Ferric cacodylate efficiently stimulates growth of rat renal glomerular epithelial cells in vitro

Rat renal glomerular epithelial cells (SGE1 cell line) can be maintained and grown continuously in serum-free medium supplemented with insulin, iron-saturated transferrin (Tr), selenium, bovine serum albumin (BSA), linoleic acid, and epidermal growth factor (EGF). Of the growth supplements used, Tr is essential for proliferation of the cells. In the present study, we describe the use of a unique iron-chelate complex, ferric cacodylate (Fe-Cac), positively charged molecules in neutral buffer, that could almost replace Tr in serum-free culture. It even stimulated the growth of SGE1 cells more efficiently than ferric chloride (FeCl₃) and other iron-chelate complexes, such as ferric nitrilotriacetate (Fe-NTA) and ferric citrate (Fe-Cit). The growth-stimulatory activity of Fe-Cac was exerted at iron concentrations of more than 0.01 g/ml, whereas a 10-fold excess of iron concentration was required with FeCl₃, Fe-NTA and Fe-Cit. We observed that SGE1 cells grew until confluent, then formed hemicysts (domes) in serum-free medium containing Fe-Cac, suggesting that Fe-Cac did not merely permit cell growth but also supported polarization and organization of the cells into a functional epithelial architecture. Moreover, since the stimulatory activity of Fe-Cac was completely abolished by desferrioxamine, a strong iron chelator, it is suggested that iron is crucial for growth of SGE1 cells. When the cells were treated with suramin, an inhibitor of cellular pinocytosis and endocytosis of a large spectrum of ligands including receptor-bound growth factors, growth-stimulatory activity of Tr was inhibited, whereas the activity of Fe-Cac was not affected. These results, taken together, strongly suggest that the growth-stimulatory activity of Fe-Cac is associated with iron delivery into the cells through the cell membrane by diffusion, which is different from Tr receptor-mediated endocytosis. The use of Fe-Cac for investigating iron-regulated cell proliferation is suggested.     

Albumin induces CD44 expression in glomerular parietal epithelial cells by activating extracellular signal-regulated kinase 1/2 pathway

De novo expression of CD44 in glomerular parietal epithelial cells (PECs) leads to a prosclerotic and migratory PEC phenotype in glomerulosclerosis. However, the regulatory mechanisms underlying CD44 expression by activated PECs remain largely unknown. This study was performed to examine the mediators responsible for CD44 induction in glomerular PECs in association with diabetes. CD44 expression and localization were evaluated in the glomeruli of Zucker diabetic rat kidneys and primary cultured PECs upon albumin stimulation. Real-time polymerase chain reaction confirmed an albuminuria-associated upregulation of the CD44 gene in the glomeruli of diabetic rats. Immunostaining analysis of diabetic kidneys further revealed an increase in CD44 in hypertrophic PECs, which often contain albumin-positive vesicles. Losartan treatment significantly attenuated albuminuria and lowered CD44 protein levels in the diabetic kidneys. In primary cultured rat PECs, rat serum albumin (0.25–1 mg/ml) caused a dose-dependent upregulation of CD44, claudin-1, and megalin protein expression, which was accompanied by an activation of extracellular signal-regulated kinase1/2 (ERK1/2) signaling. Albumin-induced CD44 and claudin-1 expression were greatly suppressed in the presence of the ERK1/2 inhibitor, U0126. In addition, knockdown of megalin by small interfering RNA interference in PECs resulted in a significant reduction of albumin-induced CD44 and claudin-1 proteins. Taken together, our results demonstrate that albumin induces CD44 expression by PECs via the activation of the ERK signaling pathway, which is partially mediated by endocytic receptor megalin.     

In search of adult renal stem cells

The therapeutic potential of adult stem cells in the treatment of chronic degenerative diseases has becoming increasingly evident over the last few years. Significant attention is currently being paid to the development of novel treatments for acute and chronic kidney diseases too. To date, promising sources of stem cells for renal therapies include adult bone marrow stem cells and the kidney precursors present in the early embryo. Both cells have clearly demonstrated their ability to differentiate into the kidney's specialized structures. Adult renal stem cells have yet to be identified, but the papilla is where the stem cell niche is probably located. Now we need to isolate and characterize the fraction of papillary cells that constitute the putative renal stem cells. Our growing understanding of the cellular and molecular mechanisms behind kidney regeneration and repair processes - together with a knowledge of the embryonic origin of renal cells - should induce us, however, to bear in mind that in the kidney, as in other mesenchymal tissues, the need for a real stem cell compartment might be less important than the phenotypic flexibility of tubular cells. Thus, by displaying their plasticity during kidney maintenance and repair, terminally differentiated cells may well function as multipotent stem cells despite being at a later stage of maturation than adult stem cells. One of the major tasks of Regenerative Medicine will be to disclose the molecular mechanisms underlying renal tubular plasticity and to exploit its biological and therapeutic potential.     

Fetal kidney stem cells ameliorate cisplatin induced acute renal failure and promote renal angiogenesis

AIM: To investigate whether fetal kidney stem cells (fKSC) ameliorate cisplatin induced acute renal failure (ARF) in rats and promote renal angiogenesis.METHODS: The fKSC were isolated from rat fetuses of gestation day 16 and expanded in vitro up to 3^rd passage. They were characterized for the expression of mesenchymal and renal progenitor markers by flow cytometry and immunocytochemistry, respectively. The in vitro differentiation of fKSC towards epithelial lineage was evaluated by the treatment with specific induction medium and their angiogenic potential by matrigel induced tube formation assay. To study the effect of fKSC in ARF, fKSC labeled with PKH26 were infused in rats with cisplatin induced ARF and, the blood and renal tissues of the rats were collected at different time points. Blood biochemical parameters were studied to evaluate renal function. Renal tissues were evaluated for renal architecture, renal cell proliferation and angiogenesis by immunohistochemistry, renal cell apoptosis by terminal deoxynucleotidyl transferase nick-end labeling assay and early expression of angiogenic molecules viz. vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α and endothelial nitric oxide synthase (eNOS) by western blot.RESULTS: The fKSC expressed mesenchymal markers viz. CD29, CD44, CD73, CD90 and CD105 as well as renal progenitor markers viz. Wt1, Pax2 and Six2. They exhibited a potential to form CD31 and Von Willebrand factor expressing capillary-like structures and could be differentiated into cytokeratin (CK)18 and CK19 positive epithelial cells. Administration of fKSC in rats with ARF as compared to administration of saline alone, resulted in a significant improvement in renal function and histology on day 3 (2.33 ± 0.33 vs 3.50 ± 0.34, P < 0.05) and on day 7 (0.83 ± 0.16 vs 2.00 ± 0.25, P < 0.05). The infused PKH26 labeled fKSC were observed to engraft in damaged renal tubules and showed increased proliferation and reduced apoptosis (P < 0.05) of renal cells. The kidneys of fKSC as compared to saline treated rats had a higher capillary density on day 3 [13.30 ± 1.54 vs 7.10 ± 1.29, capillaries/high-power fields (HPF), P < 0.05], and on day 7 (21.10 ± 1.46 vs 15.00 ± 1.30, capillaries/HPF, P < 0.05). In addition, kidneys of fKSC treated rats had an up-regulation of angiogenic proteins hypoxia-inducible factor-1α, VEGF and eNOS on day 3 (P < 0.05).CONCLUSION: Our study shows that fKSC ameliorate cisplatin induced ARF in rats and promote renal angiogenesis, which may be an important therapeutic mechanism of these stem cells in the disease.     

Biological principals and clinical potentials of limbal epithelial stem cells

In this review, we describe a population of adult stem cells that are currently being successfully used in the clinic to treat blinding ocular surface disease, namely limbal epithelial stem cells (LESC). The function and characteristics of LESC and the challenges faced in making use of their therapeutic potential will be examined. The cornea on the front surface of the eye provides our window on the world. The consistency and functionality of the outer-most corneal epithelium is essential for vision. A population of LESC are responsible for replenishing the epithelium throughout life by providing a constant supply of daughter cells that replace those constantly removed from the ocular surface during normal wear and tear and following injury. LESC deficiency results in corneal inflammation, opacification, vascularisation and severe discomfort. The transplantation of cultured LESC is one of only a few examples of the successful use of adult stem cell therapy in patients. The clinical precedence for the use of stem cell therapy and the ready accessibility of a transparent stem cell niche make the cornea a unique model for the study of adult stem cells in health and disease. The authors thank the Special Trustees of Moorfields Eye Hospital (J.T.D.) and the BBSRC (M.N.) for financial support.     

Class I HDACs specifically regulate E‐cadherin expression in human renal epithelial cells

Epithelial‐mesenchymal transition (EMT) and renal fibrosis are closely involved in chronic kidney disease. Inhibition of histone deacetylase (HDAC) has an anti‐fibrotic effect in various diseases. However, the pathophysiological role of isoform‐specific HDACs or class‐selective HDACs in renal fibrosis remains unknown. Here, we investigated EMT markers and extracellular matrix (ECM) proteins in a human proximal tubular cell line (HK‐2) by using HDAC inhibitors or by knockdown of class I HDACs (HDAC1, 2, 3 and 8). Trichostatin A (TSA), MS275, PCI34051 and LMK235 inhibited ECM proteins such as collagen type I or fibronectin in transforming growth factor β1 (TGF‐β1)‐induced HK2 cells. However, restoration of TGF‐β1‐induced E‐cadherin down‐regulation was only seen in HK‐2 cells treated with TSA or MS275, but not with PCI34051, whereas TGF‐β1‐induced N‐cadherin expression was not affected by the inhibitors. ECM protein and EMT marker levels were prevented or restored by small interfering RNA transfection against HDAC8, but not against other class I HDACs (HDAC1, 2 and 3). E‐cadherin regulation is mediated by HDAC8 expression, but not by HDAC8 enzyme activity. Thus, class I HDACs (HDAC1, 2, 3 and 8) play a major role in regulating ECM and EMT, whereas class IIa HDACs (HDAC4 and 5) are less effective.     

Establishment of epithelial and fibroblast cell cultures and cell lines from primary renal cancer nephrectomies

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan‐cytokeratin, cytokeratin 8 and E‐cadherin whereas fibroblast cells expressed high α‐smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.     

微波辐射对肾小管上皮细胞的影响以及金雀异黄素对其保护作用

目的：观察微波辐照对人近端肾小管上皮细胞（HKC）的影响及金雀异黄素对其的保护作用。方法：HKC分为对照组、微波辐照组、金雀异黄素组（n=6）。金雀异黄素组在辐照前2 h用含30μmol/L金雀异黄素的DMEM培养基进行预培养。辐照后24 h留取上清进行乳酸脱氢酶（LDH）、β-N-乙酰氨基葡萄糖苷酶（NAG）活性及丙二醛（MDA）、超氧化物歧化酶（SOD）活性检测。Hoechst 33258染色观察细胞凋亡情况。结果：与对照组比较,微波辐照组上清NAG、LDH活性明显增加（P < 0.01）,金雀异黄素预处理组则较微波辐照组明显下降（P < 0.01）;微波辐照组上清活性也较对照组明显增加（P < 0.01）。Hoechst 33258染色显示,微波辐照可导致较多量的细胞凋亡,而应用金雀异黄素预处理后细胞凋亡的比例均大大减少。微波辐照可大大提高HKC细胞中的MDA含量,SOD活性降低（P< 0.01）,应用金雀异黄素预处理后MDA的含量无明显降低,SOD的活性明显增大（P < 0.01）。结论：微波辐照可导致人近端肾小管上皮细胞出现功能损伤,金雀异黄素对其具有一定的保护作用,可能与其减轻氧化应激损伤、减少细胞凋亡有关。     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

Increased susceptibility of renal epithelial cells to TNFalpha-induced apoptosis following treatment with fumonisin B1

Previous studies have shown that tumor necrosis factor alpha (TNFalpha) is involved in the pathogenic events following exposure to fumonisin B(1) (FB(1)), a potent inhibitor of ceramide synthase and sphingolipid biosynthesis. The intimate role of sphingolipid mediators in TNFalpha signaling and cellular death suggests that FB(1) may alter the sensitivity of cells to TNFalpha-induced apoptosis. We tested the hypothesis that FB(1) treatment will increase the sensitivity of porcine renal epithelial cells to TNFalpha. Porcine renal epithelial cells (LLC-PK(1)) were treated with FB(1) for 48 h prior to treatment with TNFalpha. A dose-dependent increase in TNFalpha-induced apoptosis was observed in cells pretreated with FB(1). Cells treated with FB(1) showed increased DNA fragmentation and terminal uridine nucleotide end labeling in response to TNFalpha treatment. FB(1) increased DNA synthesis and resulted in cell cycle arrest in the G(2)/M phase of the cell cycle. Flow cytometric analysis of the cell cycle indicated that TNFalpha predominantly killed cells in the G(2)/M phase. The activation of JNK, a mitogen-activated protein kinase (MAPK), was increased following 48 h exposure to FB(1). Phosphorylation of p38 and ERK remained unchanged following treatment with FB(1). FB(1) also increased free sphingoid base levels under identical treatment conditions. Results suggest that FB(1) increased free sphingoid base levels and the population of cells in the G(2)/M phase. This population was shown to be most susceptible to TNFalpha-induced apoptosis. Phosphorylation of pro-apoptotic JNK may play an important role in these effects.     

Secretion of rat tracheal epithelial cells induces mesenchymal stem cells to differentiate into epithelial cells

Bone marrow MSCs (mesenchymal stem cells) can differentiate into various tissue cells, including epithelial cells. This presents interesting possibilities for cellular therapy, but the differentiation efficiency of MSCs is very low. We have explored specific inducing factors to improve the epithelial differentiation efficiency of MSCs. Under inducing conditions, MSCs differentiated into epithelial cells and expressed several airway epithelial markers using RTE (rat tracheal epithelial) cell secretions. Rat cytokine antibody array was used to detect cytokines of the RTE secretion components, in which 32 kinds of protein were found. Seven proteins [TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), VEGF (vascular endothelial growth factor), BDNF (brain-derived neurotrophic factor), TGFβ1 (transforming growth factor β1), MMP-2 (metalloproteinases-2), OPN (osteopontin) and activin A in RTE secretions] were assayed using ELISA kits. The four growth factors (VEGF, BDNF, TGFβ1 and activin A) were involved in regulating stem cell growth and differentiation. We speculated that these four play a vital role in the differentiation of MSCs into epithelial cells by triggering appropriate signalling pathways. To induce epithelial differentiation, MSCs were cultured using VEGF, BDNF, TGFβ1 and activin A. Differentiated MSCs were characterized both morphologically and functionally by their capacity to express specific markers for epithelial cells. The data demonstrated that MSCs can differentiate into epithelial cells induced by these growth factors.     

Sustained co-cultivation with human placenta-derived MSCs enhances ALK5/Smad3 signaling in human breast epithelial cells,leading to EMT and differentiation

The interaction between mammary epithelial cells and their surrounding microenvironment are important in the development of the mammary gland. Thus, mesenchymal stem cells (MSCs), which retain pluripotency for various mesenchymal lineages, may provide a permissive environment for the morphologic alteration and differentiation of mammary epithelial cells. To this end, we investigated whether the interactions between mammary epithelial cells and human placenta-derived MSCs (hPMSC) affect the morphology, proliferation, and differentiation of epithelial cells in a co-culture system. We show that after co-culture with hPMSCs, human mammary epithelial cell lines (MCF-10F and HEMC) underwent significant morphologic alterations and a dramatic increase in ductal–alveolar branching, which was accompanied by a decrease or loss of the epithelial marker E-cadherin and a gain of the mesenchymal markers, α-SMA and vimentin. MCF-10F and HEMC proliferation was also inhibited in the presence of hPMSCs, and this retardation in growth was due to cell cycle arrest. Furthermore, in MCF-10F and HMEC cells, hPMSCs induced the production of lipid droplets, milk fat globule protein, and milk protein lactoferrin, which are markers of functional mammary differentiation. We also noticed an elevation in ALK5 and phosphorylated Smad3 protein levels upon hPMSC co-culture. Strikingly, the changes in morphology, proliferation, and differentiation were reversed by treatment with ALK5 or Smad3 knockdown in MCF-10F/hPMSC co-cultures. Collectively, our findings suggest that co-cultivation with hPMSCs leads to epithelial to mesenchymal transition (EMT) and differentiation of human breast epithelial cells through the ALK5/Smad3 signaling pathway.     

Functional differentiation and scalable production of renal proximal tubular epithelial cells from human pluripotent stem cells in a dynamic culture system

ObjectiveTo provide a standardized protocol for large‐scale production of proximal tubular epithelial cells (PTEC) generated from human pluripotent stem cells (hPSC).MethodsThe hPSC were expanded and differentiated into PTEC on matrix‐coated alginate beads in an automated levitating fluidic platform bioLevitator. Differentiation efficacy was evaluated by immunofluorescence staining and flow cytometry, ultrastructure visualized by electron microscopy. Active reabsorption by PTEC was investigated by glucose, albumin, organic anions and cations uptake assays. Finally, the response to cisplatin‐treatment was assessed to check the potential use of PTEC to model drug‐induced nephrotoxicity.ResultshPSC expansion and PTEC differentiation could be performed directly on matrix‐coated alginate beads in suspension bioreactors. Renal precursors arose 4 days post hPSC differentiation and PTEC after 8 days with 80% efficiency, with a 10‐fold expansion from hPSC in 24 days. PTEC on beads, exhibited microvilli and clear apico‐basal localization of markers. Functionality of PTECs was confirmed by uptake of glucose, albumin, organic anions and cations and expression of KIM‐1 after Cisplatin treatment.ConclusionWe demonstrate the efficient expansion of hPSC, controlled differentiation to renal progenitors and further specification to polarized tubular epithelial cells. This is the first report employing biolevitation and matrix‐coated beads in a completely defined medium for the scalable and potentially automatable production of functional human PTEC.     

Expression profiles of mouse dendritic cell sarcoma are similar to those of hematopoietic stem cells or progenitors by clustering and principal component analyses

We isolated and screened two tumor cell clones DD1 and DG6 with different capacity of metastasis from the same parent cell line, a mouse dendritic cell (DC) sarcoma, using limited dilution method. The genome-wide expressions of DD1 and DG6 cells were detected by Affymetrix's MOE-430A microarray. The expression profiles related with mouse DC development were downloaded from GEO at NCBI and ArrayExpress at EBI database. In order to compare the expression of DC sarcoma and DC developmental arrays which was performed by MG-U74av2, we had screened the best matched probesets between MOE-430A and MG-U74av2 according to the probe identities from Affymetrix technical annotation. After the normalization of 11 housekeeping genes across the 34 arrays (2 DC sarcoma and 32 DC developmental arrays), all these expression profiles were analyzed by the methods of hierarchical clustering, principal component analysis, nearest-neighborhood, and self-organizing maps. The results indicate that expression profiles of DC sarcoma are closer to those of the DC progenitors and hematopoietic stem cells from bone marrow compared with the sorted DCs from spleen. The results support the hypothesis that cancers (tumors or sarcomas) arise from stem cells. It is suggested that the DC sarcomas are more similar to the DC progenitors and hematopoietic stem cells than the relative mature DCs in gene expressions on the large-scale.     

Enrichment and characterization of clonogenic epithelial cells from adult rat liver and initiation of epithelial cell strains

Summary A highly efficient method is described for obtaining prolifertive epithelial cells from adult rat livers for the reproducible establishment of liver epithelial cell strains. When cells were isolated from livers of 10-to 15-wk-old male Fischer 344 rats by a collagenase-perfusion method, collected by centrifugation at 50×g for 5 min, and cultured in Williams' medium E containing fetal bovine serum and dexamethasone, colonies of epithelial cells different in size and morphology from hepatocytes were obtained. Sequential perfusion with collagenase and dispase yielded numerous epithelial cell colonies. When isolated cells were fractionated by differential centrifugation, the great majority of hepatocytes were sedimented at 50 ×g for 1 min, whereas many non-hepatocytic cells remiined in the supernatant and could be sedimented by a second centrifugation at 50×g for 5 min. Culture of the two fractions revealed that almost all the epithelial cell colonies were derived from cells in the non-hepatocytic cell fraction. The epithelial cells were cytochemically negative for γ-glutamyl transpeptidase activity, whereas an increase in the activity was detected in hepatocytes with duration in culture. Ultrastructural characteristics of hepatocytes were not found in the cells of newly established cell strains. These results suggest that adult rat liver epithelial cells propagable in culture were derived from a cell type other than the hepatocyte.