Xanthogranulomatous epithelial tumor: a novel entity of uncertain biologic potential.

Xanthogranulomatous epithelial tumor (XGET) is an extremely rare and recently described mesenchymal neoplasm characterized by a distinctive histological appearance and clinical presentation. This case report describes a unique case of XGET in a 66-year-old female patient who presented with a 5 cm mass in the dorsal distal left thigh. The clinical, radiological, and pathological findings, as well as the management and follow-up, are discussed.

Protein atlas of fibroblast specific protein 1 (FSP1)/S100A4.

Fibroblast specific protein 1 (FSP1)/S100A4 is a calcium binding protein which has been linked to epithelial-mesenchymal transition, tissue fibrosis, pulmonary vascular disease, metastatic tumour development, increased tumour cell motility and invasiveness. This protein is reported to be also expressed in newly formed and differentiated fibroblasts and has been used in various studies to demonstrate epithelial-mesenchymal transition (EMT). We aimed to characterize S100A4 positive cells in different human tissue compartments, with the focus on fibroblasts/myofibroblast. We found S100A4 expression in a wide range of cells. Fibroblasts/myofibroblasts showed a broad spectrum of staining intensity, ranging from negative to strong expression of S100A4, with the strongest expression in smooth muscle actin positive myofibroblasts. Cells of haematopoietic lineage, namely CD4 and CD8 positive T-lymphocytes, but not B-lymphocytes expressed S100A4. All investigated monocytes, macrophages and specialised histiocytes were positive for S100A4. Even some epithelial cells of the kidney and bladder were positive for S100A4. Expression was also found in the vasculature. Here, cells of the subendothelial space, tunica adventitia and some smooth muscle cells of the tunica media were positive for S100A4. In summary, S100A4 is expressed in various cell types of different lineage and is not, as originally believed, specific for fibroblasts (FSP). Results attained under the premise of specificity of FSP1/S100A4 for fibroblasts, like the founding research on EMT type 2 in kidney and liver, therefore need to be reinterpreted.

[A Rare Cause of Hypercalcemia]. / Eine seltene Ursache der Hyperkalzämie.

A Rare Cause of Hypercalcemia Abstract. Sarcoidosis is a rare cause of hypercalcemia, which is in most cases caused by primary hyperparathyroidism or a malignant tumor. Other rare causes include vitamin D intoxication, other granulomatous diseases (such as tuberculosis, or fungal infection) or mutiple myeloma. Because of the broad differential diagnosis, a systematic diagnostic approach is crucial to establish the correct diagnosis. Therapeutic measures include volume replacement and subsequent administration of bisphosphonates. If hypercalcemia is caused by sarcoidosis, therapy with systemic corticosteroids is indicated.

SalvGlandDx - a comprehensive salivary gland neoplasm specific next generation sequencing panel to facilitate diagnosis and identify therapeutic targets.

Diagnosis of salivary gland neoplasms is often challenging due to their high morphological diversity and overlaps. Several recurrent molecular alterations have been described recently, which can serve as powerful diagnostic tools and potential therapeutic targets (e.g. NTRK or RET fusions). However, current sequential molecular testing can be expensive and time consuming. In order to facilitate the diagnosis of salivary gland neoplasms, we designed an all-in-one RNA-based next generation sequencing panel suitable for the detection of mutations, fusions and gene expression levels (including NR4A3) of 27 genes involved in salivary gland neoplasms. Here we present the validation of the "SalvGlandDx" panel on FFPE histological specimen including fine needle aspiration (FNA) cell block material, against the standard methods currently used at our institution. In a second part we describe selected unique cases in which the SalvGlandDx panel allowed proper diagnosis and new insights into special molecular characteristics of selected salivary gland tumors. We characterize a unique salivary gland adenocarcinoma harboring a ZCCHC7-NTRK2 fusion, a highly uncommon spindle cell and pseudoangiomatoid adenoid-cystic carcinoma with MYBL1-NFIB fusion, and a purely oncocytic mucoepidermoid carcinoma, whereas diagnosis could be made by detection of a CRTC3-MAML2 rearrangement on the cell block specimen of the FNA. Further, a rare case of a SS18-ZBTB7A rearranged low-grade adenocarcinoma previously described as potential spectrum of microsecretory adenocarcinoma, is reported. In addition, features of six cases within the spectrum of polymorphous adenocarcinoma / cribriform adenocarcinoma of salivary gland including PRKD1 p.E710D mutations and novel fusions involving PRKAR2A-PRKD1, SNX9-PRKD1 and ATL2-PRKD3, are described.

The AP1 Transcription Factor Fosl2 Promotes Systemic Autoimmunity and Inflammation by Repressing Treg Development.

Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2tg) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2tg × Rag2-/- mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2tg T cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.

Value of postmortem studies in deceased neonatal and pediatric intensive care unit patients.

Worldwide, various autopsy studies have shown a decrease in the diagnostic error rate over the last years. The cause of this positive development is mainly due to the improvement of modern medicine. However, intensive care unit patients are thought to have a higher risk for diagnostic errors, which is documented in several studies in the adult population. In contrast, there is only limited information about diagnostic errors in pediatrics, particularly in pediatric and neonatal intensive care units. The aims of this study were to analyze the spectrum of childhood death, determine the prevalence and distribution of autopsy-confirmed diagnostic errors, and describe patient characteristics that might have influenced the discordance between antemortem and postmortem findings. We analyzed 143 autopsy reports from 2004 to 2013 and correlated these with clinical reports. The overall autopsy rate during this interval was 20.3%. The leading causes of death were congenital malformations (28%), diseases closely associated with perinatal disorders (25%), disorders of the cardiovascular system (18%), and infections (15%). Additional findings were obtained in 23% of the autopsies. Major diagnostic errors were found in 6%, the lowest reported value in a developed country as yet. Most cases (75%) showed complete concordance between clinical diagnoses and postmortem findings, in line with improvements in diagnostic and therapeutic processes over the last decades. In conclusion, autopsy of neonates, infants, and children represents an important tool for monitoring the quality of pediatric and neonatal medical care.

Chromosomal aberrations of cancer-testis antigens in myeloma patients.

Cancer-testis antigens (CTAgs) play a major role in the immune response against cancer, but their biological functions in germ and cancer cells is still unclear. MAGE-C1 and MAGE-C2 are two CTAgs located at the Xq27 region of chromosome X and frequently expressed in multiple myeloma. Chromosomal rearrangements often occur in myeloma. We therefore investigated whether numerical and structural chromosomal aberrations correlate with their protein expression in primary multiple myelomas. To this aim, we designed new fluorescence in situ hybridization probes specific for the MAGE region in the Xq27 region and evaluated simultaneously aberrations of the X chromosome centromere. The comparison of MAGE copy number and chromosome X status revealed that MAGE copy number changes occurred in 6/43 (14%) cases, independent of concomitant X chromosome alterations. These numerical aberrations are less frequent than the expression of MAGE-C1 and MAGE-C2 (63% and 27% of patients, respectively) and do not always correlate with MAGE-C1 and MAGE-C2 expressions, suggesting alternative regulatory mechanisms in the expression of these genes.

eIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation.

PURPOSE: The eukaryotic translation initiation factor (eIF) 3a, the largest subunit of the eIF3 complex, is a key functional entity in ribosome establishment and translation initiation. In the past, aberrant eIF3a expression has been linked to the pathology of various cancer types but, so far, its expression has not been investigated in transitional cell carcinomas. Here, we investigated the impact of eIF3 expression on urinary bladder cancer (UBC) cell characteristics and UBC patient survival. METHODS AND RESULTS: eIF3a expression was reduced through inducible knockdown in the UBC-derived cell lines RT112, T24, 5637 and HT1197. As a consequence of eIF3a down-regulation, UBC cell proliferation, clonogenic potential and motility were found to be decreased and, concordantly, UBC tumour cell growth rates were found to be impaired in xenotransplanted mice. Polysomal profiling revealed that reduced eIF3a levels increased the abundance of 80S ribosomes, rather than impairing translation initiation. Microarray-based gene expression and ontology analyses revealed broad effects of eIF3a knockdown on the transcriptome. Analysis of eIF3a expression in primary formalin-fixed paraffin embedded UBC samples of 198 patients revealed that eIF3a up-regulation corresponds to tumour grade and that high eIF3a expression corresponds to longer overall survival rates of patients with low grade tumours. CONCLUSIONS: From our results we conclude that eIF3a expression may have a profound effect on the UBC phenotype and, in addition, may serve as a prognostic marker for low grade UBCs.

Smoking induces transcription of the heat shock protein system in the joints.

OBJECTIVES: Smoking increases the risk of developing rheumatoid arthritis (RA) and worsens the course of the disease. In the current study we analysed whether smoking can affect gene expression directly in the joints. METHODS: Synovial fibroblasts were incubated with 5% cigarette smoke extract and changes in gene expression were detected using whole genome microarrays and verified with real-time PCR. Synovial tissues were obtained from smoking and non-smoking patients with RA undergoing joint replacement surgery and from mice exposed to cigarette smoke or ambient air in a whole body exposure chamber for 3 weeks. RESULTS: Microarray and real-time PCR analysis showed a significant upregulation of the heat shock proteins DnaJA4, DnaJB4, DnaJC6, HspB8 and Hsp70 after stimulation of synovial fibroblasts with 5% cigarette smoke extract. Similarly, in synovial tissues of smokers with RA the expression of DnaJB4, DnaJC6, HspB8 and Hsp70 was significantly higher compared with non-smokers with RA. Upregulation of DnaJB4 and DnaJC6 in joints by smoking was also confirmed in mice exposed to cigarette smoke. CONCLUSIONS: Our data clearly show that smoking can change gene expression in the joints, which can lead to the activation of signalling pathways that promote development of autoimmunity and chronic joint inflammation.

αv-Integrin isoform expression in primary human tumors and brain metastases.

UNLABELLED: To determine whether metastasis to brain is associated with altered expression patterns of integrins, we investigated the expression of αvß3, αvß5, αvß6 and αvß8 integrins in primary malignancies and metastases to brain of breast, lung and renal carcinomas and in malignant melanoma. Inhibitors of αv integrins are currently in clinical trials for glioblastoma. The role of integrins in the process of brain metastasis from other human tumors is unknown. Immunohistochemistry with novel integrin subtype specific rabbit monoclonal antibodies was performed on tissue microarrays of archival material of surgical biopsies taken from primary tumors and brain metastases. Integrin αvß3 expression was increased in brain metastases compared to primary tumors of breast adenocarcinoma, non-small cell lung cancer, renal clear cell cancer and malignant cutaneous melanoma (all p < 0.01). Similarly, integrin αvß8 expression was increased in brain metastases compared to primary tumors of breast cancer (p < 0.0001), lung cancer (p < 0.01) and renal cancer (p < 0.0001), with a similar trend in metastatic melanoma. Integrin αvß5 was expressed in most primary tumors (98% breast cancer; 67% lung cancer; 90% renal cancer; 89% melanoma) and showed a stronger expression in brain metastases compared to primary tumors from lung cancer and melanoma (p < 0.05). Also integrin αvß6 expression was increased in brain metastases compared to primary breast cancer (p < 0.001). CONCLUSIONS: The stronger αv-integrin expression in brain metastases, especially of αvß3 and αvß8 integrins, suggests that certain αv integrin are involved in the process of brain metastasis. αv Integrins may be therapeutic targets for patients with metastatic cancer in brain.

MicroRNA expression array identifies novel diagnostic markers for conventional and oncocytic follicular thyroid carcinomas.

OBJECTIVE: The most difficult thyroid tumors to be diagnosed by cytology and histology are conventional follicular carcinomas (cFTCs) and oncocytic follicular carcinomas (oFTCs). Several microRNAs (miRNAs) have been previously found to be consistently deregulated in papillary thyroid carcinomas; however, very limited information is available for cFTC and oFTC. The aim of this study was to explore miRNA deregulation and find candidate miRNA markers for follicular carcinomas that can be used diagnostically. DESIGN: Thirty-eight follicular thyroid carcinomas (21 cFTCs, 17 oFTCs) and 10 normal thyroid tissue samples were studied for expression of 381 miRNAs using human microarray assays. Expression of deregulated miRNAs was confirmed by individual RT-PCR assays in all samples. In addition, 11 follicular adenomas, two hyperplastic nodules (HNs), and 19 fine-needle aspiration samples were studied for expression of novel miRNA markers detected in this study. RESULTS: The unsupervised hierarchical clustering analysis demonstrated individual clusters for cFTC and oFTC, indicating the difference in miRNA expression between these tumor types. Both cFTCs and oFTCs showed an up-regulation of miR-182/-183/-221/-222/-125a-3p and a down-regulation of miR-542-5p/-574-3p/-455/-199a. Novel miRNA (miR-885-5p) was found to be strongly up-regulated (>40-fold) in oFTCs but not in cFTCs, follicular adenomas, and HNs. The classification and regression tree algorithm applied to fine-needle aspiration samples demonstrated that three dysregulated miRNAs (miR-885-5p/-221/-574-3p) allowed distinguishing follicular thyroid carcinomas from benign HNs with high accuracy. CONCLUSIONS: In this study we demonstrate that different histopathological types of follicular thyroid carcinomas have distinct miRNA expression profiles. MiR-885-5p is highly up-regulated in oncocytic follicular carcinomas and may serve as a diagnostic marker for these tumors. A small set of deregulated miRNAs allows for an accurate discrimination between follicular carcinomas and hyperplastic nodules and can be used diagnostically in fine-needle aspiration biopsies.

Overexpression of eIF3a in Squamous Cell Carcinoma of the Oral Cavity and Its Putative Relation to Chemotherapy Response.

The eukaryotic translation initiation factor eIF3a is one of the core subunits of the translation initiation complex eIF3, responsible for ribosomal subunit joining and mRNA recruitment to the ribosome. It is known to play an important role in general translation initiation as well as in the specific translational regulation of various gene products, among which many influence tumour development, progression, and the therapeutically important pathways of DNA damage repair. Therefore, beyond its role in protein synthesis, eIF3a is emerging as regulator in tumour pathogenesis and therapy response and, therefore, a potential tumor marker. By means of a tissue microarray (TMA) for histopathological and statistical assessment, we here show eIF3a expression in 103 cases of squamous cell carcinoma of the oral cavity (OSCC), representing tissues from 103 independent patients. A subset of the study cohort was treated with platinum based therapy. Our results show that the 170 kDa protein is upregulated in OSCC and correlates with good overall survival. Overexpressing tumors respond better to platinum-based chemotherapy, suggesting eIF3a as a putative predictive as well as prognostic tumor marker in OSCC.

Cell adhesion molecules P-cadherin and CD24 are markers for carcinoma and dysplasia in the biliary tract.

P-cadherin (CDH3) and CD24 are cell adhesion molecules that control morphogenic processes, cell motility, and invasive growth of tumor cells. The aim of our study was to investigate P-cadherin and CD24 expression in carcinomas and dysplastic lesions of the biliary tract and to evaluate the potential diagnostic usefulness of these cell adhesion molecules. Using immunohistochemistry on tissue microarrays, we analyzed P-cadherin, CD24, and p53 expression in 117 carcinomas of the biliary tract (19 intrahepatic cholangiocarcinomas, 59 extrahepatic cholangiocarcinomas, and 39 gallbladder carcinomas) and correlated our findings with clinicopathologic parameters. We found P-cadherin positivity in 37% of intrahepatic cholangiocarcinomas, 73% of extrahepatic cholangiocarcinomas, and 64% of gallbladder carcinomas, respectively. CD24 reactivity was observed in 21% of intrahepatic cholangiocarcinomas, 58% of extrahepatic cholangiocarcinomas, and 42% of gallbladder carcinomas. Nuclear p53 expression was found in 37% of intrahepatic cholangiocarcinomas, 46% of extrahepatic cholangiocarcinomas, and 45% of gallbladder carcinomas. We also studied P-cadherin, CD24, and p53 expression in normal (n = 30), inflamed (n = 22), and dysplastic (n = 21) biliary epithelium of extrahepatic bile ducts. Dysplastic biliary epithelium was positive for P-cadherin in 91%, for CD24 in 71%, and for p53 in 24% of lesions, respectively. In contrast, normal and inflamed epithelia were negative for all 3 proteins. We conclude that P-cadherin and CD24 are expressed in carcinomas of the biliary tract with high frequency and at an early stage of carcinogenesis. Therefore, they may be useful markers for early detection and as targets for therapy of cholangiocarcinoma.

Pulse mTOR inhibitor treatment effectively controls cyst growth but leads to severe parenchymal and glomerular hypertrophy in rat polycystic kidney disease.

The efficacy of mammalian target of rapamycin (mTOR) inhibitors is currently tested in patients affected by autosomal dominant polycystic kidney disease. Treatment with mTOR inhibitors has been associated with numerous side effects. However, the renal-specific effect of mTOR inhibitor treatment cessation in polycystic kidney disease is currently unknown. Therefore, we compared pulse and continuous everolimus treatment in Han:SPRD rats. Four-week-old male heterozygous polycystic and wild-type rats were administered everolimus or vehicle by gavage feeding for 5 wk, followed by 7 wk without treatment, or continuously for 12 wk. Cessation of everolimus did not result in the appearance of renal cysts up to 7 wk postwithdrawal despite the reemergence of S6 kinase activity coupled with an overall increase in cell proliferation. Pulse everolimus treatment resulted in striking noncystic renal parenchymal enlargement and glomerular hypertrophy that was not associated with compromised kidney function. Both treatment regimens ameliorated kidney function, preserved the glomerular-tubular connection, and reduced proteinuria. Pulse treatment at an early age delays cyst development but leads to striking glomerular and parenchymal hypertrophy. Our data might have an impact when long-term treatment using mTOR inhibitors in patients with autosomal dominant polycystic kidney disease is being considered.

Involvement of CD252 (CD134L) and IL-2 in the expression of cytotoxic proteins in bacterial- or viral-activated human T cells.

Regulation of cytotoxic effector molecule expression in human CTLs after viral or bacterial activation is poorly understood. By using human autologous dendritic cells (DCs) to prime T lymphocytes, we found perforin only highly up-regulated in virus- (HSV-1, vaccinia virus) but not in intracellular bacteria- (Listeria innocua, Listeria monocytogenes, Mycobacterium tuberculosis, Chlamydophila pneumoniae) activated CTLs. In contrast, larger quantities of IFN-gamma and TNF-alpha were produced in Listeria-stimulated cultures. Granzyme B and granulysin were similarly up-regulated by all tested viruses and intracellular bacteria. DCs infected with HSV-1 showed enhanced surface expression of the costimulatory molecule CD252 (CD134L) compared with Listeria-infected DC and induced enhanced secretion of IL-2. Adding blocking CD134 or neutralizing IL-2 Abs during T cell activation reduced the HSV-dependent up-regulation of perforin. These data indicate a distinct CTL effector function in response to intracellular pathogens triggered via differing endogenous IL-2 production upon costimulation through CD252.

Expression of CD24, P-cadherin and S100A4 in tumors of the ampulla of Vater.

Carcinomas of the Vaterian system are rare and presumably arise from preexisting adenomas (adenoma-carcinoma-sequence). Usually, biopsies are obtained to confirm and specify endoscopic findings, but differentiating reactive atypia from dysplasia or dysplasia from invasive carcinoma can sometimes be difficult or even impossible on morphological criteria alone. In case of invasive carcinoma, furthermore, the precise classification of carcinoma subtypes needs to be established since the distinct subtypes differ significantly in terms of clinical outcome. The cell adhesion proteins CD24, P-cadherin and S100A4 were shown to be expressed in several carcinomas and in dysplastic epithelium but only rarely in normal mucosa. We therefore investigated their expression in 177 carcinoma, 114 adenoma and 152 normal mucosa specimens of the ampulla of Vater. Although the expression of the cell adhesion proteins did not differ between the carcinoma subtypes, marked differences between normal mucosa, adenoma and carcinoma samples were observed. All marker proteins were expressed in less than 7% of normal mucosa samples (S100A4 in only 1% of cases) and showed an increasing expression from adenoma to invasive carcinoma. Our findings suggest that P-cadherin and S100A4 are helpful in discriminating normal mucosa or reactive atypia from neoplastic lesions. CD24 and S100A4, furthermore, can assist in the differential diagnosis of dysplasia vs invasive carcinoma.

Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat.

Tubulo-interstitial fibrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the fibrotic kidney there exists, besides normal fibroblasts, a large population of myofibroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myofibroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from infiltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident fibroblasts into myofibroblasts in renal fibrosis. Therefore we examined the fate of resident fibroblasts in the initial phase of renal fibrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-fixed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin (alphaSMA) in resident fibroblasts was revealed by immunofluorescence and confirmed by the observation of bundles of microfilaments and webs of intermediate filaments in the electron microscope. Inversely, there was a decreased expression of 5'-nucleotidase (5'NT), a marker of renal cortical fibroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myofibroblasts, became more frequent. The mitotic activity in fibroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular fibroblasts proliferated and they showed progressive alterations, suggesting a transformation in myofibroblasts. Thus the resident fibroblasts likely play a central role in fibrosis in that model.

Proliferation capacity of the renal proximal tubule involves the bulk of differentiated epithelial cells.

We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G(1)-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G(1) in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G(1) and on the shortening of the obligatory period of quiescence that follows division.

Mitotic activation of Akt signalling pathway in Han:SPRD rats with polycystic kidney disease.

AIM: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by an imbalance between tubular epithelial cell proliferation and apoptosis. We have previously shown that the mammalian target of rapamycin (mTOR) signalling pathway is aberrantly activated in the cystic kidneys of Han:SPRD rats with ADPKD. Because the Akt kinase is an upstream regulator of mTOR, we hypothesized that the activity of Akt could be enhanced in the kidneys of Han:SPRD rats. METHODS: Reverse transcription-polymerase chain reaction, western blot, enzyme-linked immunosorbent assay and immunohistochemistry were used to analyse Akt expression in rat polycystic kidneys. RESULTS: Wild-type (+/+) and heterozygous (Cy/+) Han:SPRD rats showed constitutive expression of Akt-1, -2 and -3 mRNA by reverse transcription-polymerase chain reaction analysis with no significant difference between Cy/+ and +/+ kidneys. Western blotting and enzyme-linked immunosorbent assay showed a significant increase in phosphorylated Akt in Cy/+ compared with +/+ kidneys. The pattern of immunoreactivity for phosphorylated Akt in kidney sections was the same in +/+ and in Cy/+ rats, with very low levels in interphase cells, but extremely bright signals in mitotic cells, beginning with the onset of the prophase. The in vivo incorporation of bromo-deoxyuridine revealed approximately a ninefold higher rate of proliferation in Cy/+ cyst epithelia compared with normal tubule epithelia in +/+ rats, while the expression of the cell cycle marker Ki67 revealed approximately a sixfold higher rate of proliferation. In summary, enhanced phosphorylation of Akt can be demonstrated in Cy/+ kidneys which correlates with a markedly elevated proliferation rate of epithelial cells in cysts. Mitotic but not resting cells display strong phosphorylation of Akt. CONCLUSION: Because Akt is a proximal target of mTOR, its inhibition with specific antagonists could be useful to prevent or halt cystogenesis in ADPKD.

Proximal tubular epithelial cells are generated by division of differentiated cells in the healthy kidney.

We searched for evidence for a contribution of stem cells in growth of the proximal S3 segments of healthy rats. According to the stem cell model, stem cells are undifferentiated and slow cycling; the bulk of cycling cells are transit amplifying, rapidly cycling cells. We show the following. 1) By continuous application of a thymidine analog (ThA) for 7 days, S3 proximal epithelial cells in healthy kidneys display a high-cycling rate. 2) Slow-cycling cells, identified by lack of ThA uptake during 14 days of continuous ThA application up to death and by expression of the cell cycle protein Ki67 at death, have the same degree of differentiation as quiescent cells. 3) To detect rapidly cycling cells, rats were killed at various time points after injection of a ThA. Double immunofluorescence for ThA and a cell cycle marker was performed, with colocalization indicating successive divisions. During one week after division, daughter cells display a very low proliferation rate, indicating the absence of rapidly cycling cells. 4) Labeling with cyclin D1 showed that this low proliferation rate is due to cycle arrest. 5) More than 50% of the S3 cells entered the cell cycle 36 h after a potent proliferative stimulus (lead acetate injection). We conclude that generation of new cells in the proximal tubule relies on division of differentiated, normally slow-cycling cells. These may rapidly enter the cycle under an adequate stimulus.