Role of Leptin Deficiency in Early Acute Renal Failure during Endotoxemia in ob/ob Mice
Wei Wang,
Brian Poole,
Amit Mitra,
Sandor Falk,
Giamila Fantuzzi,
Scott Lucia and
Robert Schrier
Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado.
Correspondence to Dr. Robert Schrier, Department of Medicine, University of Colorado Health Sciences Center, 4200 East 9th Ave., Box C-281, Denver, CO 80262. Phone: 303-315-8059; Fax: 303-315-4852; E-mail: robert.schrier{at}uchsc.edu
ABSTRACT. It is known that, among human patients with sepsis,acute renal failure (ARF) dramatically increases mortality ratesto 50 to 80%. However, the pathogenesis of septic ARF is notfully understood. An increase in endotoxin-induced mortalityrates for leptin-deficient ob/ob mice was recently demonstrated.In comparison with ob/ob mice, db/db mice, which are deficientin the long isoforms of leptin receptors (Ob/Rb), demonstratelower mortality rates after exposure to the endotoxin LPS. Indb/db mice, mRNA for the short isoforms of leptin receptorsis constitutively expressed in the kidney, lung, liver, andmacrophages. It is known that plasma leptin levels increasein rodents after exposure to LPS, and this was demonstratedfor db/db mice. Because ob/ob and db/db mice are both obese,factors other than obesity must be involved in the increasedmortality rates for ob/ob mice. In this study, the hypothesisthat the short forms of leptin receptors might offer protectionagainst endotoxin-induced lethality at least in part by providingprotection against ARF was examined. Serum leptin levels weresignificantly increased with LPS treatment in wild-type anddb/db mice but not ob/ob mice. GFR decreased significantly 16h after the homozygous ob/ob mice received intraperitoneal injectionsof 0.3 mg/kg LPS (0.37 ± 0.04 ml/min per g kidney versus0.83 ± 0.06 ml/min per g kidney, n = 6, P < 0.01);the mean arterial pressure (MAP) remained unchanged. For ob/oblittermates (+/?ob), there was no significant change in eitherMAP or GFR when the mice were challenged with the same timeinterval (16 h) and dose of LPS. In contrast to ob/ob mice,there was no significant change in GFR or MAP when homozygousdb/db mice or their littermates received injections of an evenhigher dose of LPS (0.4 mg/kg). Mouse recombinant leptin hadno effect on GFR when ob/ob mice received 0.3 mg/kg LPS injections.However, renal function (serum creatinine levels, 0.4 ±0.1 mg/dl versus 0.9 ± 0.1 mg/dl, P < 0.01) and MAP(68 ± 4 mmHg versus 51 ± 2 mmHg, n = 6, P <0.01) were significantly improved with leptin replacement whenthe ob/ob mice developed hypotensive ARF with a higher doseof LPS (0.5 mg/kg). In summary, the previously reported increasedsusceptibility to LPS of ob/ob mice, compared with db/db mice,may be attributable at least in part to increased susceptibilityto ARF.
Severe sepsis and septic shock are common and are associatedwith significant mortality rates and substantial consumptionof health care resources. There are an estimated 751,000 casesof sepsis or septic shock in the United States each year, andthose conditions are responsible for as many deaths each yearas are acute myocardial infarctions (215,000 deaths) (1). Itis known that the occurrence of acute renal failure (ARF) amongseptic patients dramatically increases mortality rates to 50to 80% (2). However, the pathogenesis of septic ARF is not fullyunderstood. An increase in the endotoxin-induced mortality ratefor leptin-deficient ob/ob mice was recently demonstrated. Incomparison with ob/ob mice, db/db mice, which are deficientin the long isoforms of the leptin receptor (Ob/Rb), demonstratedlower mortality rates with exposure to the endotoxin LPS (3).In db/db mice, mRNA for other short isoforms of leptin receptorsis constitutively present in the kidney, lung, liver, and macrophages(4). It is known that plasma leptin levels increase in rodentsafter exposure to LPS, and this finding has been demonstratedin db/db mice (5,6). Because ob/ob and db/db mice are both obese,factors other than obesity must be involved in the increasedmortality rates for ob/ob mice. In this study, the hypothesisthat the short isoforms of leptin receptors might offer protectionagainst endotoxin-induced lethality at least partly by providingprotection against ARF was examined. To test our hypothesis,this study was undertaken to examine renal function during endotoxemiain ob/ob and db/db mice.
Animals
The experimental protocol was approved by the Animal EthicsReview Committee of the University of Colorado Health SciencesCenter. Wild-type (C57BL/6), ob/ob (B6.V-Lepob), and db/db (B6.Cg-m+/+leprdb) mice and their littermates were purchased from The JacksonLaboratory (Bar Harbor, ME). Male mice of 8 to 10 wk of agewere used throughout the study. Mice were maintained with standardrodent chow and had free access to water.
Materials
LPS was purchased from List Biologic Laboratories (Campbell,CA). Recombinant mouse leptin was purchased from R&D Systems(Minneapolis, MN). Other chemicals were purchased from SigmaChemical Co. (St. Louis, MO) unless otherwise specified.
Measurement of GFR and Mean Arterial Pressure
The animals were anesthetized with pentobarbital (60 mg/kg)and placed on a thermostatically controlled surgical table.A tracheotomy was performed, and a steady steam of 100% oxygenwas blown over the tracheal tube throughout the experiment.Catheters (custom-pulled from PE-250 tubing) were placed inthe jugular vein (for maintenance infusion) and the carotidartery (for BP determinations). Mean arterial pressure (MAP)was measured via a carotid artery catheter connected to a TranspacIV system (Dataq Instruments). An intravenous maintenance infusionof 2.25% BSA in normal saline solution, at a rate of 0.25 µl/gbody wt per min, was started 1 h before experimentation. FITC-inulin(0.75%) was added to the infusion solution for the determinationof GFR, as described by Lorenz and Gruenstein (7). A bladdercatheter (PE-10) was used to collect urine. Two 30-min collectionsof urine were obtained under oil and weighed for volume determination.Blood samples for plasma inulin determinations were obtainedbetween urine collections. FITC levels in plasma and urine sampleswere measured with a CytoFluor plate reader (PerSeptive Biosystems).
Measurement of Serum Leptin Levels
Serum leptin levels were measured with a Quantikine M immunoassaykit (R&D Systems). Briefly, a leptin standard and serumsamples were incubated for 2 h at room temperature in a platecoated with polyclonal antibody to mouse leptin. Antibody conjugatedto horseradish peroxidase was then added to the plate afterwashes. Finally, substrate solution was added and the OD wasdetermined with a microplate reader at 450 nm.
Measurement of Blood Glucose Levels
Blood glucose levels were measured with an Accu-Chek Advantagesystem (Boehringer Mannheim, Indianapolis, IN).
Histologic Examinations
Kidneys that had been fixed in 4% paraformaldehyde and embeddedin paraffin were sectioned at 4 µm and stained with hematoxylinand eosin, with standard methods. Histologic examinations wereperformed by a renal pathologist without knowledge of the intervention.Histologic changes attributable to tubular necrosis were quantitatedby calculation of the percentage of tubules that displayed cellularnecrosis, loss of the brush border, cast formation, and tubuledilation, as follows: 0, none; 1, 1 to 10%; 2, 11 to 25%; 3,26 to 45%. At least 10 fields (magnification, x200) were reviewedfor each slide.
Serum Leptin Levels during Endotoxemia in Mice
When C57BL/6 wild-type mice received intraperitoneal injectionsof LPS (2.5 mg/kg), leptin levels increased significantly, comparedwith the vehicle-treated control animals (21.6 ± 3.7ng/ml versus 8.8 ± 1.7 ng/ml, P < 0.01) (Figure 1).In ob/ob mice, leptin levels were almost undetectable (1.3 ±0.1 ng/ml) and there was no significant increase in responseto LPS at 0.3 mg/kg (2.0 ± 0.3 ng/ml, P = NS versus baseline)(Figure 1). In comparison with both wild-type and ob/ob mice,db/db mice demonstrated higher baseline leptin levels (70 ±4 ng/ml, P < 0.01 for both). The level increased significantlywith LPS at 0.4 mg/kg (158 ± 23 ng/ml, P < 0.01 versusbaseline) (Figure 1).
Figure 1. Serum leptin levels in wild-type, ob/ob, and db/db mice. Serum samples were obtained 16 h after intraperitoneal injection of LPS (2.5 mg/kg for wild-type mice, 0.3 mg/kg for ob/ob mice, and 0.4 mg/kg for db/db mice). Leptin levels were measured with an ELISA. Values are mean ± SEM. CON, control.
Renal Function in ob/ob and db/db Mice during Endotoxemia
When homozygous ob/ob mice received intraperitoneal injectionsof 0.3 mg/kg LPS, the GFR decreased significantly (0.37 ±0.04 ml/min per g kidney versus 0.83 ± 0.06 ml/min perg kidney, n = 6, P < 0.01), whereas the MAP was unchanged(72.0 ± 1.6 mmHg versus 63.0 ± 6.0 mmHg, n = 4,P = NS) (Figure 2, A and B). Serum albumin levels (1.53 ±0.07 g/dl versus 1.56 ± 0.09 g/dl, n = 5, P = NS) andhematocrit values (45.9 ± 1.5% versus 46.1 ± 1.5%,n = 5, P = NS) were comparable in control animals and LPS-treatedob/ob mice. Renal histologic examinations demonstrated changesindicating acute tubular injury in approximately 10% of tubulesin ob/ob mice treated with LPS, compared with no tubular changesin littermates and db/db mice before and after LPS. In contrast,when ob/ob littermates (+/?ob) received injections of the samedose of LPS, there was no significant change in MAP (71 ±3.0 mmHg versus 80 ± 3.4 mmHg, n = 4, P = NS) or GFR(0.77 ± 0.03 ml/min per g kidney versus 0.74 ±0.04 ml/min per g kidney, n = 6, P = NS) at 16 h after LPS treatment(Figure 2, C and D). To examine whether obesity plays a rolein the susceptibility to endotoxemic ARF, similarly obese db/dbmice were used in the study. In contrast to ob/ob mice, therewas no significant change in GFR (0.54 ± 0.04 ml/minper g kidney versus 0.65 ± 0.09 ml/min per g kidney,n = 4, P = NS) or MAP (88 ± 0.2 mmHg versus 87 ±1.1 mmHg, n = 4, P = NS) when homozygous db/db mice receivedinjections of an even higher dose of LPS (0.4 mg/kg) (Figure 3, A and B).There were no significant changes in GFR (0.57± 0.01 ml/min per g kidney versus 0.62 ± 0.12ml/min per g kidney, n = 5, P = NS) and MAP (78 ± 0.5mmHg versus 80 ± 0.3 mmHg, n = 4, P = NS) when db/dblittermates (db/+?) received intraperitoneal injections of LPSat 0.4 mg/kg (Figure 3, C and D).
Figure 2. GFR (A and C) and mean arterial pressure (MAP) (B and D) in ob/ob mice (A and B) and their littermates (C and D) during endotoxemia. The functional study was performed 16 h after LPS injection. GFR was measured as FITC-inulin clearance, and MAP was measured via the carotid artery. Values are mean ± SEM. CON, control.
Figure 3. GFR (A and C) and MAP (B and D) in db/db mice (A and B) and their littermates (C and D) during endotoxemia. The functional study was performed 16 h after LPS injection. GFR was measured as FITC-inulin clearance, and MAP was measured via the carotid artery. Values are mean ± SEM. CON, control.
To further clarify whether leptin deficiency was responsiblefor the susceptibility to endotoxemic ARF in ob/ob mice, recombinantmouse leptin was administered to ob/ob mice before LPS administration.Vehicle or leptin (1 µg/g per d) was injected intraperitoneallyfor 10 d before mice were challenged with LPS (0.3 mg/kg, administeredintraperitoneally). GFR were similar in the vehicle and leptingroups (0.52 ± 0.03 ml/min per g kidney versus 0.41 ±0.02 ml/min per g kidney). MAP were also comparable in the twogroups (93 ± 1 mmHg versus 83 ± 6 mmHg, P = NS).However, when mice were challenged with a higher dose of LPS(0.5 mg/kg), the mice in the vehicle-treated group became hypotensiveand anuric. Serum creatinine levels were therefore used to assessrenal function. Serum creatinine levels were significantly improvedwith leptin treatment, compared with vehicle treatment (0.4± 0.1 mg/dl versus 0.9 ± 0.1 mg/dl, P < 0.01).The MAP was significantly higher in the leptin group, comparedwith the vehicle group (68 ± 4 mmHg versus 51 ±2 mmHg, n = 6, P < 0.01) (Figure 4).
Figure 4. Effects of leptin replacement on GFR and MAP during endotoxemia in ob/ob mice. Leptin was administered at 1 µg/g per d for 10 d before LPS injection. Serum creatinine levels (A) and MAP (B) were measured 16 h after injection of LPS at 0.5 mg/kg. Values are mean ± SEM. CON, control.
Blood Glucose Levels in ob/ob and db/db Mice
There was no significant difference in blood glucose levelsbetween wild-type mice and littermates of ob/ob or db/db mice(141 ± 6, 136 ± 4, and 154 ± 15 mg/dl,respectively; P = NS). However, the blood glucose levels weresignificantly higher in ob/ob and db/db mice (223 ± 26and 442 ± 50 mg/dl, respectively; n = 8, P < 0.01versus wild-type mice and/or their littermates). Blood glucoselevels were significantly decreased in both ob/ob and db/dbmice 16 h after LPS injection (88 ± 18 and 105 ±9 mg/dl, respectively; both P < 0.01 versus the respectivebaseline values). Therefore, blood glucose levels were comparablefor ob/ob and db/db mice 16 h after LPS exposure, when renalfunction was examined.
Increased serum leptin concentrations were observed after LPSadministration in rodents (5,6), and leptin induction duringinflammation was absent in IL-1-deficient mice (8). The leptin-deficientob/ob mice are more susceptible to LPS-related lethality thanare their lean littermates (3). Although many of the cytokineresponses to LPS were observed to be comparable in the ob/obmice and their littermates, there was blunted induction of IL-10and IL-1 receptor antagonist expression (3). The db/db mice,which are deficient in the Ob/Rb isoform of the leptin receptor,are as obese as ob/ob mice but are less susceptible to LPS lethality(3). The db/db mice exhibit high basal serum leptin levels,which increase with LPS exposure.
Because ARF is known to dramatically increase mortality ratesamong human subjects during sepsis (2), this study was undertakento investigate whether there was a difference in the occurrenceof ARF during LPS exposure for ob/ob versus dbdb mice. The ob/obmice were much more sensitive to LPS-related ARF than were theirlean littermates. It was previously demonstrated, in a normotensivemouse model of LPS-related ARF, that wild-type mice must beexposed to 2.0 mg/kg (administered intraperitoneally) for adecrease in GFR of >50% to occur 16 h after LPS (9). In contrast,the ob/ob mice demonstrated a similar decrease in GFR of approximately50% with only 0.3 mg/kg LPS. The conditions and the type ofLPS were exactly the same for the wild-type and ob/ob mice.This increased renal functional sensitivity to LPS was not observedwith even a slightly higher dose (0.4 mg/kg) of LPS in db/dbmice. Only the ob/ob mice exhibited evidence of tubular necrosisafter LPS treatment. The changes were mild, however, as mostfrequently observed in human ARF. There was no evidence thatvolume depletion was a factor in the ARF in the ob/ob mice duringendotoxemia.
Although the ob/ob mice did not exhibit serum leptin levelsat baseline or after LPS treatment, the db/db mice demonstratedsignificantly higher serum leptin levels at baseline and afterLPS treatment, compared with wild-type mice. Leptin replacementfor 10 d for the ob/ob mice did not afford protection with respectto the previously demonstrated decrease in GFR 16 h after 0.3mg/kg LPS administration. The absence of protection with leptinwas not associated with an effect on MAP. However, with 0.5mg/kg LPS administration to ob/ob mice, endotoxemic shock wasobserved, with a MAP of approximately 50 mmHg. The mice wereanuric and demonstrated significantly elevated serum creatinineconcentrations. With the 10 d of leptin treatment, the ob/obmice exhibited significantly higher MAP and significantly lowerserum creatinine levels. Therefore, an effect of leptin to increaseBP may be critical for the protection observed in the endotoxemicshock model of ARF. For septic patients, it has been demonstratedthat plasma leptin levels are higher among survivors than nonsurvivors(10).
In summary, endotoxin-related ARF occurs at a much lower dosein leptin-deficient ob/ob mice than in their lean littermates.The susceptibility to endotoxemia-related ARF in ob/ob micecannot be exclusively attributable to obesity, however, becausedb/db mice, which are comparably obese, were resistant to asimilar LPS dose. In the hypotensive endotoxemia-related model,ob/ob mice were protected against ARF with prior leptin treatment.The increased MAP with leptin treatment in the LPS shock modelsuggests an effect on peripheral vascular resistance. Such aneffect is consistent with the demonstrated effect of leptinto stimulate the sympathetic nervous system (11,12). Whereasdb/db mice lack the long isoforms of the hypothalamic leptinreceptor, the short isoforms of leptin receptors are locatedin many tissues throughout the body, including the kidney andthe vasculature (4). The increased levels of endogenous leptinacting on these short isoforms of leptin receptors in db/dbmice may be critical for the observed renal protection againstendotoxemia. Renal protection with exogenous leptin in the LPS-inducedshock model in ob/ob mice may involve the same receptors. Thereare, however, other potential factors that may contribute tothe increased sensitivity of ob/ob mice to endotoxemia-relatedARF, such as blunted induction of anti-inflammatory cytokines.Finally, it should be emphasized that, although the occurrenceof ARF may contribute to the increased mortality rates amongendotoxemic ob/ob mice, deleterious effects on other organ systemsmay also be involved.
Acknowledgments
This work was supported by National Institutes of Health GrantsDK52599 and P01-HL31992.
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Received for publication August 26, 2003.
Accepted for publication December 3, 2003.
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Abstract
Introduction
-deficient mice (8). The leptin-deficient ob/ob mice are more susceptible to LPS-related lethality than are their lean littermates (3). Although many of the cytokine responses to LPS were observed to be comparable in the ob/ob mice and their littermates, there was blunted induction of IL-10 and IL-1 receptor antagonist expression (3). The db/db mice, which are deficient in the Ob/Rb isoform of the leptin receptor, are as obese as ob/ob mice but are less susceptible to LPS lethality (3). The db/db mice exhibit high basal serum leptin levels, which increase with LPS exposure. 
