1Department of Renal Medicine, University of Queensland at Princess Alexandra Hospital, Brisbane, 2Department of Medicine and 3Department of Molecular and Cellular Pathology, University of Queensland, Australia
Correspondence and offprint requests to: Dr David A. Vesey, Department of Renal Medicine, Level 2, Ambulatory Renal and Transplant Services Building, Princess Alexandra Hospital, Ipswich Road, Woolloongabba, Brisbane, Queensland 4102, Australia. Email: david_vesey{at}health.qld.gov.au
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Methods. Primary cultures of human proximal tubule cells (PTCs) were exposed to either vehicle or EPO (6.25400 IU/ml) in the presence of hypoxia (1% O2), normoxia (21% O2) or hypoxia followed by normoxia for up to 24 h. The end-points evaluated included cell apoptosis (morphology and in situ end labelling [ISEL], viability [lactate dehydrogenase (LDH release)], cell proliferation [proliferating cell nuclear antigen (PCNA)] and DNA synthesis (thymidine incorporation). The effects of EPO pre-treatment (5000 U/kg) on renal morphology and function were also studied in rat models of unilateral and bilateral ischaemiareperfusion (IR) injury.
Results. In the in vitro model, hypoxia (1% O2) induced a significant degree of PTC apoptosis, which was substantially reduced by co-incubation with EPO at 24 h (vehicle 2.5±0.5% vs 25 IU/ml EPO 1.8±0.4% vs 200 IU/ml EPO 0.9±0.2%, n = 9, P<0.05). At high concentrations (400 IU/ml), EPO also stimulated thymidine incorporation in cells exposed to hypoxia with or without subsequent normoxia. LDH release was not significantly affected. In the unilateral IR model, EPO pre-treatment significantly attenuated outer medullary thick ascending limb (TAL) apoptosis (EPO 2.2±1.0% of cells vs vehicle 6.5±2.2%, P<0.05, n = 5) and potentiated mitosis (EPO 1.1±0.3% vs vehicle 0.5±0.3%, respectively, P<0.05) within 24 h. EPO-treated rats exhibited enhanced PCNA staining within the proximal straight tubule (6.9±0.7% vs vehicle 2.4±0.5% vs sham 0.3±0.2%, P<0.05), proximal convoluted tubule (2.3±0.6% vs vehicle 1.1±0.3% vs sham 1.2±0.3%, P<0.05) and TAL (4.7±0.9% vs vehicle 0.6±0.3% vs sham 0.3±0.2%, P<0.05). The frequency of tubular profiles with luminal cast material was also reduced (32.0±1.6 vs vehicle 37.0±1.3%, P = 0.05). EPO-treated rats subjected to bilateral IR injury exhibited similar histological improvements to the unilateral IR injury model, as well as significantly lower peak plasma creatinine concentrations than their vehicle-treated controls (0.04±0.01 vs 0.21±0.08 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls.
Conclusions. The results suggest that, in addition to its well-known erythropoietic effects, EPO inhibits apoptotic cell death, enhances tubular epithelial regeneration and promotes renal functional recovery in hypoxic or ischaemic acute renal injury.
Keywords: apoptosis; hypoxia; kidney failure, acute; kidney tubular necrosis, acute; kidney tubules; mitosis; regeneration
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recently, erythropoietin (EPO), a widely available treatment for uraemic anaemia, was found to markedly attenuate experimental ischaemic brain injury via inhibition of apoptosis and/or augmentation of cellular regeneration [711]. Since EPO receptors are expressed on renal tubular epithelial cells [12], it is possible that the systemic administration of EPO may also provide protection against acute renal damage. Indeed, EPO has been found to ameliorate toxic renal injury caused by cisplatin [13,14]. However, it is critical to establish whether EPO exerts similar renoprotective action in ischaemiareperfusion renal injury, since this is the most common cause of ARF in the community [15]. The aim of the present study therefore was to determine whether EPO is renoprotective in both in vitro and in vivo models of ischaemic ARF.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cytological examination of PTC preparations from all donors failed to reveal any evidence of cellular atypia. The morphological, biochemical and functional characteristics of these cells have been studied previously in this laboratory and found reproducibly to exhibit the features of PTCs in vivo [16]. Fibroblast, mesangial and endothelial cell contamination was negligible, as evidenced by the uniform negative staining of cultures for vimentin, desmin and factor VIII, respectively.
In vitro experimental protocol
All experiments were performed on passage 2 PTCs cultured in 48-well tissue culture plates. Cells were made quiescent by two washes followed by incubation for 24 h in basic medium (DMEM/F12 containing 5 µg/ml transferrin). Cells were incubated with basic medium supplemented with various concentrations of recombinant human EPO (6.25400 IU/ml; Janssen-Cilag, Sydney) or vehicle [phosphate-buffered saline (PBS) containing 5 mg/ml glycine and 0.13 mg/ml polysorbate 80; a gift from Janssen-Cilag] and simultaneously exposed for 5 or 24 h to 1 or 21% O2, using a hypoxic tissue culture incubator. In separate hypoxiareoxygenation experiments, PTCs were exposed to 1% O2 for 16 h followed by 21% O2 for 24 h. Cellular apoptosis, proliferating cell nuclear antigen (PCNA) expression, thymidine incorporation and lactate dehydrogenase (LDH) release were then measured.
Apoptosis measurement
For in vitro apoptosis measurements, cells were grown on 13 mm Thermanox coverslips (Nalge Nunc International, Napersville, IL) in 24-well plates and treated or not with EPO, as described above, for the indicated times. At the end of the study, cells were washed with PBS, fixed in cold 4% buffered paraformaldehyde for 10 min and stored in PBS at 4°C until processed. Previously defined morphological criteria were used to identify apoptotic cells [18]. These characteristics included cellular rounding and shrinkage, eosinophilic cytoplasm, nuclear chromatin compaction (especially along the nuclear envelope in a crescentic manner), membrane-bound cellular blebbing and formation of apoptotic bodies that may appear in the tubular lumina or be phagocytosed by intrinsic renal cells or invading macrophages.
In situ end labelling (ISEL) nuclear positivity was used for quantification of apoptosis in vitro and in vivo [19]. Following the methods of Ansari and colleagues [20], paraffin sections were dewaxed, rehydrated, digested in 0.5% pepsin in 0.1 M HCl for 15 min, washed, then labelled using the Klenow fragment of DNA polymerase I (Pharmacia, Australia) and biotin-labelled dUTP (Boehringer-Mannheim, Australia). The reaction was terminated by washing in water, endogenous peroxidase activity was blocked with 0.1% H2O2, and biotin-labelled nuclei visualized with horseradish peroxidase-conjugated avidin developed in 3',3'-diaminobenzidine (DAB) and counterstained with haematoxylin. Negative controls had no Klenow polymerase, and positive controls were rat renal sections from other experiments in which high levels of ISEL were known to correlate with high levels of apoptosis assessed morphologically. A similar method was used for the fixed cultures, except that no pre-treatment with pepsin was carried out. ISEL-positive nuclei were counted at x400 microscope magnification as the percentage of total cells in a 10 x 10 eyepiece grid.
Immunohistochemistry
Immunohistochemical staining of PTC for PCNA, an auxillary protein to DNA polymerase , was used as a tool for measuring cellular proliferation. Sections were deparaffinized and rehydrated, before staining with the peroxidaseanti-peroxidase method. Non-specific binding of peroxidase or antibodies was blocked with 0.3% hydrogen peroxide (H2O2) in methanol, followed by incubation in diluted normal swine serum. The PCNA monoclonal antibody (PC10, Oncogene Sciences, 1:50 dilution) was applied at 4°C overnight, followed by buffer washes, and the secondary antibody at a dilution of 1:400 in Tris buffer, followed by the ABCperoxidase reaction for 30 min. The chromogen was DAB in 0.01% H2O2, applied for 25 min. Sections were lightly counterstained with haematoxylin and then dehydrated to xylene and mounted in Depex.
DNA synthesis
Tritiated thymidine incorporation into cellular DNA, an index of DNA synthesis, was measured according to a previously described method [21]. For the final 4 h of each experiment, 4 µCi (0.15 MBq) of [methyl-3H]thymidine (Amersham-Pharmacia-Biotech, Uppsala, Sweden) was added for each ml of medium. Cells were then washed twice with ice-cold PBS, three times with ice-cold 10% trichloroacetic acid for 20 min, and once with methanol. Monolayers were allowed to dry and then dissolved in 300 µl of 0.3 M NaOH containing 1% SDS. Aliquots of cell lysate were taken for liquid scintillation counting in a ß-counter. Results were corrected for cellular protein content. The protein content of the cell lysate was measured by a commercially available protein assay (BioRad, Hercules, CA) using bovine serum albumin as the standard.
Cell viability
PTC viability was assessed using a cytotoxicity detection kit (Roche) which measures LDH release into the culture medium. The manufacturer's protocols were followed.
Ischaemiapeperfusion (IR) renal injury model
Mature SpragueDawley rats (200220 g, n = 56 per group) were administered either recombinant human EPO (5000 U/kg) or vehicle by intraperitoneal injection. The dose of EPO used was based on that found to be maximally effective in previous studies of acute ischaemic brain injury [79]. At 30 min post-injection, the rats underwent either sham operation (controls) or unilateral or bilateral renal artery occlusion for 30 min followed by reperfusion for either 24 or 48 h. At the end of this period, aortic blood samples were collected for determination of urea, creatinine, sodium, LDH and haematocrit. Urine was also collected for measurement of urea, creatinine and sodium. Both kidneys were removed, bisected in an equatorial plane, fixed in buffered formalin at 4°C and prepared routinely for histology and immunohistochemistry. ISEL staining was performed to evaluate apoptosis. Immunohistochemical staining for PCNA was used as a tool for quantitating proliferation.
The percentages of epithelial cells in the proximal convoluted tubule (PCT), PST, distal convoluted tubule and TAL of Henle's loop demonstrating apoptosis, necrosis, mitosis or PCNA staining were determined by counting a minimum of 1000 cells and 10 high power (x400) microscope fields per kidney per experimental condition. The proportion of tubular profiles containing luminal cast material was also measured by assessing a minimum of 40 tubular profiles per section.
Statistical analysis
All in vitro studies were performed in triplicate from PTC cultures obtained from at least three separate human donors. Each experiment contained internal controls originating from the same culture preparation. For the in vivo studies, each study group consisted of 56 rats. Results are expressed as mean±SEM. Statistical comparisons between groups were made by analysis of variance (ANOVA). Pairwise multiple comparisons were made by Fisher's protected least-significant differences test. Analyses were performed using the software package, Statview version 5.0 (Abacus Concepts Inc., Berkeley, CA). P-values <0.05 were considered significant.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
EPO ameliorates renal histological damage and functional impairment following bilateral IR renal injury in rats
Compared with the unilateral IR model, rats undergoing bilateral IR demonstrated a similar pattern of renal injury (albeit with somewhat more severe PST injury) and a similar response to EPO. Compared with control (vehicle-treated and sham-operated) animals, rats subjected to bilateral IR injury demonstrated significant derangements in renal histology within 24 h, including increased PST necrosis (47.6±4.0% vs 0.0±0.0% of tubular cells, P<0.001), TAL apoptosis (3.7±0.6% vs 0.1±0.2%, P<0.01), TAL necrosis (11.4±1.2% vs 0.0±0.0%, P<0.001) and medullary tubular casts (67±4% vs 0±0%, P<0.001). EPO-treated rats exhibited significant increases in mitotic figures at 24 h compared with all other groups in both the TAL (EPO 2.8±0.3% vs vehicle 2.0±0.3% vs EPO-treated sham 0.1±0.1% vs vehicle-treated sham 0.2±0.2%, P<0.01) and cortical proximal tubule (EPO 1.5±0.2% vs vehicle 0.5±0.1% vs EPO-treated sham 0.4±0.3% vs vehicle-treated sham 0.2±0.2%, P<0.01). These changes were still observed at 48 h (TAL EPO 3.9±0.1% vs vehicle 2.7±0.4% vs EPO-treated sham 0.2±0.1% vs vehicle-treated sham 0.2±0.2%, P<0.01; cortical proximal tubule EPO 1.3±0.3% vs vehicle 0.4±0.1% vs EPO-treated sham 0.2±0.1% vs vehicle-treated sham 0.2±0.1%, P<0.001), but were less pronounced by 4 days (TAL EPO 1.5±0.1% vs vehicle 1.4±0.2% vs EPO-treated sham 0.1±0.1% vs vehicle-treated sham 0.1±0.2%, P<0.01; cortical proximal tubule EPO 0.8±0.2% vs vehicle 0.3±0.1% vs EPO-treated sham 0.2±0.1% vs vehicle-treated sham 0.2±0.1%, P<0.05). Apoptotic cell death in the outer medullary TAL was reduced similarly by EPO compared with vehicle at 24 h (EPO 1.8±0.2% vs vehicle 3.7±0.6% vs EPO-treated sham 0.0±0.0% vs vehicle-treated sham 0.1±0.2%, P<0.001), 48 h (EPO 0.9±0.2% vs vehicle 3.3±0.5% vs EPO-treated sham 0.0±0.0% vs vehicle-treated sham 0.2±0.2%, P<0.001) and 4 days following bilateral IR injury (EPO 0.3±0.1% vs vehicle 0.8±0.2% vs EPO-treated sham 0.0±0.0% vs vehicle-treated sham 0.0±0.0%, P<0.01). EPO had no significant effect on TAL apoptosis in sham-operated rats. No differences were seen in proximal tubule cell apoptosis between EPO- and vehicle-treated rats following IR injury, but overall rates of apoptotic cell death were generally low in this segment (<0.5%). PCT necrosis was significantly reduced in EPO-treated rats at 48 h (EPO 2.2±0.5% vs vehicle 4.5±0.9% vs EPO-treated sham 0.0±0.0% vs vehicle-treated sham 0.0±0.0%, P<0.05), but this difference was not observed at any other time point. By 48 h, EPO-treated rats also displayed significantly fewer casts in outer medullary (EPO 44±3% vs vehicle 67±4% vs EPO-treated sham 0±0% vs vehicle-treated sham 0±0%, P<0.05) and cortical tubular profiles (EPO 6±2% vs vehicle 37±8% vs EPO-treated sham 0±0% vs vehicle-treated sham 0±0%, P<0.001). No or very little (?0.2%) apoptosis, necrosis, mitosis or tubular cast material was detected in sham-operated control animals treated with either EPO (5000 U/kg) or vehicle for 24, 48 or 96 h.
EPO administration prior to bilateral IR renal injury in rats significantly abrogated subsequent renal functional impairment (Figure 6). Plasma creatinine elevations peaked at day 2 following bilateral IR injury and were significantly lower in EPO- compared with vehicle-treated rats (EPO 0.04±0.01 vs vehicle 0.21±0.08 mmol/l, P<0.05). EPO-treated rats subjected to bilateral IR injury had peak plasma creatinine concentrations that were not significantly different from those of sham-operated controls (EPO-treated IR injury 0.04±0.01 vs EPO-treated sham 0.02±0.00 vs vehicle-treated sham 0.02±0.00 mmol/l, P = NS). Similar results were seen for plasma urea concentrations, which also peaked on day 2 (EPO-treated IR injury 12.6±3.0 vs vehicle-treated IR injury 40.6± 10.9 mmol/l vs EPO-treated sham 4.1±0.6 vs vehicle-treated sham 4.4±0.1 mmol/l, respectively, P<0.05). EPO had no effect on renal function in sham-operated controls. No significant differences were seen between EPO- or vehicle-treated rats subjected to bilateral IR injury with respect to plasma LDH concentration, fractional sodium excretion or body weight (data not shown).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although there have been no previous published studies of the effect on EPO on tubular cellular recovery following acute ischaemic renal injury, a recent investigation by Nemoto and associates [22] found that rats treated with EPO (3000 U/kg) prior to severe IR renal injury enjoyed superior survival rates compared with controls. No changes were seen with respect to serum creatinine levels, but the follow-up was relatively short (3 days) and the number of animals studied was small (n = 3 per group). Moreover, the haematocrits of EPO-treated animals were significantly increased after 48 h, thereby making it difficult to determine whether the mortality benefitis of EPO were related to anaemia correction or to other actions.
Two studies in a model of toxin-induced ARF (cisplatin nephrotoxicity) have supported our findings that EPO enhances histological and functional recovery. Bagnis et al. [13] observed that EPO administration (100 U/kg/day i.p. for 9 days) was associated with a significantly greater number of PCNA-positive cells in the cortical and corticomedullary regions of rat kidneys following cisplatin exposure. These histological appearances, similar to those of our study, suggested enhanced tubular cell regeneration and were accompanied by significantly higher inulin clearance rates at day 9. Although apoptosis was not studied, the authors hypothesized that EPO may have directly acted as a growth factor on tubular cells in the cortex and outer medulla. Vaziri et al. [14] similarly reported enhanced renal cortical thymidine incorporation in EPO-treated rats with acute cisplatin tubulopathy. The beneficial effect appeared to be independent of haemoglobin levels, since similar results were also observed in EPO-treated rats subjected to daily phlebotomies.
In the present study, the beneficial effects of EPO on ischaemic renal histopathology were demonstrable in the absence of a significant change in haemoglobin levels, suggesting that the therapeutic effects of EPO in IR renal injury were independent of its haemopoietic actions. It was also unlikey that EPO engendered any favourable haemodynamic changes in view of the fact that acute and chronic studies of EPO therapy in animal models have not reported any measurable changes in renal blood flow or other haemodynamic parameters during the first few weeks of regular administration [23].
Since EPO receptors have recently been identified in human and rat kidney tissue (cortex, medulla and papilla) and in renal cell lines (PTCs, mesangial and collecting duct cells) [12], the possibility that EPO exerted direct renoprotective effects on tubular epithelial cells was additionally investigated in this study using primary cultures of human PTCs. In keeping with the findings of Westenfelder et al. [12], EPO stimulated PTC mitogenesis. This occurred under conditions of both hypoxia and hypoxiareoxygenation. Similarly, tubular epithelial regeneration following IR renal injury in rats was also augmented by prior EPO administration. EPO-treated PTCs additionally demonstrated a marked reduction in hypoxia-induced apoptosis compared with cells incubated with vehicle. A trend towards decreased proximal tubule apoptosis was further observed in the IR injury model, although this reduction did not achieve statistical significance. The reason for the latter negative finding was most likely to be attributable to the relatively minor degree of apoptosis observed in PTCs compared with TAL cells in this particular in vivo model, but could conceivably have reflected a difference between examining single cell vs whole animal effects.
The cytoprotective and mitogenic effects of EPO on proximal and distal tubular epithelial cells may explain the observation of a modest reduction in tubular cast formation in EPO-treated rats following IR renal injury. Decreased cell sloughing and cast formation may have, in turn, contributed to a reduction in intratubular obstruction and a subsequent amelioration of renal functional impairment.
Other studies have also shown that EPO exerts significant mitogenic and/or anti-apoptotic actions in non-renal and non-erythroid cells, including endothelial cells [24], gastric mucosal cells [25], myoblasts [26], Leydig cells [27], vascular smooth muscle cells [28], gynaecological cancers [29] and cortical neurons [30]. There is a substantial body of evidence that systemic EPO administration (5000 U/kg) markedly attenuates ischaemic cerebral injury by up to 75% [711], as well as experimental brain damage complicating blunt trauma, autoimmune encephalomyelitis and neurotoxin exposure [9,31]. The proposed mechanism responsible for the observed cytoprotective effects of EPO in the brain is Jak2 phosphorylation of IB and subsequent NF-
B-dependent transcription of neuroprotective genes [32]. Additional potential protective mechanisms that might be activated downstream from the EPOreceptorRasmitogen-activated protein kinase and EPOreceptorphosphatidylinosine 3 kinase interactions include antioxidation [8], direct neurotrophic effects [8], angiogenesis [8], activation (phosphorylation) of Bcl-xl [33,34] and inhibition of nitric oxide (NO) production [10,35]. Similar mechanisms could conceivably be operating in the renoprotective actions of EPO in ischaemic ARF. The EPO doses used in the present study were based on those previously demonstrated to provide effective neuroprotection. However, since there is no equivalent of the bloodbrain barrier in the kidney, it is possible that renoprotection could be afforded by considerably lower dosages, thereby reducing therapy costs. Nevertheless, it should be noted that the doses of EPO needed to promote maximal anti-apoptotic and mitogenic effects in vitro were quite high (200400 IU/ml).
In addition to the issue of optimal dosing, the present study did not answer the question of whether administering EPO some time after the noxious renal insult would still afford some degree of renoprotection. Extrapolation from trials of EPO administration to rats with ischaemic brain injury suggests that the drug may still be capable of ameliorating cell injury when injected some time after the injurious event [9]. However, even if EPO was only effective at preventing acute renal injury when administered prior to the inciting event, there are many common clinical situations where pre-emptive EPO treatment could potentially prove to be of enormous clinical benefit. Such clinical scenarios include renal transplantation, aortic or coronary artery surgery, and radiographic contrast administration to diabetic or chronic renal failure patients.
In conclusion, the present study demonstrated that EPO pre-treatment had a direct cytoprotective action on human PTCs in vitro and was capable of augmenting histological and functional recovery from ischaemic acute renal injury in vivo. This raises the possibility that EPO may have potential clinical applications as a novel renoprotective agent for patients at risk of ischaemic ARF. Further studies are warranted to ascertain the lowest dose and the latest dose timing which still permit effective renoprotection.
![]() |
Acknowledgments |
---|
Conflict of interest statement. Dr Johnson has received consultancy fees from Janssen-Cilag, the makers of Eprex® (erythropoietin) in Australia.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|