Genomic characterization of thymic epithelial tumors in a real-world dataset.

BACKGROUND: Thymic epithelial tumors (TETs) are rare neoplasms arising in the mediastinum, including thymic carcinomas and thymomas. Due to their rarity, little is known about the genomic profiles of TETs. Herein, we investigated the genomic characteristics of TETs evaluated in a large comprehensive genomic profiling database in a real-world setting. METHODS: We included data from two different cohorts: Foundation Medicine Inc. (FMI) in the United States and the Center for Cancer Genomics and Advanced Therapeutics (C-CAT) in Japan. Samples profiled were examined for all classes of alterations in 253 genes targeted across all assays. Tumor mutational burden (TMB) and microsatellite instability (MSI) were also evaluated. RESULTS: A total of 794 patients were collected in our study, including 722 cases from FMI and 72 cases from C-CAT. In the FMI data, CDKN2A (39.9%), TP53 (30.2%) and CDKN2B (24.6%) were frequently altered in thymic carcinoma, versus TP53 (7.8%), DNMT3A (6.8%), and CDKN2A (5.8%) in thymoma. TMB-high (≥10 mutations/Mb) and MSI were present in 7.0% and 2.3% of thymic carcinomas, and 1.6% and 0.3% of thymomas, respectively. Within C-CAT data, CDKN2A (38.5%), TP53 (36.5%) and CDKN2B (30.8%) were also frequently altered in thymic carcinoma, while alterations of TSC1, SETD2 and LTK (20.0% each) were found in thymoma. CONCLUSIONS: To the best of our knowledge, this is the largest cohort in which genomic alterations, TMB and MSI status of TETs were investigated. Potential targets for treatment previously unbeknownst in TETs are identified in this study, entailing newfound opportunities to advance therapeutic development.

Ultraconserved region-containing Transformer 2ß4 controls senescence of colon cancer cells.

Ultraconserved regions (UCRs) are >200 bp genomic segments with perfect human-to-rodent sequence identity. Transcribed UCRs constitute a new category of noncoding RNAs whose functions remain poorly understood. The human transformer 2ß (TRA2B) gene contains a 419-bp UCR spanning the 276-bp exon 2 and its neighboring introns. TRA2B exon 2 has premature stop codons, whereas an exon 2-containing splice variant (TRA2ß4) was expressed preferentially in the nuclei of human colon cancer cells. TRA2ß4 knockdown p53-independently stimulated CDKN1A transcription and increased p21, resulting in the appearance of senescent cells. Biotin pull-down and RNA immunoprecipitation assays revealed that TRA2ß4 interacted with Sp1 through a Sp1-binding sequence (485-GGGG-488) in a stem-loop structure of exon 2. Mutation of this sequence (485-AAGG-488) disrupted the stem-loop structure, blocked the interaction with Sp1 and increased CDKN1A transcription. Overexpression of TRA2ß4 significantly decreased CDKN1A mRNA levels and accelerated cell growth, but the introduction of the mutation in the Sp1-binding sequence completely canceled these effects. Taken together, TRA2ß4 may sequester Sp1 from occupying promoters of target genes including CDKN1A, promoting cell growth by interrupting the senescence-related gene expression program. This novel function of TRA2ß4 may uncover an oncogenic function of transcribed UCRs.

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1ß, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.

Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells.

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

Formation of high heat resistant coatings by using gas tunnel type plasma spraying.

UNLABELLED: Zirconia sprayed coatings are widely used as thermal barrier coatings (TBC) for high temperature protection of metallic structures. However, their use in diesel engine combustion chamber components has the long run durability problems, such as the spallation at the interface between the coating and substrate due to the interface oxidation. Although zirconia coatings have been used in many applications, the interface spallation problem is still waiting to be solved under the critical conditions such as high temperature and high corrosion environment. The gas tunnel type plasma spraying developed by the author can make high quality ceramic coatings such as Al2O3 and ZrO2 coating compared to other plasma spraying method. A high hardness ceramic coating such as Al2O3 coating by the gas tunnel type plasma spraying, were investigated in the previous study. The Vickers hardness of the zirconia (ZrO2) coating increased with decreasing spraying distance, and a higher Vickers hardness of about Hv = 1200 could be obtained at a shorter spraying distance of L = 30 mm. ZrO2 coating formed has a high hardness layer at the surface side, which shows the graded functionality of hardness. In this study, ZrO2 composite coatings (TBCs) with Al2O3 were deposited on SS304 substrates by gas tunnel type plasma spraying. The performance such as the mechanical properties, thermal behavior and high temperature oxidation resistance of the functionally graded TBCs was investigated and discussed. The resultant coating samples with different spraying powders and thickness are compared in their corrosion resistance with coating thickness as variables. Corrosion potential was measured and analyzed corresponding to the microstructure of the coatings. KEYWORDS: High Heat Resistant Coatings, Gas Tunnel Type Plasma Spraying, Hardness,

International models of investigator-initiated trials: implications for Japan.

BACKGROUND: Academic/institutional investigator-initiated clinical trials benefit individuals and society by supplementing gaps in industry-sponsored clinical trials. MATERIALS: In May 2010, experts from Japan, the Republic of Korea, the UK, and the United States, met at a symposium in Tokyo, Japan, to discuss how policies related to the conduct of clinical trials, which have been shown to be effective, may be applied to other regions of the world. RESULTS: In order to increase the availability of anticancer drugs world-wide, nations including Japan should examine the benefits of increasing the number of investigator-initiated clinical trials. These trials represent one of the most effective ways to translate basic scientific knowledge into clinical practice. These trials should be conducted under GCP guidelines and include Investigational New Drug application submissions with the ultimate goal of future drug approval. CONCLUSIONS: To maximize the effectiveness of these trials, a policy to educate health care professionals, cancer patients and their families, and the public in general on the benefits of clinical trials should be strengthened. Finally, policies that expedite the clinical development of novel cancer drugs which have already been shown to be effective in other countries are needed in many nations including Japan to accelerate drug approval.

Indoxyl sulfate induces skeletal resistance to parathyroid hormone in cultured osteoblastic cells.

Skeletal resistance to parathyroid hormone (PTH) is well known to the phenomenon in chronic renal failure patient, but the detailed mechanism has not been elucidated. In the process of analyzing an animal model of renal failure with low bone turnover, we demonstrated decreased expression of PTH receptor (PTHR) accompanying renal dysfunction in this model. In the present study, we focused on the accumulation of uremic toxins (UTx) in blood, and examined whether indoxyl sulfate (IS), a UTx, is associated with PTH resistance. We established primary osteoblast cultures from mouse calvariae and cultured the cells in the presence of IS. The intracellular cyclic adenosine 3',5' monophosphate (cAMP) production, PTHR expression, and free radical production in the primary osteoblast culture were studied. We found that the addition of IS suppressed PTH-stimulated intracellular cAMP production and decreased PTHR expression in this culture system. Free radical production in osteoblasts increased depending on the concentration of IS added. Furthermore, expression of organic anion transporter-3 (OAT-3) that is known to mediate cellular uptake of IS was identified in the primary osteoblast culture. These results suggest that IS taken up by osteoblasts via OAT-3 present in these cells augments oxidative stress to impair osteoblast function and downregulate PTHR expression. These finding strongly suggest that IS accumulated in blood due to renal dysfunction is at least one of the factors that induce skeletal resistance to PTH.

Self-association of the transmembrane domain of RET underlies oncogenic activation by MEN2A mutations.

In patients with medullary thyroid carcinoma (MTC) and type 2A multiple endocrine neoplasia (MEN2A), mutations of cysteine residues in the extracellular juxtamembrane region of the RET receptor tyrosine kinase cause the formation of covalent receptor dimers linked by intermolecular disulfide bonds between unpaired cysteines, followed by oncogenic activation of the RET kinase. The close proximity to the plasma membrane of the affected cysteine residues prompted us to investigate the possible role of the transmembrane (TM) domain of RET (RET-TM) in receptor-receptor interactions underlying dimer formation. Strong self-association of the RET-TM was observed in a biological membrane. Mutagenesis studies indicated the involvement of the evolutionary conserved residues Ser-649 and Ser-653 in RET-TM oligomerization. Unexpectedly, RET-TM interactions were also abrogated in the A639G/A641R double mutant, first identified in a sporadic case of MTC. In agreement with this, no transforming activity could be detected in full-length RET carrying the A639G and A641R mutations, which remained fully responsive to glial cell-line-derived neurotrophic factor (GDNF) stimulation. When introduced in the context of C634R - a cysteine replacement that is prevalent in MEN2A cases - the A639G/A641R mutations significantly reduced dimer formation and transforming activity in this otherwise highly oncogenic RET variant. These data suggest that a strong propensity to self-association in the RET-TM underlies - and may be required for - dimer formation and oncogenic activation of juxtamembrane cysteine mutants of RET, and explains the close proximity to the plasma membrane of cysteine residues implicated in MEN2A and MTC syndromes.

Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells.

Rho-family GTPases regulate various aspects of cell function by controlling cytoskeletal changes; however, their spatial regulation within the cells remains largely unknown. To understand this regulation, we have studied the spatiotemporal activity of Rho-family GTPases in migrating cells and growth factor-stimulated cells by using probes based on the principle of fluorescence resonance energy transfer. In migrating fibroblasts and epithelial cells, the level of RhoA activity is high both at the contractile tail and at the leading edge, whereas Rac1 and Cdc42 activities are high only at the leading edge. In cells stimulated with epidermal growth factor or nerve growth factor, activities of Rac1 and Cdc42 were transiently elevated in a broad area of the plasma membrane, followed by a localized activation at nascent lamellipodia. In contrast, on epidermal growth factor stimulation, RhoA activity decreased diffusely at the plasma membrane. Notably, RhoA activity persisted at the tip of growth factor-induced membrane ruffles and, in agreement with this finding, RhoA is required for membrane ruffling. These observations suggest that the activities of Rho-family GTPases are elaborately regulated in a time- and space-dependent manner to control cytoskeletal changes and that the basic mechanism of controlling cell shape via Rho-family GTPases is common to various cell types.

Intraoperative electrical stimulation of the pelvic splanchnic nerves during nerve-sparing radical hysterectomy.

OBJECTIVES: This study sought to determine whether intraoperative electrical stimulation (IES) of pelvic splanchnic nerves (PSNs) while monitoring bladder contraction was useful to predict postoperative bladder function during conventional nerve-sparing radical hysterectomy. METHODS: Seventeen patients with stage Ib or IIa cervical cancer underwent conventional radical hysterectomy. IES was performed in all cases, stimulating the roots of PSN, the posterior sheath of the vesicouterine ligament (PVL) and the dorsal area of the ligament. After resection of the uterus, the PSN roots were stimulated again. Bladder function was evaluated by urodynamic study (UDS) preoperatively and 3 months after surgery. RESULTS: The results of IES were consistent with bladder function evaluated by postoperative UDS. In 13 of 17 cases, an increased intravesical pressure was observed with IES of the PSN roots after uterus resection. Nine of 13 cases showed marked detrusor contraction with UDS 3 months after surgery and were able to void without using abdominal pressure except in one case. In the remaining 4 of 17 cases, no response could be detected to IES on either side. Three cases voided using abdominal pressure and one used clean intermittent self-catheterization without spontaneous voiding. CONCLUSIONS: IES while monitoring intravesical pressure during radical hysterectomy represents a technically simple and useful procedure for the prediction of postoperative bladder function.

Subacute inflammatory demyelinating polyneuropathy.

OBJECTIVE: To report the clinical, electrophysiologic, and histologic characteristics of subacute inflammatory demyelinating polyneuropathy (SIDP) and to present the diagnostic criteria of this disease. METHODS: For a diagnosis of "definite SIDP," there were four mandatory criteria: 1) progressive motor and/or sensory dysfunction consistent with neuropathy in more than one limb with time to nadir between 4 and 8 weeks, 2) electrophysiologic evidence of demyelination in at least two nerves, 3) no other etiology of neuropathy, and 4) no relapse on adequate follow-up. Supportive criteria included high spinal fluid protein level (>55 mg/dL) and inflammatory cells in the nerve biopsy. A diagnosis of "probable SIDP" required progression of demyelinating neuropathy over a 4- to 8-week period. RESULTS: Sixteen definite SIDP patients were identified among 29 probable SIDP patients. An antecedent infection was found in 38% of cases. The two most common neuropathy types were a symmetric motor-sensory neuropathy and a pure motor neuropathy. Cranial nerve deficits and respiratory failure were rare. Spinal fluid protein was high in 93% of cases. Demyelination was documented by the motor nerve conduction in 88% of cases and by the near-nerve needle sensory nerve conduction in two cases. Almost all patients were treated with prednisone and some with additional immunotherapies. Complete recovery was achieved in 69% of cases and partial recovery in others. Definite SIDP had all the characteristics of CIDP with three exceptions: a higher rate of antecedent infection, no relapse rate, and a high rate of recovery to normal. CONCLUSION: Subacute inflammatory demyelinating polyneuropathy is a definite entity bridging the gap between Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy.

Cell signalling and gene expression mediated by RET tyrosine kinase.

Germline mutations of the RET proto-oncogene cause multiple endocrine neoplasia (MEN) 2A or 2B by different mechanisms. As is the case for other receptor tyrosine kinases, mutant RET recruits a variety of signalling molecules via phosphorylated tyrosine residues present in the kinase domain and carboxy-terminal tail. As we previously reported, the signaling via phosphorylated tyrosine 1062 plays a crucial role in the transforming activities of both RET-MEN2A and RET-MEN2B mutant protein. Interestingly, this single tyrosine residue represents a binding site for several signalling molecules including SHC, Enigma, SNT/FRS2, DOK and IRS1 and is responsible for activation of the RAS/ERK, PI3-K/AKT, JNK, p38MAPK and ERK5 signalling pathways. Amongst these, the PI3-K/AKT and JNK pathways appeared to be more strongly activated in the cells expressing RET-MEN2B than in the cells expressing RET-MEN2A, suggesting the possibility that these pathways may be involved in the disease phenotype. In addition, RET is alternatively spliced to produce three isoforms and the splicing site is present just downstream of tyrosine 1062. These isoforms play different roles for the tumour development associated with MEN 2 or the development of the kidney and the enteric nervous system. Moreover, using differential display analysis, we identified several genes whose expression is highly induced by RET-MEN2B mutant proteins. The differential gene expression by RET-MEN2A and RET-MEN2B may also be important for the development of their phenotypes.

Diagnostic significance of digital rectal examination and transrectal ultrasonography in men with prostate-specific antigen levels of 4 NG/ML or less.

OBJECTIVES: To investigate the usefulness of digital rectal examination (DRE) and transrectal ultrasonography (TRUS) for prostate cancer diagnosis and to propose a diagnostic algorithm for individual-based cancer screening in subjects with prostate-specific antigen (PSA) levels of 4.0 ng/mL or less. METHODS: Between January 1992 and March 2000, 129 subjects with PSA levels of 4.0 or less and abnormal findings on DRE or TRUS underwent prostate biopsy. The subjects were divided into four groups according to the PSA range: 0 to 0.9 ng/mL, 1.0 to 1.9 ng/mL, 2.0 to 2.9 ng/mL, and 3.0 to 4.0 ng/mL. The reliability of the DRE and TRUS and the clinicopathologic features of prostate cancer were investigated among these four groups. RESULTS: Of the 129 subjects, 17 (13.2%) patients with prostate cancer were diagnosed. The detection rate was 2.2% (1 of 45), 0% (0 of 27), 20.6% (7 of 34), and 39.1% (9 of 23) in subjects with PSA levels of less than 1.0 ng/mL, 1.0 to 1.9 ng/mL, 2.0 to 2.9 ng/mL, and 3.0 to 4.0 ng/mL, respectively. The proportion of patients with Stage II, III, and IV was 58.8%, 41.2%, and 0%, respectively. The percentage with Gleason scores of 8 to 10 was 17.6%. The detection rate of abnormal findings on DRE and TRUS was 14.4% (13 of 90) and 9.5% (7 of 74), respectively. Adding TRUS to DRE in the screening program of subjects with PSA levels of 2.0 to 4.0 ng/mL, increased the detection rate of prostate cancer to 30.8% (4 of 13). CONCLUSIONS: Routine prostate biopsy should not be undertaken except for highly suspicious DRE findings in subjects with PSA levels less than 2.0 ng/mL. The additional use of TRUS in subjects with PSA levels of 2.0 to 4.0 ng/mL would improve the sensitivity of prostate cancer detection. The diagnostic algorithm proposed in the present study is useful as a screening method for prostate cancer in subjects with PSA levels of 4.0 ng/mL or less.

Malignant fibrous histiocytoma of the right spermatic cord: a case report.

The patient was a 47-year-old male, who visited Hidaka Hospital with a chief complaint of swelling in the right inguinal region and the scrotum. With a diagnosis of a right spermatic cord tumor, right high orchiectomy was performed. Since an inflammatory type of malignant fibrous histiocytoma (MFH) was diagnosed from histopathological findings, chemotherapy and radiation therapy were performed as postoperative treatment. Malignant fibrous histiocytoma with the primary focus of the spermatic cords is a rare disease. To our knowledge, this is the 20th case of MFH of the spermatic cord in Japan (the 42nd in the world) and it is the second case of inflammatory type of MFH in Japan.

Circadian characteristics of urinary leukotriene E(4) in healthy subjects and nocturnal asthmatic patients.

STUDY OBJECTIVES: Circadian rhythmicity of cysteinyl leukotrienes (LTs) and thromboxane (TX)-A(2) in healthy subjects and nocturnal asthmatic patients remains a subject of controversy. The aim of this study was to investigate the contribution of these mediators to the pathogenesis of nocturnal asthma. METHODS: We measured peak expiratory flow rate, urinary concentration of LTE(4), 11-dehydro-TXB(2), and creatinine eight times every 3 h in three groups: healthy control subjects (n = 5, group A), nocturnal asthmatic patients (n = 9, group B), and nonnocturnal asthmatic subjects (n = 9, group C). To evaluate the reproducibility of the measurement of urinary LTE(4), we measured urinary LTE(4) in group A for 3 separate days. RESULTS: The urinary LTE(4) concentrations from 3 to 6 AM were significantly (p < 0.05) higher than from 3 to 6 PM in both group A and group B, but not in group C. The mean levels of LTE(4) in group B and group C were significantly higher (p < 0.05) than those in group A. In group B, another small peak was observed from 6 to 9 PM. No significant day-to-day variation was observed in group A. Urinary 11-dehydro-TXB(2) values from 3 to 6 AM were significantly (p < 0.001) higher than those levels from 3 to 6 PM in all groups, and the mean levels in group B and group C were significantly higher than those in group A (p < 0.05). CONCLUSIONS: Circadian rhythmicity of urinary LTE(4) with a morning peak was found in healthy control subjects and nocturnal asthmatic subjects, but not in nonnocturnal asthmatic patients. It was suggested that cysteinyl LTs rather than TXA(2) might contribute to the nocturnal worsening of asthma.

Nonneurogenic hypoxia sensitivity in rat adrenal slices.

A change in the intracellular Ca(2+) ([Ca(2+)](i)) level induced by hypoxia was detected in rat adrenal slices by use of fura-2/AM. After hypoxic stress, an increase in [Ca(2+)](i) was observed only in the adrenal medulla. This increase was inhibited by nifedipine, but not modified by the cholinergic receptor blockers. The hypoxia-induced increase in [Ca(2+)](i) was observed in all postnatal developmental stages to a similar extent, whereas the nicotine and high K(+) sensitivities increased along with postnatal development. A 10 nM ryanodine enhanced the hypoxia-induced [Ca(2+)](i) increase in adult but not in neonatal rat slices. These results suggest the existence of an oxygen-sensing mechanism in adult rat adrenals even after sympathetic innervation. Hypoxic responses seemed to be similar both in neonate and in adult rat adrenals and were triggered by the influx of Ca(2+) via L-type voltage-sensitive Ca(2+) channels. However, the sustained [Ca(2+)](i) increase caused by hypoxia might depend on postnatal development and be triggered by Ca(2+)-induced Ca(2+) release (CICR).

Tubular phenotypic change in progressive tubulointerstitial fibrosis in human glomerulonephritis.

There is much debate over the origins of fibroblast-type cells that accumulate in interstitial fibrosis. A controversial hypothesis, supported by data from animal and cell-culture studies, is that fibroblast-type cells can derive from tubular epithelial cells by a process of epithelial-mesenchymal transdifferentiation. However, to date, no evidence supports this postulate in human glomerulonephritis. This study sought to provide evidence that tubular epithelial cells can undergo phenotypic change toward a fibroblast-like cell in human glomerulonephritis. One hundred twenty-seven open renal biopsy specimens from patients with minimal change disease (MCD), immunoglobulin A (IgA) nephropathy, and rapidly progressive glomerulonephritis (RPGN) were examined for tubular phenotypic change by two-color immunohistochemistry using the criteria of de novo expression of alpha-smooth muscle actin (alpha-SMA), a myofibroblast marker; loss of the epithelial marker cytokeratin; and collagen production. In normal human kidney and MCD, tubular epithelial cells expressed cytokeratin with no evidence of alpha-SMA staining. However, in 36 of 90 cases of IgA nephropathy and 9 of 18 cases of RPGN, small numbers of tubular epithelial cells in areas of fibrosis showed de novo alpha-SMA expression, accounting for 0.4% +/- 0.2% (IgA nephropathy) and 3.8% +/- 1.5% (RPGN) of cortical tubules. An intermediate stage of phenotypic change was observed in some cuboidal epithelial cells that expressed both cytokeratin and alpha-SMA. Tubules containing alpha-SMA-positive (alpha-SMA(+)) cells also stained for collagen types I and III, suggesting that tubular cells undergoing phenotypic change have an active role in the fibrotic process. There also was a marked increase in transforming growth factor-beta1 (TGF-beta1) tubular expression in areas with interstitial fibrosis, including tubules with phenotypic change. There was a highly significant correlation between tubular alpha-SMA expression and interstitial fibrosis, interstitial alpha-SMA(+) myofibroblast accumulation, deposition of collagen types I and III, tubular TGF-beta1 expression, and renal dysfunction. In conclusion, this study provides evidence that tubular epithelial cells can undergo phenotypic change toward a myofibroblast-like phenotype on the basis of de novo alpha-SMA expression, loss of cytokeratin, and de novo collagen staining. These data, although not conclusive, provide the first support for the hypothesis that transdifferentiation of tubular epithelial cells has a role in progressive renal fibrosis in human glomerulonephritis.

Specific tissue distribution of megsin, a novel serpin, in the glomerulus and its up-regulation in IgA nephropathy.

Mesangial cells play critical roles in maintaining a structure and function of the glomerulus. We previously cloned a novel mesangium-predominant gene, megsin, a new serine protease inhibitor. To clarify localization and roles of megsin protein, we raised polyclonal antibodies to megsin. By immunohistochemistry, megsin protein was specifically identified in the mesangial area. The amount of megsin protein was increased in glomeruli of patients with IgA nephropathy than in those of normal individuals and of patients with minimal change nephrotic syndrome or membranous nephropathy, suggesting a pathophysiological role of megsin as a functional modulator of mesangial functions in situ.