Renal Proximal Tubular Epithelial HK-2 Research Articles

Piperlonguminine attenuates renal fibrosis by inhibiting TRPC6

Ethnopharmacological relevanceLiuwei Dihuang (LWDH) is a classic prescription that has been used to the treatment of “Kidney-Yin” deficiency syndrome for more than 1000 years in China. Recent studies have confirmed that LWDH can prevent the progression of renal fibrosis. Numerous studies have demonstrated the critical role that TRPC6 plays in the development of renal fibrosis. Due to the complex composition of LWDH and its remarkable therapeutic effect on renal fibrosis, it is possible to discover new active ingredients targeting TRPC6 for the treatment of renal fibrosis. Aim of studyThis study aimed to identify selective TRPC6 inhibitors from LWDH and evaluate their therapeutical effects on renal fibrosis. Materials and methodsComputer-aided drug design was used to screen the biologically active ingredients of LWDH, and their affinities to human TRPC6 protein were detected by microcalorimetry. TRPC6, TRPC3, and TRPC7 over-expressed HEK293 cells were constructed, and the selective activities of the compounds on TRPC6 were determined by measuring [Ca2+]i in these cells. To establish an in vitro model of renal fibrosis, human renal proximal tubular epithelial HK-2 cells were stimulated with TGF-β1. The therapeutic effects of LWDH compounds on renal fibrosis were then tested by detecting the related proteins. TRPC6 was knocked-down in HK-2 cells to investigate the effects of LWDH active ingredients on TRPC6. Finally, a unilateral ureteral obstruction model of renal fibrosis was established to test the therapeutic effect. ResultsFrom hundreds of LWDH ingredients, 64 active components with oral bioavailability ≥30% and drug-likeness index ≥0.18 were acquired. A total of 10 active components were obtained by molecular docking with TRPC6 protein. Among them, 4 components had an affinity with TRPC6. Piperlonguminine (PLG) had the most potent affinity with TRPC6 and blocking effect on TRPC6-mediated Ca2+ entry. A 100 μM of PLG showed no detectable inhibition on TRPC1, TRPC3, TRPC4, TRPC5, or TRPC7-mediated Ca2+ influx into cells. In vitro results indicated that PLG concentration-dependently inhibited the abnormally high expression of α-smooth muscle actin (α-SMA), collagen I, vimentin, and TRPC6 in TGF-β1-induced HK-2 cells. Consistently, PLG also could not further inhibit TGF-β1-induced expressions of these protein biomarkers in TRPC6 knocked-down HK-2 cells. In vivo, PLG dose-dependently reduced urinary protein, serum creatinine, and blood urea nitrogen levels in renal fibrosis mice and markedly alleviated fibrosis and the expressions of α-SMA, collagen I, vimentin, and TRPC6 in kidney tissues. ConclusionOur results showed that PLG had anti-renal fibrosis effects by selectively inhibiting TRPC6. PLG might be a promising therapeutic agent for the treatment of renal fibrosis.

Aldehyde dehydrogenase 2 protects against acute kidney injury by regulating autophagy via Beclin-1 pathway

The mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of acetaldehyde and endogenous lipid aldehydes. Approximately 40% of East Asians, accounting for 8% of the human population, carry the E504K mutation in ALDH2 that leads to accumulation of toxic reactive aldehydes and increases the risk for cardiovascular disease, cancer, and Alzheimer disease, among others. However, the role of ALDH2 in acute kidney injury (AKI) remains poorly defined and is therefore the subject of the present study using various cellular and organismal sources. In murine models, in which AKI was induced by either the contrast agent iohexol or renal ischemia/reperfusion, KO, activation/overexpression of ALDH2 were associated with increased and decreased renal injury, respectively. In murine renal tubular epithelial cells (RTECs), ALDH2 upregulated Beclin-1 expression, promoted autophagy activation, and eliminated ROS. In vivo and in vitro, both 3-MA and Beclin-1 siRNAs inhibited autophagy and abolished ALDH2-mediated renoprotection. In mice with iohexol-induced AKI, ALDH2 knockdown in RTECs using AAV-shRNA impaired autophagy activation and aggravated renal injury. In human renal proximal tubular epithelial HK-2 cells exposed to iohexol, ALDH2 activation potentiated autophagy and attenuated apoptosis. In mice with AKI induced by renal ischemia/reperfusion, ALDH2 overexpression or pretreatment regulated autophagy mitigating apoptosis of RTECs and renal injury. In summary, our data collectively substantiate a critical role of ALDH2 in AKI via autophagy activation involving the Beclin-1 pathway.

Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

糖原合成激酶-3β/β-连环蛋白信号通路在贫铀致人肾近曲小管上皮细胞损伤中的作用

Objective To investigate the effects of glycogen synthase kinase-3β (GSK-3β) and β-catenin signaling on the human renal proximal tubular epithelial HK-2 cell injury induced by depleted uranium(DU), and provide a new enlightenment for the development of DU antidotes. Methods HK-2 cells were exposed to different concentrations of DU for 3-24 h, then the protein expressions of kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL) and nuclear β-catenin were detected by immunofluorescence staining. The protein expressions of p-GSK-3β(S9), GSK-3β and c-myc were detected by Western blot assay. HK-2 cells were transiently transfected by GSK-3β (KD) plasmid or treated by TDZD-8 to inhibit the activity of GSK-3β specifically. Other HK-2 cells were transiently transfected by β-catenin plasmid to overexpress the β-catenin protein. Results The percentages of KIM-1 and NGAL-positive cells increased with DU exposure time and concentrations from 300 and 600 μmol/L, and they were significantly higher than those of the blank control at 6-24 h of DU exposure (KIM-1-positive cells: t=11.06, 18.97, 30.49, P<0.05; t=6.79, 16.02, 85.45, P<0.05; NGAL-positive cells: t=11.78, 11.37, 34.29, P<0.05; t=7.34, 21.63, 36.84, P<0.05). In contrast, the ratio of p-GSK-3β(S9) to GSK-3β and percentage of nuclear β-catenin-positive cells were significantly higher than that of the blank control at 3-24 h of DU exposure (p-GSK-3β(S9)/GSK-3β: t=3.95, 4.69, 5.40, 3.34, P<0.05; nuclear β-catenin-positive cells: t=4.61, 6.52, 36.64, 14.93, P<0.05) with a maximum response at 9 h of DU exposure accompanied with corresponding increase of protein level of c-myc, a downstream target gene of β-catenin. Transient transfection of HK-2 cells with GSK-3β(KD) plasmid significantly inhibited the activity of GSK-3β (t=8.07, P<0.05) and reduced the DU-increased percentage of KIM-1-positive cells (t=24.77, P<0.05). Treatment cells with TDZD-8 inhibited the activity of GSK-3β and enhanced the percentage of nuclear β-catenin-positive cells, and it also significantly reduced the percentage of KIM-1-positive cells in HK-2 cells exposed to DU (t=6.25, 6.73, P<0.05). Moreover, overexpression of β-catenin significantly reduced DU-induced cell injury (t=7.48, P<0.05). Conclusions GSK-3β/β-catenin signaling plays a key role in regulating the DU-induced cytotoxicity of HK-2 cells. Inhibition of GSK-3β activity and overexpression of β-catenin can protect the HK-2 cells from DU-induced damage. Key words: Depleted uranium; GSK-3β; β-catenin; KIM-1; HK-2 cell

Atrasentan increased the expression of klotho by mediating miR-199b-5p and prevented renal tubular injury in diabetic nephropathy

Atrasentan is a promising therapy for treating diabetic nephropathy (DN). Here we evaluated whether atrasentan down-regulated the miR-199b-5p expression, thereby increasing klotho and preventing renal tubular injury in DN. One-hundred patients with type 2 diabetes mellitus (T2DM) and 40 healthy subjects were included. A DN mice model was established by an injection of streptozotocin (STZ). Human renal proximal tubular epithelial HK-2 cells were exposed to high glucose (20 mmol/L). Treated the mice and HK-2 cells with atrasentan, and we then investigated whether and how miR-199b-5p and Klotho were involved in preventing renal tubular injury in DN. In patients, the serum miR-199b-5p level increased and the klotho concentration decreased in accordance with elevated albuminuria. Atrasentan down-regulated miR-199b-5p and up-regulated klotho of the DN mice and HK-2 cells exposed to high glucose. High glucose promoted the binding of histone H3 to the miR-199b-5p promoter, and atrasentan canceled this effect. MiR-199b-5p targeted the 3′ UTR of klotho. Overexpression of miR-199b-5p canceled the effects of atrasentan on klotho expression and apoptosis of renal tubular cells in both in vivo and in vitro. The increased serum klotho, mediated by miR-199b-5p, is a possible mechanism by which atrasentan prevents renal tubular injury in DN.

Chronic alcohol exposure negatively impacts the physiological and molecular parameters of the renal biotin reabsorption process

Normal body homeostasis of biotin is critically dependent on its renal recovery by kidney proximal tubular epithelial cells, a process that is mediated by the sodium-dependent multivitamin transporter (SMVT; a product of the SLC5A6 gene). Chronic ethanol consumption interferes with the renal reabsorption process of a variety of nutrients, including water-soluble vitamins. To date, however, there is nothing known about the effect of chronic alcohol feeding on physiological and molecular parameters of the renal biotin reabsorption process. We addressed these issues using rats and transgenic mice carrying the human SLC5A6 (P1P2) 5'-regulatory region as an in vivo model systems of alcohol exposure, and cultured human renal proximal tubular epithelial HK-2 cells chronically exposed to alcohol as an in vitro model of alcohol exposure. The [(3)H]biotin uptake results showed that chronic ethanol feeding in rats leads to a significant inhibition in carrier-mediated biotin transport across both renal brush border and basolateral membrane domains. This inhibition was associated with a marked reduction in the level of expression of SMVT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Furthermore, studies with transgenic mice carrying the SLC5A6 5'-regulatory region showed that chronic alcohol feeding leads to a significant decrease in promoter activity. Studies with HK-2 cells chronically exposed to alcohol again showed a marked reduction in carrier-mediated biotin uptake, which was associated with a significant reduction in promoter activity of the human SLC5A6 5'-regulatory region. These findings demonstrate for the first time that chronic ethanol feeding inhibits renal biotin transport and that this effect is, at least in part, being exerted at the transcriptional level.