Abstract
The role of renal sympathetic nerves in the pathogenesis of ischemic acute renal failure (ARF) and the immediate changes in the renal excretory functions following renal ischemia were investigated. Two groups of male Sprague Dawley (SD) rats were anesthetized (pentobarbitone sodium, 60 mg kg−1 i.p.) and subjected to unilateral renal ischemia by clamping the left renal artery for 30 min followed by reperfusion. In group 1, the renal nerves were electrically stimulated and the responses in the renal blood flow (RBF) and renal vascular resistance (RVR) were recorded, while group 2 was used to study the early changes in the renal functions following renal ischemia. In post-ischemic animals, basal RBF and the renal vasoconstrictor reperfusion to renal nerve stimulation (RNS) were significantly lower (all p < 0.05 vs. control). Mean arterial pressure (MAP), basal RVR, urine flow rate (UFR), absolute and fractional excretions of sodium (UNaV and FENa), and potassium (UKV and FEK) were higher in ARF rats (all p < 0.05 vs. control). Post-ischemic animals showed markedly lower glomerular filtration rate (GFR) (p < 0.05 vs. control). No appreciable differences were observed in urinary sodium to potassium ratio (UNa/UK) during the early reperfusion phase of renal ischemia (p > 0.05 vs. control). The data suggest an immediate involvement of renal sympathetic nerve action in the pathogenesis of ischemic ARF primarily through altered renal hemodynamics. Diuresis, natriuresis, and kaliuresis due to impaired renal tubular functions are typical responses to renal ischemia and of comparable magnitudes.
INTRODUCTION
The kidneys are profusely innervated internal organ in which the renal adrenergic neurons exert major effects on various aspects of renal function, including renal hemodynamics, tubular sodium and water reabsorption, renin secretion, and eventually mean arterial blood pressure homeostasis.Citation[1,Citation2] Generally, the direct renal nerve stimulation (RNS) produces frequency-related renal hemodynamic changes in terms of renal blood flow (RBF) regulation.[Citation3–5]
Norepinephrine injected into the renal artery of dogsCitation[6] and ratsCitation[7] results in an ischemic type of acute renal failure (ARF), in which there are continual abnormalities of renal hemodynamics and renal tissue injury, thus suggesting that the activation of renal sympathetic nervous system plays a central role in the post-ischemic renal injury.
Renal ischemic injury is a major cause of acute renal failure (ARF), which carries a high mortality rate in humans.Citation[8] ARF is associated with renal vascular and tubular damage and decreased excretory function and involves the action of a number of autocrine and paracrine factors that may contribute to ischemic cell damage.Citation[9] Furthermore, renal sympathetic nerves and circulating catecholamines are believed to be involved in the development of the progressive renal tissue injury accompanying ischemic ARF.Citation[10,Citation11] However, little information has been obtained concerning the renal sympathetic nervous system and its contribution to the pathogenesis of the ischemia/reperfusion-induced ARF.
In the present study, in an attempt to clarify the role of renal sympathetic nervous system in the pathogenesis of the ischemia/reperfusion-induced renal injury, we have recorded the changes in renal blood flow (RBF) and renal vascular resistance (RVR) in response to the vasoconstriction produced by electrical stimulation of the renal sympathetic nerves during ischemia/reperfusion injury. We further examined the early changes in the renal functional parameters post-ischemia and carried out histological examination of the renal tissue to assess the extent of renal structural damage following ischemic renal injury.
MATERIALS AND METHODS
Animals
Male Sprague Dawley (SD) rats (250–350g) were obtained from the Animal Care Facility, Universiti Sains Malaysia (USM), Penang, Malaysia. The animals were housed in standard cages with 12:12 h light-dark cycle and were fed with normal commercial rat chow and water ad libitum. The rats were also allowed to acclimatize in the animal transit room for one week before being used for any experiment. Animal care and the experimental procedures were conducted in accordance with the policies and guidelines of the Animal Ethics Committee, USM, Malaysia. Moreover, all animals procedures was approved by the Animal Ethics Committee of USM, Penang, Malaysia, based on the international rules followed worldwide. All protocols employed had prior approval from the Animal Care and Use Committee of USM.
After acclimatization for one week, animals were randomly divided into two groups: group 1 was used to study renal hemodynamic responses to renal ischemic injury, while group 2 was used to study the changes in the renal functional responses. Each group was further subdivided into two subgroups of 5–7 animals each. These subgroups consisted of rats with ischemic ARF and control subset in which the kidney was left intact.
Surgical Preparation of Animals
Overnight fasted rats were anaesthetized with sodium pentobarbitone (Nembutal®, CAVE, France) at a dose of 60 mg/kg (i.p.). After a tracheotomy (PE250, Portex, UK), the left jugular vein was cannulated to enable the administration of an intravenous (i.v.) maintenance infusion of saline (0.9 g/L NaCl infused at a rate of 6 mL/h) and also to allow an intermittent administration of supplementary anesthetic bolus injections (10 mg/kg in saline). The right carotid artery was similarly catheterized (PE50, Portex, UK) for blood sample collection, and direct measurement of MAP using a pressure transducer (P23 ID Gould, Statham Instrument, Nottingham, UK) coupled to a computerized data acquisition system (PowerLab®, ADInstruments, Sydney, Australia). A midline abdominal incision was performed to expose the left kidney and the renal artery.
Upon completion of the surgical procedure, 2 ml of saline (i.v.) were given to the animal, after which the animal was stabilized for 1 h before the experimental protocol was begun.Citation[4,Citation12,Citation13]
Experimental Protocols
Following animal stabilization, renal ischemic insult was applied to the left kidney, where the model of ischemic ARF was employed. Ischemia was induced by clamping the left renal artery for 30 min followed by reperfusion.Citation[14] Subsequently, the animals were subjected to one of the following protocols.
Protocol 1
In this protocol basal RBF and RVR values were determined before commencing the renal nerve stimulation (RNS) experiment. The renal nerves were stimulated (Grass S 48 Stimulator, Grass Instruments, Massachusetts, USA) at frequencies of 1, 2, 4, 6, 8, and 10 Hz, at 0.2 ms duration and 15 V for a period of 15 s in ascending and then descending orders. Changes in RBF and RVR in response to electrical stimulation of the renal nerve were measured by placing a flowmeter probe (EP 100 series, Carolina Medical Instrument, King, North Carolina, USA) on the isolated renal artery. The probe was connected to a Square-Wave Electromagnetic flowmeter (Carolina Medical Instrument, King, North Carolina, USA), which was linked to a computerized data acquisition system (PowerLab®, ADInstruments, Australia).
Protocol 2
In this protocol, the left ureter was cannulated (PE10, Portex, UK), and six urine collections were performed at 20 min intervals for 2 h to measure urine volume and subsequently calculate urine flow rate (UFR), glomerular filtration rate (GFR), absolute sodium excretion (UNaV), fractional sodium excretion (FENa), absolute potassium excretion (UKV), fractional potassium excretion (FEK), and urinary sodium to potassium ratio (UNa/UK). Blood samples were obtained at the same time intervals for measurement of plasma sodium (PNa), potassium (PK), and creatinine (PCr) and then for calculating FENa, FEK, and GFR, respectively.
At the end of the experiment, the animals were euthanized by an overdose of anesthetic and the left kidney was collected. The collected kidney was cleared from any connective tissue, blotted on tissue paper, and preserved in 10% formalin for histological examination. Subsequently, the animals were disposed of in accordance with the guidelines of the Animal Ethics Committee of USM, Penang, Malaysia.
Biological Samples and Biochemical Analyses
Urine samples were collected in micro-centrifuge tubes (Eppendorf, Hamburg, Germany), and the volumes obtained were gravimetrically quantified. Blood samples were collected (0.5 ml) from the right carotid artery into a pre-cooled heparinized syringe and centrifuged (3000 rpm, 1 min), and the clear plasma was separated. The blood cells were resuspended in normal saline at an equal volume to the plasma obtained and reinfused into the animal immediately.Citation[12,Citation15] Plasma and urine samples were stored at −4°C until assayed for sodium and potassium using flame photometry (Hitachi, Japan) and creatinine by means of colorimetric method (Hitachi, Japan).
Histopathological Study of Renal Tissue
The tissues were fixed in 10% formalin before being processed using Citadel 1000 histokinette (Shandon Scientific Ltd., Cheshire, UK). After processing, the tissues were embedded in paraffin with Histo-Center II-N (Barnstead/Thermolyne Corp., Dubuque, Iowa, USA) and sectioned to a thickness of 5 μm using a Reichert-Jung Histocut 820 II (Cambridge Instrument GmbH, Nussloch, Germany). The sections were stained with hematoxylin and eosin and examined under light microscope.
Calculations
Urine flow rate was calculated by the following formula: UFR = UV / T × BW, where UFR is the urine flow rate, UV is the urine volume, T is the time, and BW is the body weight of the rat.
Absolute excretions of sodium (UNaV) and potassium (UKV) were calculated using the equation: UxV = Ux × UFR, where UxV is the absolute urinary excretion of substance x and Ux is the urine concentration of x.
Clearances were calculated using the usual formula: Cx = Ux × UFR/ Px, where Cx is the clearance of substance x and Px is the plasma concentration of x. Glomerular filtration rate (GFR) was taken to be the clearance of creatinine. Fractional excretions of sodium (FENa) and potassium (FEK) were calculated by CNa/GFR and CK/GFR, respectively, where CNa and CK are the clearances of sodium and potassium, respectively.
Urinary sodium to potassium ratio (UNa/UK) was calculated by dividing the urinary concentration of sodium (UNa) by the urinary concentration of potassium (UK).
Renal vascular resistance (RVR) was calculated by the equation: RVR = MAP/RBF, where MAP is the mean arterial pressure and RBF is the renal blood flow.
Statistical Analysis
All data are expressed in terms of mean ± SEM. The statistical analysis of the data was done using one- and two-way ANOVA followed by Bonferroni/Dunnett post hoc-test. The differences between the means were considered significant at 5% level. All statistical analysis were done using Superanova statistical package (Abacus Inc., Berkeley, California, USA).
RESULTS
General Observations
Body weights of SD rats following 14–16 h of fasting were similar in the two groups (control group: 288 ± 4g vs. ARF group: 285 ± 10g). In the ischemic kidney, the tissue damages were characterized by the presence of evident tubular injury and marked congestion and inflammation in the renal interstitium, findings that were totally absent in the control group (see Figure 1).
Published online:
01 February 2010Figure 1. Light microscopy of renal tissue (5 μm) from (a) control Sprague Dawley rats, and (b) ischemic renal failure rats. Hematoxylin and eosin staining (× 200). In ischemic renal failure rats, tubular injuries along with marked interstitial congestion and inflammation can be seen during early recovery phase of renal ischemia/reperfusion injury, as indicated by the arrow.
Figure 1. Light microscopy of renal tissue (5 μm) from (a) control Sprague Dawley rats, and (b) ischemic renal failure rats. Hematoxylin and eosin staining (× 200). In ischemic renal failure rats, tubular injuries along with marked interstitial congestion and inflammation can be seen during early recovery phase of renal ischemia/reperfusion injury, as indicated by the arrow.
Acute Effects of Ischemic Injury on Hemodynamic Responses
MAP was significantly higher (p < 0.05 vs. control) during the reperfusion phase in ischemic renal failure rats as compared to the control cohorts (see Figure 2A). Furthermore, ARF rats showed markedly lower RBF basal values (control group: 17.6 ± 1.2 mL/min/kg vs. ARF group: 10.2 ± 0.9 mL/min/kg; p < 0.05) and concomitantly higher RVR basal measurements relative to the control group (control group: 23.0 ± 2.6 mmHg/mL/min vs. ARF group: 32.9 ± 3.0 mmHg/mL/min; p < 0.05).
Published online:
01 February 2010Figure 2. Hemodynamic responses in control (○) and ischemic renal failure (▵) Sprague Dawley (SD) rats: (a) mean arterial pressure (MAP), (b) renal nerve stimulation (RNS) effect on percentage reduction in renal blood flow (RBF), and (c) renal nerve stimulation (RNS) effect on the change in renal vascular resistance (RVR). Data presented as mean ± SEM (n = 5–7). *p < 0.05: significant difference between ischemic renal failure rats and control rats. Data were analyzed by two-way ANOVA followed by Bonferonni/Dunnett post-hoc test.
Figure 2. Hemodynamic responses in control (○) and ischemic renal failure (▵) Sprague Dawley (SD) rats: (a) mean arterial pressure (MAP), (b) renal nerve stimulation (RNS) effect on percentage reduction in renal blood flow (RBF), and (c) renal nerve stimulation (RNS) effect on the change in renal vascular resistance (RVR). Data presented as mean ± SEM (n = 5–7). *p < 0.05: significant difference between ischemic renal failure rats and control rats. Data were analyzed by two-way ANOVA followed by Bonferonni/Dunnett post-hoc test.
There were frequency-related RVR and simultaneous frequency-dependent reductions in RBF in all experimental groups upon direct electrical stimulation of the renal nerves. However, it was observed that the overall mean percentage reduction in RBF and the magnitude of change in RVR were significantly lower (p < 0.05 vs. control) in ischemic renal failure animals as compared to the control counterparts (see Figures 2b and 2c).
Acute Effects of Ischemic Injury on Renal Function
A significantly (p < 0.05 vs. control) higher UFR was observed following the induction of ischemia/reperfusion injury in the left kidney (see Figure 3a). Similarly, higher (all p < 0.05) sodium and potassium excretory levels, both in absolute terms and as a fraction of the filtered load, were observed, in ischemic renal failure rats in comparison to rats not subjected to the renal ischemia challenge (see Figures 3b–3e). There was no significant alteration in UNa/UK in either experimental group (p > 0.05 vs. control) (see Figure 3f).
Published online:
01 February 2010Figure 3. Renal functional responses in control (○) and ischemic renal failure (▴) Sprague Dawley (SD) rats: (a) urine flow rate (UFR), (b) absolute sodium excretion (UNaV), (c) absolute potassium excretion (UKV), (d) fractional sodium excretion (FENa), (e) fractional potassium excretion (FEK), (f) urinary sodium to urinary potassium ratio (UNa/UK), and (g) glomerular filtration rate (GFR). Data presented as mean ± SEM (n = 5–7). *p < 0.05: significant difference between ischemic renal failure rats and control rats. Data were analyzed by two-way ANOVA followed by Bonferonni/Dunnett post-hoc test.
Figure 3. Renal functional responses in control (○) and ischemic renal failure (▴) Sprague Dawley (SD) rats: (a) urine flow rate (UFR), (b) absolute sodium excretion (UNaV), (c) absolute potassium excretion (UKV), (d) fractional sodium excretion (FENa), (e) fractional potassium excretion (FEK), (f) urinary sodium to urinary potassium ratio (UNa/UK), and (g) glomerular filtration rate (GFR). Data presented as mean ± SEM (n = 5–7). *p < 0.05: significant difference between ischemic renal failure rats and control rats. Data were analyzed by two-way ANOVA followed by Bonferonni/Dunnett post-hoc test.
In contrast, markedly lower (p < 0.05) GFR measurements were observed in ischemic renal failure rats while compared with the control counterparts (see Figure 3g).
DISCUSSION
The renal nerves are mixed nerves that consist of both afferent and efferent nerve fibers: the former acts by modulating neurotransmission toward the central nervous system, while the latter is responsible for modulating renal hemodynamics and functions. The efferent renal innervation is basically composed of postganglionic adrenergic fibers that characteristically exert their effects via norepinephrine released onto the postsynaptic adrenoceptors.Citation[2]
Renal sympathetic nerves and circulating catecholamine are believed to play a contributory role in the development of acute renal failure (ARF),Citation[10,Citation11] which can further contribute to the chronic loss of renal function over time. This can happen even under asymptomatic conditions.
Ogawa and colleaguesCitation[16] reported that renal denervation before the ischemia had attenuated the reduction in GFR after the ischemia/reperfusion. These findings support the view that that renal sympathetic nerve is directly related to the pathogenesis of ischemic renal injuries. However, there is no direct evidence on the relationships between the renal sympathetic nervous system and the post-ischemic events of ARF.
The present study provided evidence that the renal vasoconstrictor response to electrical stimulation of renal nerves, as reflected by the percentage of reduction in RBF and the magnitude of change in RVR, was noticeably decreased immediately after the reperfusion following 30-min ischemia. These findings suggest that both the ischemia itself and the following reperfusion activate the renal sympathetic nervous system and augment norepinephrine release from nerve terminals. It is known that hyperactive sympathetic nervous system and prolonged exposure to catecholamine can contribute to lower responsiveness of adrenergic receptors to endogenous as well as exogenous adrenergic vasoconstrictor stimuli, a phenomenon known as desensitization. The latter is attributed to either sequestration of the receptors that they are unavailable for interaction with the ligand, down-regulation of the receptors, or an inability of the receptor to couple to G-protein.Citation[17] Accordingly, it was likely that endogenous norepinephrine released by renal sympathetic nerve endings and increased RVR played an important role in the development of ischemia/reperfusion-induced ARF and, therefore, contributed to the observed reduction in RBF and simultaneous increase in MAP during the post-ischemic phase. Furthermore, desensitization of the α1-adrenoceptors by renal sympathetic nervous system activation may have contributed to the observed reduction in the renal vasoconstrictor response to renal nerve stimulation, as this subtype plays the major role in the regulation of renal vascular tone.Citation[13,Citation15,Citation18]
Robust sustained diuresis, natriuresis, and kaliuresis were evident in the post-ischemic period. These effects could be attributed to impaired urinary concentrating ability (at least in part), backleak of filtrate across the damaged proximal and distal tubular epithelia, and/or osmotic diuresis due to profound solutes loss. Despite the fact that the observed changes in the sodium and water excretions following renal ischemia/reperfusion have been reported in previous studiesCitation[14,Citation16,Citation19] and reaffirmed the effectiveness, reliability and reproducibility of animal model used, these reports did emphasize upon the correlation between sodium and potassium excretions following renal ischemic injury. Therefore, the present study extended previous findings to show that the rate of sodium excretion completely paralleled those of potassium and contributed to the absence of a statistically significant difference in UNa/UK in the early reperfusion phase following renal ischemia. Together, these observations would confirm that enhanced salt excretion was primarily due to impaired tubular functions following renal ischemia. This was to a degree supported by the histological evaluation of the post-ischemic kidneys.
The obvious reduction in GFR in the early reperfusion phase of renal-ischemia/reperfusion injury was most likely attributable to the marked afferent arteriolar vasoconstriction produced by the overactive sympathetic nervous system, enhanced RVR, and subsequent reduction in whole kidney RBF following renal ischemia. A similar pattern of change in GFR was observed in several earlier studies using this model.Citation[14,Citation16,Citation19–21] The possible reduction in GFR due to compromised RBF was further supported by the observation that there was a marked interstitial congestion, as was evident in the histopathological assessment of ischemic renal tissues, which would be consistent with increased renal vascular resistance.
In summary, data from these experiments strongly suggest the immediate involvement of renal sympathetic nerve action in the pathogenesis of ischemic ARF. Increased blood pressure responses and diminished RBF and GFR are most likely to be the result of enhanced vascular resistance to flow developed by the presence of hyperactive renal sympathetic nervous system following renal ischemia/reperfusion insult. Impaired sodium and potassium and water reabsorptions due to impaired renal tubular functions are typical responses to renal ischemia in the rat, whereby the extent of sodium renal loss is almost similar to that of potassium.
ACKNOWLEDGMENTS
The authors would like to express their deepest gratitude to Dr. Muhsin Abo-Shaeer for his assistance in editing this paper. The first author, Ibrahim M. Salman, gratefully acknowledges the Institute of Graduate Studies (IPS), USM, Penang, Malaysia for awarding the USM fellowship.
The authors declare that no competing financial interests exist.
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