Nephrotoxicity is a major limiting factor in the use of aminoglycoside antibiotics, the mechanisms for which are still speculative. To clarify the mechanisms of renal tubular cell death induced by aminoglycosides, we examined the renal proximal tubule-like cell line, LLC-PK1, after inducing apoptosis through a chronic treatment with gentamicin (GM). Changes in the expression of the Fas were also investigated. On flow cytometric analysis, 5.7 +/- 3.3% of the control cells appeared in a region of decreased forward light scatter and increased side light scatter, where both indices represent the characteristics of apoptotic cell death. Compared to the control, treatment with 10 mM of GM for 15 days significantly increased the proportion of cells in the apoptotic region to 23.9 +/- 8.5%. This finding was supported by electrophoretic analysis of the DNA extracted from the GM-treated cells, where a series of bands corresponding to integer multiples of 180 to 200 base pairs was visualized. However, the 15-day GM treatment did not cause a significant elevation in the expression of the 45 kD Fas protein, the cell surface molecule that stimulates apoptosis, by Western blot analysis. In conclusion, long-term exposure to GM induces apoptosis of the renal tubular epithelial cells, and this process may contribute to some of the aminoglycoside nephrotoxicities. Further studies are needed on the mechanism(s) of apoptosis induced by GM.
Animal ; Antibiotics, Aminoglycoside/toxicity* ; Antigens, CD95/analysis ; Apoptosis/drug effects* ; Cell Line ; Gentamicins/toxicity* ; Kidney Tubules, Proximal/pathology ; Kidney Tubules, Proximal/drug effects* ; Swine

The present study was designed to investigate the effect of administration of adrenomedullin (ADM) into subfornical organ (SFO) on renal tubular Na(+),K(+)-ATPase activity in rats. Rats under anesthesia were injected with ADM 0.1 mL (20 ng/mL) via an implanted cannula into SFO (n=6). Plasma ADM and serum endogenous digitalis-like factor (EDLF) levels were assayed with radioimmunoassay, and urine samples were collected via a canoula intubated in bladder. Urinary sodium concentration was assayed with flame spectrophotometry. Single proximal renal tubule segments were obtained by hand under stereomicroscope and its Na(+),K(+)-ATPase activity was measured by liquid scintillation counting. In addition, single proximal renal tubule segments from normal rats (n=6) were incubated with serum from animals administered with ADM into SFO, and then the Na(+),K(+)-ATPase activity was determined. The results showed that both urinary volume and sodium excretion amounted to the peak value at 30 min after ADM administration, and sustained a significant high level at 60 min (P<0.01). At 30 min after ADM administration, there was a significant increase in serum EDLF and a decrease in Na(+),K(+)-ATPase activity of proximal tubule (P<0.01, respectively), but not in plasma ADM level. Na(+),K(+)-ATPase activity was decreased significantly in single proximal renal tubule segments from normal rats incubated with serum from rats administered with ADM into SFO (P<0.01). These results suggest that the diuretic and natriuretic responses following administration of ADM into SFO are associated with the inhibition of renal tubule Na(+),K(+)-ATPase activity. The inhibition of renal tubule Na(+),K(+)-ATPase activity is related to the increase in the serum level of EDLF.
Adrenomedullin ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Rats ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

OBJECTIVETo explore the toxic effects of lead acetate on the apoptosis and ultrastructure of human renal tubular epithelial cells (HK-2).

METHODSAfter HK-2 cells were exposed to 5, 10 and 20 µmol/L lead acetate for 24 h, the morphological changes of HK-2 cells were observed by Hochest 33342-PI staining, and the ultrastructure changes of HK-2 cells were examined under a electron microscope, LDH activity and MDA content in supernatant of HK-2 cellular culture were detected by spectrophotometer, DNA damage of HK-2 was determined by DNA ladder and the apoptotic rates of HK-2 cells were measured by flow cytometry.

RESULTSThe morphological changes of apoptotic HK-2 cells in exposure group were observed by Hochest 33342-PI staining. The cytoplasm vacuoles, karyopycnosis, nuclear membrane vague and apoptotic bodies in HK-2 cells of exposure group were found under electron microscopy. LDH activity and MDA contents in exposure group increased significantly, as compared to control group (P < 0.01). The results of DNA Ladder showed that DNA damage of HK-2 cells in exposure group appeared. The apoptotic rates of HK-2 cells exposed to 5, 10, 20 µmol/L lead acetate were 14.16% ± 2.94%, 19.45% ± 2.73%, 25.01% ± 3.97%, respectively, which were significantly higher than that (5.81% ± 2.18%) in control group (P < 0.05).

CONCLUSIONLead acetate could remarkably induce the apoptosis of HK-2 cells and affect the kidney.

Apoptosis ; drug effects ; Cell Line ; Epithelial Cells ; cytology ; drug effects ; ultrastructure ; Humans ; Kidney Tubules, Proximal ; cytology ; drug effects ; ultrastructure ; Organometallic Compounds ; toxicity

OBJECTIVETo study effects of Yishen Kangxian Compound (YKC) and benazepril containing serums on HK-2 cells (human renal proximal tubule epithelial cells) in the process of renal tubular epithelial cells to mesenchymal myofibroblasts transdifferentiation (TEMT) by gene chip.

METHODSYKC and benazepril containing serums were prepared. Their inhibitory effects on HK-2 cells in the transforming growth factor-beta1 (TGF-beta1)-induced TEMT process were observed. HK-2 cells were randomly divided into four groups, i.e., the blank control group, the model group, the benazepril group, and the YKC group. The gross RNAs were extracted and purified by taking advantage of the HumanHT-12 v4 of IlluminaBeadChip. Differentially expressed genes were obtained after they were reversely transcribed to cDNA, incorporating biotin labeling probe, hybridized with GeneChip, picture signals of fluorescence in gene array scanned and compared with differential genes by computer analysis.

RESULTSDifferentially expressed genes were successfully identified by gene chip. Compared with the model group, there were 227 differentially expressed genes in the benazepril group, including 118 up-regulated genes and 109 downregulated genes. Compared with the model group, there were 97 differentially expressed genes in the YKC group, including 69 up-regulated genes and 28 down-regulated genes. The Gene Ontology (GO) analysis indicated that YKC was more actively involved in the regulatory process than benazepril in terms of cell damage, apoptosis, growth, NF-KB, protein kinase, neuron, and blood vessel growth.

CONCLUSIONSYKC and benazepril could inhibit the TEMT process of HK-2 cells. But YKC also had taken part in cell damage, apoptosis, growth,and more pathways of early stage TEMT.

Cell Line ; Cell Transdifferentiation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; Genomics ; Humans ; Kidney Tubules, Proximal ; cytology ; pathology

OBJECTIVETo investigate the effects of microRNA-145 (miR-145) on epithelial-mesenchymal transition (EMT) of TGF-β1-induced human renal proximal tubular epithelial (HK-2) cells.

METHODSThe gene sequence of miR-145 was synthesized and cloned into pCMV-myc to construct recombinant plasmid pCMV-miR-145. HK-2 cells were divided into four groups: control (untreated), TGF-β1 (treated with TGF-β1), blank+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with blank plasmid) and miR-145+TGF-β1 (treated with TGF-β1 after HK-2 cells transfected with pCMV-miR-145 recombinant plasmid). Expression of miR-145 was detected by real-time PCR (RT-PCR). TGF-β1, Smad3, Smad2/3, p-Smad2/3, α-SMA, FN and type I collagen (Col I) protein levels were detected by Western blot. Concentrations of fibronectin (FN) and Col I in cell culture supernatants were measured using ELISA.

RESULTSpCMV-miR-145 recombinant plasmid was successfully transfected into HK-2 cells. Compared with the control group, the miR-145+TGF-β1 group showed a significant up-regulation in the expression level of miR-145 (P<0.01). However, the TGF-β1 and blank+TGF-β1 groups showed a significant down-regulation in the expression level of miR-145 compared with that in the control and miR-145+TGF-β1 groups (P<0.01). Compared with the TGF-β1 and blank+TGF-β1 groups, the miR-145+TGF-β1 group showed significantly reduced levels of the signal proteins TGF-β1, Smad3, Smad2/3 and p-Smad2/3 (P<0.05), as well as significantly reduced levels of the biomarkers α-SMA, FN and Col I (P<0.05). Meanwhile, concentrations of FN and Col I in cell culture supernatants also decreased (P<0.05).

CONCLUSIONSmiR-145 modulates the EMT of HK-2 cells treated with TGF-β1, possibly by inhibition of the activation of TGF-β-dependent Smad signaling pathway.

Cells, Cultured ; Epithelial Cells ; drug effects ; pathology ; Epithelial-Mesenchymal Transition ; Humans ; Kidney Tubules, Proximal ; drug effects ; pathology ; MicroRNAs ; physiology ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVETo observe the effect of bone morphogenetic protein-7 (BMP-7) on aristolchic acid induced renal tubular epithelial cell trans-differentiation to look for new therapeutic approach for aristolchic acid nephropathy (AAN).

METHODSIn vitro cultured human proximal renal tubular epithelial cell line HK-2 cells were treated with different concentrations of BMP-7 (75 ng/mL, 150 ng/mL and 300 ng/mL) after trans-differentiation of the cells was induced by AA (10 microg/mL). Levels of alpha-SMA mRNA and protein expressions were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively.

RESULTSBMP-7 reversed the AA inducing alpha-SMA expressions in HK-2 cells in a dose-dependent manner.

CONCLUSIONBMP-7 can inhibit the trans-differentiation of human renal tubular epithelial cell induced by AA, thereby might be a new potential drug for AAN prevention and treatment.

Actins ; metabolism ; Aristolochic Acids ; adverse effects ; Bone Morphogenetic Protein 7 ; pharmacology ; Cell Line ; Cell Transdifferentiation ; drug effects ; Epithelial Cells ; cytology ; Humans ; Kidney Tubules, Proximal ; cytology

AIMTo investigate the effect of micro-injection Ang II into the subfornical organ (SFO) on the proximal tubules (PT) Na+, K(+)-ATPase activity in rats and its mechanism.

METHODSSFO in SD rats was administrated respectively with Ang II (20 ng), or losartan (5 microg) and AngII (20 ng) successively. The levels of serum EDLS and plasm AngII were assessed with radioimmunoassay (RIA). The PT segments were microdissected freehand and their Na+, K(+)-ATPase activities were assessed by liquid scintillation counter (LSC).

RESULTSThe serum EDLS levels increased significantly compared with a CSF group after SFO administration with Ang II; The Na+, K(+)-ATPase activities in PT segments decreased significantly at 30 min and 60 min after SFO administration with Ang II. There was a negative linear correlation between serum EDLS level and the Na+, K(+)-ATPase activity of PT segments in rats administrated with Ang II (r = -0.938).

CONCLUSIONInhibition of the Na+, K(+)-ATPase activity in PT as a result of administration of Ang II in SFO is mediated by AT1 receptors. The increase in EDLS release may play an important role in this inhibition.

Angiotensin II ; administration & dosage ; pharmacology ; Animals ; Kidney Tubules, Proximal ; drug effects ; enzymology ; Male ; Rats ; Rats, Sprague-Dawley ; Sodium-Potassium-Exchanging ATPase ; metabolism ; Subfornical Organ

Animals ; Cell Line ; Diabetic Nephropathies ; metabolism ; Epithelial-Mesenchymal Transition ; drug effects ; Eukaryotic Initiation Factor-2 ; metabolism ; Glucose ; pharmacology ; Humans ; Kidney ; drug effects ; metabolism ; pathology ; Kidney Tubules, Proximal ; drug effects ; metabolism ; Rats ; Signal Transduction ; drug effects

OBJECTIVETo observe the expressions of Wnt/beta-catenin and the effects of tanshinone IIA (TII A) on Wnt/beta-catenin signaling pathway in high glucose induced renal tubular epithelial cell transdifferentiation.

METHODSHuman kidney proximal tubular epithelial cells (HK-2) were divided into three groups, i. e., the normal glucose group, the high glucose group, and the high glucose plus tanshinone IIA group. The expression of beta-catenin was observed using immunocytochemical staining. The protein expression of beta-catenin, E-cadherin, and alpha-smooth muscle actin (alpha-SMA) were detected by Western blot. The mRNA levels of beta-catenin and E-cadherin were detected by RT-PCR.

RESULTSCompared with the normal glucose group, both the protein and the mRNA expressions of beta-catenin were significantly enhanced (P < 0.01), the expression of E-cadherin significantly decreased (P < 0.01), the expression of beta-catenin increased in the cytoplasm and nucleus in the high glucose group. TIIA at the final concentration of 100 micromol/L significantly reduced the ectopic expression of beta-catenin. At that concentration, the protein and mRNA expressions of beta-catenin in the nucleus significantly decreased, while the protein and mRNA expressions of E-cadherin were up-regulated. Meanwhile, the expression of alpha-SMA obviously decreased.

CONCLUSIONSWnt/beta-catenin signaling pathway participated in the high glucose induced renal tubular epithelial cell transdifferentiation. TIIA inhibited the transdifferentiation process possibly through down-regulating the activities of Wnt/beta-catenin signaling pathway, thus further playing a role in renal protection.

Cadherins ; metabolism ; Cell Line ; Cell Transdifferentiation ; drug effects ; Diterpenes, Abietane ; pharmacology ; Epithelial Cells ; cytology ; drug effects ; metabolism ; Glucose ; adverse effects ; Humans ; Kidney Tubules, Proximal ; cytology ; drug effects ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo study whether aristololactam I (AL-I) can enter renal proximal tubular epithelial cells and the situation of intracellular distribution and accumulation.

METHODCultured human renal proximal tubular epithelial cell line (HK-2) was used as the subject. Intracellular fluorescence from AL-I and its distribution are examined by fluorescence microscopy after a treatment with different concentration of AL-I, the intracellular accumulation of AL-I was also investigated by incubated cells in AL-I -free medium for 48 h after washing-out the media containing AL-I.

RESULTAfter treatment of AL-I (concentration from 5 microg x mL(-1) to 20 microg x mL(-1)), glaucous fluorescence could be observed inside renal proximal tubular epithelial cells at 0.5 h, and the fluorescence distributed only in cytoplasm while not be observed in nuclei. Moreover, the fluorescence of AL-I could be kept in cytoplasm for more than 48 h after washing out the media containing AL-I .

CONCLUSIONAL-I is able to enter renal proximal tubular epithelial cells in short time and accumulate in cytoplasm, but not enter nuclei. This property may contribute to the cytotoxic mechanism of renal injury induced by AL-I, which may partially explain the persistent renal toxicity of AAs and its metabolites in the development of aristolochic acid nephropathy.

Animals ; Aristolochic Acids ; metabolism ; toxicity ; Cell Line ; Cell Nucleus ; drug effects ; metabolism ; Cytoplasm ; drug effects ; metabolism ; Epithelial Cells ; cytology ; drug effects ; metabolism ; pathology ; Humans ; Kidney Diseases ; metabolism ; pathology ; Kidney Tubules, Proximal ; cytology ; pathology ; Microscopy, Fluorescence