK was involved in ROS-induced autophagy. In addition, using tissue from patients with obstructive nephropathy, excessive autophagy was observed by an increased LC3-II/LC3-I ratio. CONCLUSION: NaHS-treatment may exert a protective effect on mouse kidney against UUO by suppressing the ROS-AMPK pathway. ROS-AMPK-mediated autophagy may represent a promising therapeutic target for obstructive nephropathy.

Kidney injury molecule-1 identifies antemortem injury in postmortem adult and fetal kidney.

There is currently no technique to unambiguously diagnose antemortem kidney injury on postmortem examination since postmortem tissue damage and autolysis are common. We assessed the ability to detect kidney injury molecule-1 (KIM-1) expression in adult and fetal kidneys examined at autopsy. In adult kidneys ( n = 52 subjects), we found that the intensity of KIM-1 staining significantly correlated with the antemortem level of serum creatinine, and this was independent of the extent of tissue autolysis. In addition, kidneys from a total of 52 fetal/neonatal subjects, 30 stillborns and 22 liveborns, were assessed for KIM-1 staining. Given that serum creatinine is unreliable and often unavailable in fetuses and newborns, we assessed preterminal hypoxia in fetuses by the presence of squames in pulmonary alveoli and by required intubation. KIM-1 expression correlated with these clinical indexes of hypoxia. The expression of KIM-1 was seen in a majority of the fetal and neonatal autopsy kidneys (77%, 40/52) as early as 16 wk of gestation, even in the presence of autolysis. Thus KIM-1 is a specific and stable marker of antemortem tubular injury in kidneys of adults and fetuses despite postmortem autolysis.

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Non-Proximal Renal Tubule-Derived Urinary Exosomal miR-200b as a Biomarker of Renal Fibrosis.

BACKGROUND: Renal fibrosis is a common outcome of nearly all kinds of chronic kidney disease (CKD) and eventually progresses to end-stage renal disease. The identification of an optimal biomarker of renal fibrosis to replace the invasive renal biopsy will have important clinical implications. METHODS: We isolated urinary exosomes from 50 participants and examined the exosomal protein content and particle number in 38 CKD patients with different degrees of renal fibrosis and in 12 normal individuals. We examined the levels of exosomal microRNAs (miRNAs), namely, miR-200a, miR-200b, miR-200c, miR-141, miR-429, miR-29a, miR-29b, miR-29c, miR-192, and miR-21, by sorting the exosomes and comparing the levels of proximal tubular, non-proximal tubular, and total exosomal miR-200b. RESULTS: The exosome content was higher in the CKD group, but no differences were evident among the mild, moderate, and severe fibrosis groups. Among the 10 exosomal miRNAs, miR-200b was lower in the CKD group than in the normal group and decreased more significantly with fibrosis progression as well as in IgA nephropathy and diabetic kidney disease. CD13+ CD63+ exosomes constituted 18.6% of all urinary exosomes. Sorting the proximal tubular exosomes with the CD13 protein marker revealed that miR-200b in the CD13+ group was extremely low; however, the result was significantly different in the CD13- group but not in the CD13+ group. The magnitude of the decline was greater in the CD13- groups than in the non-sorted whole groups between the fibrosis and normal patients. CONCLUSIONS: Non-proximal renal tubule-derived urinary exosomal miR-200b is a biomarker of renal fibrosis. Exosomes can be used as a liquid biopsy and may replace the traditional invasive renal biopsy in the diagnosis of renal fibrosis.

Quantification of cystine in human renal proximal tubule cells using liquid chromatography-tandem mass spectrometry.

Nephropathic cystinosis is characterized by abnormal intralysosomal accumulation of cystine throughout the body, causing irreversible damage to various organs, particularly the kidneys. Cysteamine, the currently available treatment, can reduce lysosomal cystine and postpone disease progression. However, cysteamine poses serious side effects and does not address all of the symptoms of cystinosis. To screen for new treatment options, a rapid and reliable high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed to quantify cystine in conditionally immortalized human proximal tubular epithelial cells (ciPTEC). The ciPTEC were treated with N-ethylmaleimide, lysed and deproteinized with 15% (w/v) sulfosalicylic acid. Subsequently, cystine was measured using deuterium-labeled cystine-D4, as the internal standard. The assay developed demonstrated linearity to at least 20 µmol/L with a good precision. Accuracies were between 97.3 and 102.9% for both cell extracts and whole cell samples. Cystine was sufficiently stable under all relevant analytical conditions. The assay was successfully applied to determine cystine levels in both healthy and cystinotic ciPTEC. Control cells showed clearly distinguishable cystine levels compared with cystinotic cells treated with or without cysteamine. The method developed provides a fast and reliable quantification of cystine, and is applicable to screen for potential drugs that could reverse cystinotic symptoms in human kidney cells.

Unique proximal tubular cell injury and the development of acute kidney injury in adult patients with minimal change nephrotic syndrome.

BACKGROUND: Adult patients with minimal change nephrotic syndrome (MCNS) are often associated with acute kidney injury (AKI). To assess the mechanisms of AKI, we examined whether tubular cell injuries unique to MCNS patients exist. METHODS: We performed a retrospective analysis of clinical data and tubular cell changes using the immunohistochemical expression of vimentin as a marker of tubular injury and dedifferentiation at kidney biopsy in 37 adult MCNS patients. AKI was defined by the criteria of the Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guidelines for AKI. RESULTS: Thirteen patients (35.1%) were designated with AKI at kidney biopsy. No significant differences in age, history of hypertension, chronic kidney disease, diuretics use, proteinuria, and serum albumin were noted between the AKI and non-AKI groups. Urinary N-acetyl-ß-D-glucosaminidase (uNAG) and urinary alpha1-microglobulin (uA1MG) as markers of tubular injury were increased in both groups, but the levels were significantly increased in the AKI group compared with the non-AKI group. The incidence of vimentin-positive tubules was comparable between AKI (84.6%) and non-AKI (58.3%) groups, but vimentin-positive tubular area per interstitial area was significantly increased in the AKI group (19.8%) compared with the non-AKI group (6.8%) (p = 0.011). Vimentin-positive injured tubules with tubular simplification (loss of brush-border of the proximal tubule/dilated tubule with flattening of tubular epithelium) were observed in the vicinity of glomeruli in both groups, suggesting that the proximal convoluted tubules were specifically injured. Two patients exhibited relatively severe tubular injuries with vimentin positivity and required dialysis within 2 weeks after kidney biopsy. The percentage of the vimentin-positive tubular area was positively correlated with uNAG but not with uA1MG in the non-AKI group. CONCLUSIONS: Proximal tubular injuries with increased uNAG exist in MCNS patients without renal dysfunction and were more severe in the AKI group than they were in the non-AKI group. The unique tubular injuries probably due to massive proteinuria might be a predisposing factor for the development of severe AKI in adult MCNS patients.

Urinary DcR2 is a novel biomarker for tubulointerstitial injury in patients with diabetic nephropathy.

Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients who were diagnosed by renal biopsy. uDcR2 levels were measured by ELISA, and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2 and renal functional parameters were evaluated. Receiver operating characteristics (ROC) curve analyzed area under the curve (AUC) of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers; senescent markers p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal); and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics and were associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was coexpressed with p16 and p21, and nearly more than one-half of tubular DcR2 was positive for SA-ß-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.

Diurnal and Long-term Variation in Plasma Concentrations and Renal Clearances of Circulating Markers of Kidney Proximal Tubular Secretion.

BACKGROUND: The renal proximal tubule is essential for removing organic solutes and exogenous medications from the circulation. We evaluated diurnal, prandial, and long-term biological variation of 4 candidate endogenous markers of proximal tubular secretion. METHODS: We used LC-MS to measure plasma and urine concentrations of hippurate (HA), cinnamoylglycine (CMG), indoxyl sulfate (IS), and p-cresol sulfate (PCS) in 25 healthy adults. We measured plasma concentrations of secreted solutes at 13 time points over a 24-h period, and again after 2 weeks and 14 weeks of follow-up. We further measured 24-h renal clearances of secreted solutes at baseline, 2 weeks, and 14 weeks. RESULTS: Plasma concentrations of secreted solutes varied over the 24-h baseline period. Diurnal variation was greatest for HA, followed by CMG, IS, and PCS. Plasma concentrations of HA (P = 0.002) and IS (P = 0.02), but not CMG and PCS, increased significantly following meals. Long-term intraindividual biological variation (CVI) in plasma concentrations of secreted solutes over 14 weeks varied from 21.8% for IS to 67.3% for PCS, and exceeded that for plasma creatinine (CVI, 7.1%). Variation in 24-h renal clearances was similar among the secreted solutes [intraindividual variation (CVA+I), 33.6%-47.3%] and was lower using pooled plasma samples from each study visit. CONCLUSIONS: Plasma concentrations of HA, CMG, IS, and PCS fluctuate within individuals throughout the day and over weeks. Renal clearances of these secreted solutes, which serve as estimates of renal proximal tubule secretion, are also subject to intraindividual biological variation that can be improved by additional plasma measurements.

Activation of angiotensin II type 1 receptors increases D4 dopamine receptor expression in rat renal proximal tubule cells.

Both the dopaminergic and renin-angiotensin systems play important roles in the regulation of blood pressure. Our previous study showed that the stimulation of dopaminergic D4 receptors reduced angiotensin II type 1 (AT1) receptor expression in renal proximal tubule (RPT) cells. In this study, we tested whether AT1 receptors, in return, would regulate D4 receptor expression and function in RPT cells. Expression of the D4 receptor from Wistar-Kyoto (WKY) or spontaneously hypertensive rats (SHRs) RPT cells and renal cortex tissues were determined by western blot, and Na+-K+ ATPase activity was determined using an enzyme assay. Urine volume and urine sodium of WKY rats and SHRs treated with or without D4 receptor stimulation were measured. Thus, activation of AT1 receptors with angiotensin II (Ang II) increased D4 receptor protein expression in RPT cells, and this increase was blocked by nicardipine, a calcium influx blocker. The D4 receptor agonist PD168077 inhibited Na+-K+ ATPase activity in WKY RPT cells but not in SHR RPT cells. Ang II pre-treatment promoted D4 receptor-mediated inhibition of Na+-K+ ATPase in RPT cells in WKY rats but not in SHRs. Meanwhile, Ang II pre-treatment augmented the natriuretic effect of PD168077 in WKY rats but not in SHRs. In conclusion, AT1 stimulation can regulate the expression and natriuretic function of dopaminergic D4 receptors in RPT cells and might be involved in the pathogenesis of essential hypertension.

Expression of sodium-dependent dicarboxylate transporter 1 (NaDC1/SLC13A2) in normal and neoplastic human kidney.

Regulated dicarboxylate transport is critical for acid-base homeostasis, prevention of calcium nephrolithiasis, regulation of collecting duct sodium chloride transport, and the regulation of blood pressure. Although luminal dicarboxylate reabsorption via NaDC1 (SLC13A2) is believed to be the primary mechanism regulating renal dicarboxylate transport, the specific localization of NaDC1 in the human kidney is currently unknown. This study's purpose was to determine NaDC1's expression in normal and neoplastic human kidneys. Immunoblot analysis demonstrated NaDC1 expression with an apparent molecular weight of ~61 kDa. Immunohistochemistry showed apical NaDC1 immunolabel in the proximal tubule of normal human kidney tissue; well-preserved proximal tubule brush border was clearly labeled. Apical NaDC1 expression was evident throughout the entire proximal tubule, including the initial proximal convoluted tubule, as identified by origination from the glomerular tuft, and extending through the terminal of the proximal tubule, the proximal straight tubule in the outer medulla. We confirmed proximal tubule localization by colocalization with the proximal tubule specific protein, NBCe1. NaDC1 immunolabel was not detected other than in the proximal tubule. In addition, NaDC1 immunolabel was not detected in tumors of presumed proximal tubule origin, clear cell and papillary renal cell carcinoma, or in tumors of nonproximal tubule origin, oncocytoma and chromophobe carcinoma. In summary, 1) in the human kidney, apical NaDC1 immunolabel is present throughout the entire proximal tubule, and is not detectable in other renal cells; and 2) NaDC1 immunolabel is not present in renal tumors. These studies provide important information regarding NaDC1's role in human dicarboxylate metabolism.

Effect of D3 dopamine receptor on dopamine D4 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto rats and spontaneously hypertensive rats.

BACKGROUND: Dopamine receptors induce natriuresis in kidney. Previous studies have shown interactions between different subtypes of dopamine receptors in renal proximal tubule (RPT) cells. We hypothesize that D3 receptors have an interaction with D4 receptors in RPT cells from normotensive rats (Wistar-Kyoto, WKY) and spontaneously hypertensive rats (SHRs). METHODS: Immunoblotting and reverse transcriptase-polymerase chain reaction (RT-PCR) were used to examine the expression of D3 and D4 receptors. Na-K-ATPase activity was used to measure the function of receptors. The distribution and colocalization of D3 and D4 receptors were detected by confocal microscopy and co-immunoprecipitation. RESULTS: D3 receptor agonist PD128907 increased the mRNA and protein expression of D4 receptors in RPT cells from WKY rats, but decreased that from SHRs. In the presence of PLC blocker (U73122, 10-mol/l) or PKC inhibitor 19 -31 (10-mol/l), the up-regulation of D3 receptor on D4 receptor was lost in WKY cells. Moreover, stimulation with PD128907 for 30 minutes decreased D4 receptor degradation in WKY cells, not in SHR cells. D3 and D4 receptors colocalized and co-immunoprecipitated in RPT cells. PD128907 increased co-immunoprecipitation of D3 and D4 receptors in WKY RPT cells, but not in SHR RPT cells. Pre-treatment with D3 receptor agonist also increases D4 receptor mediated inhibitory effect on Na-K-ATPase activity in WKY cells, but not in SHR cells. CONCLUSION: Renal D3 receptor regulates the expression and function of D4 receptor in RPT cells via PLC /PKC signaling pathway, the loss of this interaction might be involved in the pathogenesis of hypertension.

The stress response of human proximal tubule cells to cadmium involves up-regulation of haemoxygenase 1 and metallothionein but not cytochrome P450 enzymes.

Enzymes of the cytochrome P450 (CYP) super-family are implicated in cadmium (Cd) -induced nephrotoxicity, however, direct evidence is lacking. This study investigated the endogenous expression of various CYP proteins together with the stress-response proteins, heme oxygenase-1 (HO-1) and metallothionein (MT) in human kidney sections and in cadmium-exposed primary cultures of human proximal tubular epithelial cells (PTC). By immunohistochemistry, the CYP members 2B6, 4A11 and 4F2 were prominently expressed in the cortical proximal tubular cells and to a lesser extent in distal tubular cells. Low levels of CYPs 2E1 and 3A4 were also detected. In PTC, in the absence of Cd, CYP2E1, CYP3A4, CYP4F2 and MT were expressed, but HO-1, CYP2B6 and CYP4A11 were not detected. A range of cadmium concentrations (0-100µM) were utilized to induce stress conditions. MT protein was further induced by as little as 0.5µM cadmium, reaching a 6-fold induction at 20µM, whereas for HO-1, a 5µM cadmium concentration was required for initial induction and at 20µM cadmium reached a 15-fold induction. The expression of CYP2E1, CYP3A4, and CYP4F2 were not altered by any cadmium concentrations tested at 48h. Cadmium caused a reduction in cell viability at concentrations above 10µM. In conclusion although cultured PTC, do express CYP proteins, (CYP2E1, CYP3A4, and CYP4F2), Cd-induced cell stress as indicted by induction of HO-1 and MT does not alter expression of these CYP proteins at 48h.

Screening for Potential Bioactive Components in Ginkgo biloba Extract by the Rat Renal Tubular Epithelial Cell Extraction and LC-MS/MS.

Rat renal tubular epithelial cell (RTEC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN). The compounds in GBE binding with cell membrane or entering into cell are still unknown, which may be potential bioactive components. In this paper, a powerful method for screening and analyzing the potential bioactive components from GBE was developed using cell extraction coupled with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 8 prototype compounds and 5 metabolites were obtained, among which 6 prototype compounds and 1 metabolite were identified or tentatively characterized as rutin, bilobalide, ginkgolide B, ginkgolide C, genkwanin, apigenin and diosmetin by comparing their retention times and MS spectra with those of authentic standards or literature data. The 6 prototype compounds were further quantitatively analyzed using electrospray ionization in negative mode multiple reaction monitoring (MRM). The results showed that high glucose changed the Tmax, MRT(0-t), Cmax and AUC(0-t) of all observed compounds and decreased the t1/2 of genkwanin and apigenin, significantly. The overall findings indicate that 8 prototype compounds may be the potential bioactive components of GBE with preventive effect against DN and the method of RTEC extraction coupled with LC-MS/MS technology screening method we developed is a feasible, rapid, and useful tool for screening and analyzing potential bioactive components.

Mesoscale nanoparticles selectively target the renal proximal tubule epithelium.

We synthesized "mesoscale" nanoparticles, approximately 400 nm in diameter, which unexpectedly localized selectively in renal proximal tubules and up to 7 times more efficiently in the kidney than other organs. Although nanoparticles typically localize in the liver and spleen, modulating their size and opsonization potential allowed for stable targeting of the kidneys through a new proposed uptake mechanism. Applying this kidney targeting strategy, we anticipate use in the treatment of renal disease and the study of renal physiology.

Histochemical and immunoelectron microscopic analysis of ganglioside GM3 in human kidney.

BACKGROUND: Gangliosides are amphipathic lipids ubiquitously expressed in all vertebrate cells. They have been reported to play pivotal roles in cell morphology, cell adhesion, signal transduction, and modulation of immune reaction. Although human kidney contains various kinds of ganglioside, their physiological and pathophysiological roles have not been elucidated yet. As ganglioside GM3 is the most abundant ganglioside in human kidney, we tried to reveal the distribution of GM3 using histological analysis. METHODS: Macroscopically normal parts of operatively resected kidney from renal cell carcinoma patients were used for analyses. Immunohistochemical and immunoelectron microscopic analyses were performed with anti-GM3 antibody. RESULTS: Immunohistochemical analyses showed that GM3 was observed in glomeruli and renal proximal tubules. Immunoelectron microscopy demonstrated that GM3 was localized on the foot process of podocyte and also in Golgi region of renal proximal tubule cells. CONCLUSIONS: Ganglioside GM3 might take a part of the negative electric charge on the surface of podocyte and its multiple physiological actions may play pivotal roles for maintaining glomerular function.

Tetramethylpyrazine (TMP) protects against sodium arsenite-induced nephrotoxicity by suppressing ROS production, mitochondrial dysfunction, pro-inflammatory signaling pathways and programed cell death.

Although kidney is a target organ of arsenic cytotoxicity, the underlying mechanisms of arsenic-induced nephrotoxicity remain poorly understood. As tetramethylpyrazine (TMP) has recently been found to be a renal protectant in multiple kidney injuries, we hypothesize that TMP could suppress arsenic nephrotoxicity. In this study, human renal proximal tubular epithelial cell line HK-2 was used to elucidate the precise mechanisms of arsenic nephrotoxicity as well as the protective mechanism of TMP in these cells. Sodium arsenite exposure dramatically increased cellular reactive oxygen species (ROS) production, decreased levels of cellular glutathione (GSH), decreased cytochrome c oxidase activity and mitochondrial membrane potential, which indicated mitochondrial dysfunction. On the other hand, sodium arsenite activated pro-inflammatory signals, including ß-catenin, nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor alpha and cyclooxygenase-2 (COX-2). Small molecule inhibitors of NF-κB and p38 MAPK blocked arsenic-induced COX-2 expression, suggesting arsenic-induced COX-2 up-regulation was NF-κB- and p38 MAPK-dependent. Finally, sodium arsenite induced autophagy in HK-2 cells at early phase (6 h) and the subsequent apoptosis at 24 h. Treatment by TMP or by the antioxidant N-acetylcysteine decreased arsenic-induced ROS production, enhanced GSH levels, prevented mitochondria dysfunction and suppressed the activation of pro-inflammatory signals and the development of autophagy and apoptosis. Our results suggested that TMP may be used as a new potential therapeutic agent to prevent arsenic-induced nephrotoxicity by suppressing these pathological processes.