Prostaglandin E₂ promotes cellular recovery from established nephrotoxic serum nephritis in mice, prosurvival, and regenerative effects on glomerular cells

Abstract

We postulated that prostaglandin E₂ (PGE₂), which exhibits regulatory functions to control immune-mediated inflammation, fibrosis, oxidative stress, and tissue/cellular regeneration, has the potential to improve the course of nephritis. Therefore, the therapeutic potential of prostanoid on established nephritis in mice was evaluated focusing on its role on renal cellular recovery, with emphasis on its cytoprotecting and growth-promoting effects. Acute nephritis was induced in mice by single injection of nephrotoxic serum (NTS), followed by PGE₂ administration with severity of nephritis evaluated over time. Mice injected with PGE₂ recovered promptly with normalization of blood urea nitrogen and urine protein levels and histology. Recovery was observed with dosing of prostanoid at day 1, as well as day 4. With the use of selective EP1–4 receptor agonists, EP3 receptor has been identified as important in mediating beneficial effects of PGE₂ in our system. PGE₂ normalized glomerular cell losses during nephrotoxic serum-induced nephritis, restored synaptopodin distribution and F-actin filaments arrangement in glomeruli. In cell culture, PGE₂ reduced nephrotoxim serum (NTS)-induced apoptosis of glomerular cells and promoted cell reproliferation after NTS-mediated injury. In conclusion, PGE₂ treatment promotes resolution of glomerular inflammation. Consistent with this observation, the regenerative and cytoprotective effects of prostanoid on glomerular cells in culture were observed, suggesting that PGE₂ may be beneficial in the treatment of glomerulonephritis.

much progress has been made in understanding the mechanisms involved in the pathogenesis of glomerulonephritis; however, efforts to understand and promote cellular and tissue recovery have been less forthcoming. In humans, most therapies are directed at alleviating either the underlying systemic disease process or preventing cellular events that lead to fibrosis within the kidney. Success at repair or reversal of disease has been limited. In this context, understanding how renal tissue recovers/regenerates after insult is especially relevant, since it has the potential to provide insights into strategies to improve outcomes.

Recovery from injury is a highly organized process, influenced by an array of cytokines, growth factors, and prostaglandins, produced by damaged cells and infiltrating leukocytes (6). In this study we investigated the efficacy of prostaglandin E₂ (PGE₂), a potent lipid mediator and major product of arachidonic acid metabolism, in restoration of nephrotoxic serum (NTS)-induced clinical and pathological changes in mouse kidneys and in cultured mouse glomerular cells. In nephrotoxic serum-induced nephritis (NTN), Fc receptor (FcR) and complement-mediated processes initiate inflammation, with subsequent cellular infiltration and glomerular cell injury, which results in proteinuria and reduced glomerular filtration rate (10, 33, 36). NTS targets include α₃(IV) collagen and other basement membrane, cell surface, and matrix proteins (e.g., collagens, α₃β₁-integrin, aminopeptidase A, and laminin; Refs. 26, 28). Whether FcR or complement-mediated events dominate depends on the NTS preparation and context, although both pathways contribute to disease. PGE₂ is produced in nearly all cell types in response to various stimuli and is synthesized from membrane lipids via the sequential actions of phospholipase A2, cyclooxygenases (PLA2, COXs), and prostaglandin (PG) terminal synthases (13, 35). PGE₂ plays a role in maintaining the physiological homeostasis and is implicated in many physiologic functions (17, 2, 40). Differential expression of four distinct PGE₂ receptors (EP1–4) defines the intracellular pathways activated by PGE₂, and differential pathway activation results in specific functions that either promote or inhibit inflammation. The initial view that PGE₂ promotes inflammation, like other PGs, has been countered, and the evidence supports its anti-inflammatory nature (8, 34, 23). Nevertheless, the mechanisms of PGE₂ activation are complex and context dependent.

A wide range of regulatory actions of PGE_2, particularly on immune-mediated inflammation, developmental processes, and tissue/cellular regeneration have been demonstrated on different cell types and diseases. Earlier studies have shown beneficial effect of PGE₂ related to its general anti-inflammatory activity and effects on leukocytes (7, 32). More recent studies have focused on its role in tissue remodeling and regeneration. For example, renal PGE₂-EP4 interactions inhibited the development of tubulointerstitial fibrosis (27). Additionally, PGE₂ signaling via EP2 receptors facilitated growth/regeneration of articular cartilage and bone marrow cells (29), whereas COX-2-deficient systems have shown impaired muscle healing, implying a role for PGs in muscle regeneration (26). In other studies, PGE₂ promoted blood cell recovery after injury in zebrafish and mice, and the stable analog of PGE₂, dimethyl-PGE₂ (dmPGE₂), significantly increased engraftment of unfractionated and CD34⁺, human cord blood cells after xenotransplantation (7, 23). Nevertheless, a role of PGE₂ in kidney recovery and regeneration has not been yet elucidated.

Given these considerations, we postulated that PGE₂ would improve the course of established nephritis by cumulative effects on limiting inflammation, blunting immune responses, alleviating oxidative stress, and enhancing renal cellular recovery. Our main focus was on the role of PGE₂ in renal cellular recovery during NTN, with emphasis on its cytoprotecting and growth-promoting effects. The experiment approach involved administration of PGE₂ after cellular injury was established, using both whole animal and culture models. This strategy is distinguished from other experimental approaches, where prevention has been utilized to examine the role of participants. Herein we take advantage of the potential restorative properties of PGE_2.

MATERIALS AND METHODS

Animals, Cells, and Reagents

Female C57BL/6 mice were purchased from The Jackson Laboratory. All experiments were performed in compliance with federal laws and institutional guidelines. The animal protocol was approved by the Georgia Health Sciences University Institutional Animal Care and Use Committee (no. A3307-01).

Eight- to-ten-week-old mice (18–20 g) were used for all experiments. Established cloned immortalized mouse podocyte and glomerular endothelial cell clones were employed as described previously (1). For passage, the cells were grown under “growth permissive” conditions (33°C), whereas to acquire a differentiated and quiescent phenotype for use in experiments, the cells were grown under “restrictive conditions” at 37°C in 95% air-5% CO₂. EP1–4 receptor antagonists (SC 19220, AH 6809, L-798106, and L-161982; Tocris Bioscience), annexin V apoptosis kit (Phoenix Flow Systems), DeadEnd Fluorometric TUNEL System (Promega), anti-synaptopodin antibodies (Santa Cruz Biotechnology), PGE₂ (Sigma), and FITC-conjugated anti-rabbit antibodies were purchased (Abcam).

Nephrotoxic Nephritis

Sheep nephrotoxic sera was prepared and administered as described previously (1, 19). In brief, glomeruli were isolated from normal C57BL/6 mice by differential sieving and used to immunize sheep. The nephrotoxic sheep serum was heat inactivated and absorbed with an excess amount of murine blood cells before use. Nephrotoxic sera were aliquoted and frozen at −80°C. For disease induction, NTS was administered as a single dose intraperitoneally (13.5 μl of serum per g mouse weight). Mice were then followed with measurement of proteinuria (BCA; Pierce) and serum urea level using the urea nitrogen direct kit (Stanbio Laboratory). At days 2, 4, 7, 9, kidneys were removed and either fixed in 10% buffered formalin or frozen in OCT medium. Four-micrometer sections from paraffin-embedded samples were stained with hematoxylin and eosin. Pathological evaluation by light microscopy was done in a blinded manner by M. P. Madaio.

PGE₂ (5 μg/g) was injected intraperitoneally on days 2 and 4 after NTS dosing. In other experiments, different doses of PGE₂ (10, 5, and 1 μg/g) were given daily to mice the day after NTS treatment (day 2). EP1–4 receptor antagonists (5 and 25 μg/g of body wt) were injected in separate groups of mice 4 h in advance of PGE₂ infusion. Proteinuria and blood urea nitrogen (BUN) levels were measured as described previously (9).

Histological Evaluation

Kidneys were removed and either fixed in 10% buffered formalin or frozen in OCT medium. Light microscopy and immunofluorescence analysis were performed and evaluated in a blinded manner for severity of nephritis (22). Indirect immunofluorescence was used to evaluate synaptopodin expression using anti-synaptopodin and FITC conjugated anti-rabbit Abs. F-actin filaments were visualized with CytoPainter F-actin stainig kit (Abcam) according to the manufacturer's instructions. Images were acquired using Zeiss 7 Live Duoscan Confocal Microscope. Mean fluorescence intensity of stained glomeruli was quantified using Zen 2009 Software for laser scanning microscopes. In brief, by using a two-dimensional grid, the fluorescence intensity was quantified from the same pixel sized area of specimen, which related to the fluorophores present at the corresponding area and therefore to the local concentration of studied protein. Mean fluorescent intensity was determined by average value of intensities calculated from three similar images.

Electron microscopy.

Cells/tissue were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate (NaCac) buffer, pH 7.4, postfixed in 2% osmium tetroxide in NaCac, stained with 2% uranyl acetate, dehydrated with a graded ethanol series, and embedded in Epon-Araldite resin. Thin sections were cut with a diamond knife on a Leica EM UC6 ultramicrotome (Leica Microsystems, Bannockburn, IL), collected on copper grids, and stained with uranyl acetate and lead citrate. Cells/tissue were observed in a JEM 1230 transmission electron microscope (JEOL, Peabody, MA) at 110 kV and imaged with an UltraScan 4000 CCD camera and First Light Digital Camera Controller (Gatan, Pleasanton, CA).

Apoptosis of Cultured Glomerular Endothelial Cells and Podocytes: Regeneration Treatment Conditions

Glomerular epithelial cells (GEC) and podocytes were detached by mild enzymatic treatment from tissue flasks, placed in 15-ml conical tubes, 1 × 10⁶, incubated with NTS (6%) and NTS and PGE₂ (0.01–1 μg/ml) for 5 h. Apoptotic cells were enumerated using annexin V assay (Phoenix Flow Sytems), and the DeadEnd Fluorometric Tunel System (Promega) according to manufacturer's instructions. For regeneration experiments, podocytes and GEC were placed in Petri dishes overnight, starved for 24 h, and then 4% NTS was added for 9 h, after which the NTS removed, fresh media with/without PGE₂ were added, and the cells were cultured for and additional 12 h. The cells were gently detached, and absolute cell enumeration was carried out by flow cytometry. Glomerular cell enumeration in kidney sections was carried out as described previously (37).

Synaptopodin Detection in Cultured Podocytes

Podocytes in four-well chamber slides were pretreated with PGE₂ (0.01–1 μg/ml) for 1 h, followed by addition of NTS (6%) for 5 h. Distribution of synaptopodin was evaluated by indirect immunofluorescence using anti-synaptopodin and FITC-conjugated anti-rabbit antibodies. F-actin filaments were visualized with CytoPainter F-actin stainig kit (Abcam) according to manufacturer's instructions.

Microarray Analysis

Following RNA isolation (Trizol extraction) from the kidneys of control, NTS-, and NTS/PGE₂-treated mice, the RNAs were processed for microarray analysis at the Georgia Health Sciences University Genomic Core Facility. Affymetrix Gene Chip: Mouse Gene 1.0 ST Array was used for gene expression profiling. Further statistical analysis and biological processes were generated through the use of iReport (Ingenuity Systems).

Accession Number

The gene array data discussed in the publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series Accession No. GSE27126.

Statistical Analysis

All values are presented as means ± SD. A Student's t-test was used for significant differences between two data sets and ANOVA for multiple data sets (proteinurea and BUN).

RESULTS

PGE₂ improves Clinical and Pathologic Parameters in Mice with Established NTN

Animals who received NTS without PGE₂, uniformly developed severe nephritis with proteinuria, ascites, weight gain, and azotemia, whereas PGE₂ limited these elevations (Fig. 1, A and B). Among several doses (1, 5, and 10 μg/g) of PGE₂ tested, 5 μg/g of animal body wt was the most effective (data not shown). Particularly noteworthy, initiation of therapeutic administration of PGE₂ on day 4, after nephritis was well established, resulted in normalization and significant decrease in urine protein and BUN levels on day 9 (Fig. 1C). At days 7 and 9, the kidneys of the NTS animals showed signs of severe nephritis with profound glomerular and tubulointerstital injury, necrosis, and heavy proteinuria. By contrast, NTN mice treated with PGE₂ demonstrated recovery, with no or limited evidence of nephritis (Fig. 2, A and C). The NTN mice had a reduced life expectancy: on day 9 one mouse died and the other four appeared moribund with oliguia, which limited evaluation of proteinuria in these animals, whereas all mice in NTS/PGE₂ group acted normally in their cages.

Fig. 1.

Prostaglandin E₂ (PGE₂) administration limits clinical evidence of nephrotoxic serum-induced nephritis (NTN). Time-dependent development of proteinuria and elevation in blood urea nitrogen (BUN; A and B) in NTN mice with inhibition after PGE₂ administration [started either on day 2 (A and B) or day 4 (C) following nephrotoxic serum (NTS) administration] Initially, NTS (13.5 μl/g wt) was injected into C57/Bl6 mice, followed by daily PGE₂ administrations (5 μg/g ip) starting on days 2 or 4, following the NTS injection. Urine protein levels could not be evaluated in the NTS, day 9 mice, due to oliguria or death. Even late administration of PGE₂ started on day 4 resulted in decrease in BUN and proteinuria on day 9 (C). Results are shown as means ± SD; 5 mice were used in each group. *P ≤ 0.05, **P ≤ 0.01.

Fig. 2.

PGE₂ treatment limits histologic evidence of nephritis. Representative microscopies (hematoxylin and eosin) from day 7 mice are shown (A). Kidneys of the NTS animals showed signs of severe nephritis with profound glomerular and tubulointerstital injury, necrosis, and heavy proteinuria. No or minimal evidence of nephritis was observed in NTS mice treated with PGE_2. (top and bottom: ×100 magnification; middle: ×400). Morphometric evaluation of glomerular cells (B). Glomerular cell numbers were significantly higher in NTS/PGE₂-treated mice. Cells were enumerated per glomeruli in 6 representative glomeruli. Representative transmission electron microscopy of glomeruli from an NTS-treated mouse after 24 h on day 7 and NTS/PGE₂-treated mouse on day 7 and PBS-injected control mouse (C). After NTS administration, foot processes were variably shortened and widened on day 1 and completely effaced on day 7 (C). By contrast in NTS and PGE₂-treated mice, foot processes were mostly intact on day 7. Results are shown as means ± SD. **P ≤ 0.01; ***P ≤ 0.001.

To determine which of four PGE₂ receptors (EP1–4) were responsible for the observed beneficial effect, EP1–4 receptor antagonists (5 μg/g wt) were injected daily, beginning 4 h in advance of PGE₂ administration to the NTN mice. Only the EP3 receptor antagonist fully inhibited the beneficial effects of PGE₂ (Fig. 3). To further confirm that EP1, 2, and 4 receptors do not participate in PGE₂-mediated recovery, higher concentrations (25 μg/g wt) of either EP1, EP2, and EP4 receptor antagonists were infused in separate groups of mice. By contrast with the EP3 receptor antagonist, they did not reverse the beneficial effects of PGE_2.

Fig. 3.

EP3 receptor antagonist inhibits the beneficial effect of PGE₂. EP1–4 receptor antagonists (SC 19220, AH 6809, L-798106, and L-161982, respectively) were injected in groups of mice (5 and 25 μg/g) 4 h in advance of PGE₂ infusion starting on day 2 after NTS treatment. BUN levels were measured in mice on day 7. Results are shown as means ± SD. *P < 0.05.

PGE₂ Limits Podocyte Injury in Established NTN

Since PGE₂ treatment limited NTS-induced proteinuria, we reasoned that improvement in podocyte injury and restoration of actin associated proteins were involved. Therefore, we investigated modulation of expression of synaptopodin, which regulates the actin cytoskeleton and is expressed in foot processes as the Synpo-long isoform (25, 39). On initial analysis, there were differences in the pattern and intensity of synaptopodin staining, where normal animals displayed a “fine linear” staining pattern with intervals between lines, likely corresponding to intact foot processes in podocytes (Fig. 4A). By contrast, in the NTN mice, the lines and intervals disappeared, presumably due to foot process effacement. This normal pattern was restored in the NTS/PGE₂ mice. Consistent with this conclusion, assessment of synaptopodin expression between the groups revealed quantitative differences: the mean fluorescence intensity of stained synaptopodin (evaluated from equal pixel sized area of specimens, see materials and methods) from the NTS-treated mice was reduced (487.4 ± 128.211), while the NTS/PGE₂ mice had significantly higher values (760.05 ± 65.02; P < 0.05). Modulation in podocyte ultrastructure and foot processes was also observed by electron microscopy, where extensive loss of foot processes in nephritic mice was present (Fig. 2C). By contrast, foot process fusion had been largely restored after PGE₂ administration.

Fig. 4.

PGE₂ treatment restores alterations in synaptopodin and F-actin staining in NTS-treated mice and cultured podocytes. A: in mice, the “fine linear” pattern of synaptopodin and F-actin staining in normal glomeruli was mostly eliminated in NTS mice, presumably due to foot process effacement. This normal pattern was restored in the NTS/PGE₂ mice. B: in culture, podocytes were pretreated with PGE₂ (0.01–1 μg/ml) 1 h, followed by addition of NTS (6%) for 5 h. Following NTS treatment of cultured podocytes, synaptopodin localization became less cytoplasmic and more perinuclear and PGE₂ treatment limited synaptopodin distribution and F-actin filaments to nearly the untreated condition (C). Results are shown as means ± SD. *P < 0.05.

Alterations in cytoskeleton and associated proteins were evaluated in cultured podocytes by assessment of synaptopodin staining and F-actin arrangement after NTS/ ± PGE₂ treatment conditions. Disarrangement of F-actin cytoskeleton, perinuclear dislocation, and loss of cytoplasmic staining of synapotopodin were observed after NTS treatment (Fig. 4B). PGE₂ treatment maintained synaptopodin distribution and F-actin filaments nearly to the untreated condition (Fig. 4B).

PGE₂ Upregulates Cell Cycle Progression Genes in the Kidney Following NTS Exposure

To begin to define genes/pathways responsible for PGE₂-mediated recovery, microarray gene expression and pathway analysis (Ingenuity Systems) was carried out. Kidneys from NTS- and NTS/PGE₂-exposed animals were analyzed. The most significant biological processes affected by prostanoid treatment are listed in Table 1. Proliferation of cells was ranked highest based on P value enrichment score and the number of differentially expressed genes, consistent with the in vivo results. Notably, genes specific for cell cycle/growth-promoting pathways were upregulated: cyclin-dependent kinase 1 (CDK1; 5.568-fold change), FOXM1 (3.882-fold), S100A6 (8.484-fold), CCNA2 (2.003-fold), CCNF (2.014-fold), and ANLN (8.698-fold) in PGE₂-treated animals. These findings are consistent with the reduction in glomerular cell numbers observed in NTS-injected mice as evaluated by morphometric quantification on days 4 and 7. By contrast glomerular cell numbers were higher in the PGE₂-treated mice (Fig. 2B) consistent with the conclusion that enhanced glomerular cell proliferation contributed to the improvement.

Table 1.

Ingenuity iReport reveals most significant biological processes affected by PGE₂ treatment

Biological Process	DEG	P Value
Proliferation of cells	272	6.52879E-14
Tissue development	232	1.38971E-07
Growth of cells	195	6.83175E-10
Cell movement	183	9.61159e-09
Development of organ	146	0.000503125
Immune response	138	1.97659E-05
Transport of molecules	133	4.16644E-08
Organismal death	106	0.000186372
Cell death of organs	89	9.45585E-05
Cell movement of leukocytes	71	0.000250124
Survival of organism	67	1.92577E-07
Cell movement of phagocytes	52	0.000250844
Kidney development	26	0.000354856

PGE₂, prostaglandin E₂; DEG, differentially expressed genes.

To further address this issue, cellular recovery was investigated in cultured glomerular cells following NTS treatment. In brief, the cells were exposed to NTS, which time and dose-dependently reduced glomerular cell numbers. The NTS was then removed, and the cells were either treated with PGE₂ or allowed to grow without it in normal media. Cellular numbers were then evaluated. After removal of NTS, PGE₂ increased cell proliferation rates (Fig. 5, A and B), and more of the PGE₂-treated cells regained a normal morphology (Fig. 5, C and D). Noteworthy, PGE₂ had no effect on cells under normal conditions (data not shown); the growth-promoting effect was observed only after following NTS exposure.

Fig. 5.

PGE₂ enhances mouse glomerular epithelial cell (GEC) proliferation after NTS-induced cell injury. Podocytes were treated with NTS for 9 h followed by media change to remove NTS; thereafter cultures were with or without PGE₂ (0.01–1 μg/ml, overnight). Cells were detached and cell numbers assessed by FACS (A and B). Endothelial cells were incubated with NTS overnight, followed by changing media to remove NTS, with addition of fresh media with or without PGE₂ 0.1 μg/ml (C and D).

PGE₂ Has Direct Effects on Cultured Mouse Glomerular Cells

To further evaluate the effects of PGE₂ on glomerular cells, the capacity of PGE₂ to limit NTS-induced cellular injury was evaluated. In culture NTS treatment reduced murine podocyte and endothelial cell numbers in a time- and dose-dependent manner, whereas normal sheep serum had no effect (data not shown). This effect was modulated by PGE₂, as the number of viable cells after NTS exposure was increased. Furthermore, the cells appeared more confluent in the presence of PGE₂, and their morphology and adherence appeared normal. To determine whether inhibition of cell death and/or apoptosis contributed to the benefit, podocytes were preincubated with PGE₂ before NTS and the number of apoptotic cells was compared by FACS. NTS induced an increase in annexin V-positive cells, whereas, preincubation with PGE₂ significantly reduced the NTS-mediated increase (Fig. 6A). Similar results were obtained with endothelial cells (data not shown). Apoptosis was also examined by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling (TUNEL); PGE₂ reduced TUNEL-positive staining in a dose-dependent manner (Fig. 6B).

Fig. 6.

PGE₂ attenuates apoptotic actions of NTS on glomerular cells. Cultured podoctyes (POD) or GEC were pretreated with or without PGE₂. Thereafter, NTS was added for 5 h. The number of apoptotic cells were determined using annexin V assay by FACS. Pretreatment with PGE₂ suppressed NTS induced increase of phosphatidyl serine (A) and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling-positive/apoptotic cells (B). Results are shown as means ± SD. *P < 0.05.

DISCUSSION

Our results confirm the hypothesis that PGE₂ promotes recovery during the course of established nephritis in mice. The experimental approach involved treating animals after disease induction, with evaluation of the course of disease following PGE₂ administration. PGE₂ reversed the clinical and pathologic parameters of nephritis. This was not likely due to an effect on prevention of newly established disease (i.e., with ongoing NTS deposition), since nephritis was well established at 24 h and most of the nephritogenic antibodies are no longer circulating at this time (22). Thus the improvement was most likely due to a restorative effect of PGE₂ on disease by promoting tissue repair after injury.

The effects are likely mediated through the EP3 receptor, as its selective antagonist, but not other selective EP receptor antagonists limited the PGE₂ benefit. PGE₂ signaling through EP3 receptors has been shown to mediate reorganization of damaged neuronal connections (16), attenuate myocardial injury during ischemia reperfusion (24), and regulate COX-2 expression in the kidney (38). This is a well-characterized G protein-coupled receptor (4), which exists in multiple isoforms, and each of them can be involved in variety of signaling pathways, including the prosurvival pathways of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (11, 12), and in the modulation of Ca²⁺/calmodulin reaction and Rho kinases (16), which all play important role in maintaining/regulating podocyte ultrastructure (20).

Improvement in proteinuria by PGE₂ is likely explained by a direct effect on podocytes, as restoration of synaptopodin and podocyte foot processes were observed. Synaptopodin is essential for the integrity of the podocyte actin cytoskeleton, and when phosphorylated, it is protected from degradation (25, 39). This particular benefit was likely due, at least in part, by a PGE₂-mediated increase in cAMP; this pathway is known to activate cAMP dependent PKA, inhibit Ca immobilization, and reduce inositol 3-phosphate turnover (35). These effects diminish the activity of calcineurin, thereby protecting synaptopodin from degradation (31).

The cell culture experiments provide further insights. PGE₂ decreased NTS-induced apoptosis of both podocytes and endothelial cells and enhanced GEC proliferation rates following NTS-mediated damage. Although we cannot eliminate the possibility that a protective effect on adhesion may have contributed, modulation of apoptosis/survival was definitely observed. These findings are consistent with the Ingeniuty gene expression profiling/results, where 272 differentially expressed genes were identified as responsive to PGE₂ treatment and related to cell proliferation processes, Among them FOXM1 was upregulated (3.882-fold); its expression is known to be restricted to proliferative tissues and has been detected in regenerative tissues and various malignancies (30). A number of cyclins and cyclin dependent kinases were also significantly elevated, such as CDK1 (5,588-fold), cyclin A2 (CCN2, 2.003-fold), and cyclin F (2.014-fold), consistent with their role in cell cycle progression and activators of FOXM 1 gene. Particularly noteworthy, cyclins have been shown to interact with DNA repair proteins and contribute to repair processes (33, 18). Enhanced S100A6 gene expression, known to be preferentially expressed in proliferating cells (21), is also consistent with the beneficial effect of PGE₂. Collectively, the results indicate benefit mediated at least in part, by eliciting growth-promoting genes regulating cell cycle progression and recovery processes.

In conclusion, regenerative and renoprotective effects of PGE₂ during established NTN and after injury to glomerular cells in culture were observed. The results of these studies provide the rational to further define the cellular pathways involved in the process and to investigate the efficacy of PGE₂ during the course of human glomerulonephritis. Ideally, regeneration-promoting therapeutics will be small molecule/s that can easily be produced in large amounts, facilitate regeneration, and have limited adverse effects in other tissues. PGE₂ is an excellent candidate, given its physiologic and restorative effects.

GRANTS

The work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R21-DK-081140.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: N.K., M.B., and M.P.M. conception and design of research; N.K., M.M., and K.C. performed experiments; N.K., M.M., K.C., and M.P.M. analyzed data; N.K. and M.P.M. interpreted results of experiments; N.K. prepared figures; N.K. drafted manuscript; M.P.M. edited and revised manuscript; M.P.M. approved final version of manuscript.