Ferroptosis is involved in cisplatin sensitivity of the S3 segment of immortalized proximal tubule cells.

Cisplatin (CDDP) is administered as an anticancer drug across a broad spectrum of cancer treatments, but it causes severe renal damage. Several studies have attempted to elucidate the cause of CDDP-induced renal injury, but the detailed mechanism remains unclear. We previously found that S3 cells are more sensitive to CDDP than S1 and S2 cells by using immortalized cells derived from S1, S2, and S3 segments of proximal tubules. In this study, we investigated the potential contribution of reactive oxygen species (ROS) to the sensitivity of S3 cells to CDDP. The results showed that S3 cells have high sensitivity to CDDP, paraquat (PQ) and three ROS substances. To examine the mechanisms underlying the sensitivity to ROS in S3 cells, we compared the cellular responses of CDDP- and PQ-exposed S3 cells. The results indicated that the levels of intracellular ROS and lipid peroxides were increased in S3 cells after CDDP and PQ exposure. The intracellular levels of antioxidant proteins such as thioredoxin, thioredoxin reductase 1 and glutathione peroxidase 4 were also increased by exposure to PQ, but these proteins were decreased by CDDP exposure in S3 cells. Furthermore, the levels of intracellular free Fe2+ were increased by CDDP exposure only in S3 cells but not S1 or S2 cells, and cytotoxicity by exposure to CDDP in S3 cells was suppressed by ferroptosis inhibitors. These results suggested that the induction of ferroptosis due to the ROS production through attenuation of the antioxidant system and elevated free Fe2+ is partly responsible for the sensitivity of S3 cells to CDDP.

Reduced Mn uptake of pleiotropic ZIP8 SNP is caused by its loss of Mn-responsive accumulation on the cell-surface.

Zrt/Irt-like protein 8 (ZIP8), which is a zinc transporter, plays a pivotal role as a manganese transporter. Recent studies have shown that a ZIP8 SNP (rs13107325 C→T, A391T) is associated with multiple diseases, likely by causing systemic Mn deficiency. However, the underlying molecular mechanisms remain unclear. We attempted to address this issue in cell-based experiments using Madin-Darby canine kidney cells stably expressing ZIP8 WT or the A391T SNP mutant under the control of the Tet-regulatable promoter. We showed that the A391T mutant lost the property of Mn-responsive accumulation on the cell surface, which was observed in WT ZIP8. We also showed that the loss of Mn-responsive accumulation of A391T mutant was associated with its reduced Mn uptake, compared to WT ZIP8, in the Mn uptake assay using the radioisotope 54Mn. Our results potentially explain how the ZIP8 A391T substitution is associated with disease pathogenesis caused by Mn deficiency.

Effects of methylation of arginine residue 83 on the enzymatic activity of human arsenic (+3 oxidation state) methyltransferase.

Arsenic (+3 oxidation state) methyltransferase is an enzyme responsible for arsenic methylation, and it requires S-adenosyl-methionine (SAM) as a coenzyme. We here generated two mutants to clarify the role of the highly conserved 83rd arginine residue (Arg83) in Motif I, the SAM-binding domain, of human AS3MT. When the AS3MT activity was compared between the mutants and the wild type (WT) recombinant protein, little activity was detected in the glycine mutant (Arg83Gly) or lysine mutant (Arg83Lys). When we examined the ability of transfected HEK293 cells exposed to arsenite to methylate arsenic, the methylation ability was significantly reduced in Arg83Gly compared to the WT, but was not significantly different between Arg83Lys and WT. Western blot analysis of the recombinant WT and Arg83Gly with an antibody that recognizes methylated Arg showed that an Arg residue in the WT was mono- and di-methylated, but not in Arg83Gly. Furthermore, a peptide containing dimethylated Arg83 was detected by MALDI-TOF/MS of the WT digested with chymotrypsin. These results indicate that AS3MT maintains its enzymatic activity through the methyl modification of Arg83.

CHAC1 exacerbates arsenite cytotoxicity by lowering intracellular glutathione levels.

We here examined whether CHAC1 is implicated in arsenite (As(III))-induced cytotoxicity in HaCaT cells. We found that HaCaT cells in which the intracellular GSH levels were elevated by transfection with CHAC1 siRNA showed decreased sensitivity to As(III) compared to the control cells. Treatment with BSO (an inhibitor of GSH biosynthesis) abolished the decrease in sensitivity to As(III), suggesting that an increase in intracellular GSH levels was involved in the decrease in sensitivity to As(III) due to the decrease in the levels of CHAC1 expression. When we examined the expression of CHAC1 after exposure of HaCaT cells to As(III), the levels of CHAC1 were increased. Since CHAC1 is a proapoptotic factor, we examined appearance of apoptotic cells and cleavage of caspase-3 after exposure to As(III) to determine whether As(III)-induced CHAC1 up-regulation was involved in apoptosis induction. The results showed that induction of apoptosis by As(III) exposure was not detected in CHAC1 siRNA-transfected cells. Together, our findings indicate that CHAC1 is involved in the sensitivity of HaCaT cells to As(III) by regulating the intracellular GSH levels, and in particular, CHAC1 is involved in As(III)-induced apoptosis.

Anatomical study of the bone morphology of the anterior talofibular ligament attachment.

Anterior talofibular ligament (ATFL) injuries are the most common cause of ankle sprains. To ensure anatomically accurate surgery and ultrasound imaging of the ATFL, anatomical knowledge of the bony landmarks around the ATFL attachment to the distal fibula is required. The purpose of the present study was to anatomically investigate the ATFL attachment to the fibula with respect to bone morphology and attachment structures. First, we analyzed 36 feet using microcomputed tomography. After excluding 9 feet for deformities, the remaining 27 feet were used for chemically debrided bone analysis and macroscopic and histological observations. Ten feet of living specimens were observed using ultrasonography. We found that a bony ridge was present at the boundary between the attachments of the ATFL and calcaneofibular ligament (CFL) to the fibula. These two attachments could be distinguished based on a difference in fiber orientation. Histologically, the ATFL was attached to the anterodistal part of the fibula via fibrocartilage anterior to the bony ridge indicating the border with the CFL attachment. Using ultrasonography in living specimens, the bony ridge and hyperechoic fibrillar pattern of the ATFL could be visualized. We established that the bony ridge corresponded to the posterior margin of the ATFL attachment itself. The ridge was obvious, and the superior fibers of the ATFL have directly attached anteriorly to it. This bony ridge could become a valuable and easy-to-use landmark for ultrasound imaging of the ATFL attachment if combined with the identification of the fibrillar pattern of the ATFL.

Spatial localization of cadmium and metallothionein in the kidneys of mice at the early phase of cadmium accumulation.

Chronic exposure to cadmium (Cd) leads to an accumulation of Cd in the kidneys. Metallothionein (MT) is a low-molecular-weight protein having a high affinity for Cd. Cd bound to MT in serum is filtered through the glomeruli of kidney nephrons and reabsorbed by endocytosis into the proximal tubules from the luminal side. Accumulation of Cd in renal cells induces MT synthesis, leading to long-term deposition of Cd and the suppression of Cd toxicity. Recently, many studies have investigated the tissue distribution of metals using laser ablation ICP-MS (LA-ICP-MS). However, little information has been available regarding renal Cd distribution. Hence, we dually investigated the renal distribution of Cd by LA-ICP-MS and that of MT by immunohistochemistry to clarify the dose- and time-dependent changes in the distributions of Cd and MT in mice exposed to Cd from drinking water for 1, 2, and 4 months. Both Cd and MT exhibited characteristic heterogeneous distribution patterns in the renal cortex. The accumulation of Cd and MT near the surface of the cortex suggests a preferential accumulation of Cd in the surface nephrons. MT distribution was more pronounced in the proximal tubules than in the distal tubules, and there were clear differences in MT immunostaining even among the proximal tubules. The heterogeneous localization of MT may reflect the nephron-specific accumulation of Cd. Combining elemental imaging of Cd with immunostaining of MT proved a successful strategy to reveal the characteristic renal Cd distribution, especially in the early stages of Cd accumulation.

Effects of individual amino acid mutations of zinc transporter ZIP8 on manganese- and cadmium-transporting activity.

Zinc (Zn) transporter ZIP8, encoded by SLC39A8, is a unique transporter that can transport divalent manganese (Mn) and cadmium (Cd) in addition to Zn. Recently, associations between various human diseases and variant forms of ZIP8 have been reported. Four amino acid residues, V33, G38, S335, and I340, of human ZIP8 (hZIP8) are mutated in patients with congenital disorders of glycosylation (CDG), whose blood Mn levels are extremely low. Many genome-wide association studies have reported that the A391T mutation of hZIP8 caused by rs13107325 is associated with a wide range of diseases. However, the roles of individual mutations of hZIP8 on metal-transporting activity remain elusive. We established DT40 cells respectively expressing the four mutant hZIP8s and compared the Mn- and Cd-transporting activity between the mutants and wild-type hZIP8. Among the four mutations observed in the ZIP8-mutated CDG patients, the S335T and I340 N mutations in the predicted transmembrane domain 5 (TMD5) completely abolished Mn- and Cd-transporting activity, while V33 M or G35R mutations at the N-terminus did not. We also examined the A391T mutation, which slightly reduced metal transporting activity. Finally, we examined the effects of artificial mutations in the metal-binding motif EEXXH in the TMD5. Replacing EEXXH with HEXXH, which exists in most ZIP transporters, abolished the Mn- and Cd-transporting activity of hZIP8, indicating that glutamic acid in this motif plays a critical role in the unique affinity of ZIP8 for Mn and Cd. Thus, the utilization of DT40 cells enabled us to clarify the different functions of each residue of hZIP8 on metal transport.

Lateral Ulnar Collateral Ligament of the Elbow Joint: Reconsideration of Anatomy in Terms of Connection with Surrounding Fibrous Structures.

BACKGROUND: To improve the clinical results of lateral ulnar collateral ligament (LUCL) reconstruction of the elbow joint, better understanding of the anatomy of the aponeuroses and joint capsule could be relevant. This study considers the previously described anatomy of the LUCL in relation to the related aponeuroses and joint capsule rather than as a discrete ligament. We hypothesized that the deep aponeuroses of the superficial extensor muscles and supinator form a relevant portion of the joint capsule previously defined as the LUCL. METHODS: Twenty-four elbows (12 right) from 21 embalmed cadavers (age at the time of death, 54 to 99 years) were included in the study. Twenty elbows were studied macroscopically and 4, histologically. The joint capsule was detached from the bones, and local thickness was quantitatively analyzed using micro-computed tomography (micro-CT). RESULTS: The supinator aponeurosis and joint capsule intermingled to form a thick membrane (mean and standard deviation, 4.8 ± 1.2 mm), which we termed "the capsulo-aponeurotic membrane." It was thicker than the anterior (1.3 ± 0.4 mm) and posterior (2.5 ± 0.9 mm) parts of the capsule of the humeroradial joint (p < 0.001). The capsulo-aponeurotic membrane had a wide attachment on the distal part of the extensor digitorum communis and extensor digiti minimi (EDC/EDM) origin of the humerus, the lateral part of the coronoid process, and the posterior part of the radial notch of the ulna. The humeral attachment had a fibrocartilaginous structure. The deep aponeuroses of the EDC and extensor carpi ulnaris (ECU) were connected to the capsulo-aponeurotic membrane. CONCLUSIONS: The capsulo-aponeurotic membrane was composed of the supinator aponeurosis and joint capsule and was attached to the lateral epicondyle of the humerus, radial side of the coronoid process, and posterior part of the radial notch on the ulna. The entire structure appeared identical to the commonly defined lateral collateral ligament. The most posterior part was connected to the EDC and ECU aponeuroses, which is commonly labeled the LUCL but does not exist as a discrete ligament. CLINICAL RELEVANCE: Consideration of the accurate anatomy of the extensive attachment of the capsulo-aponeurotic membrane could provide useful clues for improvement in techniques of LUCL reconstruction and lateral epicondylitis pathology.

Manganese transport in mammals by zinc transporter family proteins, ZNT and ZIP.

Manganese (Mn) is an essential trace element required for various biological processes. However, excess Mn causes serious side effects in humans, including parkinsonism. Thus, elucidation of Mn homeostasis at the systemic, cellular, and molecular levels is important. Many metal transporters and channels can be involved in the transport and homeostasis of Mn, and an increasing body of evidence shows that several zinc (Zn) transporters belonging to the ZIP and ZNT families, specifically, ZNT10, ZIP8, and ZIP14, play pivotal roles in Mn metabolism. Mutations in the genes encoding these transporter proteins are associated with congenital disorders related to dysregulated Mn homeostasis in humans. Moreover, single nucleotide polymorphisms of ZIP8 are associated with multiple clinical phenotypes. In this review, we discuss the recent literature on the structural and biochemical features of ZNT10, ZIP8, and ZIP14, including transport mechanisms, regulation of expression, and pathophysiological functions. Because a disturbance in Mn homeostasis is closely associated with a variety of phenotypes and risk of human diseases, these transporters constitute a significant target for drug development. An understanding of the roles of these key transporters in Mn metabolism should provide new insights into pharmacological applications of their inhibitors and enhancers in human diseases.

[Roles of Zinc Transporters That Control the Essentiality and Toxicity of Manganese and Cadmium].

Cellular transport systems for both essential and toxic trace elements remain elusive. In our studies on the transport systems for cadmium (Cd), we found that the cellular uptake of Cd is mediated by the transporter for manganese (Mn). We identified ZIP8 and ZIP14, members of the ZIP zinc (Zn) transporter family, as transporters having high affinities for both Cd and Mn. Notably, the uptake of Cd into rice root from soil is mediated by a transporter for Mn as well. We found that ZIP8 is highly expressed at the S3 segment of the kidney proximal tubule and can transport glomerulus-filtered Cd and Mn ions in the lumen into epithelial cells of the proximal tubule, suggesting that ZIP8 has an important role in the renal reabsorption of both toxic Cd and essential Mn. Mutations in ZIP8 and ZIP14 genes were found in humans having congenital disorders associated with the disturbed transport of Mn, although ZIP8 mutation causes whole-body Mn deficiency while ZIP14 mutation causes Mn accumulation in the brain. Mutations in ZnT10, a Zn transporter responsible for Mn excretion, also cause hyperaccumulation of Mn in the brain. Results of genome-wide association studies have indicated that ZIP8 SNPs are involved in a variety of common diseases. Thus, ZIP8, ZIP14, and ZnT10 play crucial roles in the transport of Mn and thereby control Mn- and Cd-related biological events in the body.

Comparisons of segment-specific toxicity of platinum-based agents and cadmium using S1, S2, and S3 cells derived from mouse kidney proximal tubules.

Renal toxicants such as cisplatin and cadmium cause segment-specific damages in kidney proximal tubules. Recently, we established an in vitro experimental system for evaluating segment-specific toxicity and transport of chemicals using immortalized S1, S2, and S3 cells derived from the S1, S2, and S3 regions of mouse kidney proximal tubules. In the present study, we examined the toxicity and accumulation of cisplatin, carboplatin, oxaliplatin, and cadmium in S1, S2, and S3 cells. We found that not only cisplatin but also carboplatin and oxaliplatin exhibited higher lethal toxicity in S3 cells than in S1 and S2 cells. At sublethal doses, cisplatin showed delayed induction of Kim-1 and clusterin on days 3 and 6, which may reflect the latent renal toxicity of cisplatin in vivo. The high sensitivities of S3 cells to the platinum-based agents were not due to the high accumulation of Pt in S3 cells. Exposure to cadmium resulted in similar toxicity among these cells, suggesting that S3 cells were not sensitive to any renal toxicants. Thus, the utilization of S1, S2, and S3 cells may provide a useful tool for the in vitro evaluation of the proximal tubule segment-specific toxicity of chemicals.

In vitro Evaluation of The Effects of Cadmium on Endocytic Uptakes of Proteins into Cultured Proximal Tubule Epithelial Cells.

Cadmium (Cd) is an environmental pollutant known to cause dysfunctions of the tubular reabsorption of biomolecules in the kidney. Elevated levels of urinary excretion of low-molecular-weight proteins such as ß2-microglobulin (ß2-MG) have been used as an indicator of Cd-induced renal tubular dysfunctions. However, very few studies have examined the direct effects of Cd on the reabsorption efficiency of proteins using cultured renal cells. Here, we developed an in vitro assay system for quantifying the endocytic uptakes of fluorescent-labeled proteins by flow cytometry in S1 and S2 cells derived from mouse kidney proximal tubules. Endocytic uptakes of fluorescent-labeled albumin, transferrin, ß2-MG, and metallothionein into S1 cells were confirmed by fluorescence imaging and flow cytometry. The exposure of S1 and S2 cells to Cd at 1 and 3 µM for 3 days resulted in significant decreases in the uptakes of ß2-MG and metallothionein but not in those of albumin or transferrin. These results suggest that Cd affects the tubular reabsorption of low-molecular-weight proteins even at nonlethal concentrations. The in vitro assay system developed in this study to evaluate the endocytic uptakes of proteins may serve as a useful tool for detecting toxicants that cause renal tubular dysfunctions.

Toxicometallomics of Cadmium, Manganese and Arsenic with Special Reference to the Roles of Metal Transporters.

The transport systems for metals play crucial roles in both the physiological functions of essential metals and the toxic effects of hazardous metals in mammals and plants. In mammalian cells, Zn transporters such as ZIP8 and ZIP14 have been found to function as the transporters for Mn(II) and Cd(II), contributing to the maintenance of Mn homeostasis and metallothionein-independent transports of Cd, respectively. In rice, the Mn transporter OsNramp5 expressed in the root is used for the uptake of Cd from the soil. Japan began to cultivate OsNramp5 mutant rice, which was found to accumulate little Cd, to prevent Cd accumulation. Inorganic trivalent arsenic (As(III)) is absorbed into mammalian cells via aquaglyceroporin, a water and glycerol channel. The ortholog of aquaporin in rice, OsLsi1, was found to be an Si transporter expressed in rice root, and is responsible for the absorption of soil As(III) into the root. Since rice is a hyperaccumulator of Si, higher amounts of As(III) are incorporated into rice compared to other plants. Thus, the transporters of essential metals are also utilized to incorporate toxic metals in both mammals and plants, and understanding the mechanisms of metal transports is important for the development of mitigation strategies against food contamination.

Segment-specific and direction-dependent transport of cadmium and manganese in immortalized S1, S2, and S3 cells derived from mouse kidney proximal tubules.

The kidney proximal tubule is a target of many renal toxicants, including cadmium (Cd), and also a place of reabsorption of essential metals in glomerular filtrate to systemic circulation. Although the mechanisms of metal transport in the convoluted proximal tubule (S1 and S2 segments) and the straight proximal tubule (S3 segment) may differ, little is known about the segment-specific modes of metal transport. Here, we utilized immortalized cell lines derived from the S1, S2, and S3 segments of mouse kidney proximal tubules, and examined the segment-specific and direction-dependent transport of Cd and manganese (Mn) using a trans-well culture system. The results showed that the uptakes of Cd2+ and Mn2+ from apical sides were the highest in S3 cells, and Cd2+, Mn2+, and Zn2+ mutually inhibited the apical uptake of each metal. As the expression of ZIP8, a zinc transporter having affinities for Cd2+ and Mn2+, was the highest in S3 cells, ZIP8 may contribute largely to the apical uptakes of these metals. The efficient uptake of Mn2+ from apical side of S3 cells may suggest an important role of ZIP8 in proximal tubule in reabsorption of Mn, an essential metal. Our study demonstrated that S1, S2, and S3 cells provide a useful tool for studying the segment-specific and direction-dependent transport of both toxic and essential metals in the kidney's proximal tubules.

New Insights into the Roles of ZIP8, a Cadmium and Manganese Transporter, and Its Relation to Human Diseases.

ZIP8, a Zrt-/Irt-related protein encoded by Slc39A8, was originally discovered as a zinc transporter, but since then its roles as a transporter for cadmium (Cd) and manganese (Mn) have also been well characterized. ZIP8 is highly expressed in the S3 segment of the proximal tubules of the mouse kidney and may play a significant role in reabsorption of both toxic Cd and essential Mn from the lumen to the epithelial cells of the proximal tubule. In recent years, associations between various human diseases and genetic variations of ZIP8 have been reported. Missense mutations in the human SLC39A8 gene are associated with serious disorders of Mn metabolism, showing symptoms similar to congenital glycosylation deficiency. Enhanced excretion of Mn via bile or urine might be the cause of extremely low blood Mn levels in ZIP8-mutated patients, leading to the defects in Mn-dependent glycosylation. Several genome-wide association studies have demonstrated the associations of multiple diseases and ZIP8 SNPs constituting missense mutations. These findings suggest that ZIP8 plays more important roles than previously expected as a modulator of Mn homeostasis in the body. Elucidation of biochemical mechanisms regarding the metal-transporting ability of ZIP8 and its alteration by mutation is required for better understanding of the role of ZIP8 in human diseases.

Arsenite suppresses NO production evoked by lipopolysaccharide and poly(I:C) via the suppression of interferon-ß expression in RAW264.7 cells.

Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-ß/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-ß, the expression of the components of IFN-α/ß receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-ß were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-ß, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-ß on the disturbances in immunological defense against both bacteria and viruses.

Anatomic analysis of the whole articular capsule of the shoulder joint, with reference to the capsular attachment and thickness.

BACKGROUND: Although conventional Bankart repair has been the accepted procedure for traumatic anterior glenohumeral instability, the humeral avulsion of the glenohumeral ligament or an elongation of the capsule remains challenging to decide the appropriate treatment. The anatomical knowledge regarding the whole capsule of glenohumeral joint is necessary to accurately treat for the capsular disorders. The aims of the current study were to investigate the anatomical features of capsular attachment and thickness in a whole capsule of glenohumeral joint. METHODS: We used 13 shoulders in the current study. In 9 shoulders, we macroscopically measured the attachment widths of the capsulolabrum complex on the scapular glenoid, and the attachment widths of the capsule on the humerus in reference to the scapular origin of the long head of triceps brachii, and the humeral insertion of the rotator cuff tendons. We additionally used 4 cadaveric shoulders, which were embalmed using Thiel's method, for the analysis of the thickness in a whole capsule by using micro-CT. RESULTS: The glenoidal attachment of the articular capsule appeared to have a consistent width except for the superior part of the origin of the long head of triceps brachii. On the humerus, the articular capsule was widely attached to areas without overlying rotator cuffs, with the widest width (17.3 ± 0.9 mm) attached to the axillary pouch. The inferior part of the capsule, which was consistently thicker than the superior part, continued to the superior part along the glenoid and humeral side edge. CONCLUSIONS: The current study showed that the inferior part of the glenohumeral capsule had a wide humeral attachment from the inferior edge of the subscapularis insertion to the inferior edge of the teres minor insertion via the anatomical neck of the humerus, and the thickness of it was thicker than the superior part of the capsule.

An anatomic study on the attachment of the joint capsule to the tibia in the lateral side of the knee.

PURPOSE: The purpose of the current study was to examine the width, area, and histological characteristics of the capsular attachment to the tibia in the lateral side of the knee. METHODS: A total of 27 knees were used in this study. The joint capsule of the knee was peeled away from the tibia and the width of the capsular attachment to the tibia was measured by two independent observers using a caliper. Interclass correlation coefficients for each value were calculated to evaluate the validity of the measurement. The capsular attachment to the tibia of the seven knees was histologically analyzed using Masson's trichrome staining. RESULTS: At the posterior border of Gerdy's tubercle, the capsular attachment was wide; the average width was 8.6 mm (SD 3.0). Toward the posterolateral aspect of the knee, the capsular attachment gradually tapered. Finally, the capsular attachment was linear at the apex of the head of the fibula. Histological analysis at the posterior border of Gerdy's tubercle revealed developed uncalcified fibrocartilage on the capsular attachment. In contrast, at the apex of the head of the fibula, the joint capsule was adhered to the capsule of the proximal tibiofibular joint. Fibrous connective tissue was directly attached to the calcified fibrocartilage. CONCLUSIONS: The attachment width of the knee joint capsule at the lateral side varied according to location. We consider that this finding on the capsular attachment will facilitate an understanding of the pathology or mechanism of diseases on the lateral side of the knee joint.

Protective effect of cadmium-induced autophagy in rat renal mesangial cells.

Cadmium damages renal cells, and in particular may cause mesangial cell death by necrosis or apoptosis, depending on exposure conditions in cultured cells. However, there is an uncertainty as to whether Cd2+-induced autophagy can protect mesangial cells against these other mechanisms of cell death. We have used autophagy-incompetent mouse embryonic fibroblast (MEF) cells lacking the Atg16 gene, as well as cultured rat mesangial cells (RMC) in which Atg16 has been silenced, to examine this issue. Measuring the processing of LC3-I to LC3-II and expression of sequestosome-1 (p62), we define conditions under which RMC can be induced to undergo autophagy in response to 0-20 µM CdCl2. Similarly, Cd2+ can initiate autophagy in MEF cells. However, when autophagy is compromised, either by gene knockout in MEF cells or by RNA silencing in RMC, cell viability is decreased, and concomitantly a Cd2+ dose-dependent increase in pro-caspase-3 cleavage indicates the initiation of apoptotic cell death. In contrast to some previous reports, Cd2+-induced autophagy is not correlated with increased levels of cellular reactive oxygen species but, among a panel of kinases investigated, is suppressed by inhibition of the Jun kinase. We conclude that concentrations of Cd2+ that initiate autophagy may afford renal mesangial cells some degree of protection against other modes (apoptosis, necrosis) of cell death.

Concentration-dependent roles of DMT1 and ZIP14 in cadmium absorption in Caco-2 cells.

Intestinal absorption of cadmium (Cd) is considered to be mediated mainly by the ferrous iron transporter DMT1, or the calcium transporter CaT1. The roles of zinc transporters such as ZIP8 and ZIP14 remain unclear, and the roles of these four transporters in the intestinal uptake of Cd under physiological conditions have not been compared. Here, we used a trans-well cell culture system to investigate the effects of the down-regulation of these four transporters on the uptake of Cd from the apical side of enterocytes. We used a Caco-2-kh cell line that can form tight junctions within a few days. The transfection of DMT1 siRNA significantly decreased the Cd uptake from the apical side at 5 µM, but not at 0.1 or 1 µM. The transfection of ZIP14 siRNA markedly decreased the Cd uptake at 0.1 and 1 µM, but not at 5 µM. The transfection of siRNA of CaT1 or ZIP8 did not alter the Cd uptake at any concentrations of Cd examined. These results suggest that DMT1 and ZIP14 play different roles in intestinal Cd absorption depending on the concentration of Cd.