Membrane traffic after inhibition of endocytosis in renal proximal tubules

This study was performed to examine quantitatively the cellular organelles involved in membrane recycling after inhibition of luminal endocytosis in renal proximal tubules. Paraffin oil was microinfused into rat renal proximal convoluted tubules to prevent luminal endocytosis. After 1-2 hr the kidneys were fixed by perfusion and prepared for electron microscopy. Segment 1 proximal tubules infused with paraffin oil and control tubules from the same kidney were studied. In addition we examined proximal tubules from kidneys fixed by immersion 30 sec after removal of the kidney. In the oil-infused tubules the large endocytic vacuoles (greater than 0.5 micron) disappeared, the amount of small endocytic vacuoles (less than 0.5 micron) was reduced to about 10%, and the amount of dense apical tubules was significantly increased. The dense apical tubules were very seldom seen connected to the apical plasma membrane in controls but this was occasionally observed in tubules fixed by immersion and relatively often in oil-infused tubules. An ultrastructural morphometric analysis substantiated and extended the qualitative observations and provided quantitative estimates of volumes and surface areas for large endocytic vacuoles, lysosomes, mitochondria, small endocytic vacuoles, and dense apical tubules in control and experimental tubules. The results strongly support the suggestion that the dense apical tubules located in the apical cytoplasm represent the vehicle for the recycling of membrane from endocytic vacuoles back to the plasma membrane, and show that in renal proximal tubule cells small and large endocytic vacuoles are transformed into dense apical tubules when endocytosis is stopped.     

Aurothiomalate as an ultrastructural marker. Electron microscopy and x-ray microanalysis of various tissues after in vivo gold injections.

Rabbits and rats were given single injections of aurothiomalate by different routes. The animals were killed at progressive intervals, and sections from various organs were examined by electron microscopic x-ray microanalysis. Ultrastructurally, characteristic material was regularly found in vacuoles, dense and heterogeneous bodies of macrophages, hepatocytes and renal epithelial cells. Occasionally, other mesenchymal cells also contained gold. Histochemical and analytical tests showed that the gold-containing organelles were devoid of acid phosphatase activity. The generalized rapid spread, retention and selectivity of localization after a single small dose make aurothiomalate a useful marker substance for ultrastructural studies.     

Light and electron microscopic observations of fabrication, release, and fate of biphasic secretion granules produced by epididymal epithelial principal cells of the fan-throated lizard Sitana ponticeriana cuvier

The epididymis of the fan-throated lizard Sitana ponticeriana was examined with light and transmission electron microscopy to understand the cellular mechanisms of fabrication of secretion granules in epithelial principal cells, granule release into the lumen, and the fate of the dense structured granules after reaching the lumen. Principal cells of the ductus epididymis, except at the cauda, secrete electron-dense biphasic granules copiously, which decrease in abundance from the initial segment to corpus. The principal cell possesses a prominent Golgi apparatus and all versions of endoplasmic reticulum (ER), rough, smooth, and sparsely granulated. The material of the dense portion of the secretion granules, after processing at the Golgi apparatus, appears to accumulate in large ER cisternae in the supranuclear cytoplasm. It undergoes condensation when the cisternae become condensing vacuoles. Mitochondria appear to play a role in dense granule formation. The condensing vacuoles are displaced toward the apical cytoplasm when the material of the less dense portion is added to the condensing vacuoles at the Golgi area. Thus, the less dense and dense portions of the secretion granules are secreted and added to the condensing vacuoles separately. The composite granules are released into the lumen by exocytosis when the less dense portion merges with the luminal content, whereas the dense portion maintains its structured identity. The latter, initially measuring 1-2 microm in diameter, increases in size several times. It is inferred that these granules release their content gradually, resulting in the appearance of vacuoles, and suggesting that the granules have an insoluble matrix in which there is a sparingly soluble material. The substance leaching out of the granules appears to contribute to keeping the sperm quiescent and alive during storage in the male reproductive tract.     

Effect of estrophilic platinum complex on the mouse uterus

Resistance of hormone-dependent mammary carcinoma to cisplatin as a potent antitumor agent led to the synthesis of other estrophilic platinum complexes. In this investigation, the effects of a newly synthesized estrogen-receptor affine platinum complex on the mouse uterus were studied using light and electron-microscopy. The results have been compared with Tamoxifen, cisplatin and the estrophilic ligand. Both estrophilic ligand and estrophilic platinum complex produced strong estrogenic effects as well as features characteristic of the uterine epithelial cell in the luteal phase of the cycle, corresponding to a massive stimulation of the surface and glandular epithelial cells. The uteri showed large glandular lumina. An increase in the number of multivesicular and residual bodies, accompanied by a proliferation of eosinophilic granulocytes, was also seen. The appearance of inter- and intracellular lumina and the activation of smooth muscle cells represent further characteristic effects of the estrophilic ligand and estrophilic platinum complex. Anticipated increases in the incidence of cell death and/or deviant cyto-nuclear architecture in the uteri treated with cisplatin or platinum complex, were not observed.     

Contributions of the Epidermal Growth Factor Receptor to Acquisition of Platinum Resistance in Ovarian Cancer Cells

Acquisition of platinum resistance following first line platinum/taxane therapy is commonly observed in ovarian cancer patients and prevents clinical effectiveness. There are few options to prevent platinum resistance; however, demethylating agents have been shown to resensitize patients to platinum therapy thereby demonstrating that DNA methylation is a critical contributor to the development of platinum resistance. We previously reported the Epidermal Growth Factor Receptor (EGFR) is a novel regulator of DNA methyltransferase (DNMT) activity and DNA methylation. Others have shown that EGFR activation is linked to cisplatin treatment and platinum resistance. We hypothesized that cisplatin induced activation of the EGFR mediates changes in DNA methylation associated with the development of platinum resistance. To investigate this, we evaluated EGFR signaling and DNMT activity after acute cisplatin exposure. We also developed an in vitro model of platinum resistance to examine the effects of EGFR inhibition on acquisition of cisplatin resistance. Acute cisplatin treatment activates the EGFR and downstream signaling pathways, and induces an EGFR mediated increase in DNMT activity. Cisplatin resistant cells also showed increased DNMT activity and global methylation. EGFR inhibition during repeated cisplatin treatments generated cells that were more sensitive to cisplatin and did not develop increases in DNA methylation or DNMT activity compared to controls. These findings suggest that activation of EGFR during platinum treatment contributes to the development of platinum resistance. Furthermore, EGFR inhibition may be an effective strategy at attenuating the development of platinum resistance thereby enhancing the effectiveness of chemotherapeutic treatment in ovarian cancer.     

Epidermal growth factor receptor pathway analysis identifies amphiregulin as a key factor for cisplatin resistance of human breast cancer cells

The use of platinum complexes for the therapy of breast cancer is an emerging new treatment modality. To gain insight into the mechanisms underlying cisplatin resistance in breast cancer, we used estrogen receptor-positive MCF-7 cells as a model system. We generated cisplatin-resistant MCF-7 cells and determined the functional status of epidermal growth factor receptor (EGFR), MAPK, and AKT signaling pathways by phosphoreceptor tyrosine kinase and phospho-MAPK arrays. The cisplatin-resistant MCF-7 cells are characterized by increased EGFR phosphorylation, high levels of AKT1 kinase activity, and ERK1 phosphorylation. In contrast, the JNK and p38 MAPK modules of the MAPK signaling pathway were inactive. These conditions were associated with inactivation of the p53 pathway and increased BCL-2 expression. We investigated the expression of genes encoding the ligands for the ERBB signaling cascade and found a selective up-regulation of amphiregulin expression, which occurred at later stages of cisplatin resistance development. Amphiregulin is a specific ligand of the EGFR (ERBB1) and a potent mitogen for epithelial cells. After exposure to cisplatin, the resistant MCF-7 cells secreted amphiregulin protein over extended periods of time, and knockdown of amphiregulin expression by specific short interfering RNA resulted in a nearly complete reversion of the resistant phenotype. To demonstrate the generality and importance of our findings, we examined amphiregulin expression and cisplatin resistance in a variety of human breast cancer cell lines and found a highly significant correlation. In contrast, amphiregulin levels did not significantly correlate with cisplatin resistance in a panel of lung cancer cell lines. We have thus identified a novel function of amphiregulin for cisplatin resistance in human breast cancer cells.     

GDF15基因在卵巢上皮性癌组织中的表达及其临床意义

目的:探讨生长分化因子GDF15(Growth Differentiation Factor 15)基因在卵巢上皮性癌组织中的表达及其与铂类耐药的相关性。方法:应用免疫组化、western blot、RT-PCR等方法对80例原发性卵巢癌组织和卵巢癌顺铂敏感/耐药株A2780和CP70、SKOV3和SKOV3/DDP中生长分化因子GDF15表达水平进行测定。结果:生长分化因子GDF15的表达强度与卵巢癌铂类耐药性显著相关。在卵巢癌顺铂耐药株CP70、SKOV3/DDP中GDF15表达水平较顺铂敏感株A2780、SKOV3明显增高。结论:GDF15表达水平与卵巢癌发生发展及铂类耐药相关,对于卵巢癌患者早期筛选、预测预后具有一定的临床指导价值。     

Mechanism of cisplatin nephrotoxicity

cis-Diamminedichloroplatinum II (cisplatin) is widely used in cancer treatments. Renal dysfunction is the major toxic effect of this drug. Micropuncture studies suggest that cisplatin reduces single-nephron glomerular filtration rate (GFR) and causes a significant backleak of inulin across the renal tubule. Pathological alterations are localized to the S3 segment of the proximal tubule situated in the outer stripe of the outer medulla. Renal clearance studies in humans demonstrate that the free platinum clearance exceeds the GFR, which suggests that cisplatin or a platinum metabolite is actively secreted by the kidney. Studies with renal cortex slices indicate that platinum is accumulated by renal tissue against a concentration gradient. This uptake is blocked by metabolic inhibitors and the organic base triethanolamine. Heavy metals are thought to produce renal damage because of interaction with renal sulfhydryl (SH) groups. After cisplatin administration to rats, total renal SH groups decreased by 14% owing to a decrease of protein-bound SH groups. The greatest decline of SH groups occurred in the mitochondrial and cytosolic fractions. These fractions also had the highest platinum concentrations. These results suggest that the nephrotoxic effects of cisplatin may be related to depletion of SH groups, but a cause and effect relationship has not been definitively established.     

Edaravone inhibits acute renal injury and cyst formation in cisplatin-treated rat kidney

Background: Although cis-diamminedichloroplatinum (II) (cisplatin) is an effective anticancer agent, its clinical use is highly limited predominantly due to its adverse effects on renal functions. The present work examined the therapeutic potential of edaravone, a free radical scavenger, for inhibiting cisplatin-induced renal injury.

Methods: Edaravone, 3-methyl-1-phenyl-pyrazolin-5-one, was administrated intravenously at a dose of 30 mg/kg of body weight to male Wistar rats (200-220 g). After 30 min, cisplatin was injected intraperitoneally at a dose of 5 mg/kg of body weight. At the indicated times after the treatment, functions and histological changes of the kidney were analyzed. To test the therapeutic potential of edaravone in chemotherapy, its effect on the anticancer action of cisplatin was examined in ascites cancer-bearing rats.

Results: We found that cisplatin rapidly impaired the respiratory function and DNA of mitochondria in renal proximal tubules, thereby inducing apoptosis of tubular epithelial cells within a few days and chronic renal dysfunction associated with multiple cysts one-year after the administration. Administration of edaravone inhibited the cisplatin-induced acute injury of mitochondria and their DNA and renal epithelial cell apoptosis as well as the occurrence of chronic renal dysfunction and multiple cyst formation. The anticancer effect of cisplatin remained unaffected by intravenous administrating of edaravone.

Conclusions: These results indicate that edaravone may have therapeutic potential for inhibiting the acute and chronic injury of the kidney induced by cisplatin.     

Electron microscopic study on the epiplexus (Kolmer) cells of the cat choroid plexus

Summary A light and electron microscopic study was made of the epiplexus (Kolmer) cells of the cat choroid plexus. These polymorphic, motile cells were typically found juxtaposed to the ventricular surface of the choroidal epithelium. They have many ultrastructural features in common with free macrophages of other systems, namely, an indented nucleus with condensed chromatin, sparse mitochondria and endoplasmic reticulum, free ribosomes, multiple Golgi elements, microtubules, coated surface invaginations and microvesicles, and numerous membrane-limited vacuoles and lysosomal dense bodies. A unique feature of epiplexus cells is the manner in which they are anchored to the choroidal epithelium by the invagination of their surfaces by epithelial cell microvilli and cilia.Electron dense tracer particles (biological India ink, Thorotrast, ferritin) injected into the cerebral ventricles were ingested rapidly by epiplexus cells. Uptake of the particles was by way of coated surface invaginations which produced coated cytoplasmic microvesicles. Particle-containing microvesicles subsequently fused with each other and presumably also with pre-existent cytoplasmic vacuoles and lysosomal dense bodies to form storage vacuoles (phagosomes phagolysosomes and residual bodies).Present evidence suggests that epiplexus cells are of hematogenous origin. Under certain conditions these cells may detach from the surface of the choroid plexus to become free-floating cells in the various cerebrospinal fluid compartments of the brain.This investigation was supported by USPHS research grants 1-K04 HD20871, 5 R01 HD 02616 and NB-04456.     

Renal toxicity studies of protein-bound platinum(cis)

Incubation of dialyzed rat serum with cisplatin at a concentration of 908 ppm results in binding of platinum to the proteins and electrophoretic evidence of protein alterations.Intravenous administration of protein-bound platinum(cis), containing the same platinum content as an 8.5-mg/kg dose of cisplatin, fails to produce elevated serum BUN and creatinine levels as do equivalent doses of ‘free’ cisplatin in 0.9% saline.Light microscopy of the right side kidneys of rats receiving protein-bound platinum(cis) revealed no obvious pathology while the kidneys of rats receiving ‘free’ cisplatin showed consistent pathological alterations including hydropic degeneration and pyknotic nuclei.These observations suggest that a protein-bound form of platinum will be unlikely to contribute to the renal toxicity observed during cisplatin chemotherapy.     

Herpes simplex virus 1 envelopment follows two diverse pathways

Herpesvirus envelopment is assumed to follow an uneconomical pathway including primary envelopment at the inner nuclear membrane, de-envelopment at the outer nuclear membrane, and reenvelopment at the trans-Golgi network. In contrast to the hypothesis of de-envelopment by fusion of the primary envelope with the outer nuclear membrane, virions were demonstrated to be transported from the perinuclear space to rough endoplasmic reticulum (RER) cisternae. Here we show by high-resolution microscopy that herpes simplex virus 1 envelopment follows two diverse pathways. First, nuclear envelopment includes budding of capsids at the inner nuclear membrane into the perinuclear space whereby tegument and a thick electron dense envelope are acquired. The substance responsible for the dense envelope is speculated to enable intraluminal transportation of virions via RER into Golgi cisternae. Within Golgi cisternae, virions are packaged into transport vacuoles containing one or several virions. Second, for cytoplasmic envelopment, capsids gain direct access from the nucleus to the cytoplasm via impaired nuclear pores. Cytoplasmic capsids could bud at the outer nuclear membrane, at membranes of RER, Golgi cisternae, and large vacuoles, and at banana-shaped membranous entities that were found to continue into Golgi membranes. Envelopes originating by budding at the outer nuclear membrane and RER membrane also acquire a dense substance. Budding at Golgi stacks, designated wrapping, results in single virions within small vacuoles that contain electron-dense substances between envelope and vacuolar membranes.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

NLRP3/Caspase-1通路对半夏厚朴汤干预慢性间歇性低氧小鼠肾脏炎性损伤中的作用*

目的: 观察半夏厚朴汤对慢性间歇性低氧小鼠肾脏NLRP3/Caspase-1/IL-1β信号通路的影响。方法: 将C57BL/6小鼠随机分为3组,每组10只,分别是正常对照组(Control)、慢性间歇性低氧组(CIH)、半夏厚朴汤治疗组(BHD)。CIH组与BHD组小鼠置于低氧舱内,先充入氮气,使氧气浓度在90 s内从21%下降到9%,后充入氧气,使氧气浓度在90 s内逐渐上升到21%,Control组小鼠置于舱内,充入正常空气,每天处理8 h,持续21 d。BHD组于每日入舱前灌胃给予半夏厚朴汤,Control组和CIH组同时灌胃等体积的生理盐水。造模结束检测各组小鼠肾功能变化和观察肾脏病理情况;通过蛋白印迹法和免疫组织化学染色法检测NOD样受体3(NLRP3)、天冬氨酸特异性半胱氨酸蛋白1(Caspase-1)、白介素 (IL-1β) 的表达水平。结果: 与Control组比较,CIH组小鼠血清尿酸(UA)、尿素氮(BUN)及肌酐(SCr)的含量显著升高(P<0.01),与CIH组比较,BHD组均显著下降(P<0.01)。病理染色结果显示,与对照组相比,CIH组肾小管管腔增大,肾小管上皮细胞肿胀和出现大小不等的空泡,少量细胞脱落、坏死;BHD组有少量的肾小管上皮细胞脱落、坏死。Masson染色结果显示,与对照组比较,CIH组肾小球周围出现明显的纤维化, BHD组纤维化明显减少。Western blot显示,与对照组比较,CIH组肾组织NLRP3、caspase-1、IL-1β及IL-18的蛋白水平显著升高(P<0.01,P<0.05),与CIH组比较,BHD组显著下降(P<0.01,P<0.05)。免疫组织化学染色显示,与对照组比较,CIH组NLRP3、caspase-1和IL-1β的蛋白表达显著增多(P<0.01),且NLRP3在肾小管上皮细胞和间质巨噬细胞内表达最多,caspase-1和IL-1β主要见于肾小管上皮细胞胞浆,经BHD治疗后,与CIH组比较,BHD组NLRP3、caspase-1和IL-1β表达显著下降(P<0.05)。结论: 半夏厚朴汤可通过抑制NLRP3/Casapse-1/IL-1β信号通路相关分子的表达,减轻肾脏炎性损伤。     

A STUDY OF NUCLEOLAR VACUOLES IN CULTURED TOBACCO CELLS USING RADIOAUTOGRAPHY, ACTINOMYCIN D, AND ELECTRON MICROSCOPY

Previously it has been found that in tobacco callus cells nucleolar vacuoles repeatedly form and contract. In this study, nucleolar vacuoles were investigated by using radioautography, actinomycin D, and electron microscopy. It was found, from grain counts of nucleoli labeled with uridine-³H, that nucleoli containing vacuoles had more than three times as many grains/µ² of nucleolar substance as did nucleolei without vacuoles. Treatment of tobacco callus cells with various concentrations of actinomycin D caused the percentage of cells containing nucleolar vacuoles to decrease; with the highest concentration the percentage of these cells dropped from the normal level of about 70% to less than 10%. However, after removal of actinomycin D the cells regained nucleolar vacuoles up to the control level. When radioautography was used with actinomycin D, it was found that the actinomycin D inhibited the uptake of uridine-³H, i.e. inhibited RNA synthesis, in those nucleoli which lost their nucleolar vacuoles. In addition, after removal of the cells from actinomycin D, it was found that as the cells regained nucleolar vacuoles the nucleoli also began to incorporate uridine-³H. Electron micrographs showed the nucleoli to be composed of a compact, finely fibrous central portion surrounded by a layer of dense particles 100–150 A in diameter. Nucleolar vacuoles occurred in the fibrous central portion. Dense particles similar to those in the outer layer of the nucleoli were found scattered throughout the vacuoles and in a dense layer at their outer edge. These data suggest that in cultured tobacco callus cells the formation and contraction of nucleolar vacuoles is closely related to RNA synthesis in the nucleolus.     

Histochemical and ultrastructural changes in the haustorium of date (Phoenix dactylifera L.)

Summary During imbibition ofPhoenix dactylifera embryos, all cotyledon cells show the same changes: protein and lipid bodies degrade, smooth endoplasmic reticulum (ER) increases in amount, and dictyosomes appear. At germination, the distal portion of the cotyledon expands to form the haustorium. At this time, epithelial cells have a dense cytoplasm with many extremely small vacuoles. Many ribosomes are present along with ER, dictyosomes, and mitochondria. The parenchyma cells have large vacuoles and a small amount of peripheral cytoplasm. Between 2 and 6 weeks after germination, epithelial cells still retain the dense cytoplasm and many organelles appear: glyoxysomes, large lipid bodies, amyloplasts, large osmiophilic bodies, and abundant rough and smooth ER which appear to merge into the plasmalemma. A thin electron-transparent inner wall layer with many small internal projections is added to the cell walls. Starch grains appear first in the subsurface and internal parenchyma and subsequently in the epithelium. Lipid bodies, glyoxysomes, protein, and osmiophilic bodies occur in the epithelial and subepithelial cell layers but not in the internal parenchyma. At 8 weeks after germination, the cytoplasm becomes electron transparent, vacuolation occurs, lipid bodies and osmiophilic bodies degrade, and the endomembranes disassemble. After 10 weeks, the cells are empty. These data support the hypothesis that the major functions of the haustorium are absorption and storage.     

The classic Golgi apparatus and vacuoles

The dense vacuoles, considered to be the classic Golgi apparatus in the root meristem ofFagopyrum, were studied by the following methods: 1. Impregnation methods for the demonstration of the Golgi apparatus, 2. cytochemical methods, 3. electron microscopic methods in the light microscope and 4. the electron microscope. A comparison was made with the classic Golgi apparatus in animal cells in the light and electron microscope. Dense vacuoles inFagopyrum and also evidently in other plants, were taken for the classic Golgi apparatus on account of their morphological similarity to the Golgi apparatus in animal cells on impregnation with silver and osmium and their staining preperties with lipoid methods. Dense vacuoles differ from the classic Golgi apparatus in other chemical properties, such as content of phenol substances, etc. No formations were found in animal cells which were similar to dense vacuoles on investigating by electron microscopy. In the electron microscope dense vacuoles have the appearance of derivatives of the normal light vacuoles known in plant cells. They therefore belong to vacuome of plant cell and cannot be analogous to the classic Golgi apparatus in animal cells. Thus the use of the term Golgi apparatus for dense vacuoles is not well founded. A comparison was made of fixation and impregnation used in the light microscope with fixation in the electron microscope. After fixation with permanganate, dense vacuoles have the same shape as after impregnation. After fixation with permanganate, they stain an intense black in the same way as after impregnation with silver and osmium. The form of the vacuoles is dependent on the fixation used. The comparison was made in the light microscope.     

Autophagocytosis in the larval midgut cells of Pieris brassicae during metamorphosis

Summary We observed three types of cells in the epithelial layer of the midgut of last instars of Pieris brassicae. The columnar and goblet cells degenerate during the second part of the last larval stage while the undifferentiated basal cells proliferate during this period and create the epithelium of the pupal midgut. The first morphological sign of involution is the formation of autophagic vacuoles and dense bodies in the cytoplasm of columnar and goblet cells which begins on day 4 of the stage. The number and size of autophagic vacuoles and dense bodies increase during the spinning period (85–96 h). Finally, at the end of the stage, the columnar and goblet cells become displaced by the growing pupal epithelium and reach the lumen where they disintegrate.Autophagocytosis was not seen in the cells during the feeding period (0–72 h). However, we observed many autophagic vacuoles in the columnar and goblet cells of 50-h-old instars 3 h after the administration of 30 g/g body weight of 20-hydroxyecdysone. The hormone treatment elevated by 100% the incorporation of ³H-leucine into the proteins of the midgut. Inhibitors of protein synthesis, cycloheximide and puromycin, in doses that supressed the incorporation of the amino acid by 60–70% either in hormone treated or untreated larvae, exerted diverse effects on the autophagic process. Puromycin did not block the hormone-induced formation of autophagic vacuoles while cycloheximide prevented it. Possible explanations for this diversity are discussed.     

TLR2 protects cisplatin-induced acute kidney injury associated with autophagy via PI3K/Akt signaling pathway

Toll-like receptors (TLRs), which are essential components of the innate immune response, play an important role in acute kidney injury (AKI). Toll-like receptor 2 (TLR2) is constitutively expressed in tubular epithelial cells of the kidney and participates in cisplatin-induced AKI. The autophagy is a dynamic catabolic process that maintains intracellular homeostasis, which is involved in the pathogenesis of AKI. Recent studies demonstrate that PI3K/Akt signaling pathway regulates autophagy in response to various stimuli. Therefore, we propose that cisplatin might activate TLR2, which subsequently phosphorylates PI3K/Akt, leading to enhanced autophagy of renal tubular epithelial cells and protecting cisplatin-induced AKI. We found that TLR2 expression was significantly increased in the kidney after the cisplatin treatment. TLR2-deficient mice exacerbated renal injury in cisplatin-induced AKI, with higher serum creatinine and blood urea nitrogen, more severe morphological injury compared with that of wild-type mice. In vitro, we found that inhibition of TLR2 reduced tubular epithelial cell autophagy after the cisplatin treatment. Mechanistically, TLR2 inhibited autophagy via activating PI3K/Akt signaling pathway in renal tubular epithelial cells after the cisplatin treatment. Take together, these results suggest that TLR2 may protect cisplatin-induced AKI by activating autophagy via PI3K/Akt signaling pathway.     

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation, and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.

Addedum to: Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy and apoptosis are associated in cisplatin injury to renal tubular epithelial cell injury. Am J Physiol Renal Physiol 2008; 294:F777-87.