HK-2 cell response to TGF-ß highly depends on cell culture medium formulations.

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-ß) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-ß-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-ß1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-ß1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

Pioglitazone Protects Tubular Epithelial Cells during Kidney Fibrosis by Attenuating miRNA Dysregulation and Autophagy Dysfunction Induced by TGF-ß.

Excessive renal TGF-ß production and pro-fibrotic miRNAs are important drivers of kidney fibrosis that lack any efficient treatment. Dysfunctional autophagy might play an important role in the pathogenesis. We aimed to study the yet unknown effects of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone (Pio) on renal autophagy and miRNA dysregulation during fibrosis. Mouse primary tubular epithelial cells (PTEC) were isolated, pre-treated with 5 µM pioglitazone, and then stimulated with 10 ng/mL TGF-ß1 for 24 h. Male 10-week-old C57Bl6 control (CTL) and TGF-ß overexpressing mice were fed with regular chow (TGF) or Pio-containing chow (20 mg/kg/day) for 5 weeks (TGF + Pio). PTEC and kidneys were evaluated for mRNA and protein expression. In PTEC, pioglitazone attenuated (p < 0.05) the TGF-ß-induced up-regulation of Col1a1 (1.4-fold), Tgfb1 (2.2-fold), Ctgf (1.5-fold), Egr2 (2.5-fold) mRNAs, miR-130a (1.6-fold), and miR-199a (1.5-fold), inhibited epithelial-to-mesenchymal transition, and rescued autophagy function. In TGF mice, pioglitazone greatly improved kidney fibrosis and related dysfunctional autophagy (increased LC3-II/I ratio and reduced SQSTM1 protein content (p < 0.05)). These were accompanied by 5-fold, 3-fold, 12-fold, and 2-fold suppression (p < 0.05) of renal Ccl2, Il6, C3, and Lgals3 mRNA expression, respectively. Our results implicate that pioglitazone counteracts multiple pro-fibrotic processes in the kidney, including autophagy dysfunction and miRNA dysregulation.

Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression.

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-ß1) under the control of a mouse albumin promoter (Alb/TGF-ß1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-ß1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFß and CBAxB6-TGFß (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFß mice developed severe kidney fibrosis and premature death, while B6-TGF-ß mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFß mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-ß induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.

Condition-dependent functional shift of two Drosophila Mtmr lipid phosphatases in autophagy control.

Myotubularin (MTM) and myotubularin-related (MTMR) lipid phosphatases catalyze the removal of a phosphate group from certain phosphatidylinositol derivatives. Because some of these substrates are required for macroautophagy/autophagy, during which unwanted cytoplasmic constituents are delivered into lysosomes for degradation, MTM and MTMRs function as important regulators of the autophagic process. Despite its physiological and medical significance, the specific role of individual MTMR paralogs in autophagy control remains largely unexplored. Here we examined two Drosophila MTMRs, EDTP and Mtmr6, the fly orthologs of mammalian MTMR14 and MTMR6 to MTMR8, respectively, and found that these enzymes affect the autophagic process in a complex, condition-dependent way. EDTP inhibited basal autophagy, but did not influence stress-induced autophagy. In contrast, Mtmr6 promoted the process under nutrient-rich settings, but effectively blocked its hyperactivation in response to stress. Thus, Mtmr6 is the first identified MTMR phosphatase with dual, antagonistic roles in the regulation of autophagy, and shows conditional antagonism/synergism with EDTP in modulating autophagic breakdown. These results provide a deeper insight into the adjustment of autophagy.Abbreviations: Atg, autophagy-related; BDSC, Bloomington Drosophila Stock Center; DGRC, Drosophila Genetic Resource Center; EDTP, Egg-derived tyrosine phosphatase; FYVE, zinc finger domain from Fab1 (yeast ortholog of PIKfyve), YOTB, Vac1 (vesicle transport protein) and EEA1 cysteine-rich proteins; LTR, LysoTracker Red; MTM, myotubularin; MTMR, myotubularin-related; PI, phosphatidylinositol; Pi3K59F, Phosphotidylinositol 3 kinase 59F; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(3,5)P2, phosphatidylinositol-3,5-bisphosphate; PtdIns5P, phosphatidylinositol-5-phosphate; ref(2)P, refractory to sigma P; Syx17, Syntaxin 17; TEM, transmission electron microscopy; UAS, upstream activating sequence; Uvrag, UV-resistance associated gene; VDRC, Vienna Drosophila RNAi Center; Vps34, Vacuolar protein sorting 34.

Developmentally regulated autophagy is required for eye formation in Drosophila.

The compound eye of the fruit fly Drosophila melanogaster is one of the most intensively studied and best understood model organs in the field of developmental genetics. Herein we demonstrate that autophagy, an evolutionarily conserved selfdegradation process of eukaryotic cells, is essential for eye development in this organism. Autophagic structures accumulate in a specific pattern in the developing eye disc, predominantly in the morphogenetic furrow (MF) and differentiation zone. Silencing of several autophagy genes (Atg) in the eye primordium severely affects the morphology of the adult eye through triggering ectopic cell death. In Atg mutant genetic backgrounds however genetic compensatory mechanisms largely rescue autophagic activity in, and thereby normal morphogenesis of, this organ. We also show that in the eye disc the expression of a key autophagy gene, Atg8a, is controlled in a complex manner by the anterior Hox paralog Lab (Labial), a master regulator of early development. Atg8a transcription is repressed in front of, while activated along, the MF by Lab. The amount of autophagic structures then remains elevated behind the moving MF. These results indicate that eye development in Drosophila depends on the cell death-suppressing and differentiating effects of the autophagic process. This novel, developmentally regulated function of autophagy in the morphogenesis of the compound eye may shed light on a more fundamental role for cellular self-digestion in differentiation and organ formation than previously thought. ABBREVIATIONS: αTub84B, α-Tubulin at 84B; Act5C, Actin5C; AO, acridine orange; Atg, autophagy-related; Ato, Atonal; CASP3, caspase 3; Dcr-2; Dicer-2; Dfd, Deformed; DZ, differentiation zone; eGFP, enhanced green fluorescent protein; EM, electron microscopy; exd, extradenticle; ey, eyeless; FLP, flippase recombinase; FRT, FLP recognition target; Gal4, gene encoding the yeast transcription activator protein GAL4; GFP, green fluorescent protein; GMR, Glass multimer reporter; Hox, homeobox; hth, homothorax; lab, labial; L3F, L3 feeding larval stage; L3W, L3 wandering larval stage; lf, loss-of-function; MAP1LC3, microtubule-associated protein 1 light chain 3; MF, morphogenetic furrow; PE, phosphatidylethanolamine; PBS, phosphate-buffered saline; PI3K/PtdIns3K, class III phosphatidylinositol 3-kinase; PZ, proliferation zone; Ref(2)P, refractory to sigma P, RFP, red fluorescent protein; RNAi, RNA interference; RpL32, Ribosomal protein L32; RT-PCR, reverse transcription-coupled polymerase chain reaction; S.D., standard deviation; SQSTM1, Sequestosome-1, Tor, Target of rapamycin; TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay; UAS, upstream activation sequence; qPCR, quantitative real-time polymerase chain reaction; w, white.

The small molecule AUTEN-99 (autophagy enhancer-99) prevents the progression of neurodegenerative symptoms.

Autophagy functions as a main route for the degradation of superfluous and damaged constituents of the cytoplasm. Defects in autophagy are implicated in the development of various age-dependent degenerative disorders such as cancer, neurodegeneration and tissue atrophy, and in accelerated aging. To promote basal levels of the process in pathological settings, we previously screened a small molecule library for novel autophagy-enhancing factors that inhibit the myotubularin-related phosphatase MTMR14/Jumpy, a negative regulator of autophagic membrane formation. Here we identify AUTEN-99 (autophagy enhancer-99), which activates autophagy in cell cultures and animal models. AUTEN-99 appears to effectively penetrate through the blood-brain barrier, and impedes the progression of neurodegenerative symptoms in Drosophila models of Parkinson's and Huntington's diseases. Furthermore, the molecule increases the survival of isolated neurons under normal and oxidative stress-induced conditions. Thus, AUTEN-99 serves as a potent neuroprotective drug candidate for preventing and treating diverse neurodegenerative pathologies, and may promote healthy aging.

AUTEN-67 (Autophagy Enhancer-67) Hampers the Progression of Neurodegenerative Symptoms in a Drosophila model of Huntington's Disease.

BACKGROUND: Autophagy, a lysosome-mediated self-degradation process of eukaryotic cells, serves as a main route for the elimination of cellular damage [1-3]. Such damages include aggregated, oxidized or misfolded proteins whose accumulation can cause various neurodegenerative pathologies, including Huntington's disease (HD). OBJECTIVE: Here we examined whether enhanced autophagic activity can alleviate neurophatological features in a Drosophila model of HD (the transgenic animals express a human mutant Huntingtin protein with a long polyglutamine repeat, 128Q). METHODS: We have recently identified an autophagy-enhancing small molecule, AUTEN-67 (autophagy enhancer 67), with potent neuroprotective effects [4]. AUTEN-67 was applied to induce autophagic activity in the HD model used in this study. RESULTS: We showed that AUTEN-67 treatment interferes with the progressive accumulation of ubiquitinated proteins in the brain of Drosophila transgenic for the pathological 128Q form of human Huntingtin protein. The compound significantly improved the climbing ability and moderately extended the mean life span of these flies. Furthermore, brain tissue samples from human patients diagnosed for HD displayed increased levels of the autophagy substrate SQSTM1/p62 protein, as compared with controls. CONCLUSIONS: These results imply that AUTEN-67 impedes the progression of neurodegenerative symptoms characterizing HD, and that autophagy is a promising therapeutic target for treating this pathology. In humans, AUTEN-67 may have the potential to delay the onset and decrease the severity of HD.