Search

* Context.--Testing reproducibility is critical for the development of methodologies for morphologic assessment. Our previous study using the descriptor-based Nephrotic Syndrome Study Network Digital Pathology Scoring System (NDPSS) on glomerular images revealed variable reproducibility. Objective.--To test reproducibility and feasibility of alternative scoring strategies for digital morphologic assessment of glomeruli and explore use of alternative agreement statistics. Design.--The original NDPSS was modified (NDPSS1 and NDPSS2) to evaluate (1) independent scoring of each individual biopsy level, (2) use of continuous measures, (3) groupings of individual descriptors into classes and sub-classes prior to scoring, and (4) indication of pathologists' confidence/uncertainty for any given score. Three and 5 pathologists scored 157 and 79 glomeruli using the NDPSS1 and NDPSS2, respectively. Agreement was tested using conventional (Cohen [kappa]) and alternative (Gwet agreement coefficient 1 [[AC.sub.1]]) agreement statistics and compared with previously published data (original NDPSS). Results.--Overall, pathologists' uncertainty was low, favoring application of the Gwet [AC.sub.1]. Greater agreement was achieved using the Gwet [AC.sub.1] compared with the Cohen [kappa] across all scoring methodologies. Mean (standard deviation) differences in agreement estimates using the NDPSS1 and NDPSS2 compared with the single-level original NDPSS were -0.09 (0.17) and -0.17 (0.17), respectively. Using the Gwet [AC.sub.1], 79% of the original NDPSS descriptors had good or excellent agreement. Pathologist feedback indicated the NDPSS1 and NDPSS2 were time-consuming. Conclusions.--The NDPSS1 and NDPSS2 increased pathologists' scoring burden without improving reproducibility. Use of alternative agreement statistics was strongly supported. We suggest using the original NDPSS on whole slide images for glomerular morphology assessment and for guiding future automated technologies. (Arch Pathol Lab Med. 2018;142:613-625; doi: 10.5858/arpa.2017-0181-OA), In the setting of clinical trials and translational research, the morphologic evaluation of renal biopsies has progressively transitioned from use of conventional light microscopy to digital pathology on whole slide [...]

Production, recycling, and elimination of urea are important to maintain nitrogen balance. Adaptation to varying loads of urea due to different protein intake or in renal failure may involve changes in urea transport and may possibly affect urea transporters. In this study, we examined the expression of the UT-B urea transporter in rats fed a low-protein diet (LPD), a high-protein diet (HPD), and a 20% urea-supplemented diet. In the kidney, UT-B protein abundance increased in the outer medulla of both LPD-fed rats and 20% urea-fed rats, without changes in the inner medulla of either group compared with controls. In HPD-fed rats, UT-B protein decreased significantly in both the outer and inner medulla. We identified expression of UT-B in the rat colon, as a 2-kb mRNA transcript and as an ~45-kDa protein, with apical localization in superficial colon epithelial cells. UT-B also is expressed in rat small intestine. In rat colon, UT-B protein abundance was mildly, but significantly, decreased in LPD-fed and 20% urea-fed rats. UT-B abundance also was examined in the colon of 7/8 nephrectomized, uremic rats and in HPD-fed rats and was not significantly different from that in control rats. These findings indicate that UT-B expression is regulated in response to different loads of urea, with a pattern that suggests involvement of tissue-specific regulatory mechanism in kidney and colon. kidney; urea transport; colon; uremia

EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides -4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning -1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately -1.5 kb nor a pair of putative Tone elements at approximately -3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure. osmotic; cell volume regulation; receptor tyrosine kinase; signal transduction; rat; kidney

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of ~50- and ~98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of [sup.14]C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa. urea transport; Caco-2

Urea plays various roles in the biology of diverse organisms. The past decade has produced new information on the molecular structure of several urea transporters in various species. Availability of DNA probes has revealed that the presence of urea transporters is not confined to the mammalian kidney but is also evident in testis and brain, raising new questions about the possible physiological role of urea in these organs. Cloning of the genes encoding the two closely related mammalian urea transporters UT-A and UT-B has helped in identifying molecular mechanisms affecting expression of urea transporters in the kidney, such as transcriptional control for UT-A abundance. On the basis of analysis of genomic sequences of individuals lacking the UT-B transporter, mutations have been found that explain deficits in their capacity to concentrate urine. More urea transporters are being characterized in marine organisms and lower vertebrates, and studying the role and regulation of urea transport from an evolutionary perspective can certainly enrich our understanding of renal physiology. urea; kidney; Kidd antigen; concentrating mechanism; osmoregulation

Dexamethasone treatment increases urea excretion and decreases urea permeability and urea transporter UT-A1 protein abundance in the inner medullary collecting duct (IMCD) of adrenalectomized rats. We examined the effect of dexamethasone treatment for 3 days on the abundance of several UT-A mRNA transcripts in rat renal medulla. By Northern blot analysis, a significant decrease in mRNA expression was observed in the inner medulla of dexamethasone-treated rats compared with controls for UT-A1 (71%), UT-A3 (75%), and UT-A3b (75%), but not for UT-A2. We then tested the effect of 100 nM dexamethasone on the activity of promoter I in the UT-A gene, using LLC-P[K.sub.1]-GR101 cells that express the glucocorticoid receptor. Dexamethasone significantly decreased the activity of rat UT-A promoter I (72%) but did not affect UT-A promoter II. Deletion analysis and site-directed mutagenesis demonstrated that sequences between -423 and -244 are important for this inhibition and that a 10-bp sequence at -363, which binds a nuclear protein in a gel shift assay, is necessary for basal promoter activity. The specific factors involved in repression of UT-A promoter I activity by glucocorticoids remain to be determined. Slc14a2 gene; kidney

Genetic cloning and analysis of the betaine gamma-amino-n-butyric acid transporter gene (BGT1) indicates the presence of eight different types of mRNA divided into three types on the basis of their 5' end sequence. Hypertonicity induces the sequence variations in these tissue-specifically expressed isoforms, as revealed by Northern blot analysis. 1B exons are present in the 28 kb BGT1 gene, of which the 1A, 1B and 1C+D exons possess separate sites of transcription initiation.

Changes in the function of intracellular calcium due to cytochrome P-450 oxidase inhibitors during the hypertonic to isotonic medium shifts in Madin- Darby canine kidney cells were examined. Myo-inositol and betaine efflux, intracellular calcium and intracellular myo-inositol were measured by transepithelial fluxing, inverted fluoresence microscopy and by an enzymatic method, respectively. The importance of extracellular calcium efflux through cell swelling-activated channels in the formation of osmolyte efflux was demonstrated.