Transcriptomes of Major Proximal Tubule Cell Culture Models

BackgroundCultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line.MethodsFourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures.ResultsTranscripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins.ConclusionsNo cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.

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Nitric oxide activates PKCα and inhibits Na+-K+-ATPase in opossum kidney cells

AJP Renal Physiology ◽

10.1152/ajprenal.1999.277.6.f859 ◽

1999 ◽

Vol 277 (6) ◽

pp. F859-F865 ◽

Cited By ~ 15

Author(s):

Mingyu Liang ◽

Franklyn G. Knox

Keyword(s):

Nitric Oxide ◽

Protein Kinase ◽

Proximal Tubule ◽

Cell Line ◽

Soluble Guanylate Cyclase ◽

Inhibitory Effect ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Opossum Kidney ◽

Ok Cells

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCα from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCα in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCα in OK, but not in LLC-PK1, cells. The activation of PKCα in OK cells by NO is associated with inhibition of Na+-K+-ATPase.

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Metabolic effects of fatty acid-bearing albumin on a proximal tubule cell line

AJP Renal Physiology ◽

10.1152/ajprenal.1995.268.6.f1177 ◽

1995 ◽

Vol 268 (6) ◽

pp. F1177-F1184 ◽

Cited By ~ 7

Author(s):

M. E. Thomas ◽

A. R. Morrison ◽

G. F. Schreiner

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Proximal Tubule ◽

Cell Line ◽

Metabolic Effects ◽

Proximal Tubule Cell ◽

Tubule Cell ◽

Ok Cells ◽

Triglyceride Accumulation ◽

Cells Cultured

In glomerular disease, fatty acids carried on albumin are taken up by the proximal tubule with filtered albumin. We postulate that the fatty acids carried on filtered albumin could contribute to the deleterious effects of proteinuria. The effects of fatty acid-albumin complexes on lipid metabolism have been studied in opossum kidney (OK) cells, a proximal tubule cell line. OK cells transported two-thirds of [14C]palmitate-albumin (5 mg/ml) intracellularly within 16 h. [14C]palmitate-albumin was distributed into phosphatidylcholines, phosphatidylinositols, and tri- and diglycerides. 14C-labeled unsaturated fatty acid albumins (oleate, linoleate, and arachidonate) showed preferential incorporation into triglycerides, with lesser incorporation into phospholipids. Studies of total lipid pools showed that fatty acid-albumin uptake produced a particularly marked increase in total triglyceride levels (approximately 10-fold). Oil red O staining of OK cells cultured with oleate-albumin showed a marked increase in intracellular lipid droplets, compared with cells cultured with delipidated albumin, consistent with triglyceride accumulation. Less than 1% of [14C]palmitate taken up was isolated as intracellular free fatty acid. Less than 5% of [14C]palmitate internalized was oxidized to 14CO2. Different fatty acids, when taken up by the OK cell, have distinct metabolic fates. Each fatty acid is incorporated in a characteristic fashion into certain complex lipids, possibly dependent on the presence or absence of double bonds. We propose that this may have functional consequences for the proximal tubule in the human nephrotic syndrome.

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The actin cytoskeleton and integrin expression in the recovery of cell adhesion after oxidant stress to a proximal tubule cell line (JTC-12).

Journal of the American Society of Nephrology ◽

10.1681/asn.v9101787 ◽

1998 ◽

Vol 9 (10) ◽

pp. 1787-1797

Author(s):

S Nigam ◽

C E Weston ◽

C H Liu ◽

E E Simon

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Proximal Tubule ◽

Cell Line ◽

Oxidant Stress ◽

Proximal Tubule Cell ◽

Integrin Expression ◽

Tubule Cell ◽

Essentially Normal ◽

Atp Levels

This study examines the role of the actin cytoskeleton and integrin expression in the recovery of cell adhesion in the proximal tubule cell line JTC-12 after peroxide injury. The cells were exposed to 10, 20, or 50 mM hydrogen peroxide for 10 min and then allowed to recover. Viability measurements by trypan blue exclusion confirmed that the injury was largely nonlethal with 85% viability at 1 h even at 50 mM peroxide. ATP levels fell immediately after the peroxide incubation in all groups to approximately 10% of normal, but already showed some recovery by 1 h and full recovery in the 10 and 20 mM groups by 24 h. Cell adhesion to extracellular matrix immediately after injury was depressed at 20 and 50 mM peroxide, but by 12 h was abnormal only at 50 mM peroxide and at 24 h was essentially normal at all peroxide concentrations. Immediately after exposure to 10 mM peroxide, there were subtle abnormalities in the actin cytoskeleton (thickening of fibrils) as assessed by phalloidin staining, with more pronounced effects at 20 and 50 mM. At 1 h, many cells showed collapse of the actin cytoskeleton to the periphery. There was some recovery at 4 h; by 12 h, the actin cytoskeleton showed further recovery, although was still abnormal (coarsened microfilaments), especially at 20 and 50 mM peroxide. By 24 h, the actin cytoskeleton showed only subtle coarsening. Integrin surface expression was assessed by flow cytometry. The alpha6 subunit on cells exposed to 20 mM peroxide was unchanged at 1 h and 4 h, but by 12 h had increased to 118.5+/-4.5% and by 24 h to 146+/-13.4% of control levels. The expression of the beta1 and alphaVbeta3 integrins remained unchanged. Thus, despite coarsening of the actin cytoskeleton and depressed ATP levels, cell adhesion recovered from oxidant stress. Abnormal cell adhesion after injury was not a consequence of a decrease in integrin expression, and recovery of cell adhesion was not a consequence of the modest and selective increase in integrin expression.

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