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Abstract Background Microarray analysis provides a powerful approach to identify gene expression alterations following transplantation. In patients the heterogeneity of graft specimens, co-morbidity, co-medications and the challenges in sample collection and preparation complicate conclusions regarding the underlying mechanisms of graft injury, rejection and immune regulation. Results We used a rat kidney transplantation model with strict transplant and sample preparation procedures to analyze genome wide changes in gene expression four days after syngeneic and allogeneic transplantation. Both interventions were associated with substantial changes in gene expression. After allogeneic transplantation, genes and pathways related to transport and metabolism were predominantly down-regulated consistent with rejection-mediated graft injury and dysfunction. Up-regulated genes were primarily related to the acute immune response including antigen presentation, T-cell receptor signaling, apoptosis, interferon signaling and complement cascades. We observed a cytokine and chemokine expression profile consistent with activation of a Th1-cell response. A novel finding was up-regulation of several regulatory and protective genes after allogeneic transplantation, specifically IL10, Bcl2a1, C4bpa, Ctla4, HO-1 and the SOCS family. Conclusion Our data indicate that in parallel with the predicted activation of immune response and tissue injury pathways, there is simultaneous activation of pathways for counter regulatory and protective mechanisms that would balance and limit the ongoing inflammatory/immune responses. The pathophysiological mechanisms behind and the clinical consequences of alterations in expression of these gene classes in acute rejection, injury and dysfunction vs. protection and immunoregulation, prompt further analyses and open new aspects for therapeutic approaches.

Objective: Na+/- and H2O-reabsorption, as well as the reninangiotensin- aldosterone system are altered in renal transplant recipients after transplantation. However, cellular mechanisms underlying those changes remain unclear. Methods: We examined control and transplanted rats (Lewis- Brown-Norway kidneys transplanted into uninephrectomized Lewis rats) under immunosuppressive therapy (CsA 5 mg/kg/day i.p.). Transporters and receptors were analysed by means of microfluorimetry, semiquantitative RT-PCR and Western Blot in whole kidney and isolated nephron segments. Blood and urine samples were collected and body weight and blood pressure were recorded. Results: Previously we reported a reduction of Na+/H+ exchanger type 3 (NHE3) activity and expression after transplantation and acute rejection (Velic et al.: JASN 15:967;2004). These effects were not significant anymore under immunosuppression. Angiotensin II (10 pM)-mediated activation of NHE3 which was diminished at day 4 after transplantation with rejection (-48 ± 65%, n = 8), was preserved under CsA, as was the expression of the epithelial Na+/- channel (ENaC) and aquaporin 2 (AQP2), which were downregulated in the acute rejection model. Application of CsA did not prevent transient increases in plasma aldosterone and renin levels, again mirrored by decreased fractional excretions of Na+ and K+. Under CsA, urine volume increase after transplantation was less pronounced compared to the rejection model without CsA (without CsA 27.1 ± 4 ml/24 h, n = 17 at day 4 after transplantation and with CsA 20.6 ± 2 ml/24 h, n = 1 at day 4 after transplantation, control 14.4 ± 0.6 ml/24 h, n = 32). Proteinuria was completely abolished. In control animals, the immunosuppressive therapy did not affect expression and activity of the transport proteins. Conclusions: We conclude that with the applied dose of CsA rejection was reduced while activity and expression of transporters which were down-regulated with rejection was preserved. The conserved expression of AQP2 under CsA may be a cause of the decreased diuresis in comparison to animals with rejection. [ABSTRACT FROM AUTHOR]

The hormone renin is produced in the kidney by the juxtaglomerular cells. It is the rate-limiting factor in the circulating renin-angiotensin-aldosterone system (RAAS), which contributes to electrolyte, water, and blood pressure homeostasis. In the kidneys, the distal tubule and the collecting duct are the key target segments for RAAS. The collecting duct is important for urine production and also for salt, water, and acid-base homeostasis. The critical functional role of the collecting duct is mediated by the principal and the intercalated cells and is regulated by different hormones like aldosterone and vasopressin. The collecting duct is not only a target for hormones but also a place of hormone production. It is accepted that renin is produced in the collecting duct at a low level. Several studies have described that the cells in the collecting duct exhibit plasticity properties because the ratio of principal to intercalated cells can change under specific circumstances. This narrative review focuses on two aspects of the collecting duct that remain somehow aside from mainstream research, namely the cell plasticity and the renin expression. We discuss the link between these collecting duct features, which we see as a promising area for future research given recent findings.

T-cell lymphomas are heterogeneous and rare lymphatic malignancies with unfavorable prognosis. Consequently, new therapeutic strategies are needed. The enhancer of zeste homologue 2 (EZH2) is the catalytic subunit of the polycomb repressive complex 2 and responsible for lysine 27 trimethylation of histone 3. EZH2 is overexpressed in several tumor entities including T-cell neoplasms leading to epigenetic and consecutive oncogenic dysregulation. Thus, pharmacological EZH2 inhibition is a promising target and its clinical evaluation in T-cell lymphomas shows favorable results. We have investigated EZH2 expression in two cohorts of T-cell lymphomas by mRNA-profiling and immunohistochemistry, both revealing overexpression to have a negative impact on patients' prognosis. Furthermore, we have evaluated EZH2 inhibition in a panel of leukemia and lymphoma cell lines with a focus on T-cell lymphomas characterized for canonical EZH2 signaling components. The cell lines were treated with the inhibitors GSK126 or EPZ6438 that inhibit EZH2 specifically by competitive binding at the S -adenosylmethionine (SAM) binding site in combination with the common second-line chemotherapeutic oxaliplatin. The change in cytotoxic effects under pharmacological EZH2 inhibition was evaluated revealing a drastic increase in oxaliplatin resistance after 72 h and longer periods of combinational incubation. This outcome was independent of cell type but associated to reduced intracellular platinum. Pharmacological EZH2 inhibition revealed increased expression in SRE binding proteins, SREBP1/2 and ATP binding cassette subfamily G transporters ABCG1/2. The latter are associated with chemotherapy resistance due to increased platinum efflux. Knockdown experiments revealed that this was independent of the EZH2 functional state. The EZH2 inhibition effect on oxaliplatin resistance and efflux was reduced by additional inhibition of the regulated target proteins. In conclusion, pharmacological EZH2 inhibition is not suitable in combination with the common chemotherapeutic oxaliplatin in T-cell lymphomas revealing an EZH2-independent off-target effect.

The corticomedullary osmotic gradient between renal cortex and medulla induces a specific spatial gene expression pattern. The factors that controls these differences are not fully addressed. Adaptation to hypertonic environment is mediated by the actions of the nuclear factor of activated T-cells 5 (NFAT5). NFAT5 induces the expression of genes that lead to intracellular accumulation of organic osmolytes. However, a systematical analysis of the NFAT5-dependent gene expression in the kidneys was missing. We used primary cultivated inner medullary collecting duct (IMCD) cells from control and NFAT5 deficient mice as well as renal cortex and inner medulla from principal cell specific NFAT5 deficient mice for gene expression profiling. In primary NFAT5 deficient IMCD cells, hyperosmolality induced changes in gene expression were abolished. The majority of the hyperosmolality induced transcripts in primary IMCD culture were determined to have the greatest expression in the inner medulla. Loss of NFAT5 altered the expression of more than 3000 genes in the renal cortex and more than 5000 genes in the inner medulla. Gene enrichment analysis indicated that loss of NFAT5 is associated with renal inflammation and increased expression of kidney injury marker genes, like lipocalin-2 or kidney injury molecule-1. In conclusion we show that NFAT5 is a master regulator of gene expression in the kidney collecting duct and in vivo loss of NFAT function induces a kidney injury like phenotype., (© 2022. The Author(s).)

Water homeostasis is tightly regulated by the kidneys via the process of urine concentration. During reduced water intake, the antidiuretic hormone arginine vasopressin (AVP) binds to the vasopressin receptor type II (V2R) in the kidney to enhance countercurrent multiplication and medullary osmolality, and increase water reabsorption via aquaporin-2 (AQP2) water channels. The importance of this AVP, V2R, and AQP2 axis is highlighted by low urine osmolality and polyuria in people with various water balance disorders, including nephrogenic diabetes insipidus (NDI). ELF5 and nuclear factor of activated T cells 5 (NFAT5) are two transcription factors proposed to regulate Aqp2 expression, but their role is poorly defined. Here we generated two novel mouse lines with principal cell (PC)-specific deletion of ELF5 or NFAT5 and phenotyped them in respect to renal water handling. ELF5-deficient mice (ELF5 PC-KO ) had a very mild phenotype, with no clear differences in AQP2 abundance, and mild differences in renal water handling and maximal urinary concentrating capacity. In contrast, NFAT5 (NFAT5 PC-KO ) mice had significantly higher water intake and their 24 h urine volume was almost 10-fold greater than controls. After challenging with dDAVP or 8 h water restriction, NFAT5 PC-KO mice were unable to concentrate their urine, demonstrating that they suffer from NDI. The abundance of AQP2, other AQPs, and the urea transporter UT-A1 were greatly decreased in NFAT5 PC-KO mice. In conclusion, NFAT5 is a major regulator of not only Aqp2 gene transcription, but also other genes important for water homeostasis and its absence leads to the development of NDI., (© 2022 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

αKlotho is a transmembrane protein acting as a co-receptor for FGF23, a bone hormone regulating renal phosphate and vitamin D metabolism. αKlotho expression is controlled by PPARγ. Soluble αklotho (sKL) regulates cellular signaling impacting stress resistance and death. αKlotho deficiency causes early onset of aging-associated diseases while its overexpression markedly increases lifespan. Cellular stress due to cytotoxic therapeutics or apoptosis induction through caspase activation or serum deficiency may result in cell death. Owing to αklotho's role in cellular stress and aging, this study explored the effect of cytotoxic agents or apoptosis stimulants on cellular αklotho expression. Experiments were performed in renal MDCK, NRK-52E and HK-2 cells. Gene expression was determined by qRT-PCR, sKL by ELISA, apoptosis and necrosis by annexin V binding and a fluorescent DNA dye, and cell viability by MTT assay. Cytostatic drugs cisplatin, paclitaxel, and doxorubicin as well as apoptosis induction with caspase 3 activator PAC-1 and serum deprivation induced αklotho and PPARG gene expression while decreasing viability and proliferation and inducing apoptosis of MDCK and NRK-52E cells to a variable extent. PPARγ antagonism attenuated up-regulation of αklotho in MDCK cells. In HK-2 cells, αklotho gene expression and sKL protein were down-regulated by chemotherapeutics. SKL serum levels in patients following chemotherapy were not significantly changed. In summary, potentially fatal stress results in up-regulation of αKlotho gene expression in MDCK and NRK-52E cells and down-regulation in HK-2 cells. These results indicate that different renal cell lines may exhibit completely different regulation of αklotho.

Fibroblast growth factor 23 (FGF23) controls vitamin D and phosphate homeostasis in the kidney and has additional paracrine effects elsewhere. As a biomarker, its plasma concentration is associated with progression of inflammatory, renal, and cardiovascular diseases. Major stimuli of FGF23 synthesis include active vitamin D and inflammation. Antineoplastic chemotherapy treats cancer by inducing cellular damage ultimately favoring cell death (apoptosis and necrosis) and causing inflammation. Our study explored whether chemotherapeutics and other apoptosis inducers impact on Fgf23 expression. Experiments were performed in osteoblast-like UMR106 cells, Fgf23 gene expression and protein synthesis were determined by qRT-PCR and ELISA, respectively. Viability was assessed by MTT assay and NFκB activity by Western Blotting. Antineoplastic drugs cisplatin and doxorubicin as well as apoptosis inducers procaspase-activating compound 1 (PAC-1), a caspase 3 activator, and serum depletion up-regulated Fgf23 transcripts while reducing cell proliferation and viability. The effect of cisplatin on Fgf23 transcription was paralleled by Il-6 up-regulation and NFκB activation and attenuated by Il-6 and NFκB signaling inhibitors. To conclude, cell viability-decreasing chemotherapeutics as well as apoptosis stimulants PAC-1 and serum depletion up-regulate Fgf23 gene expression. At least in part, Il-6 and NFκB may contribute to this effect.

In the context of transplantation, complement activation is associated with poor prognosis and outcome. While complement activation in antibody-mediated rejection is well-known, less is known about complement activation in acute T cell-mediated rejection (TCMR). There is increasing evidence that complement contributes to the clearance of apoptotic debris and tissue repair. In this regard, we have analysed published human kidney biopsy transcriptome data clearly showing upregulated expression of complement factors in TCMR. To clarify whether and how the complement system is activated early during acute TCMR, experimental syngeneic and allogeneic renal transplantations were performed. Using an allogeneic rat renal transplant model, we also observed upregulation of complement factors in TCMR in contrast to healthy kidneys and isograft controls. While staining for C4d was positive, staining with a C3d antibody showed no C3d deposition. FACS analysis of blood showed the absence of alloantibodies that could have explained the C4d deposition. Gene expression pathway analysis showed upregulation of pro-apoptotic factors in TCMR, and apoptotic endothelial cells were detected by ultrastructural analysis. Monocytes/macrophages were found to bind to and phagocytise these apoptotic cells. Therefore, we conclude that early C4d deposition in TCMR may be relevant to the clearance of apoptotic cells.

Background: Renal cell carcinomas (RCC) are characterized by the deregulation of several hundred hyperosmolality-responsive genes. High expression of a subset of these genes including the Ran binding protein 3 like (RANBP3L) is linked to a favorable prognostic outcome in RCC. However, the cellular function of RANBP3L remains largely unknown., Methods: We used CRISPR/Cas9-mediated gene editing to generate functional deletions of the Ranbp3l and nuclear factor of activated T cells 5 (Nfat5) gene loci in a murine renal cell line. The NFAT5-KO cells were used to assess the regulation of Ranbp3l by NFAT5 using immunofluorescence, RNA-Seq and promoter assays. RANBP3L-deficient cells were analyzed for changes in cell morphology, proliferation, migration and colony-forming capacity using immunofluorescence and live cell imaging. RANPB3L-dependent changes in gene expression were identified by RNA-Seq., Results: We show that NFAT5 directly regulates Ranpb3l under hyperosmotic conditions by binding its promoter. Functional analysis of RANBP3L-deficient cells revealed a loss of epithelial structure, an increased cell migration behavior and colony forming capacity, accompanied by massive alterations in gene expression, all of which are hallmarks for tumor cells. Strikingly, a RANBP3L dependent signature of 60 genes separated samples with clear cell carcinoma (KIRC) from papillary (KIRP), chromophobe renal carcinoma (KICH) and healthy tissue., Conclusions: Loss of RANBP3L induces a tumor like phenotype resembles RCC, especially KIRC, on the morphological and gene expression level and might promote tumor development and progression. Therapeutic reconstitution or elevation of osmoregulated RANBP3L expression might represent a novel treatment strategy for RCC or KIRC.

Aquaporin-2-4 (AQP) are expressed in the principal cells of the renal collecting duct (CD). Beside their role in water transport across membranes, several studies showed that AQPs can influence the migration of cells. It is unknown whether this also applies for renal CD cells. Another fact is that the expression of these AQPs is highly modulated by the external osmolality. Here we analyzed the localization of AQP2-4 in primary cultured renal inner medullary CD (IMCD) cells and how osmolality influences the migration behavior of these cells. The primary IMCD cells showed a collective migration behavior and there were no differences in the migration speed between cells cultivated either at 300 or 600 mosmol/kg. Acute increase from 300 to 600 mosmol/kg led to a marked reduction and vice versa an acute decrease from 600 to 300 mosmol/kg to a marked increase in migration speed. Interestingly, none of the analyzed AQPs were localized at the leading edge. While AQP3 disappeared within the first 2-3 rows of cells, AQP4 was enriched at the rear end. Further analysis indicated that migration induced lysosomal degradation of AQP3. This could be prevented by activation of the protein kinase A, inducing localization of AQP3 and AQP2 at the leading edge and increasing the migration speed.

Many melanomas are associated with activating BRAF mutation. Targeted therapies by inhibitors of BRAF and MEK (BRAFi, MEKi) show marked antitumor response, but become limited by drug resistance. The mechanisms for this are not fully revealed, but include miRNA. Wishing to improve efficacy of BRAFi and knowing that certain miRNAs are linked to resistance to BRAFi, we wanted to focus on miRNAs exclusively associated with response to BRAFi. We found increased expression of miR-129-5p during BRAFi treatment of BRAF- mutant melanoma cells. Parallel to emergence of resistance we observed mir-129-5p expression to become suppressed by BRAF/EZH2 signaling. In functional analyses we revealed that miR-129-5p acts as a tumor suppressor as its overexpression decreased cell proliferation, improved treatment response and reduced viability of BRAFi resistant melanoma cells. By protein expression analyses and luciferase reporter assays we confirmed SOX4 as a direct target of mir-129-5p. Thus, modulation of the miR-129-5p-SOX4 axis could serve as a promising novel strategy to improve response to BRAFi in melanoma.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide underlining the urgent need for new biomarkers and therapeutic targets for this disease. Long noncoding RNAs are critical players in NSCLC but the role of small RNA species is not well understood. In the present study, we investigated the role of H/ACA box small nucleolar RNAs (snoRNAs) and snoRNA-bound ribonucleoproteins (snoRNPs) in the tumorigenesis of NSCLC. H/ACA box snoRNPs including the NOP10 core protein were highly expressed in NSCLC. High levels of either NOP10 mRNA or protein were associated with poor prognosis in NSCLC patients. Loss of NOP10 and subsequent reduction of H/ACA box snoRNAs and rRNA pseudouridylation inhibited lung cancer cell growth, colony formation, migration, and invasion. A focused CRISPR/Cas9 snoRNA knockout screen revealed that genomic deletion of SNORA65, SNORA7A, and SNORA7B reduced proliferation of lung cancer cells. In line, high levels of SNORA65, SNORA7A, and SNORA7B were observed in primary lung cancer specimens with associated changes in rRNA pseudouridylation. Knockdown of either SNORA65 or SNORA7A/B inhibited growth and colony formation of NSCLC cell lines. Our data indicate that specific H/ACA box snoRNAs and snoRNA-associated proteins such as NOP10 have an oncogenic role in NSCLC providing new potential biomarkers and therapeutic targets for the disease.

The body homeostasis is maintained mainly by the function of the kidneys, which regulate salt and water balance and excretion of metabolism waste products and xenobiotics. This important renal function is determined by the action of many transport systems, which are specifically expressed in the different parts of the nephron, the functional unit of the kidneys. These transport systems are involved, for example, in the reabsorption of sodium, glucose, and other important solutes and peptides from the primary urine. They are also important in the reabsorption of water and thereby production of a concentrated urine. However, several studies have shown the importance of transport systems for different tumor entities. Transport systems, for example, contributed to the proliferation and migration of cancer cells and thereby on tumor progression. They could also serve as drug transporters that could enable drug resistance by outward transport of, for example, chemotherapeutic agents and other drugs. Although many renal transporters have been characterized in detail with respect to the significance for proper kidney function, their role in renal cancer progression is less known. Here, we describe the types of renal cancer and review the studies that analyzed the role of organic cation transporters of the SLC22-family and of the aquaporin water channel family in kidney tumors., (© 2020. Springer Nature Switzerland AG.)

The information derived from next generation sequencing technology allows the identification of deregulated genes, gene mutations, epigenetic modifications, and other genomic events that are associated with a given tumor entity. Its combination with clinical data allows the prediction of patients' survival with a specific gene expression pattern. Organic anion transporters and organic cation transporters are important proteins that transport a variety of substances across membranes. They are also able to transport drugs that are used for the treatment of cancer and could be used to improve treatment. In this study, we have made use of publicly available data to analyze if the expression of organic anion transporters or organic cation transporters have a prognostic value for a given tumor entity. The expression of most organic cation transporters is prognostic favorable. Within the organic anion transporters, the ratio between favorable and unfavorable organic anion transporters is nearly equal for most tumor entities and only in liver cancer is the number of unfavorable genes two times higher compared to favorable genes. Within the favorable genes, UNC13B , and SFXN2 cover nine cancer types and in the same way, SLC2A1 , PLS3 , SLC16A1 , and SLC16A 3 within the unfavorable set of genes and could serve as novel target structures.

Noncoding RNAs, including small nucleolar RNAs (snoRNAs), play important roles in leukemogenesis, but the relevant mechanisms remain incompletely understood. We performed snoRNA-focused CRISPR-Cas9 knockout library screenings that targeted the entire snoRNAnome and corresponding host genes. The C/D box containing SNORD42A was identified as an essential modulator for acute myeloid leukemia (AML) cell survival and proliferation in multiple human leukemia cell lines. In line, SNORD42A was consistently expressed at higher levels in primary AML patient samples than in CD34+ progenitors, monocytes, and granulocytes. Functionally, knockout of SNORD42A reduced colony formation capability and inhibited proliferation. The SNORD42A acts as a C/D box snoRNA and directs 2'-O-methylation at uridine 116 of 18S ribosomal RNA (rRNA). Deletion of SNORD42A decreased 18S-U116 2'-O-methylation, which was associated with a specific decrease in the translation of ribosomal proteins. In line, the cell size of SNORD42A deletion carrying leukemia cells was decreased. Taken together, these findings establish that high-level expression of SNORD42A with concomitant U116 18S rRNA 2'-O-methylation is essential for leukemia cell growth and survival., (© 2020 by The American Society of Hematology.)

Lithium chloride (LiCl) is a widely used drug for the treatment of bipolar disorders, but as a side effect, 40% of the patients develop diabetes insipidus. LiCl affects the activity of the glycogen synthase kinase 3 (GSK3), and mice deficient for GSK3β showed a reduction in the urine concentration capability. The cellular and molecular mechanisms are not fully understood. We used primary cultured inner medullary collecting duct cells to analyze the underlying mechanisms. LiCl and the inhibitor of GSK3 (SB216763) induced a decrease in the aquaporin-2 (Aqp2) protein level. LiCl induced downregulation of Aqp2 mRNA expression while SB216763 had no effect and TWS119 led to increase in expression. The inhibition of the lysosomal activity with bafilomycin or chloroquine prevented both LiCl- and SB216763-mediated downregulation of Aqp2 protein expression. Bafilomycin and chloroquine induced the accumulation of Aqp2 in lysosomal structures, which was prevented in cells treated with dibutyryl cyclic adenosine monophosphate (dbcAMP), which led to phosphorylation and membrane localization of Aqp2. Downregulation of Aqp2 was also evident when LiCl was applied together with dbcAMP, and dbcAMP prevented the SB216763-induced downregulation. We showed that LiCl and SB216763 induce downregulation of Aqp2 via different mechanisms. While LiCl also affected the mRNA level, SB216763 induced lysosmal degradation. Specific GSK3β inhibition had an opposite effect, indicating a more complex regulatory mechanism.

Loss of von Hippel-Lindau (VHL) protein function can be found in more than 90% of patients with clear cell renal carcinoma (ccRCC). Mice lacking Vhl function in the kidneys have urine concentration defects due to postulated reduction of the hyperosmotic gradient. Hyperosmolality is a kidney-specific microenvironment and induces a unique gene expression pattern. This gene expression pattern is inversely regulated in patients with ccRCC with consequences for cancer-specific survival. Within this study, we tested the hypothesis if Vhl function influences the hyperosmolality induced changes in gene expression. We made use of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology to inhibit functional Vhl expression in murine collecting duct cell line. Loss of Vhl function induced morphological changes within the cells similar to epithelial to mesenchymal transition like phenotype. Vhl-deficient cells migrated faster and proliferated slower compared to control cells. Gene expression profiling showed significant changes in gene expression patterns in Vhl-deficient cells compared to control cells. Several genes with unfavorable outcomes showed induced and genes with favorable outcomes for patients with renal cancer reduced gene expression level. Under hyperosmotic condition, the expression of several hyperosmolality induced genes, with favorable prognostic value, was downregulated in cells that do not express functional Vhl. Taken together, this study shows that Vhl interferes with hyperosmotic signaling pathway and hyperosmolality affected pathways might represent new promising targets.

The inner medullary collecting duct (IMCD) is subject to severe changes in ambient osmolality and must either allow water transport or be able to seal the lumen against a very high osmotic pressure. We postulate that the tight junction protein claudin-19 is expressed in IMCD and that it takes part in epithelial adaptation to changing osmolality at different functional states. Presence of claudin-19 in rat IMCD was investigated by Western blotting and immunofluorescence. Primary cell culture of rat IMCD cells on permeable filter supports was performed under different osmotic culture conditions and after stimulation by antidiuretic hormone (AVP). Electrogenic transepithelial transport properties were measured in Ussing chambers. IMCD cells cultivated at 300 mosm/kg showed high transepithelial resistance, a cation selective paracellular pathway and claudin-19 was mainly located in the tight junction. Treatment by AVP increased cation selectivity but did not alter transepithelial resistance or claudin-19 subcellular localization. In contrast, IMCD cells cultivated at 900 mosm/kg had low transepithelial resistance, anion selectivity, and claudin-19 was relocated from the tight junctions to intracellular vesicles. The data shows osmolality-dependent transformation of IMCD epithelium from tight and sodium-transporting to leaky, with claudin-19 expression in the tight junction associated to tightness and cation selectivity under low osmolality.

Tyrosine kinase inhibitors have emerged as a therapeutic option for rheumatic diseases such as systemic sclerosis (SSc). Because tyrosine kinases like c-Abl kinase are important for fibroblast activation and fibrosis development in SSc, the c-Abl inhibitor imatinib was proposed for SSc treatment. Transporters for organic cations have become increasingly recognized as an important determinant for uptake and efficacy of tyrosine kinase inhibitors. Therefore, we investigated the role of organic cation transporters in the uptake of imatinib. Moreover, the influence of important SSc pathogenetic factors, like PDGF and Notch pathway activation on these uptake processes, has been studied. We showed that organic cation transporters OCT1-3, novel organic cation transporters OCTN1/2, and the multidrug and toxin extrusion protein MATE1 are expressed in healthy dermal and SSc fibroblasts. Decreased expression levels of MATE1 and decreased imatinib uptake were measured in SSc fibroblasts. In small interfering RNA experiments, MATE1 was identified as key transporter for imatinib uptake and biological effect in dermal fibroblasts. Furthermore, PDGF reduced imatinib uptake by decreasing MATE1 expression in SSc fibroblasts, but not in healthy fibroblasts. Blocking the Notch pathway in SSc fibroblasts increased MATE1 transporter expression and imatinib uptake. In conclusion, MATE1-mediated transport governs therapeutic efficacy of imatinib in SSc., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Reactivation of Notch signaling in kidneys of animal models and patients with chronic kidney disease (CKD) has been shown to contribute to epithelial injury and fibrosis development. Here, we investigated the mechanisms of Notch-induced injury in renal epithelial cells. We performed genome-wide transcriptome analysis to identify Notch target genes using an in vitro system of cultured tubular epithelial cells expressing the intracellular domain of Notch1. One of the top downregulated genes was Disabled-2 ( Dab2). With the use of Drosophila nephrocytes as a model system, we found that Dab (the Drosophila homolog of Dab2) knockdown resulted in a significant filtration defect, indicating that loss of Dab2 plays a functional role in kidney disease development. We showed that Dab2 expression in cultured tubular epithelial cells is involved in endocytic regulation and that it also protects cells from TGF-β-induced epithelial-to-mesenchymal transition. In vivo correlation studies indicated its additional role in renal ischemia-induced injury. Together, these data suggest that Dab2 plays a versatile role in the kidney and may impact on acute and CKDs.-Schütte-Nütgen, K., Edeling, M., Mendl, G., Krahn, M. P., Edemir, B., Weide, T., Kremerskothen, J., Michgehl, U., Pavenstädt, H. Getting a Notch closer to renal dysfunction: activated Notch suppresses expression of the adaptor protein Disabled-2 in tubular epithelial cells.

Recent therapeutic approaches of rheumatoid arthritis (RA) address the use of small molecules such as tyrosine kinase inhibitors (TKIs). However, the TKIs developed to date have important side effects and/or scarce efficacy in inflammatory diseases such as RA. Since intracellular effective TKIs must enter the cell to reach their intracellular targets, here we investigated the interaction of the TKI saracatinib, a dual inhibitor of c-Src and c-Abl signaling, with transporters for organic cations as well as the role of these transporters for the biological effect of saracatinib in human RA-synovial fibroblasts (hRASF). Saracatinib significantly reduced proliferation of hRASF. The cellular saracatinib uptake was mainly dependent on the human novel organic cation transporter 1 (hOCTN1), which showed the highest apparent affinity for saracatinib among all other transporters for organic cations analyzed here. In hRASF, saracatinib biologic function was dependent on hOCTN1. Further analysis showed that disease specific factors (pH, inflammatory cytokines such as TNFα) regulated saracatinib uptake in hRASF. The knowledge of which transporters mediate the specific uptake of TKIs in target cells and of how the expression and function of such transporters are regulated in RA is of highest priority to develop effective drugs for successful therapy with minimal side-effects.

With this study, we wanted to prove the hypothesis that the unique extracellular osmolality within the renal medulla modulates a specific gene expression pattern. The physiologic functions of the kidneys are mediated by the segment-specific expression of key proteins. So far, we have limited knowledge about the mechanisms that control this gene expression pattern. The hyperosmolality in the renal medullary interstitium is of major importance as a driving force for urine concentration. We made use of primarily cultured rat renal inner medullary collecting-duct cells and microarray analysis to identify genes affected by the environmental osmolality of the culture medium. We identified hundreds of genes that were either induced or repressed in expression by hyperosmolality in a time- and osmolality-dependent fashion. Further analysis demonstrated that many of them, physiologically, showed a kidney- and even collecting-duct-specific expression, including secreted proteins, kinases, and transcription factors. On the other hand, we identified factors, down-regulated in expression, that have a diuretic effect. In conclusion, the kidney is the only organ that has such a hyperosmotic environment, and study provides an excellent method for controlling tissue-specific gene expression.-Schulze Blasum, B., Schröter, R., Neugebauer, U., Hofschröer, V., Pavenstädt, H., Ciarimboli, G., Schlatter E., Edemir, B. The kidney-specific expression of genes can be modulated by the extracellular osmolality., (© FASEB.)

The solute carrier family 45 a3 member (SLC45A3), known also as prostein, has been implicated with prostate cancer and the regulation of lipid metabolism in oligodendrocytes. Recently, we expressed SLC45A3 in yeast cells and characterised it as a proton-coupled sucrose symporter. However, the physiological functions of SLC45A3 were still unknown. Here, we report that SLC45A3 occurs in the kidney and is highly expressed in the medullary collecting duct (IMCD), a part of the kidney responsible for final urine concentration and faced to hyperosmotic environment. Moreover, messenger RNA (mRNA) expression of endogenous SLC45A3 in rat IMCD cells as well as in NRK52E cells increased up to four-fold under hyperosmotic conditions at 600 mOsmol/kg. Using NRK52E cells as an experimental model, we investigated the proton-coupled sugar transport and found that the uptake of sucrose or glucose was enhanced by hyperosmolarity. Down-regulation of expression by small interfering RNA (siRNA) decreased the osmotically inducible part of sucrose uptake and confirmed the involvement of SLC45A3 in this process. Furthermore, we observed an up to four-fold elevation of sucrose uptake triggered by hyperosmolarity across the apical membrane of NRK52E cells, while uptake across the basolateral membrane was not affected. Due to this finding, we conclude that SLC45A3 may occur at the luminal side of kidney epithelial cells and thus may take up solutes from the tubular fluid. Altogether, we show that SLC45A3 is a novel sugar transporter in kidney and hypothesise that the disaccharide sucrose, and probably the monosaccharides glucose and fructose, may serve as compatible osmolytes in urine.

Mammalian class IX myosin Myo9a is a single-headed, actin-dependent motor protein with Rho GTPase-activating protein activity that negatively regulates Rho GTPase signaling. Myo9a is abundantly expressed in ciliated epithelial cells of several organs. In mice, genetic deletion of Myo9a leads to the formation of hydrocephalus. Whether Myo9a also has essential functions in the epithelia of other organs of the body has not been explored. In the present study, we report that Myo9a-deficient mice develop bilateral renal disease, characterized by dilation of proximal tubules, calyceal dilation, and thinning of the parenchyma and fibrosis. These structural changes are accompanied by polyuria (with normal vasopressin levels) and low-molecular-weight proteinuria. Immunohistochemistry revealed that Myo9a is localized to the circumferential F-actin belt of proximal tubule cells. In kidneys lacking Myo9a, the multiligand binding receptor megalin and its ligand albumin accumulated at the luminal surface of Myo9a-deficient proximal tubular cells, suggesting that endocytosis is dysregulated. In addition, we found, surprisingly, that levels of murine diaphanous-related formin-1, a Rho effector, were decreased in Myo9a-deficient kidneys as well as in Myo9a knockdown LLC-PK1 cells. In summary, deletion of the Rho GTPase-activating protein Myo9a in mice causes proximal tubular dilation and fibrosis, and we speculate that downregulation of murine diaphanous-related formin-1 and impaired protein reabsorption contribute to the pathophysiology., (Copyright © 2015 the American Physiological Society.)

Chromatin-modifying enzymes are frequently altered in acute myeloid leukemia (AML). In the current study, we identified MYST2, a core histone acetyltransferase, to be suppressed in blast cells from AML patients compared with nonmalignant hematopoietic progenitor cells. Functionally, loss of MYST2 accelerated leukemic growth and colony formation, while forced expression of MYST2 induced H4K5 acetylation (H4K5Ac) and suppressed hematopoietic progenitor cell growth. Consistently, global H4K5Ac levels were frequently decreased in AML blasts. Low levels of H4K5Ac were most prominent in patients with complex karyotype AML and were associated with inferior overall survival in univariate but not multivariate analysis. ChIP-seq experiments in primary AML patients' blasts revealed widespread H4K5Ac deregulation, most prominent at gene promoters. Taken together, MYST2 is a repressed growth suppressor in AML mediating reduced acetylation of histone 4 at residue 5 and is associated with inferior AML patient survival., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)