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Original Article
Year : 2019   |  Volume : 11   |  Issue : 4   |  Page : 117-123  

Effects of pre-ischemic prolyl-hydroxylase inhibitor on long-term renal functions in rats

Gaaminepreet Singh, Rimpi Arora, Pawan Krishan

Correspondence Address:Department of Pharmacology, ISF College of Pharmacy, Moga, Punjab, India, Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala, Punjab, India

Source of Support: Nil, Conflict of Interest: None Declared

DOI: 10.4103/2231-4040.197331


Background: Ischemia leads to acute kidney injury (AKI) and associated with adverse hospital outcomes. It is known that AKI resolves in few days, but the progressive kidney disease does not halt. Induction of ischemia-reperfusion (I/R) injury results in oxygen metabolism defects resulting in hypoxia, which activates fibrogenic responses. The hypoxia inducible factors (HIFs) system comprising of HIF-1α and HIF-1β subunits is degraded by prolyl-hydroxylase (PHD) enzyme. Several evidences have suggested that inhibition of HIFs degradation prevents renal I/R induced AKI in rodents. However, the effects of HIF pre-induction on renal I/R induced chronic kidney disease (CKD) in rodents has not been investigated. Materials and Methods: Bilateral renal I/R injury was performed by clamping both renal pedicels for 45 min. Modulation of HIFs was done by administering PHD inhibitor cobaltous chloride prior to I/R. CKD was evaluated by proteinuria, oxidative stress, HIF-1α protein levels and histological studies. Results: Renal I/R injury induced AKI as indicated by elevated serum creatinine levels post 1 day. Various CKD features such as proteinuria, reduced catalase activity, glomerular hypertrophy, glomerulosclerosis, fibrosis were evidenced 90 days post I/R injury. Pre-treatment with PHD inhibitor reduced the incidence of AKI in I/R rats. Besides up-regulation of renal HIF-1α protein levels after 90 days, development of CKD was not retarded in pre-treated I/R rats. Conclusion: Thus, we conclude that early HIF modulation is insufficient in attenuating I/R induced CKD in rats.

Keywords: Acute kidney injury, chronic kidney disease, hypoxia inducible factors, prolyl-hydroxylase enzyme, proteinuria

How to cite this article:
Singh G, Arora R, Krishan P. Effects of pre-ischemic prolyl-hydroxylase inhibitor on long-term renal functions in rats. Pharmaspire 2019;11(4):117-123.


An acute kidney injury (AKI) episode is common among patients in intensive care units and is clearly associated with the increased hospital mortality rate. AKI is defined as abrupt decline of kidney function or kidney damage which ranges from hours to few days.[1] The most prominent causes of AKI includes predisposition to certain risk factors stroke, arterial hypertension, diabetes mellitus, septicemia, cardiac surgeries, and contrast agents.[2-5]
Sudden onset of reperfusion followed by ischemia induces renal oxygenation defects by generating reactive oxygen species (ROS), promoting inflammation, and the reduction of peritubular capillary density.[6-9] Renal tissue in-turn begins repair mechanisms to neutralize these pathogenic mechanisms. Indeed, it was presumed earlier that renal dysfunction resolves completely after an AKI episode. However, several evidences from experimental models and clinical studies have demonstrated that AKI leads to development of progressive proteinuria, structural damage, and tubular-interstitial fibrosis ensuing chronic kidney disease (CKD).[8,10,11] In response to low oxygen tension hypoxia inducible factors (HIFs) are activated and transcriptionally regulate various renoprotective genes involved in oxygen metabolism, antioxidant defense, and inflammatory response.[12,13] The two subunits HIF-1α and HIF-1β form a heterodimer complex under hypoxic conditions and thus escape degradation by prolyl-hydroxylase (PHD) enzyme, while in normal conditions the alpha subunit undergoes ubiquitylation and destruction by PHD enzyme.
Previously a study has shown that expression of HIF-1α and HIF-1β subunits in kidneys was absent 6 h after renal ischemia-reperfusion (I/R) injury in mice. Moreover, in this study, pre-treatment with different PHD inhibitors prevented renal I/R induced renal dysfunction and histological abnormalities by upregulating HIF-1α and HIF-1β subunits in kidneys.[14] Similarly, other studies have also demonstrated that pre-treatment with PHD inhibitor abrogates renal I/R induced AKI, probably by induction of HIF target genes heme oxygenase-1, and erythropoietin.[15,16] Although the HIFs activation has been well documented to pre-condition against I/R induced AKI, the effects on the development of progressive kidney injury were not studied. Thus, the present study aims to investigate the effects of preischemic PHD inhibitor treatment on long-term renal functional and histological alterations in rats.


Animals and experimental procedures

All the experimental work involving animals was approved by the institutional animal ethics committee of the Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala (Reg. No-107/99/CPCSEA/2016-22). Male Wistar rats weighing 190 ± 10 g were included in the study. For induction of CKD, rats were exposed to bilateral renal I/R injury for 45 min using non-traumatic vascular clamps under sodium thiopentone (Thiosol, Neon Laboratory India) anesthesia (40 mg/kg., i.p).[17] The incisions were closed using absorbable vicryl suture for both inner muscular layer and outer skin. A local anesthetic lignocaine gel (2%, Neon Laboratory India) was applied topically on wounds to relieve postsurgical pain and neosporin powder was sprinkled on dried wounds. PHD inhibitor cobalt chloride (SD Fine-Chem Ltd., India) at a dose of 20 body wt−1 per 24 h, was given to rats in drinking water.[18] Each rat in cobalt control and cobalt pre-treated I/R groups received a dose of 3–4 mg of cobalt chloride per day for 3 consecutive days in drinking water (daily water intake of each rat: 30–50 ml).
Male Wistar rats (n = 6/each group) used in the present study were divided as (1) normal control which did not underwent any surgical procedure, (2) sham surgery (sham surg): Rats which underwent laparotomy and exposure of renal pedicle under anesthesia without clamping, (3) renal I/R group: Subjected to 45 min of bilateral renal I/R injury, (4) cobalt control (cob cntrl): 3 days treatment with cobalt chloride (HIFs activator) only, and 5) cobalt + I/R (cob+I/R) group: 3 days cobalt pre-treatment in rats subjected to bilateral renal I/R injury. Urine collection was done by keeping each rat from each group separately in metabolic cages (24 h), and then urine was centrifuged at 3000 rpm for 5 min. The assessment of progressive renal function was done by estimation of serum creatinine (Jaffe’s method) concentration at 1, 7, and 28 days and proteinuria (24 h urine, biuret method) at 30, 60, and 90 days post-renal I/R injury in rats using diagnostic kits (Transasia Bio-Medical Ltd.) on semi autoanalyzer. After 90 days, rats were anesthetized and both kidneys were isolated, cleaned, and weighed to calculate kidney/body weight ratio, thereafter rats were sacrificed by cervical dislocation.

Renal tissue anti-oxidant assays and HIF-1α protein levels

Renal homogenate was prepared by homogenizing left kidney in cold phosphate buffer saline (pH 7.4, 50mM) at 3000 rpm (10 min, 4°C). Thereafter, supernatants were collected and immediately used for anti-oxidant assays and protein estimation. The reduced glutathione (GSH) content was checked in renal tissue homogenate according to the method of Boyne and Ellman.[19] A standard curve was plotted using various known micro-molar concentrations of reduced glutathione, which was further used to calculate renal tissue GSH content. Catalase enzyme activity in renal homogenate was determined according to the analytical method described by Gwinner et al. and expressed as units/mg protein.[20] The assay for total protein content in renal tissue homogenate was performed using bovine serum albumin as standard.[21] The estimation of HIF-1α protein content in the renal tissue homogenate was done by rat HIF-1α ELISA kit (Wuhan Fine Biological Technology Co., Ltd. Wuhan, China) using i-Mark Microplate reader, BIO-RAD.

Histological examination of kidney sections

For histological analyses, right kidney was fixed in 10% formaldehyde solution. Post-fixation blocks of kidney tissues were prepared with paraffin wax and kidney sections of 5 μm size were cut for hematoxylin and eosin staining and observed at 100× (Leica DM 4000 microscope) for the assessment of glomerular diameter. Periodic acid Schiff (×400) staining was performed to evaluate the extent of mesangial expansion, glomerulosclerosis, basement membrane damage, and picrosirius red (×100) staining which was done to evaluate renal fibrosis.[22,23] Glomerular diameter (an index of hypertrophy) in kidney sections was determined using NIH image J software by expert blinded to the study.[11,17] The percentage of glomerulosclerosis and tubulo-interstitial fibrosis was quantified in various kidney sections as described previously.[24]

Statistical analysis

Data from animals in each group are shown as Mean ± SD. The results were analyzed using GraphPad prism software-5 version by applying one-way or two-way ANOVA followed by Bonferroni post hoc test for multiple comparison between groups, P < 0.05 was considered as statistically significant. The sizes of Glomeruli diameter from various study groups were compared with corresponding glomeruli size range in sham surgery group by two-way ANOVA followed by Bonferroni test.


Effect of cobalt pre-treatment on body weight changes in ischemic rats

Body weight in I/R and I/R+Cob groups was not observed to be different compared to normal, sham surg, and cob cntrl groups at 0 day and 30, 60 days post-renal ischemia injury. However, a significant decline in the I/R and I/R+Cob groups body weight was seen against normal, sham surg, and cob cntrl groups (P < 0.001) at 90 days period of renal ischemia injury [Figure 1]. Importantly, cobalt pre-treated ischemic rats did not exhibit difference in body weight than the corresponding I/R group during the entire study period.

Effects of cobalt pre-treatment on kidney weight/ body weight ratio in ischemic rats

I/R group rats displayed significant elevation of k.wt/b.wt ratio (45.39%, 41.86%, and 23.61%) compared to normal, sham surg, and cob cntrl rat (P < 0.05) groups [Figure 2]. Cobalt pre-treatment in I/R group rats did not produce any difference in k.wt/b.wt ratio than the corresponding I/R group.

Effects of cobalt pre-treatment on serum creatinine levels in ischemic rats

Serum creatinine concentration was significantly elevated (189.32%, 210.12%, and 201.2%) in I/R Group 1 day post-renal ischemia compared to normal, sham surg, and cobalt cntrl groups (P < 0.0001), but normalized at 7th and 28th days post-renal ischemia injury in I/R group [Table 1]. Cobalt pre-treatment in ischemic rats (Cob+I/R) resulted in significant reduction (P < 0.0001) of serum creatinine concentration (40.8%) compared to I/R group 1 day post-renal ischemia injury. However, these values remained significantly elevated than the corresponding normal, sham surg, and cobalt cntrl groups, post 1 day injury, respectively.

Effects of cobalt pre-treatment on I/R induced changes in urinary protein excretion

Renal I/R injury resulted in significant elevation of urinary protein excretion by 102%, 106%, and 108% compared to normal, sham, and cobalt control groups after 90 days (P < 0.001) [Figure 3]. However, cobalt pre-treated I/R rats (I/R+Cob) did not exhibit any significant reduction of proteinuria than the corresponding I/R group 90 days post-renal I/R injury.

Effects of cobalt pre-treatment on kidney reduced glutathione content, catalase activity, and HIF-1α protein concentration in ischemic rats

In the present study, renal ischemic injury did not produce any difference of reduced glutathione content in the various study groups [Figure 4a]. However, catalase enzyme activity was observed to be reduced in both ischemic (I/R) rat by 18.72%, 19.54%, and 23.37%, (P < 0.001) and cobalt pre-treated ischemic rat 31.5%, 31.81%, and 35.06%, (P < 0.0001) groups compared to normal, sham surg, and cobalt cntrl groups, respectively. No difference in catalase enzyme activity was noted between I/R and cobalt pre-treated I/R groups [Figure 4b]. Moreover, HIF-1α protein concentration was not different in I/R group compared to normal, sham surg, and cob cntrl groups. Interestingly, HIF-1α protein levels were significantly elevated in cobalt pre-treated I/R (cob+I/R) group by 90.83%, 90.41%, and 78.04% (P < 0.01) than the corresponding normal, sham surg, and cobalt cntrl groups and 81.79% (P < 0.001) compared to I/R group [Figure 4c], respectively.

Effects of cobalt pre-treatment on histological changes in ischemic rats

Hematoxylin and eosin staining of ischemic rats (I/R) kidney sections showed reduced glomerular spacing and presence of hypercellularity [Figure 5]. Cobalt pre-treatment in ischemic rats (Cob+I/R) reduced cellular infiltration but did not influence the glomerular enlargement.



AKI predisposes to progressive kidney disease which results in tubulointerstitial fibrosis and end stage renal failure. Indeed, the recovery after AKI episode may be incomplete which could easily translate to CKD in surviving patients.[25,26] Experimental studies in rodents have demonstrated the recovery of renal functional defects (serum creatinine, and proteinuria) within 10 days of ischemic insult but the vascular rarefaction, urinary concentrating defects, massive proteinuria, glomerulosclerosis, chronic inflammation, and tubulointerstitial fibrosis were observed in the later months.[8,11] Renal hypoxia has been recognized as critical mediator for AKI to CKD transition.[27] In addition, the pre-induction of HIFs by PHD inhibitor was found to exert renoprotective effects against renal ischemia induced AKI in rats.[16,28] However, the consequences of pre-ischemic PHD inhibitor treatment on long-term renal outcomes in rats were not studied.
In the present study, bilateral renal I/R injury induced AKI as indicated by elevated serum creatinine levels after 24 h, which resolved at 7th day post-I/R injury. Cobalt pre-treatment improved AKI severity by reducing the elevated serum creatinine levels post 1 day I/R. These findings are in line with the previous studies which have also shown that pre-induction of HIFs attenuated acute renal dysfunction in I/R model.[15,16] Furthermore, in our study, I/R rats exhibited progressive proteinuria toward 90 days of study period. This indicates that the repair mechanisms after an AKI episode might be incomplete, predisposing the kidney to CKD. Similarly, a previous study has demonstrated impaired regenerative mechanisms in ischemic kidneys which exhibited normal serum creatinine levels after 1 week and development of proteinuria at 16 weeks that progressed to chronic levels at 40 weeks period.[8] A recent study has reported the establishment of CKD features after 5 months of bilateral renal I/R injury along with the development of significant proteinuria at 3 months period.[29] Whereas, another study has documented the development of proteinuria in bilateral renal I/R model at 30 days, which raised consistently in subsequent months.[11] The damage of capillary microvasculature in CKD has been proposed to induce a hypoxia environment, which may translate initial glomerular injury to tubulointerstitial damage and reduce the number of viable nephrons.[30] Thus, the development of progressive proteinuria after ischemia injury could partially be explained by the enhanced workload of surviving nephrons.[8] From our findings, we speculate that early cobalt treatment was insufficient in preserving the population of functional nephrons after I/R injury and thereby lead to damage of glomerular filtration barrier in remaining nephrons and protein leakage.
Oxidative stress is a result of reduced anti-oxidant capacity against the increased oxidative response in tissues. Indeed, the generation of ROS has been reported to increase with the progression of CKD stages.[31] The free radical H2O2 is a strong oxidizer agent which is generated during the catalytic conversion of superoxide O2 enzyme superoxide dismutase and is further degraded in the presence of enzyme catalase.[32] Previously in renal bilateral I/R model, CKD was associated with reduced catalase enzyme activity and increased urinary H2O2 excretion.[11] In the present study, renal catalase activity was also found to be reduced 90 days post-I/R injury in rats indicating development of oxidative stress. The reperfusion followed by complete blockade of renal blood flow results in generation of excessive ROS. In the low oxygen environment, the imbalance of cellular aerobic respiration process leads to leakage of electrons and formation of oxidative radicals. In response to hypoxia, the activation of HIF system shifts the cellular respiration from aerobic to anaerobic glycolysis and thereby reduces the formation of ROS.[33] In our study, renal HIF-1α protein levels were elevated after 90 days but catalase activity was not improved in I/R rats pre-treated with PHD inhibitor cobalt chloride. Importantly, a recent study has demonstrated the absence of low oxygen tension (PO2) in renal tissue 5 days (sub-acute phase) after severe renal I/R injury in rats.[34] Whereas, another study has shown that renal HIF-1α protein levels not changed 15 days postinduction of renal bilateral I/R injury in rats.[17] Nevertheless, our findings suggest that the upregulated HIF-1α protein levels failed to reduce the development of oxidative stress in the advance stage of CKD (90 days).
Cobalt pre-treatment did not prevent ischemia induced glomerular hypertrophy (increased glomerular diameter), mesangial expansion, glomerulosclerosis, tubular lining degenerative changes, and tubulointerstitial fibrosis. Atubular nephrons have been documented to be widely present in renal ischemic injury.[35] The loss of functional nephrons post-ischemic insult could result in compensatory increase of renal function by remaining nephrons to preserve the renal function. Thus, reduced functional nephrons after ischemia injury may undergo the maladaptive changes such as mesangial expansion and glomerular hypertrophy due to hyperfiltration. Previously, a study has indicated that glomerular hypertrophy along with reduced cell density accelerates the development of glomerulosclerosis.[36] In present study, both glomerular enlargement and glomerulosclerosis were evident in I/R group and PHD inhibitor pre-treatment did not attenuate these histological abnormalities. This finding suggests that early HIF pathway modulation might not improve the density of functional nephrons post-I/R injury in rats.
According to the “chronic hypoxia hypothesis” initial glomerular injury can induce renal hypoxia by capillary rarefaction, which triggers fibrogenic cascades in the tubulointerstitium.[30] The occurrence of tubulointerstitial fibrosis involves recruitment of inflammatory cells and their differentiation to fibroblasts causing extra cellular matrix deposition deposition and transforming growth factor-β activation.[37,38] In I/R-induced CKD model renal dysfunction was found to be normalize in 10 days, but the pro-inflammatory and profibrotic mediators were upregulated which could reach chronic levels after months.[11] Indeed, the presence of tubulointerstitial fibrosis is considered as a hallmark of CKD. The HIF signaling is considered as a key regulator of genes involved in angiogenesis, iron and glucose metabolism, antioxidant defense, inflammation, and fibrosis.[39,40]
In earlier study, activation of HIF system was demonstrated to exert renoprotective effects and reduce renal fibrosis 3 days post- I/R injury.[16,28] Here, in this study, HIF-1α protein upregulation was observed at 90 days post cobalt pre-treatment in I/R rats, but the incidence of tubulointerstitial fibrosis was not reduced. Our results indicate that HIF1α protein upregulation in the advance stage (90 days) of CKD is insufficient in ameliorating the various manifestations of CKD. We speculate that persistent inflammation during the progressive kidney disease promoted renal fibrosis (detected by sirius-red staining), which might have abrogated the beneficial effects of PHD inhibitor pre-treatment in advance stage of CKD. However, the exact underlying mechanisms to explain the observed findings need to be elucidated.


Early treatment with PHD inhibitor can effectively reduce the severity of AKI, but might not determine the long-term renal outcomes in I/R model. Further studies are required to test the impact of shortterm and long-term PHD inhibitor post-treatment on I/R induced progressive kidney disease in rats.


Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the Department of Pharmaceutical Sciences and drug research, Punjabi University, Patiala (Punjab), India (Reg. No-107/99/CPCSEA/2016-22).


Gaaminepreet Singh received Basic Science Research fellowship from University Grant Commission, New Delhi, India, during his Ph.D study. Authors thank Mr. S P Jindal, Rajindra Hospital Patiala (India) for help with histological studies.

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