Role of HRTPT in kidney proximal epithelial cell regeneration: Integrative differential expression and pathway analyses using microarray and scRNA-seq.

Damage to proximal tubules due to exposure to toxicants can lead to conditions such as acute kidney injury (AKI), chronic kidney disease (CKD) and ultimately end-stage renal failure (ESRF). Studies have shown that kidney proximal epithelial cells can regenerate particularly after acute injury. In the previous study, we utilized an immortalized in vitro model of human renal proximal tubule epithelial cells, RPTEC/TERT1, to isolate HRTPT cell line that co-expresses stem cell markers CD133 and CD24, and HREC24T cell line that expresses only CD24. HRTPT cells showed most of the key characteristics of stem/progenitor cells; however, HREC24T cells did not show any of these characteristics. The goal of this study was to further characterize and understand the global gene expression differences, upregulated pathways and gene interaction using scRNA-seq in HRTPT cells. Affymetrix microarray analysis identified common gene sets and pathways specific to HRTPT and HREC24T cells analysed using DAVID, Reactome and Ingenuity software. Gene sets of HRTPT cells, in comparison with publicly available data set for CD133+ infant kidney, urine-derived renal progenitor cells and human kidney-derived epithelial proximal tubule cells showed substantial similarity in organization and interactions of the apical membrane. Single-cell analysis of HRTPT cells identified unique gene clusters associated with CD133 and the 92 common gene sets from three data sets. In conclusion, the gene expression analysis identified a unique gene set for HRTPT cells and narrowed the co-expressed gene set compared with other human renal-derived cell lines expressing CD133, which may provide deeper understanding in their role as progenitor/stem cells that participate in renal repair.

Characterization and determination of cadmium resistance of CD133+/CD24+ and CD133-/CD24+ cells isolated from the immortalized human proximal tubule cell line, RPTEC/TERT1.

Stem/progenitor cells are involved in the regeneration of the renal tubules after damage due to a toxic insult. However, the mechanism involved in the regeneration of the tubules by the stem cells is not well understood due to the lack of immortal cell lines that represent the stem/progenitor cells of the kidney. A previous study from our laboratory has shown that the immortalized cell line RPTEC/TERT1 contains two populations of cells, one co-expressing CD24 and CD133, the other expressing CD24 only. The goal of the present study was to determine if both these populations could be sorted into separate independent cultures and if so, determine their characteristic features and response to the nephrotoxicant cadmium. The results of our study show that both the populations of cells could grow as independent cultures and maintain their phenotype after extended sub-culture. The CD133+/CD24+ co-expressing cells formed multicellular spheroids (nephrospheres), a characteristic feature of stem/progenitor cells, and formed branched tubule-like structures when grown on the surface of matrigel, whereas the CD133-/CD24+ cells were unable to form these structures. The CD133+/CD24+ cells were able to grow and undergo neurogenic, adipogenic, osteogenic, and tubulogenic differentiation, whereas the CD133-/CD24+ cells expressed some of the differentiation markers but were unable to grow in some of the specialized growth media. The CD133+/ CD24+ co-expressing cells had a shorter doubling time compared to the cells that expressed only CD24, and were more resistant to the toxic effects of the heavy metal, cadmium. In conclusion, the isolation and characterization of these two cell populations form the RPTEC/TERT1 cell line will facilitate the development of studies that determine the mechanisms involved in tubular damage and regeneration particularly after a toxic insult.

Primary and Immortalized Cultures of Human Proximal Tubule Cells Possess Both Progenitor and Non-Progenitor Cells That Can Impact Experimental Results.

Studies have reported the presence of renal proximal tubule specific progenitor cells which co-express PROM1 and CD24 markers on the cell surface. The RPTEC/TERT cell line is a telomerase-immortalized proximal tubule cell line that expresses two populations of cells, one co-expressing PROM1 and CD24 and another expressing only CD24, identical to primary cultures of human proximal tubule cells (HPT). The RPTEC/TERT cell line was used by the authors to generate two new cell lines, HRTPT co-expressing PROM1 and CD24 and HREC24T expressing only CD24. The HRTPT cell line has been shown to express properties expected of renal progenitor cells while HREC24T expresses none of these properties. The HPT cells were used in a previous study to determine the effects of elevated glucose concentrations on global gene expression. This study showed the alteration of expression of lysosomal and mTOR associated genes. In the present study, this gene set was used to determine if pure populations of cells expressing both PROM1 and CD24 had different patterns of expression than those expressing only CD24 when exposed to elevated glucose concentrations. In addition, experiments were performed to determine whether cross-talk might occur between the two cell lines based on their expression of PROM1 and CD24. It was shown that the expression of the mTOR and lysosomal genes was altered in expression between the HRTPT and HREC24T cell lines based on their PROM1 and CD24 expression. Using metallothionein (MT) expression as a marker demonstrated that both cell lines produced condition media that could alter the expression of the MT genes. It was also determined that PROM1 and CD24 co-expression was limited in renal cell carcinoma (RCC) cell lines.

First Report of Lecanicephalidean Tapeworms (Eucestoda) from Freshwater, Including Description of Three New Species of Tetragonocephalum Shipley and Hornell, 1905.

The lecanicephalidean cestodes parasitizing the spiral intestine of the endangered giant freshwater whipray, Urogymnus polylepis (Bleeker), are investigated for the first time. Eight host specimens were collected between 2002 and 2008 at 2 collecting sites off the eastern coast of Borneo: 6 from the Kinabatangan River (Malaysia) and 2 from a fish market in Tarakan (Indonesia). Two of these individuals were found to be infected with a total of 3 new species of TetragonocephalumShipley and Hornell, 1905. Tetragonocephalum georgei n. sp. and Tetragonocephalum opimum n. sp. were recovered from a host specimen from the Kinabatangan River, and Tetragonocephalum levicorpum n. sp. was found parasitizing a host specimen purchased at a fish market in Tarakan. Specimens of each of the new species were prepared for light microscopy; specimens of 2 of the new species were prepared for scanning electron microscopy, and histological sections were prepared for 1 of the new species. The 3 new species are distinct from the 9 valid species of Tetragonocephalum and the 1 species inquirendum based on, for example, total length, number of proglottids and testes, and size of the scolex and acetabula. Tetragonocephalum georgei n. sp. and T. levicorpum n. sp. are unusual among their congeners in that they are euapolytic (i.e., gravid proglottids were not observed) rather than apolytic. They differ from one another in scolex and acetabula size. Tetragonocephalum opimum n. sp. is unusual among its congeners in its possession of vitelline follicles arranged in 2, rather than 3, regions in the proglottid. These new species increase the total number of valid species of Tetragonocephalum to 12 and the total number of known cestodes from U. polylepis to 13 species across 6 genera in 4 orders. This is the first account of lecanicephalideans reported from freshwater. The taxonomic status of each of the 32 nominal taxa historically associated with Tetragonocephalum is re-assessed. Type host identities of all valid species are revised and discussed in light of recent taxonomic efforts in the Dasyatidae Jordan and Gilbert.

Elevated glucose represses lysosomal and mTOR-related genes in renal epithelial cells composed of progenitor CD133+ cells.

Hyperglycemia is one of the major health concern in many parts of the world. One of the serious complications of high glucose levels is diabetic nephropathy. The preliminary microarray study performed on primary human renal tubular epithelial (hRTE) cells exposed to high glucose levels showed a significant downregulation of mTOR as well as its associated genes as well as lysosomal genes. Based on this preliminary data, the expression of various lysosomal genes as well as mTOR and its associated genes were analyzed in hRTE cells exposed to 5.5, 7.5, 11 and 16 mM glucose. The results validated the microarray analysis, which showed a significant decrease in the mRNA as well as protein expression of the selected genes as the concentration of glucose increased. Co-localization of lysosomal marker, LAMP1 with mTOR showed lower expression of mTOR as the glucose concentration increased, suggesting decrease in mTOR activity. Although the mechanism by which glucose affects the regulation of lysosomal genes is not well known, our results suggest that high levels of glucose may lead to decrease in mTOR expression causing the cells to enter an anabolic state with subsequent downregulation of lysosomal genes.

Xylaria necrophora, sp. nov., is an emerging root-associated pathogen responsible for taproot decline of soybean in the southern United States.

Taproot decline (TRD) is a disease of soybean that has been reported recently from the southern United States (U.S.). Symptoms of TRD include foliar interveinal chlorosis followed by necrosis. Darkened, charcoal-colored areas of thin stromatic tissue are evident on the taproot and lateral roots along with areas of necrosis within the root and white mycelia within the pith. Upright stromata typical of Xylaria can be observed on crop debris and emerging from infested roots in fields where taproot decline is present, but these have not been determined to contain fertile perithecia. Symptomatic plant material was collected across the known range of the disease in the southern U.S., and the causal agent was isolated from roots. Four loci, ⍺-actin (ACT), ß-tubulin (TUB2), the nuclear rDNA internal transcribed spacers (nrITS), and the RNA polymerase subunit II (RPB2), were sequenced from representative isolates. Both maximum likelihood and Bayesian phylogenetic analyses showed consistent clustering of representative TRD isolates in a highly supported clade within the Xylaria arbuscula species complex in the "HY" clade of the family Xylariaceae, distinct from any previously described taxa. In order to understand the origin of this pathogen, we sequenced herbarium specimens previously determined to be "Xylaria arbuscula" based on morphology and xylariaceous endophytes collected in the southern U.S. Some historical specimens from U.S. herbaria collected in the southern region as saprophytes as well as a single specimen from Martinique clustered within the "TRD" clade in phylogenetic analyses, suggesting a possible shift in lifestyle. The remaining specimens that clustered within the family Xylariaceae, but outside of the "TRD" clade, are reported. Both morphological evidence and molecular evidence indicate that the TRD pathogen is a novel species, which is described as Xylaria necrophora.

Morphological variation in the hyperapolytic lecanicephalidean species Anteropora japonica (Yamaguti, 1934) (Eucestoda).

In November of 2013, a specimen of Japanese sleeper ray, Narke japonica (Temminck et Schlegel), caught off Nanfang-ao, Taiwan was found to be parasitised by the cestode Anteropora japonica (Yamaguti, 1934). Specimens comprised whole worms and free proglottids, both of varying degrees of maturity. This material allowed for the opportunity to examine in detail the developmental progression of this hyperapolytic lecanicephalidean species with regard to overall size, scolex dimensions, and microthrix pattern. Complete immature worms ranged in size from 2.4 mm to 14 mm. The smallest scoleces were half as wide as larger scoleces and exhibited a much smaller ratio of apical organ width to bothridial width. Proglottids more than quadrupled in length during maturation from terminal attached immature to detached proglottids. In addition, a change in microthrix pattern was observed on the anterior region of the proglottids from immature to gravid proglottids; the anterior region of attached immature proglottids is covered with gladiate to coniform spinitriches with capilliform filitriches only rarely visible, whereas this region in detached proglottids is covered with gladiate to coniform spinitriches and conspicuous capilliform filitriches. This is the first report of A. japonica from outside Japan expanding its distribution south to Taiwan. In addition, a preliminary phylogenetic analysis of the genus is presented that suggests congeners from the same host species are not each other's closest relatives, nor is there an apparent phylogenetic signal for apical organ type or reproductive strategy (apolysis). However, reproductive strategy does seem to be correlated with host group such that euapolytic species parasitise dasyatid stingrays while hyperapolytic species parasitise either torpediniform rays or orectolobiform sharks.