| 1. |
- Krawczyk, Krzysztof M., et al.
(author)
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Injury induced expression of caveolar proteins in human kidney tubules - role of megakaryoblastic leukemia 1
- 2017
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In: BMC Nephrology. - : Springer Science and Business Media LLC. - 1471-2369. ; 18:1
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Journal article (peer-reviewed)abstract
- BACKGROUND: Caveolae are membrane invaginations measuring 50-100 nm. These organelles, composed of caveolin and cavin proteins, are important for cellular signaling and survival. Caveolae play incompletely defined roles in human kidneys. Induction of caveolin-1/CAV1 in diseased tubules has been described previously, but the responsible mechanism remains to be defined.METHODS: Healthy and atrophying human kidneys were stained for caveolar proteins, (caveolin 1-3 and cavin 1-4) and examined by electron microscopy. Induction of caveolar proteins was studied in isolated proximal tubules and primary renal epithelial cells. These cells were challenged with hypoxia or H2O2. Primary tubular cells were also subjected to viral overexpression of megakaryoblastic leukemia 1 (MKL1) and MKL1 inhibition by the MKL1 inhibitor CCG-1423. Putative coregulators of MKL1 activity were investigated by Western blotting for suppressor of cancer cell invasion (SCAI) and filamin A (FLNA). Finally, correlative bioinformatic studies of mRNA expression of caveolar proteins and MKL1 were performed.RESULTS: In healthy kidneys, caveolar proteins were expressed by the parietal epithelial cells (PECs) of Bowman's capsule, endothelial cells and vascular smooth muscle. Electron microscopy confirmed caveolae in the PECs. No expression was seen in proximal tubules. In contrast, caveolar proteins were expressed in proximal tubules undergoing atrophy. Caveolar proteins were also induced in cultures of primary epithelial tubular cells. Expression was not enhanced by hypoxia or free radical stress (H2O2), but proved sensitive to inhibition of MKL1. Viral overexpression of MKL1 induced caveolin-1/CAV1, caveolin-2/CAV2 and SDPR/CAVIN2. In kidney tissue, the mRNA level of MKL1 correlated with the mRNA levels for caveolin-1/CAV1, caveolin-2/CAV2 and the archetypal MKL1 target tenascin C (TNC), as did the MKL1 coactivator FLNA. Costaining for TNC as readout for MKL1 activity demonstrated overlap with caveolin-1/CAV1 expression in PECs as well as in atrophic segments of proximal tubules.CONCLUSIONS: Our findings support the view that MKL1 contributes to the expression of caveolar proteins in healthy kidneys and orchestrates the induction of tubular caveolar proteins in renal injury.
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| 2. |
- Gapińska, Marta, et al.
(author)
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Structure-functional characterization of Lactococcus AbiA phage defense system
- 2024
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In: Nucleic Acids Research. - 1362-4962. ; 52:8, s. 4723-4738
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Journal article (peer-reviewed)abstract
- Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.
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| 3. |
- Kha, Michelle, 1994, et al.
(author)
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The injury-induced transcription factor SOX9 alters the expression of LBR, HMGA2, and HIPK3 in the human kidney
- 2023
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In: American Journal of Physiology - Renal Physiology. - : American Physiological Society. - 1931-857X .- 1522-1466. ; 324:1
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Journal article (peer-reviewed)abstract
- Induction of SRY box transcription factor 9 (SOX9) has been shown to occur in response to kidney injury in rodents, where SOX9-positive cells proliferate and regenerate the proximal tubules of injured kidneys. Additionally, SOX9-positive cells demonstrate a capacity to differentiate toward other nephron segments. Here, we characterized the role of SOX9 in normal and injured human kidneys. SOX9 expression was found to colocalize with a proportion of so-called scattered tubular cells in the uninjured kidney, a cell population previously shown to be involved in kidney injury and regeneration. Following injury and in areas adjacent to inflammatory cell infiltrates, SOX9-positive cells were increased in number. With the use of primary tubular epithelial cells (PTECs) obtained from human kidney tissue, SOX9 expression was spontaneously induced in culture and further increased by transforming growth factor-b1, whereas it was suppressed by interferon-c. siRNA-mediated knockdown of SOX9 in PTECs followed by analysis of differential gene expression, immunohistochemical expression, and luciferase promoter assays suggested lamin B receptor (LBR), high mobility group AT-hook 2 (HMGA2), and homeodomain interacting protein kinase 3 (HIPK3) as possible target genes of SOX9. Moreover, a kidney explant model was used to demonstrate that only SOX9-positive cells survive the massive injury associated with kidney ischemia and that the surviving SOX9-positive cells spread and repopulate the tubules. Using a wound healing assay, we also showed that SOX9 positively regulated the migratory capacity of PTECs. These findings shed light on the functional and regulatory aspects of SOX9 activation in the human kidney during injury and regeneration.
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| 4. |
- Krawczyk, Krzysztof M., et al.
(author)
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Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model
- 2018
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In: Cytotechnology. - : Springer Science and Business Media LLC. - 0920-9069 .- 1573-0778. ; 70:2, s. 761-782
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Journal article (peer-reviewed)abstract
- The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco’s Modified Eagle’s Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability.
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| 5. |
- Krawczyk, Krzysztof M., et al.
(author)
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Localization and Regulation of Polymeric Ig Receptor in Healthy and Diseased Human Kidney
- 2019
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In: American Journal of Pathology. - : Elsevier BV. - 0002-9440. ; 189:10, s. 1933-1944
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Journal article (peer-reviewed)abstract
- The polymeric Ig receptor (PIgR) constitutes an important part of the immune system by mediating transcytosis of dimeric IgA into mucosal fluids. Although well studied in organs such as the intestine, the regulation and localization of PIgR in human kidney are incompletely characterized. Herein, using immunohistochemistry, we show that in healthy human kidneys, PIgR is expressed by the progenitor-like tubular scattered cells of the proximal tubules and by parietal epithelial cells of glomeruli. We further show that proximal tubular expression of PIgR becomes widespread during kidney disease, correlating to elevated levels of urinary secretory IgA. Urinary secretory IgA levels also correlated to the degree of tubular fibrosis, plasma creatinine, and urea levels. In addition, primary tubular cells were cultured to study the function and regulation of PIgR in vitro. Cellular PIgR expression was induced by conditioned medium from activated human leukocytes, as well as by inflammatory cytokines, whereas transforming growth factor-beta 1 caused decreased expression. Furthermore, interferon-gamma increased the transcytosis of dimeric IgA in cultured tubular cells. Finally, a correlation study of mRNA data from the Genotype-Tissue Expression portal indicated that PIGR mRNA expression in kidney correlates to the expression of TNFSF13, a cytokine involved in plasma cell class switching to IgA. These results indicate that PIgR induction is an integral part of the injury phenotype of renal tubular cells.
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| 6. |
- Krawczyk, Krzysztof M., et al.
(author)
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Papillary renal cell carcinoma-derived chemerin, IL-8, and CXCL16 promote monocyte recruitment and differentiation into foam-cell macrophages
- 2017
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In: Laboratory Investigation. - : Elsevier BV. - 0023-6837. ; 97:11, s. 1296-1305
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Journal article (peer-reviewed)abstract
- Papillary renal cell carcinoma (pRCC) is the second most common type of renal cell carcinoma. The only curative treatment available for pRCC is radical surgery. If the disease becomes widespread, neither chemo- nor radiotherapy will have therapeutic effect, hence further research on pRCC is of utmost importance. Histologically, pRCC is characterized by a papillary growth pattern with focal aggregation of macrophages of the foam cell phenotype. In other forms of cancer, a clear role for tumor-associated macrophages during cancer growth and progression has been shown. Although the presence of foamy macrophages is a histological hallmark of pRCC tumors, little is known regarding their role in pRCC biology. In order to study the interaction between pRCC tumor and myeloid cells, we established primary cultures from pRCC tissue. We show that human pRCC cells secrete the chemokines IL-8, CXCL16, and chemerin, and that these factors attract primary human monocytes in vitro. RNAseq data from The Cancer Genome Atlas confirmed a high expression of these factors in pRCC tissue. Conditioned medium from pRCC cultures induced a shift in human monocytes toward the M2 macrophage phenotype. In extended cultures, these macrophages became enlarged and loaded with lipids, adopting the foam cell morphology found in pRCC tissue. These results show for the first time that pRCC primary tumor cells secrete factors that attract and differentiate monocytes into anti-inflammatory tumor-associated macrophages with foam cell histology.
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| 7. |
- Krawczyk, Krzysztof
(author)
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Role of tubular scattered cells of the kidney in disease and regeneration
- 2018
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Doctoral thesis (other academic/artistic)abstract
- With well over 700 000 deaths every year worldwide, kidney disease constitutes an immense health problem for patients and society. The total global number of people with kidney disease regardless of severity amounts to a staggering 600 000 000. Not surprisingly, the total health care expenditure associated with kidney disease is second to no other costs, including oncology/cancer. If acute or chronic kidney disease is allowed to progress they may eventually lead to end-stage renal disease. This is unfortunately not an unusual development, since specific treatments for renal disease are virtually none, and current treatments mostly focus on symptoms rather than specifically altering disease progression. The end-stage renal disease is a lethal condition if renal replacement therapy is not amenable in the form of either dialysis or more seldom renal transplantation. Obviously, kidney injury only causes clinically relevant organ damage if the regenerative capacity of the kidney is overwhelmed. Surprisingly the basis for and regulation of kidney regeneration is still not unequivocally established. This is why it is so important to understand the processes behind kidney injury and regeneration.This thesis focuses on the cellular basis for kidney regeneration by investigating the so-called tubular scattered cells (TSCs). These are of central importance for renal injury and regeneration. Whether these cells are stem or progenitor cells or represent cellular reactions to injury is an unsettled issue, despite the intense investigation. First, we established a novel protocol based on fluorescence activated sell sorting (FACS) for rare cell isolatation, allowing us to isolate good quality RNA from cell suspensions after fixation, permeabilization, and intracellular antibody labelling.Next, we focused on mechanistic aspects of the kidney injury and regeneration by studying the transcription factor MKL1. It was shown to control proximal tubular cell expression of caveolin-1, a protein not present in healthy kidney but highly expressed post-renal injury.In the third paper, we investigated the carrier protein polymeric immunoglobulin receptor (pIgR). In normal kidney, we showed this protein to be expressed by TSCs and expression increases significantly after kidney injury and we postulate that this serves to protect the kidney from, among others, ascending bacterial infections by facilitating excretion of IgA into the urine.Next, we analyzed the expression pattern of the transcription factor SOX9 in a normal and diseased kidney. We and others have found SOX9 to be associated with TSCs. We show that SOX9 expression is increased in injured kidney tissue and that the cellular expression is modulated by chemokines secreted from secondary inflammatory infiltrates attracted by tubular injury.Last, we investigated another aspect of TSC pathology. We suggest that papillary renal cell carcinoma (pRCC) is derived from TSCs and that pRCC might use regenerative programs associated with TSCs. We studied the most characteristic feature of the pRCC tumor, which is the presence of foamy macrophages in the papillary fronds. We identified a set of cytokines with the capacity to attract monocytes into the tumor tissue and convert these into M2 phenotype foam cells resulting in the characteristic histology.
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| 8. |
- Lindgren, David, et al.
(author)
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Cell-Type-Specific Gene Programs of the Normal Human Nephron Define Kidney Cancer Subtypes
- 2017
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In: Cell Reports. - : Elsevier BV. - 2211-1247. ; 20:6, s. 1476-1489
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Journal article (peer-reviewed)abstract
- Comprehensive transcriptome studies of cancers often rely on corresponding normal tissue samples to serve as a transcriptional reference. In this study, we performed in-depth analyses of normal kidney tissue transcriptomes from the TCGA and demonstrate that the histological variability in cellularity, inherent in the kidney architecture, lead to considerable transcriptional differences between samples. This should be considered when comparing expression profiles of normal and cancerous kidney tissues. We exploited these differences to define renal-cell-specific gene signatures and used these as a framework to analyze renal cell carcinoma (RCC) ontogeny. Chromophobe RCCs express FOXI1-driven genes that define collecting duct intercalated cells, whereas HNF-regulated genes, specific for proximal tubule cells, are an integral part of clear cell and papillary RCC transcriptomes. These networks may be used as a framework for understanding the interplay between genomic changes in RCC subtypes and the lineage-defining regulatory machinery of their non-neoplastic counterparts.
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| 9. |
- Nilsson, Helén, et al.
(author)
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High salt buffer improves integrity of RNA after fluorescence-activated cell sorting of intracellular labeled cells.
- 2014
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In: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 192:Sep 30, s. 62-65
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Journal article (peer-reviewed)abstract
- Over the past years, massive progress has been made in the ability to collect large-scale gene expression data from a limited sample size. Combined with improvements in multiplex flow cytometry-based techniques, this has made it possible to isolate and characterize specific cellular subtypes within heterogeneous populations, with a great impact on our understanding of different biological processes. However, sorting based on intracellular markers requires fixation and permeabilization of samples, and very often the integrity of RNA molecules is compromised during this process. Many attempts have been made to improve the quality of nucleic acids from such samples, but RNA degradation still remains a limiting factor for downstream analyses. Here we present a method to isolate high quality RNA from cells that have been fixed, permeabilized, intracellularly labeled and sorted. By performing all incubation steps in the presence of a high salt buffer, RNA degradation was avoided and samples with remarkable integrity were obtained. This procedure offers a straightforward and very affordable technique to retrieve high quality RNA from isolated cell populations, which increases the possibilities to characterize gene expression profiles of subpopulations from mixed samples, a technique with implications in a broad range of research fields.
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