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Endothelial nitric oxide synthase-deficient mice exhibit increased susceptibility to endotoxin-induced acute renal failure

Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262

Submitted 16 April 2004 ; accepted in final form 21 May 2004

	ABSTRACT

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Acute renal failure (ARF) in septic patients drastically increases the mortality to 50–80%. Nitric oxide (NO) has been shown to be increased in sepsis. Endothelial nitric oxide synthase (eNOS) is one of the major regulators of arterial blood pressure and regional blood flow; however, its in vivo role in septic ARF is still unclear. We hypothesized that eNOS affords a protective effect against the renal vasoconstriction during endotoxemia. Because there are no specific inhibitors for eNOS, the study was therefore undertaken in eNOS knockout mice. There was no significant difference in baseline glomerular filtration rate (GFR) between the wild-type mice and the eNOS knockout mice (140 ± 10 vs. 157 ± 18 µl/min, n = 9, P = not significant). However, renal blood flow (RBF) was significantly decreased in eNOS knockout mice compared with the wild-type controls (0.62 ± 0.05 ml/min, n = 6 vs. 0.98 ± 0.13 ml/min, n = 8, P < 0.05). Mean arterial pressure (MAP) was significantly higher in eNOS knockout mice than the wild-type controls (109 ± 5 vs. 80 ± 1 mmHg, n = 10, P < 0.01). Thus renal vascular resistance (RVR) was much higher in eNOS knockout mice than in the wild-type mice (176 ± 2, n = 6 vs. 82 ± 1 mmHg·ml^–1·min^–1, n = 8, P < 0.01). When 1.0 mg/kg LPS was injected, there was no change in MAP in either the wild-type (84 ± 3 mmHg, n = 10) or the eNOS knockout mice (105 ± 5 mmHg, n = 10). Although GFR (154 ± 22 µl/min, n = 8) and RBF (1.19 ± 0.05 ml/min, n = 9) remained unchanged with the 1.0-mg/kg dose of LPS in the wild-type mice, GFR (83 ± 18 vs. 140 ± 10 µl/min, n = 6, P < 0.01) and RBF (0.36 ± 0.04 vs. 0.62 ± 0.05 ml/min, n = 6, P < 0.01) decreased significantly in the eNOS knockout mice. Fractional excretion of sodium increased significantly in eNOS knockout mice during endotoxemia (3.61 ± 0.78, n = 7 vs. 0.95 ± 0.14, n = 6, P < 0.01), whereas it remained unchanged in the wild-type mice (0.59 ± 0.16, n = 9 vs. 0.42 ± 0.05, n = 6, P = not significant). In summary, eNOS knockout mice have increased RVR and are more susceptible to endotoxemic ARF than wild-type mice despite higher MAP.

fractional excretion of sodium; vasoconstriction; renal vascular resistance

A hallmark of sepsis is the finding that cytokines such as TNF- induce inducible nitric oxide synthase (iNOS). The role of iNOS in septic shock is supported by the observation that a hypotensive dose of endotoxin in wild-type mice does not lower blood pressure in iNOS knockout mice (2). Such results led to a prospective clinical trial to examine the role of NOS inhibitor on mortality (10). The nonspecific NOS inhibitor used blocked the effect not only of iNOS but also of constitutive endothelial NOS (eNOS). This nonspecific NOS inhibitor not only did not decrease but actually increased mortality (10). One explanation is that more specific iNOS inhibition with preservation of eNOS would be necessary to demonstrate protection during endotoxemia.

Studies in isolated rat tubules from iNOS, but not eNOS, knockout mice demonstrate protection against hypoxia-induced tubular injury (9). Endotoxemia in rats has also been shown to decrease in vitro renal eNOS activity (13, 14), thus suggesting a potential role of eNOS in sepsis-related ARF. Because a specific eNOS inhibitor is not available, the in vivo role of eNOS in endotoxemic ARF has not been established.

In the present study, the hypothesis was tested that eNOS knockout mice would demonstrate significant renal dysfunction with a minimal dose of endotoxin (LPS) that does not alter renal function in wild-type mice.

	MATERIALS AND METHODS

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Animals. The experimental protocol was approved by the Animal Ethics Review Committee at the University of Colorado Health Sciences Center. C57BL/6 and eNOS knockout mice were purchased from Jackson Laboratories (Bar Harbor, ME). Male mice aged 8–10 wk were used throughout the study. Mice were maintained on a standard rodent chow and had free access to water.

Materials. Chemicals were purchased from Sigma (St. Louis, MO) unless otherwise specified.

Animal protocol. In preliminary studies, 5 mg/kg of LPS were fatal within 24 h in the eNOS knockout mice. However, with a 1-mg/kg LPS dose, the glomerular filtration rate (GFR) decreased in only the eNOS knockout mice. Mice were therefore intraperitoneally injected with a 1.0-mg/kg dose of LPS (LIST Biological Laboratories, Campbell, CA). Renal blood flow (RBF), GFR, and mean arterial pressure (MAP) were examined at 16 h after LPS (1.0 mg/kg) injection.

Measurement RBF, GFR, and MAP. The animals were anesthetized with pentobarbital sodium (60 mg/kg) and placed on a thermostatically controlled surgical table. A tracheotomy was performed in all mice. Catheters (custom pulled from PE-250) were placed in the jugular vein for maintenance infusion and in the carotid artery for blood pressure measurement. The kidney was exposed by a left subcostal incision and was dissected free from perirenal tissue, and the renal arteries were isolated for the determination of RBF using a blood flowmeter and probe (Transonic Systems, Ithaca, NY) as described by Traynor and Schnermann (16). MAP was measured via a carotid artery catheter connected to a TranspacIV transducer and monitored continuously using Windaq Waveform recording software (Dataq Instruments). An intravenous maintenance infusion of 2.25% BSA in normal saline (NS) at a rate of 0.25 µl·g body wt^–1·min^–1 was started 1 h before experimentation; 0.75% FITC-inulin was added to the infusion solution for the determination of GFR as described by Lorenz and Gruenstein (11). A bladder catheter (PE-10) was used to collect urine. Two 30-min collections of urine were obtained under oil and weighed for volume determination. Blood for plasma inulin determination was drawn between urine collections. FITC in plasma and urine samples was measured using CytoFluor plate reader (PerSeptive Biosystems, Foster City, CA).

Measurement of serum NO levels. Serum NO levels were determined by measuring serum NO₂/NO₃ levels using nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI).

Histological examination. Mice kidneys were harvested after functional measurements. They were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin and periodic acid-Schiff standard methods. Histological examinations were performed by renal pathologists (S. L. and S. T.) without knowledge of the intervention. Histological changes due to tubular necrosis were quantitated by calculation of the percent of tubules that displayed cell necrosis, loss of brush border, cast formation, and tubule dilatation as follows: 0, none; 1, 1–10%; 2, 11–25%; 3, 26–45%. At least 10 fields (x200) were reviewed for each slide.

The renal pathologists quantitatively assessed neutrophil infiltration in a blinded fashion by counting the number of neutrophils per high-power field. At least 10 fields were counted in the cortex and outer medulla on slides stained with hematoxylin and eosin.

Morphological criteria were used to count apoptotic cells on hematoxylin and eosin staining. These characteristics included cellular rounding and shrinkage, nuclear chromatin compaction, and formation of apoptotic bodies (4). Apoptotic tubular cells were quantitatively assessed per 10 high-power fields by the renal pathologists in a blinded fashion.

Measurement of serum creatinine and fractional excretion of sodium. Urine was collected during GFR measurement and blood was collected through cardiac punctuation. Fractional excretion of sodium (FE_Na) is calculated as FE_Na = [(urine sodium x serum creatinine)/(serum sodium x urine creatinine)] x 100.

Statistical analysis. Values are expressed as means ± SE. Multiple comparisons were assessed by ANOVA using the post hoc Newman-Keuls test.

	RESULTS

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Baseline renal function in eNOS knockout vs. wild-type mice. There was no significant difference in baseline GFR between the wild-type mice and eNOS knockout mice (140 ± 10 vs. 157 ± 18 µl/min, n = 9, P = not significant; Fig. 1A). However, MAP was significantly higher in eNOS knockout mice than in the wild-type controls (109 ± 5 vs. 80 ± 1 mmHg, n = 10, P < 0.01; Fig. 1B). RBF was significantly decreased in eNOS knockout mice compared with the wild-type controls (0.62 ± 0.05 ml/min, n = 6 vs. 0.98 ± 0.13 ml/min, n = 8, P < 0.05; Fig. 1C). Thus renal vascular resistance (RVR) was much higher in eNOS knockout mice than in the wild-type mice (176 ± 2 vs. 82 ± 1 mmHg·ml^–1·min^–1, P < 0.01; Fig. 1D).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Baseline renal function in endothelial nitric oxide synthase (eNOS) knockout (ko) and wild-type (WT) mice. Glomerular filtration rate (GFR; A) was measured by inulin clearance, mean arterial pressure (MAP; B) was measured through carotid artery, and renal blood flow (RBF; C) was measured by blood flowmeter. Renal vascular resistance (RVR; D) was calculated as MAP/RBF. Con, control; NS, not significant.

View larger version (10K): [in this window] [in a new window]   Fig. 2. Serum NO levels in wild-type (A) and eNOS knockout (B) mice during endotoxemia. Serum was collected at 16 h after intraperitoneal LPS (1.0 mg/kg) injection. L-NIL was given 30 min before LPS injection. Serum NO level was measured using nitrate/nitrite colorimetric assay.

View larger version (21K): [in this window] [in a new window]   Fig. 3. MAP, GFR, and RBF during endotoxemia in wild-type (A, C, E) and eNOS knockout (B, D, F) mice. MAP, GFR, and RBF were measured at 16 h after intraperitoneal injection of LPS (1.0 mg/kg). MAP was measured through carotid artery, GFR was measured by inulin clearance, and RBF was measured by blood flowmeter.

Serum creatinine and FE_Na. Sixteen hours after LPS injection, urine and serum were collected for sodium and creatinine measurement. There was no significant difference in FE_Na between the LPS- and vehicle-treated mice (0.59 ± 0.16, n = 9 vs. 0.42 ± 0.05, n = 6, P = not significant; Fig. 4A). However, FE_Na increased significantly in LPS-treated eNOS knockout mice compared with vehicle-treated eNOS knockout mice (3.61 ± 0.78, n = 7 vs. 0.95 ± 0.14, n = 6, P < 0.01; Fig 4B).

View larger version (8K): [in this window] [in a new window]   Fig. 4. Fractional excretion of sodium (FENa) in wild-type and eNOS knockout mice during endotoxemia. Serum and urine were collected during GFR measurement at 16 h after LPS (1.0 mg/kg) injection. FENa was calculated as [(urine sodium x serum creatinine)/(serum sodium x urine creatinine)] x 100.

	DISCUSSION

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In the renal vasoconstrictor phase of endotoxemic ARF, we hypothesized that renal constitutive eNOS is important in attenuating the effects of vasoconstrictor agents. Moreover, in contrast to the injurious effect of iNOS, eNOS has not been implicated in the deleterious effect of hypoxia on proximal tubules in vitro (9). Stimulation of iNOS by endotoxin also has been shown in the rat to downregulate renal eNOS in vitro and could therefore be pivotal in predisposing to the renal vasoconstriction phase of endotoxemic ARF (14). There is however no specific eNOS inhibitor to test this hypothesis in vivo. Nonspecific NOS inhibitors that block both iNOS and eNOS activity have been shown not to protect but rather to worsen endotoxemic ARF (2). Use of knockout mice to examine the role of eNOS during endotoxemia was therefore undertaken in the present study.

The eNOS knockout mice were shown to have a significant increase in MAP, confirming an earlier finding (5). The increase in MAP, and thus renal arterial pressure, could potentially afford protection against any renal insult. On the other hand, the eNOS knockout mice exhibited a profound and significant increase in RVR and decrease in RBF. This renal vasoconstriction in the eNOS knockout mice was in excess of any increase in RVR expected with autoregulation of RBF secondary to the increase in renal perfusion pressure. Specifically, such renal autoregulation would be expected to maintain but not decrease total RBF.

In preliminary studies, a search was undertaken for an endotoxin dose that did not affect renal function in wild-type mice, as assessed by GFR or RBF at 16 h of intraperitoneal endotoxin in wild-type mice. A LPS dose of 5 mg/kg was fatal at 24 h in the eNOS knockout mice. However, a lower endotoxin dose of 1 mg/kg was found at 16 h in eNOS knockout mice to decrease GFR and RBF and caused mild tubular necrosis. This 1-mg/kg endotoxin dose however did not alter these same parameters in wild-type mice. The increase in serum NO concentration was comparable in the wild-type and eNOS knockout mice. The increase in serum NO was attributed to iNOS, because it was normalized with L-NIL, a specific iNOS inhibitor, in both groups of animals. This observation was consistent with the findings in iNOS knockout mice in which 5 mg/kg ip of endotoxin did not increase serum NO concentration (7). Thus the rise in serum NO during endotoxemia appears not to be dependent on the presence of eNOS. Because serum NO concentrations were not different in the wild-type and eNOS knockout mice 16 h after 1 mg/kg ip of endotoxin, the decrease in GFR in the eNOS knockout mice must have been due to another factor(s). In this regard, the significant fall in RBF during endotoxemia in the eNOS knockout mice was the most likely pathogenetic factor, because the wild-type mice with intact eNOS did not demonstrate a fall in RBF with the 1-mg/kg LPS dose.

The combination of rise in serum creatinine, decrease in GFR and RBF, mild tubular necrosis, and increased FE_Na in these eNOS knockout mice during endotoxemia are characteristics similar to the clinical ARF associated with sepsis.

In summary, the present in vivo results support the hypothesis that the eNOS is an important determinant of the renal response to endotoxemia. This conclusion is compatible with the previous suggestions that desensitization of renal guanylate cyclase and thus cGMP, the secondary messenger of eNOS, may be a determinant of the vasoconstriction phase of ARF during endotoxemia in mice (6).

	GRANTS

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This work was supported by National Institutes of Health Grants DK-52599 and P01-HL-31992.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. Schrier, Dept. of Medicine, Univ. of Colorado Health Sciences Center, 4200 East 9th Ave., Box C-281, Denver, CO 80262 (E-mail: robert.schrier{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

	REFERENCES

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