Human Renal Proximal Tubular HK-2 Research Articles

Telomerase reverse transcriptase protects against diabetic kidney disease by promoting AMPK/PGC-1a-regulated mitochondrial energy homeostasis

Disordered glucose and lipid metabolism, coupled with disturbed mitochondrial bioenergetics, are pivotal in the initiation and development of diabetic kidney disease (DKD). While the essential role of telomerase reverse transcriptase (TERT) in regulating mitochondrial function in the cardiovascular system has been recognized, its specific function in maintaining mitochondrial homeostasis in DKD remains unclear. This study aimed to explore how TERT regulates mitochondrial function and the underlying mechanisms. In vitro, human renal proximal tubular HK-2 cells exposed to high glucose/high fat (HG/HF) presented significant downregulation of TERT and AMPK dephosphorylation. This led to decreased ATP production, altered NAD+/NADH ratios, reduced mitochondrial complex activities, increased mitochondrial dysfunction, lipid accumulation, and reactive oxygen species (ROS) production. Knockdown of TERT (si-TERT) further exacerbated mitochondrial dysfunction, decreased mitochondrial membrane potential, and lowered levels of cellular oxidative phosphorylation and glycolysis, as determined via a Seahorse X24 flux analyzer. Conversely, mitochondrial dysfunction was significantly alleviated after pcDNA-TERT plasmid transfection and adeno-associated virus (AAV) 9-TERT gene therapy in vivo. Notably, treatment with an AMPK inhibitor, activator, and si-PGC-1a (peroxisome proliferator-activated receptor γ coactivator-1α), resulted in mitochondrial dysfunction and decreased expression of genes related to energy metabolism and mitochondrial biogenesis. Our findings reveal that TERT protects mitochondrial function and homeostasis by partially activating the AMPK/PGC-1a signaling pathway. These results establish a crucial foundation for understanding TERT's critical role inmitochondrial regulation and its protective effect on DKD.

Exploring the Metabolic Differences between Cisplatin- and UV Light-Induced Apoptotic Bodies in HK-2 Cells by an Untargeted Metabolomics Approach.

Among the extracellular vesicles, apoptotic bodies (ABs) are only formed during the apoptosis and perform a relevant role in the pathogenesis of different diseases. Recently, it has been demonstrated that ABs from human renal proximal tubular HK-2 cells, either induced by cisplatin or by UV light, can lead to further apoptotic death in naïve HK-2 cells. Thus, the aim of this work was to carry out a non-targeted metabolomic approach to study if the apoptotic stimulus (cisplatin or UV light) affects in a different way the metabolites involved in the propagation of apoptosis. Both ABs and their extracellular fluid were analyzed using a reverse-phase liquid chromatography-mass spectrometry setup. Principal components analysis showed a tight clustering of each experimental group and partial least square discriminant analysis was used to assess the metabolic differences existing between these groups. Considering the variable importance in the projection values, molecular features were selected and some of them could be identified either unequivocally or tentatively. The resulting pathways indicated that there are significant, stimulus-specific differences in metabolites abundancies that may propagate apoptosis to healthy proximal tubular cells; thus, we hypothesize that the share in apoptosis of these metabolites might vary depending on the apoptotic stimulus.

Effect of sulfur fumigation on quality and safety of Lilii Bulbus

Lilii Bulbus is a commonly used Chinese herbal medicine with both medicinal and edible values, while the market products usually has the problem of sulfur fumigation. Therefore, the quality and safety of Lilii Bulbus products deserve attention. In this study, ultra-high performance liquid chromatography-time of flight-tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was combined with principal component analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA) to analyze the differential components of Lilii Bulbus before and after sulfur fumigation. We identified ten markers generated after sulfur fumigation, summarized their mass fragmentation and transformation patterns, and verified the structures of phenylacrylic acid markers of sulfur fumigation. At the same time, the cytotoxicity of the aqueous extracts of Lilii Bulbus before and after sulfur fumigation was evaluated. The results showed that in the concentration range of 0-800 mg·L~(-1), the aqueous extract of Lilii Bulbus after sulfur fumigation had no significant effect on the viability of human liver LO2 cells, human renal proximal tubular HK-2 cells, and rat adrenal pheochromocytoma PC-12 cells. Moreover, the viability of the cells exposed to the aqueous extract of Lilii Bulbus before and after sulfur fumigation showed no significant difference. This study identified phenylacrylic acid and furostanol saponins as markers of sulfur-fumigated Lilii Bulbus for the first time, and made clear that proper sulfur fumigation of Lilii Bulbus would not produce cytotoxicity, providing a theoretical basis for the rapid identification and quality and safety control of sulfur-fumigated Lilii Bulbus.

Morin has protective potential against ER stress induced apoptosis in renal proximal tubular HK-2 cells

ER stress is an early event of acute kidney injury and has been linked to accelerate the development of chronic kidney disease. Therefore, the compounds that can mimic ER stress inhibitor may confer regulatory effects on ER stress induced apoptosis. In this study, we investigated the protective effects of flavonoid morin against ER stress induced apoptosis in human renal proximal tubular HK-2 cells. Morin downregulated the expression of GRP78, central regulator of ER stress response, induced by ER stress inducer tunicamycin. Interestingly, morin selectively inhibited the IRE1 pathway among the three major arms of the ER stress responses. The increased expression of XBP1-sp, phosphor-IRE-1α, and phosphor-JNK by TM were markedly suppressed by the pretreatment of morin. Morin also decreased the intracellular ROS production and the apoptosis induced by TM in HK-2 cells. Taken together, our finding show that morin acts as an ER stress inhibitor, and can be a good candidate in various ER-stress associated kidney diseases.

Transactivation of EGFR by prostaglandin E2 receptors: a nuclear story?

The pharmacological modulation of hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. Although it is assumed that prostaglandin E(2) (PGE(2)) exerts its biological effects from the extracellular medium through activation of EP receptors located at the cell membrane, we have shown in human renal proximal tubular HK-2 cells (and other cell lines) that intracellular PGE(2) regulates the expression of HIF-1α expression and the production of VEGF-A. Here, we have found--through experiments involving EP receptors agonists, EP receptor gene silencing and inhibition of the prostaglandin uptake transporter--that these biological effects of PGE(2) are mediated by intracellular EP(2) receptors. In sharp contrast with cell membrane EP(2), whose activation results in increased production of cAMP, intracellular EP(2) signaling was independent of cAMP. Instead, it involved c-src-dependent transactivation of epidermal growth factor receptor, which led to p38/ERK1/2-dependent activation of mitogen- and stress-activated kinase-1 (MSK-1) and to MSK-1-dependent-histone H3 phosphorylation and transcriptional up-regulation of retinoic acid receptor-β. Even more important, this signaling pathway was fully reproduced in nuclei isolated from HK-2 cell, which highlights the relevance of nuclear EP receptors in the up-regulation of HIF-1α. These results open the possibility that signal cascades that proceed entirely in the cell nucleus might be responsible for several PGE(2) effects that are assumed to be due to cell membrane EP receptors.

Prostaglandin E2 induces retinoic acid receptor-β up-regulation through MSK1

The pharmacological modulation of putative renoprotective factors hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. In human renal proximal tubular HK2 cells, prostaglandin E2 (PGE2) up-regulates HIF-1α and VEGF-A through epidermal growth factor receptor (EGFR)-dependent up-regulation of retinoic acid receptor-β (RARβ). Here we studied the role of mitogen-activated protein kinases (MAPKs) ERK1/2 and p38 and their target kinase, mitogen- and stress activated kinase-1 (MSK1), in the signaling cascade. Treatment of HK2 cells with PGE2 resulted in increased phosphorylation of EGFR, the three studied kinases and the histone H3 (Ser10) at the RARβ gene promoter (the latter has been proposed as a molecular signature of the activated RARβ gene promoter). Prevention of the phosphorylation of EGFR, ERK1/2, p38 MAPK or MSK1 is by incubating, respectively, with AG1478, PD98059, SB203580 or H89 allowed to elucidate the precise phosphorylation order in the signaling cascade triggered by PGE2: first, EGFR; then, ERK1/2 and p38 MAPK and, finally, MSK1. Phosphorylation of MSK1 led to that of Ser10 in histone H3 and to activation of RARβ gene transcription (and the consequent increase in the expression of HIF-1α and VEGF-A), which was suppressed by H89 or by transfecting cells with a vector encoding for a dominant-negative mutant of MSK1. These results highlight the relevance of MSK1 in the up-regulation of RARβ by PGE2. They also may contribute to new therapeutic approaches based upon the pharmacological control of HIF-1α/VEGF-A in the proximal tubule through the modulation of the PGE2/EGFR/MAPK/MSK1/RARβ pathway.

Microparticles released by vascular endothelial cells increase hypoxia inducible factor expression in human proximal tubular HK-2 cells

Microparticles are produced by vesiculation of the cell plasma membrane and serve as vectors of cell-to-cell communication. Co-culture experiments have shown that hypoxia-inducible factor-α (HIF-α)-regulated-genes are up-regulated in human renal proximal tubular HK-2 cells by endothelial cell factors which might be transported inside endothelial microparticles (EMP). Here we aimed to study in HK-2 cells the effect of EMP, produced by activated endothelial cells, on HIF-α and HIF-α-regulated vascular endothelial growth factor-A (VEGF-A). EMP, at a concentration much lower than that found in plasma, increased the expression of HIF-α/VEGF-A in a COX-2/EP2 receptor dependent manner. Since the EMP/cells ratio was ∼1/1000, we hypothesized that paracrine mediators produced by HK-2 cells amplified the initial signal. This hypothesis was confirmed by two facts which also suggested that the mediators were conveyed by particles released by HK-2 cells: (i) HIF-α was up-regulated in HK-2 cells treated with the pellet obtained from the conditioned medium of the EMP-treated HK-2 cells. (ii) In transwell experiments, EMP-treated cells increased the expression of HIF-α in untreated HK-2 cells. Interestingly, we detected these cells, particles that were released by EMP-treated HK-2 cells. Depending on the pathological context, activation of HIF-α and VEGF-A signaling in renal tissue/cells may have either beneficial or harmful effects. Therefore, our results suggest that their presence in the urinary space of EMP produced by activated endothelial cells may influence the outcome of a number of renal diseases.

Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug-induced osteomalacia

Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (-10%; p = 0.03), and a nonsignificant change in 24R,25(OH)2D3 (-8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.

Intracrine prostaglandin E2 signalling regulates hypoxia-inducible factor-1α expression through retinoic acid receptor-β

We have previously found in human renal proximal tubular HK-2 cells that hypoxia- and all-trans retinoic acid-induced hypoxia-inducible factor-1α up-regulation is accompanied by retinoic acid receptor-β up-regulation. Here we first investigated whether hypoxia-inducible factor-1α expression is dependent on retinoic acid receptor-β and our results confirmed it since (i) hypoxia-inducible factor-1α-inducing agents hypoxia, hypoxia-mimetic agent desferrioxamine, all-trans retinoic acid and interleukin-1β increased retinoic acid receptor-β expression, (ii) hypoxia-inducible factor-1α up-regulation was prevented by retinoic acid receptor-β antagonist LE-135 or siRNA retinoic acid receptor-β and (iii) there was direct binding of retinoic acid receptor-β to the retinoic acid response element in hypoxia-inducible factor-1α promoter upon treatment with all-trans retinoic acid and 16,16-dimethyl-prostaglandin E2. Since intracellular prostaglandin E2 mediates hypoxia-inducible factor-1α up-regulation in normoxia in HK-2 cells, we next investigated and confirmed, its role in the up-regulation of retinoic acid receptor-β in normoxia by hypoxia-inducible factor-1α-inducing agents all-trans retinoic acid, interleukin-1β and 16,16-dimethyl-prostaglandin E2 by inhibiting cyclooxygenases, prostaglandin influx transporter or EP receptors. Interestingly, the hypoxia-induced increase in retinoic acid receptor-β expression and accumulation of hypoxia-inducible factor-1α was also blocked by the inhibitors tested. This is the first time, to our knowledge, that retinoic acid receptor-β signalling is involved in the control of the expression of transcription factor hypoxia-inducible factor-1α in both normoxia and hypoxia and that retinoic acid receptor-β expression is found to be strictly regulated by intracellular prostaglandin E2. Given the relevance of hypoxia-inducible factor-1α in the kidney in terms of tumorigenesis, progressive renal failure, production of erythropoietin and protection in several models of renal disease, our results open new therapeutic opportunities on the control of hypoxia-inducible factor-1α based upon the pharmacological modulation of retinoic acid receptor-β, either directly or through the control of intracellular prostaglandin E2 levels/signalling.

Melamine Induces Human Renal Proximal Tubular Cell Injury via Transforming Growth Factor-β and Oxidative Stress

Although several reports have failed to observe adverse subchronic renal effects following relatively high melamine exposure, the safety of low and continuous melamine exposure is still debatable. Recent studies suggest that long-term, low-dose melamine exposure is associated with an increased risk of urolithiasis, which has been linked to chronic kidney disease (CKD). CKD is a consequence of nephron loss and is associated with the interaction of inflammation, oxidative stress, and transforming growth factor-β (TGF-β), which increases extracellular matrix genes and cell apoptosis with progression to fibrosis and end-stage renal disease. Thus far, information is still lacking regarding the influence of melamine at the gene and protein levels, which are activated at a much earlier phase than the occurrence of the renal morphological change. In this study, we stimulated human renal proximal tubular HK-2 cells with melamine (0, 125, 250, 500, or 1000 µg/ml) for different time intervals and observed its effects on several well-documented molecular mechanisms of CKD. Here, we demonstrate that melamine can activate mitogen-activated protein kinases, NFκB, and reactive oxygen species, which results in the upregulation of interleukin-6, monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and TGF-β1 in HK-2 cells. The melamine-stimulated overexpression of TGF-β1 not only promotes fibronectin production but also leads to decreased antiapoptotic (bcl-2, bcl-xl)/proapoptotic (bad, bax) protein ratio, increased caspase-3 and caspase-9 activities, and eventually HK-2 cell apoptosis. Our study suggests that melamine exposure may be a risk factor for the chronic loss of tubular cells and may ultimately lead to tubulointerstitial damage.