Establishment and characterization of an immature epithelial cell line (ENU-T-1) derived from a rat nephroblastoma

A new cell line designated ENU-T-1 has been established from a xenotransplanted experimental rat nephroblastoma. The cultured cells are spindle-shaped or polygonal and are arranged in a wavy fashion morphologically similar to cultured embryonal renal epithelial cells. The cells exhibit a number of epithelial characteristics. Enzyme histochemistry gives positive reactions for gamma-glutamyltranspeptidase and alkaline phosphatase, both of which are present in renal tubular epithelial cells. Immunofluorescence studies show positive reactions for vimentin and cytokeratin. When inoculated into athymic nude mice, the cultured cells form tumors composed of sheets of epithelial cells with focal tubular formation. This cell line may be of value in studying differentiation of nephroblastoma, and possibly normal nephrogenesis.     

p~(38)MAPK在IL-18诱导肾小管上皮细胞转分化中的作用

目的:白细胞介素18(IL-18)可诱导肾小管上皮细胞转分化,本研究探讨其是否是通过p38MAPK途径而起作用。方法:应用不同浓度的p38MAPK通路特异性阻断剂SB203580(0、5、10、20μmol/L)预孵育人近端肾小管上皮细胞(HK-2细胞)30min后,加入IL-18(100ng/ml)共培养24、48、72h。应用RT-PCR法检测α-平滑肌肌动蛋白(α-SMA)mRNA的表达水平;应用ELISA法测定细胞浆中α-SMA蛋白质含量。结果:SB203580呈剂量依赖性地抑制IL-18诱导的HK-2细胞α-SMA基因表达(P0.05)。结论:p38MAPK通路是调控IL-18诱导肾小管上皮细胞转分化的主要信号通路之一。     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

Caspase 3/GSDME-dependent pyroptosis contributes to chemotherapy drug-induced nephrotoxicity

Chemotherapy drug-induced nephrotoxicity limits clinical applications for treating cancers. Pyroptosis, a newly discovered programmed cell death, was recently reported to be associated with kidney diseases. However, the role of pyroptosis in chemotherapeutic drug-induced nephrotoxicity has not been fully clarified. Herein, we demonstrate that the chemotherapeutic drug cisplatin or doxorubicin, induces the cleavage of gasdermin E (GSDME) in cultured human renal tubular epithelial cells, in a time- and concentration-dependent manner. Morphologically, cisplatin- or doxorubicin-treated renal tubular epithelial cells exhibit large bubbles emerging from the cell membrane. Furthermore, activation of caspase 3, not caspase 9, is associated with GSDME cleavage in cisplatin- or doxorubicin-treated renal tubular epithelial cells. Meanwhile, silencing GSDME alleviates cisplatin- or doxorubicin-induced HK-2 cell pyroptosis by increasing cell viability and decreasing LDH release. In addition, treatment with Ac-DMLD-CMK, a polypeptide targeting mouse caspase 3-Gsdme signaling, inhibits caspase 3 and Gsdme activation, alleviates the deterioration of kidney function, attenuates renal tubular epithelial cell injury, and reduces inflammatory cytokine secretion in vivo. Specifically, GSDME cleavage depends on ERK and JNK signaling. NAC, a reactive oxygen species (ROS) inhibitor, reduces GSDME cleavage through JNK signaling in human renal tubular epithelial cells. Thus, we speculate that renal tubular epithelial cell pyroptosis induced by chemotherapy drugs is mediated by ROS-JNK-caspase 3-GSDME signaling, implying that therapies targeting GSDME may prove efficacious in overcoming chemotherapeutic drug-induced nephrotoxicity.Subject terms: Apoptosis, Acute kidney injury     

Uric acid induced epithelial−mesenchymal transition of renal tubular cells through PI3K/p-Akt signaling pathway

The phenotypic changes of tubular epithelial cell are hallmark features of renal diseases caused by abnormal uric acid levels. We hereby intend to investigate whether PI3K/p-Akt signaling plays a role in uric-acid induced epithelial−mesenchymal transition process. The normal rat kidney cell line (NRK-52E) was used as a proximal tubular cell model in this study. NRK-52E cells were exposed to different concentrations of uric acid, or PI3K inhibitor LY294002, or both, respectively. The effects of uric acid on cell morphology were examined by phase contrast microscopy, while molecular alternations were assessed by western blot analysis and immunofluorescence staining. We found that uric acid induced visible morphological alterations in NRK-52E cells accompanied by increased expression of α-smooth muscle actin and reduced expression of E-cadherin. Moreover, phosphorylation of Akt protein was obviously increased, whereas Akt level remained stable. Furthermore, the above effects were abolished when PI3K/p-Akt pathway was blocked by the PI3K inhibitor. These findings demonstrated that high uric acid could induce phenotypic transition of cultured renal tubular cells, which was probably via activating PI3K/p-Akt signaling pathway.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

高糖背景下白蛋白抑制肾小管上皮细胞的自噬表达

目的:探讨高糖背景下白蛋白造成肾小管间质损伤的作用及其机制。方法:体外培养大鼠近端肾小管上皮细胞系NRK-52E细胞,观察高糖培养环境下细胞自噬表达的改变;同时观察低浓度牛血清白蛋白(BSA)单独刺激,对肾小管上皮细胞自噬蛋白表达的影响以及细胞凋亡蛋白的表达改变;接着在高糖培养环境下加入低浓度的白蛋白刺激,观察肾小管上皮细胞的损伤效应及自噬表达情况。结果:高糖培养条件下肾小管上皮细胞自噬蛋白Beclin-1表达增加(P0.05),低浓度白蛋白也诱导肾小管上皮细胞自噬蛋白Beclin-1、Atg12表达增加(P0.05),以及细胞凋亡蛋白cleaved caspase3的轻度增加,乳酸脱氢酶活性增加(P0.05);但高糖培养下,少量白蛋白却抑制了肾小管上皮细胞自噬蛋白Beclin-1、Atg12的表达,并且显著增加了肾小管上皮细胞的凋亡蛋白cleaved caspase3的表达(P0.05)。结论:自噬是细胞自身的一种保护机制。在高糖背景下,白蛋白通过影响自噬的自身调节机制,促进了肾小管间质的损害作用。     

Hypoxic stress-enhanced expression and release of adrenomedullin (AM) and up-regulated AM receptors, while glucose starvation reduced AM expression and release and down-regulated AM receptors in monkey renal cells

The proliferative peptide adrenomedullin (AM) has a wide distribution in a variety of tissues and cells. The mechanism how the AM gene is regulated in cells is not yet known. The renal cortex, renal vascular smooth muscles, glomeruli and tubular epithelial cells are very sensitive to hypoxia. Renal hypoxia produces acute renal tubular necrosis and markedly induces AM expression in damaged cells. However, little information is available regarding the possible pathophysiological production and release of renal tubular AM. Regulation of membrane-bound AM receptors in renal cells has not yet been systematically studied. To elucidate the potential pathological role of human AM we examined the production and release of AM, as well as the characteristics of surface membrane AM receptors in cultured monkey renal tubular epithelial cells (RC) exposed to hypoxia, induced with endothelin-1, and subjected to glucose deprivation. Exposure of RC to hypoxia (1 % O(2), 5 % CO(2) in N(2)), and to phorbol 12-myristate 13-acetate (PMA) increased production and secretion of AM and increased specific [(125)I]AM binding on RC. Metabolic stress (1 % glucose in the cultivation medium) and preincubation of RC with rival peptide endothelin-1 significantly reduced immunoreactive-AM in a conditioned medium and whole cell surface membrane AM binding on RC. Altogether, our data suggest that the AM is involved in the adaptation of renal tubular cells to hypoxia. Increased expression of AM mRNA and regulation of AM receptors in metabolic stress may function as an important autocrine/paracrine regulator(s) of renal tubular epithelial cells.     

Role of P-glycoprotein in the renal transport of dideoxynucleoside analog drugs.

P-glycoprotein (P-gp), the MDR1 multidrug transporter, is known to be expressed in several human organs and tissues, including the apical membrane of the renal proximal tubular cells. It has been reported that human immunodeficiency virus 1 (HIV-1) can trigger the expression of P-gp in cultured cells (i.e., H9, a T-lymphocyte cell line, and U937, a monocyte cell line), which may render the cells resistant to antiretrovirals. Since multiple membrane transport systems (i.e., organic cation, organic anion, and nucleoside systems) can be involved in the renal tubular transport of dideoxynucleoside analog drugs (DADs) (i.e., zidovudine and zalcitabine), we have questioned if P-gp is involved in the renal transport of DADs. Chinese hamster ovary colchicine-resistant cells (CH(R)C5), a cell line that is well known to highly express P-gp, and continuous renal epithelial cell lines (LLC-PK1 and OK), which have also been shown to express P-gp, were used. The accumulation of [3H]vinblastine (20 nM), an established P-gp substrate, by the monolayer cells was significantly enhanced in the presence of two P-gp inhibitors (i.e., verapamil and cyclosporin A) and nucleoside transport inhibitors (i.e., dipyridamole and dilazep). In contrast, DADs (i.e., zidovudine, lamivudine, didanosine, and zalcitabine) did not significantly affect vinblastine accumulation by these cell lines. These data suggest that P-gp does not play a significant role in the renal tubular transport of DADs. Dipyridamole and dilazep, two nucleoside membrane transport inhibitors, appear to be P-gp inhibitors.     

Haematopoietic lineage-committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl 2 -induced acute tubular injury

Abstract.   Objective : Our previous studies have demonstrated that endogenous bone marrow cells (BMCs) contribute to renal tubular regeneration after acute tubular injury. The aim of this study was to examine which fraction of BMCs, haematopoietic lineage marrow cells (HLMCs) or mesenchymal stem cells (MSCs), are effective. Materials and methods : Six-week-old female mice were lethally irradiated and were transplanted with female enhanced green fluorescent protein-positive (GFP⁺), plastic non-adherent marrow cells (as a source of HLMCs) plus cloned cultured male GFP⁻ MSCs. Four weeks later, they were assigned into two groups: control mice with vehicle treatment and mice treated with HgCl₂. Tritiated thymidine was given 1 h before animal killing which occurred at intervals over 2 weeks. Kidney sections were stained for a tubular epithelial marker, cell origin indicated by GFP immunohistochemistry or Y chromosome in situ hybridization; periodic acid-Schiff staining was performed, and samples were subjected to autoradiography. One thousand consecutive renal tubular epithelial cells per mouse, in S phase, were scored as either female (indigenous) GFP⁺ (HLMC-derived) or male (MSC-derived). Results : Haematopoietic lineage marrow cells and MSCs stably engrafted into bone marrow and spleen, but only HLMC-derived cells, not MSCs, were found in the renal tubules and were able to undergo DNA synthesis after acute renal injury. A few MSCs were detected in the renal interstitium, but their importance needs to be further explored. Conclusion : Haematopoietic lineage marrow cells, but not cloned cultured MSCs, can play a role not only in normal wear-and-tear turnover of renal tubular cells, but also in repair after tubular injury.     

改构型酸性成纤维细胞生长因子对顺铂肾损害保护作用的实验研究

目的 研究改构型酸性成纤维细胞生长因子(MaFGF)对顺铂(DDP)引起的体外培养的肾小管上皮细胞损害的保护作用。方法 将原代培养的肾小管上皮细胞接种于96孔培养板：(1)培养72h后加入一系列浓度的DDP,实验组在DDP作用12h后加入不同浓度MaFGF,再培养36h后用WST-8法检测细胞存活率。（2）以DDP建立损伤模型,并在加药后12小时加入一定量的MaFGF,观察MaFGF对肾小管上皮细胞的保护作用。结果 (1) MaFGF能使DDP对肾小管上皮细胞的半数抑制浓度（IC50）升高。（2）DDP组与对照组比较,各生化和酶学指标差异均有统计学意义;而MaFGF + DDP组与对照组比较,SOD、GSH-Px酶活性差异无统计学意义,MDA、NO升高差异仍有统计学意义。结论 MaFGF对DDP损伤的肾小管上皮细胞有明显的保护作用。     

Urinary cytodiagnosis of renal involvement in disseminated histiocytic lymphoma

Two cases of patients with disseminated histiocytic lymphoma are presented in which positive urine cytology provided evidence of renal involvement. In addition to malignant cells, the urine sediment characteristics included renal tubular epithelial cell exfoliation, renal epithelial fragments and pathologic cast formation, which distinguished renal from lower urinary tract involvement. Such cytologic observations may be useful in differentiating among the various causes of renal disease in patients with malignant lymphomas.     

Clusterin/Apolipoprotein J Attenuates Angiotensin II-Induced Renal Fibrosis

The blockade of angiotensin II (Ang II) is a major therapeutic strategy for diabetic nephropathy. The main roles of Ang II in renal disease are mediated via the Ang type 1 receptor (AT1R). Upregulation of clusterin/apolipoprotein J has been reported in nephropathy models, suggesting it has a protective role in nephropathogenesis. Here, we studied how clusterin acts against Ang II-induced renal fibrosis. Levels of AT1R and fibrotic markers in clusterin-/- mice and Ang II infused rats transfected with an adenovirus encoding clusterin were evaluated by immunoblot analysis, real time RT-PCR, and immunohistochemical staining. The effect of clusterin on renal fibrosis was evaluated in NRK-52E cells, a cultured renal tubular epithelial cell line, using immunoblot analysis and real time RT-PCR. Nuclear localization of NF-κB was evaluated using immunofluorecence and co-immunoprecipitation. Renal fibrosis and expression of AT1R was higher in the kidneys of clusterin^-/- mice than in those of wild-type mice. Furthermore, loss of clusterin accelerated Ang II-stimulated renal fibrosis and AT1R expression. Overexpression of clusterin in proximal tubular epithelial cells decreased the levels of Ang II-stimulated fibrotic markers and AT1R. Moreover, intrarenal delivery of clusterin attenuated Ang II-mediated expression of fibrotic markers and AT1R in rats. Fluorescence microscopy and co-immunoprecipitation in conjunction with western blot revealed that clusterin inhibited Ang II-stimulated nuclear localization of p-NF-κB via a direct physical interaction and subsequently decreased the AT1R level in proximal tubular epithelial cells. These data suggest that clusterin attenuates Ang II-induced renal fibrosis by inhibition of NF-κB activation and subsequent downregulation of AT1R. This study raises the possibility that clusterin could be used as a therapeutic target for Ang II-induced renal diseases.     

Regulation of epithelial cell morphology and functions approaching to more in vivo-like by modifying polyethylene glycol on polysulfone membranes

Cytocompatibility is critically important in design of biomaterials for application in tissue engineering. However, the currently well-accepted "cytocompatible" biomaterials are those which promote cells to sustain good attachment/spreading. The cells on such materials usually lack the self-assembled cell morphology and high cell functions as in vivo. In our view, biomaterials that can promote the ability of cells to self-assemble and demonstrate cell-specific functions would be cytocompatible. This paper examined the interaction of polyethylene glycol (PEG) modified polysulfone (PSf) membranes with four epithelial cell types (primary liver cells, a liver tumor cell line, and two renal tubular cell lines). Our results show that PSf membranes modified with proper PEG promoted the aggregation of both liver and renal cells, but the liver cells more easily formed aggregates than the renal tubular cells. The culture on PEG-modified PSf membranes also enhanced cell-specific functions. In particular, the cells cultured on F127 membranes with the proper PEG content mimicked the in vivo ultrastructure of liver cells or renal tubules cells and displayed the highest cell functions. Gene expression data for adhesion proteins suggest that the PEG modification impaired cell-membrane interactions and increased cell-cell interactions, thus facilitating cell self-assembly. In conclusion, PEG-modified membrane could be a cytocompatible material which regulates the morphology and functions of epithelial cells in mimicking cell performance in vivo.     

Effect of the extracellular matrix molecules fibronectin and laminin on the adhesion and growth of primary renal cortical epithelial cells

Primary tubular epithelial cells were isolated from renal cortex following enzymatic dissociation with collagenase. These cells were then grown in chemically defined media containing insulin, transferrin, selenium, tri-iodothyronine and either fibronectin or laminin. The tubular epithelial cells were studied ultrastructurally and compared to another epithelial cell type present in the renal cortex, the glomerular epithelial cell. In contrast to the constant morphology of glomerular epithelial cells grown in chemically defined media, tubular epithelial cell morphology depended on whether the cells were placed in fibronectin or laminin and on the age of the donor animal used for culture. Primary tubular cells grown in laminin formed colonies; cells grown from young animals were rounded, whereas cells grown from adult animals were flattened. Primary tubular cells grown in fibronectin were flattened regardless of age, but cells from young animals formed colonies while those from adult animals formed a monolayer. Despite these differences in gross morphology, scanning and transmission electron microscopy revealed similar ultrastructural features in primary tubular cells from young and adult animals grown in fibronectin or laminin. Quantitative adhesion studies demonstrated that secondary subcultured tubular cells adhered equally well to dimeric and multimeric forms of fibronectin, but not to laminin. Quantitative colony growth studies of subcultured secondary tubular cells showed that laminin supports colony formation of trypsinized tubular cells, while previous work has demonstrated that fibronectin supports colony formation of glomerular cells. These results are consistent with the hypothesis that different extracellular matrix molecules are involved in colony formation of different cell types, with fibronectin stimulating growth of glomerular cells and laminin supporting growth of tubular cells.     

Transforming growth factor-beta-like activity is secreted by rabbit renal cortical tubular cells in primary culture.

The activity of an autocrine growth factor in a medium conditioned by cultured rabbit renal cortical tubular cells was investigated. Little stimulatory growth activity for tubular cells was observed in the conditioned medium, and inhibitory activity was seen only in acidified conditioned medium. This factor stimulated the colony formation of NRK 49F cells in soft agar only with epidermal growth factor and inhibited the DNA synthesis of primary cultured rat hepatocytes, and its molecular weight was about 25 kDa. The factor was neutralized by the specific antibody to transforming growth factor (TGF)-beta 1. These results indicate that renal tubular epithelial cells can produce latent TGF-beta in primary culture.     

Negative Regulation of TGFβ Signaling by Stem Cell Antigen-1 Protects against Ischemic Acute Kidney Injury

Acute kidney injury, often caused by an ischemic insult, is associated with significant short-term morbidity and mortality, and increased risk of chronic kidney disease. The factors affecting the renal response to injury following ischemia and reperfusion remain to be clarified. We found that the Stem cell antigen-1 (Sca-1), commonly used as a stem cell marker, is heavily expressed in renal tubules of the adult mouse kidney. We evaluated its potential role in the kidney using Sca-1 knockout mice submitted to acute ischemia reperfusion injury (IRI), as well as cultured renal proximal tubular cells in which Sca-1 was stably silenced with shRNA. IRI induced more severe injury in Sca-1 null kidneys, as assessed by increased expression of Kim-1 and Ngal, rise in serum creatinine, abnormal pathology, and increased apoptosis of tubular epithelium, and persistent significant renal injury at day 7 post IRI, when recovery of renal function in control animals was nearly complete. Serum creatinine, Kim-1 and Ngal were slightly but significantly elevated even in uninjured Sca-1-/- kidneys. Sca-1 constitutively bound both TGFβ receptors I and II in cultured normal proximal tubular epithelial cells. Its genetic loss or silencing lead to constitutive TGFβ receptor—mediated activation of canonical Smad signaling even in the absence of ligand and to KIM-1 expression in the silenced cells. These studies demonstrate that by normally repressing TGFβ-mediated canonical Smad signaling, Sca-1 plays an important in renal epithelial cell homeostasis and in recovery of renal function following ischemic acute kidney injury.     

In vitro modulation of antioxidant enzymes in normal and malignant renal epithelium

Summary The activities of three antioxidant enzymes, superoxide dismutase, catalase, and glutathione peroxidase, were monitored in isolated human renal adenocarcinoma tissues and in cultured human renal adenocarcinoma cells. The results were compared to the activities of these enzymes in the proposed cell of origin, isolated human proximal tubular tissues, and cultured proximal tubular epithelial cells. Strong modulation of these enzymes by culture conditions was observed in normal cells but not in carcinoma cells. Low levels of cellular lipid peroxidation, as assessed by levels of malondialdehyde (MDA), were observed in adenocarcinoma cells under the culture conditions tested with one exception: greatly elevated MDA was observed in renal adenocarcinoma cells growth on plastic in serum-free, chemically defined medium. This increased lipid peroxidation correlated with a loss of cell viability under these conditions. This work was supported by a grant from the Veterans Adminsitration (T. D. O.) and by grant 1 R01 CA 41267 from the National Institutes of Health (L. W. O.), Bethesda, MD.     

Cross talk between primary human renal tubular cells and endothelial cells in cocultures

Interactions between renal tubular epithelial cells and adjacent endothelial cells are essential for normal renal functions but also play important roles in renal disease and repair. Here, we investigated cocultures of human primary renal proximal tubular cells (HPTC) and human primary endothelial cells to address the cross talk between these cell types. HPTC showed improved proliferation, marker gene expression, and enzyme activity in cocultures. Also, the long-term maintenance of epithelia formed by HPTC was improved, which was due to the secretion of transforming growth factor-β1 and its antagonist α2-macroglobulin. HPTC induced endothelial cells to secrete increased amounts of these factors, which balanced each other functionally and only displayed in combination the observed positive effects. In addition, in the presence of HPTC endothelial cells expressed increased amounts of hepatocyte growth factor and vascular endothelial growth factor, which have well-characterized effects on renal tubular epithelial cells as well as on endothelial cells. Together, the results showed that HPTC stimulated endothelial cells to express a functionally balanced combination of various factors, which in turn improved the performance of HPTC. The results give new insights into the cross talk between renal epithelial and endothelial cells and suggest that cocultures could be also useful models for the analysis of cellular communication in renal disease and repair. Furthermore, the characterization of defined microenvironments, which positively affect HPTC, will be helpful for improving the performance of this cell type in in vitro applications including in vitro toxicology and kidney tissue engineering.