Evidence for the Role of Reactive Nitrogen Species in Polymicrobial Sepsis-Induced Renal Peritubular Capillary Dysfunction and Tubular Injury
Liping Wu*,
Neriman Gokden and
Philip R. Mayeux*
Departments of * Pharmacology and Toxicology and Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas
Address correspondence to: Dr. Philip R. Mayeux, Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, 4301 West Markham Street, #611, Little Rock, AR 72205. Phone: 501-686-8895; Fax: 501-686-5521; E-mail: prmayeux{at}uams.edu
Received for publication December 26, 2006.
Accepted for publication March 22, 2007.
Acute kidney injury (AKI) remains a frequent and serious complicationof human sepsis that contributes significantly to mortality.For better understanding of the development of AKI during sepsis,the cecal ligation and puncture (CLP) murine model of sepsiswas studied using intravital video microscopy (IVVM) of thekidney. IVVM with FITC-dextran was used to determine the percentageof capillaries with continuous, intermittent or no flow at 0(sham), 10, 16, and 22 h after CLP. There was a dramatic fallin capillary perfusion as early as 10 h after CLP that persistedthrough 22 h. The percentage of vessels with continuous flowat 16 h decreased from 73 ± 2% in shams to 16 ±2% (P < 0.05), whereas the percentage of vessels with noflow increased from 4 ± 1% in shams to 42 ± 2%(P < 0.05). The capillary perfusion defect preceded the risein serum creatinine. IVVM with dihydrorhodamine-123 was usedto quantify in real time reactive nitrogen species (RNS) generationby renal tubules, and the inducible nitric oxide synthase inhibitorl-iminoethyl-lysine (mg/kg) was used to examine the role ofinducible nitric oxide synthase inhibitor on capillary dysfunctionand RNS generation. Tubular generation of RNS was significantlyelevated at 10 h after CLP and was associated with tubules thatwere bordered by capillaries with reduced perfusion. L-Iminoethyl-lysinesignificantly reversed the capillary perfusion defect, blockedRNS generation, and reduced AKI. These data show that capillarydysfunction and RNS generation contribute to tubular injuryand suggest that RNS should be considered a potential therapeutictarget in the treatment of sepsis-induced AKI.
Acute kidney injury (AKI) remains a frequent and serious complicationof human sepsis that contributes significantly to mortality.It is estimated to occur in 20 to 50% of patients with sepsis,and the mortality rate for these patients with renal failureapproaches 70% (1,2). Sepsis-induced AKI is most common in theelderly patient in the intensive care unit, for whom currenttherapy is, for the most part, still primarily supportive (3).Therefore, there is a critical need to uncover new therapeuticapproaches because the incidence of sepsis-induced AKI is predictedto increase as the population ages (4).
The timing of therapy in the patient with sepsis clearly affectsoutcome (3,5), particularly the development of AKI (1). A majorhindrance to the advance of new therapeutic approaches is alack of understanding regarding the temporal relationships betweenthe evolution of the inflammatory response, mediator signaling,and end-organ failure. This is especially important becausetreatment in the patient with sepsis is generally begun onlyafter the onset of symptoms (e.g., systemic inflammatory responsesyndrome, hypotension) (3).
Endothelial cells are a well-established target for bacterialtoxins (6), and although endothelial injury and microvascularfailure are thought to play important roles in the developmentof organ failure during sepsis, direct evidence of renal capillarydysfunction during sepsis has only recently begun to emerge(7). We recently reported that peritubular capillary perfusionis dramatically reduced before the development of renal injuryin a murine model of lipopolysaccharide (LPS)-induced AKI (8).These findings suggest a possible relationship between capillarydysfunction and the development of renal injury during sepsis.However, LPS models of sepsis-induced AKI typically do not toreplicate the hyperdynamic changes that are observed in humansepsis. Therefore, it is important to examine peritubular capillaryperfusion in more clinically relevant models of sepsis-inducedAKI. The cecal ligation and puncture (CLP) murine model of polymicrobialsepsis is generally accepted to exhibit more key pathogenicfeatures that are observed in humans than does LPS administration(9), including a hyperdynamic phase that is initiated by fluidresuscitation (10). In this model, older rather than youngeradult mice are used because sepsis-induced renal failure ismore commonly a disease of the elderly, and antibiotics andfluids are administered to mimic standard care for the patientwith sepsis (7,11). An understanding of the development of renalmicrovascular injury after sepsis and its relationship to tubularinjury could lead to new, more targeted therapies (12).
It is generally agreed that inducible nitric oxide synthase(iNOS)-derived NO is an important mediator of the hemodynamicfailure that is associated with sepsis, but its role in CLP-inducedAKI has never been examined. It has also been proposed thatreactive nitrogen species (RNS) derived from iNOS may contributeto the development of sepsis-induced AKI. This notion is supportedby the appearance of nitrated plasma proteins in the patientwith sepsis and kidney injury (13) and the appearance of nitratedproteins and oxidation products in the kidney during LPS-inducedrenal injury in rats (14,15). Although the generation of RNSin the kidney after CLP has never been examined, the protectiveeffects of anti-inflammatory antioxidants in CLP suggest thatoxidants may play a role in CLP-induced AKI (10,16). Accordingly,these studies were undertaken to monitor, in real time, peritubularcapillary perfusion using intravital videomicroscopy (IVVM)and to establish whether RNS generation occurs in the peritubularcapillary/tubular microenvironment during the development ofCLP-induced AKI.
FITC-dextran 500,000-Da conjugate was purchased from Sigma-Aldrich(St. Louis, MO). Dihydrorhodamine-123 (DHR) was purchased fromInvitrogen (Eugene, OR). Rabbit polyclonal anti-nitrotyrosineand anti-iNOS antibodies were purchased from Upstate Cell SignalingSolutions (Lake Placid, NY). L-N6-(1-iminoethyl lysine) (L-NIL)was purchased from Axxora (San Diego, CA).
CLP
CLP-induced AKI was established essentially as described byMiyaji et al. (10). All animals were housed and killed in accordancewith the National Institutes of Health Guide for the Care andUse of Laboratory Animals and with approval of the Universityof Arkansas for Medical Sciences Institutional Animal Care andUse Committee. Male C57/BL6 mice (Harlan, Indianapolis, IN)at 39 to 40 wk of age were acclimated for 1 wk with free accessto food and water. At the time of surgery, the mice were anesthetizedusing isoflurane inhalation. After laparotomy, a 4-0 silk ligaturewas placed 1.5 cm from the cecal tip. The cecum was puncturedtwice with a 21-G needle and gently squeezed to express approximatelya 1-mm column of fecal material. In sham-operated mice, thececum was located but neither ligated nor punctured. The abdominalincision was closed in two layers with 4-0 silk sutures. Aftersurgery, 1 ml of prewarmed normal saline was given intraperitoneally.At 6 h after surgery, all mice received imipenem/cilastatin(14 mg/kg, subcutaneously) in 1.5 ml of two thirds normal saline(40 ml/kg). Mice were placed in individual cages and set ona warming pad.
IVVM
At 10, 16, or 22 h after CLP or 16 h after sham surgery, micewere anesthetized with isoflurane and underwent laparotomy toexpose the left kidney. The kidney was positioned on a glassstage above an inverted fluorescence microscope (Zeiss Axiovert200; Jena, Germany) equipped with a digitizing camera (Hamamatsu,Bridgewater, NJ) and kept moist with saline and covered. Theentire IVVM procedure, including surgery, was completed within30 min. During this time, core temperature was monitored andmaintained at 36 to 37°C using an infrared heat lamp. Atthe conclusion of IVVM, blood was collected by cardiac punctureand the mouse was killed by cervical dislocation. Kidneys werefixed in 10% phosphate-buffered formalin. Serum creatinine concentrationwas determined using a Roche Cobas Mira Clinical Analyzer (RocheDiagnostic Systems, Branchburg, NJ) using the picric acid-basedcolorimetric assay.
Evaluation of Peritubular Capillary Dysfunction and Reactive Oxygen Species/RNS Generation
Approximately 15 min before IVVM, FITC-dextran (500,000 Da,0.13 µmol/kg) and DHR (0.3 mg/kg) were administered viatail vein in a volume of 70 µl. FITC-dextran was usedto visualize the renal intravascular space and red blood cell(RBC) movement at an excitation of 470 nm and emission of 520nm. For each mouse, five videos of 10 s each were captured atapproximately 15 frames per second from five randomly selectedfields of view (x200). Capillary perfusion was analyzed as describedpreviously (17). Briefly, approximately 150 randomly selectedvessels per mouse were classified into three categories of bloodperfusion: "Continuous flow," whereby RBC movement in the vesselwas not interrupted during the video; "intermittent flow," wherebyRBC movement stopped or reversed any time during the video;and "no flow," whereby no RBC movement was detected. Data wereexpressed as the percentage of vessels in each of the threecategories.
Under the same fields of view that were used to assess capillaryperfusion, oxidation of DHR to rhodamine was visualized at 535nm excitation and 590 nm emission. For minimization of photobleaching, a <3-s exposure was used to capture videos ofthe same five randomly selected fields of view per mouse thatwere used to assess RBC movement. Gain and other contrast enhancementsettings were identical for all fields of view. Fluorescenceintensity was quantified densitometrically using AxioVisionImaging Software (Zeiss). Data were expressed as arbitrary unitsper µm2.
Dosing Protocol for L-NIL
The iNOS inhibitor L-NIL (18) was administered as two dosesof 3 mg/kg (intraperitoneally) each. The first dose was administeredat the end of surgery, and the second dose was administeredat 6 h.
Assessment of Kidney Morphology
Paraffin-embedded sections (3 µm) were prepared from kidneysthat were fixed in 10% phosphate-buffered formalin. The periodicacid-Schiff (PAS) stain was used for the analysis of morphologywith light microscopy (Nikon E800; Melville, NY) by a blindedobserver. A semiquantitative score for tubular injury and acutetubular necrosis was assigned as described by Wang et al. (19).For each mouse, at least 10 high-power fields were examined.The percentage of tubules that displayed cellular necrosis,loss of brush border, cast formation, vacuolization, and tubuledilation were scored as follows: 0, none; 1, <10%; 2, 11to 25%; 3, 26 to 45%; 4, 46 to 75%; and 5, >76%.
Immunohistochemistry
Paraffin-embedded tissue sections were cleared in xylene, rehydrated,and washed in PBS. Endogenous peroxidase activity and nonspecificprotein binding were blocked using reagents that were suppliedin the DakoCytomation LSAB+ System-HPR kit (Dako, Carpinteria,CA). Sections were then incubated with rabbit anti-iNOS antibody(1:1000 dilution) or anti-nitrotyrosine antibody (1:400 dilution)overnight at 4°C. After washing in PBS, sections were incubatedwith the second antibody supplied by the kit at room temperaturefor 30 min. Sections were then incubated with streptavidin peroxidasefor 30 min, then with chromagen solution as described by themanufacturer. Gill's hematoxylin II was used as a counterstain.The negative control for detection of nitrotyrosine was preincubationof the antibody with 10 mM nitrotyrosine for 1 h before use.
Serum Nitrate/Nitrite Levels
Serum nitrate/nitrite (NOx) levels were determined using a KamiyaBiomedical assay kit (Seattle, WA) as directed by the manufacturer.Briefly, nitrate in serum samples was first converted to nitriteusing nitrate reductase. Data were expressed as serum NOx concentrationin µM.
Statistical Analyses
Data were analyzed with Prism 4.0 software for Mac (GraphPadSoftware, San Diego, CA). Data are expressed as means ±SEM. A one-way ANOVA followed by the Student-Newman-Keuls testwas used to compare groups. For analysis of tissue injury scores,a one-way ANOVA followed by the Kruskal-Wallis test was usedto compare groups. P < 0.05 was considered significant. Eachn represents data from one mouse.
Time Course of Kidney Injury
Representative images of PAS-stained sections from sham-treatedmice and mice at 10, 16, and 22 h after CLP are shown in Figure 1,A through D, respectively. Morphologic changes including lossof brush border, tubular cell sloughing, tubular dilation, andtubule vacuolization increased in severity over time. Serumcreatinine concentration increased over time to a significantlevel at 16 h and remained elevated through 22 h (Figure 1E),indicating the development of functional renal injury afterCLP.
Figure 1. Time course of renal injury after cecal ligation and puncture (CLP). Representative histology images of kidney stained with periodic acid-Schiff (PAS) are shown for sham mice (A) and mice at 10 h (B), 16 h (C), and 22 h (D) after CLP. CLP caused morphologic changes, including loss of brush border (1), tubular cell sloughing (2), tubular dilation (3), and tubule vacuolization (4) that increased in severity over time. The time course of renal dysfunction (E) showed significantly elevated serum creatinine concentration by 16 h. Data are means ± SEM (n = 6 to 7 mice per time point). *P < 0.05 versus time 0 h (sham group). Magnification, x400.
Time Course of Disruption of Capillary Perfusion
Mice were subjected to IVVM with FITC-dextran to assess corticalperitubular capillary perfusion at 10, 16, or 22 h after CLP.These data are presented in Figure 2. Sham controls are indicatedas 0 h. The percentage of vessels with continuous flow decreasedfrom 73 ± 2% in sham controls to 49 ± 8% at 10h (P < 0.05 compared with sham) and decreased further to16 ± 2% at 16 h (P < 0.05 compared with 10 h). Thedecrease in the percentage of vessels with continuous flow wasaccompanied by a significant increase in the percentage of vesselswith no flow. At 16 h after CLP, the percentage of vessels withintermittent flow was significantly elevated also. The decreasein cortical peritubular capillary perfusion persisted through22 h.
Figure 2. Time course of the change in functional peritubular capillary density after CLP. At the indicated time, intravital videomicroscopy (IVVM) using FITC-labeled dextran was carried out to assess changes in cortical peritubular capillary perfusion. Sham mice are represented by time 0 h. Plotted are the percentage of vessels designated as having continuous flow, intermittent flow, or no flow at each time point (see the Materials and Methods section for details). Data are means ± SEM (n = 6 to 7 mice per time point). *P < 0.05 versus the sham group. P < 0.05 versus 10 h.
Imaging of Reactive Oxygen Species/RNS Generation
DHR is oxidized to fluorescence rhodamine by hydroxyl radical,nitrogen dioxide, peroxynitrite, and peroxidase-derived species(20). Therefore, the oxidation of DHR to rhodamine was usedas an indicator of reactive oxygen species (ROS)/RNS generation.Rhodamine fluorescence was visualized in real time and localizedusing IVVM. Representative images of FITC-dextran and rhodaminefluorescence from the same field of view from the kidney ofa mouse 16 h after CLP are shown in Figure 3. Capillaries withno flow were defined as having no detectable RBC movement duringthe 10-s video capture. In single-frame images, as in Figure 3,these capillaries may appear white (because of the lack of RBCmovement), collapsed, or to contain stacked RBC. Single-frameimages of capillaries with continuous perfusion generally appearas dark because of the movement of RBC that obscure FITC-dextranfluorescence. Rhodamine fluorescence was increased in tubulesthat were bordered by capillaries with compromised perfusion.Moreover, rhodamine fluorescence seemed to be most intense indiscrete areas within the tubular epithelium. For further examinationof this, the same fields of view from the kidneys of mice 16h after CLP were subjected to IVVM to detect rhodamine fluorescenceand epi-illumination to reveal surface contour. Shown in Figure 4are representative images from the same field of view of rhodaminefluorescence (Figure 4A) and epi-illumination (Figure 4B) at16 h after CLP. Increased rhodamine fluorescence was observedin what seemed to be surface tubule vacuoles. Figure 4C showsa representative PAS-stained kidney section from a mouse 16h after CLP. The circle encloses to vacuoles in the tubularepithelium.
Figure 3. Imaging of reactive oxygen species/reactive nitrogen species (ROS/RNS) generation in renal tubules. Shown are representative images of perfusion (A) and rhodamine fluorescence (B) captured from videos of the same field of view from the kidney of a mouse 16 h after CLP. Arrows indicate capillaries with no perfusion. A pseudocolored image of B is shown in C to highlight changes in pixel intensity. Intense regions of rhodamine fluorescence are localized to discrete regions of the tubular epithelium bordered by capillaries with reduced perfusion.
Figure 4. ROS/RNS generation in injured tubules after CLP. Rhodamine fluorescence in A is localized in tubules with vacuoles and possible blebbing observed in the same field of view (B) using epi-illumination to reveal surface contour. Light microscopy in C from the PAS-stained kidney cortex at 16 h after CLP shows extensive vacuolization (within circle). Magnification, x400.
Time Course of Systemic NO Generation and Tubular ROS/RNS Generation
The time course of changes in serum NOx concentration and tubularrhodamine fluorescence are presented in Figure 5. Serum NOxconcentration, an indicator of systemic NO synthesis, was significantlyelevated at 10 h after CLP and remained elevated through 22h (Figure 5A). Tubular rhodamine fluorescence was also significantlyelevated at 10 h after CLP, paralleling systemic NO generation(Figure 5B). Values for mice that were subjected to sham surgeryat each time point are presented to rule out the effects ofsurgery trauma on these parameters. Immunohistochemistry wasperformed to detect iNOS protein and nitrotyrosine-protein adducts,a marker of peroxynitrite generation, in kidney section fromsham mice and mice at 16 h after CLP. Specific staining foriNOS and nitrotyrosine was very weak in sham mice (Figure 5,C and E, respectively). In contrast, iNOS and nitrotyrosinestaining were observed in the kidneys from mice at 16 h afterCLP (Figure 5, D and F, respectively) in both glomeruli andtubules. Sections from the kidneys of mice at 16 h after CLPdid not show staining for iNOS in the absence of primary antibodyor staining for nitrotyrosine when the anti-nitrotyrosine antibodywas preincubated with 10 mM nitrotyrosine before use (data notshown). Both IVVM and immunohistochemistry indicated that proximaltubules were the major site of RNS generation. Although ourIVVM studies were not capable of imaging murine cortical glomerulibecause they reside too deep within the cortex, immunohistochemistryrevealed some staining for nitrotyrosine in the glomeruli andvery weak staining in some distal tubules of the cortex.
Figure 5. Time course of serum nitrate/nitrite (NOx) levels and tubular ROS/RNS generation. Serum NOx levels increased at 10 h after CLP and remained constant through 22 h (A). At the indicated time, IVVM using Dihydrorhodamine-123 (DHR) was carried out to assess changes in cortical tubular ROS/RNS generation (B). Rhodamine fluorescence was significantly elevated at 10 h and remained elevated through 22 h after CLP. Neither NOx levels nor rhodamine fluorescence changed over time in sham mice. Data are means ± SEM (n = 6 to 7 mice per time point). *P < 0.05 versus time 0 h. Shown in C through F are representative photographs of immunohistochemistry performed in kidney section from sham 16 h (C and E) and CLP 16 h (D and F) for inducible nitric oxide synthase (iNOS; C and D) and nitrotyrosine (E and F). Brown staining indicates location of antigen. Staining in the absence of the iNOS primary antibody or with anti-nitrotyrosine antibody preincubate with 10 mM tyrosine served as nonspecific controls. Neither of these conditions resulted in tissue staining (data not shown). Magnification, x200.
L-NIL Protects Against Capillary Dysfunction and Tubular ROS/RNS Generation
To assess the effects of pharmacologic inhibition of iNOS oncapillary perfusion and ROS/RNS generation after CLP, mice weretreated with L-NIL (3 mg/kg, intraperitoneally) at the timeof surgery and received a second dose at 6 h. Four groups ofmice (sham, sham + L-NIL, CLP 16 h, and CLP 16 h + L-NIL) underwentIVVM with FITC-dextran to assess peritubular capillary perfusionand DHR to assess ROS/RNS generation. The effects of L-NIL onperitubular capillary flow at 16 h after CLP is presented inFigure 6. The percentage of vessels with continuous flow inthe CLP + L-NIL group was significantly higher compared withthe CLP group (42 ± 8 versus 18 ± 3; P < 0.05).L-NIL treatment also significantly reduced the percentage ofvessels with no flow compared with the CLP group (21 ±4 versus 40 ± 1; P < 0.05) but did not affect thepercentage of vessels with intermittent flow. L-NIL treatmentdid not affect the distribution of flow in sham mice.
Figure 6. Effects of L-N6-(1-iminoethyl lysine (L-NIL) on functional peritubular capillary density after CLP. At 16 h after CLP, the percentage of vessels with continuous flow (A), intermittent flow (B), and no flow (C) were determine for sham, sham + L-NIL, CLP, and CLP + L-NIL groups. L-NIL treatment significantly reduced the fall in the percentage of vessels with continuous flow and significantly reduced the increase in vessels with no flow. Data are means ± SEM (n = 4 to 6). *P < 0.05 versus sham + L-NIL; P < 0.05 versus CLP.
For confirmation that the dosing regimen for L-NIL inhibitedNO synthesis, serum NOx levels were measured. At 16 h afterCLP, L-NIL significantly blocked the rise in serum NOx levels(Figure 7A), indicating induction of iNOS activity and the effectivenessof L-NIL treatment. The effects of L-NIL on rhodamine fluorescenceare presented in Figure 7B. L-NIL treatment blocked the increasein rhodamine fluorescence that was observed in CLP-treated mice.These data suggest that after CLP, DHR oxidation (rhodaminefluorescence) is predominantly due to iNOS-derived RNS.
Figure 7. Effects of L-NIL on tubular serum NOx levels and tubular ROS/RNS generation. L-NIL treatment prevented the rise in serum NOx concentration at 16 h after CLP, suggesting that increased NO synthesis was, at least in part, mediated by iNOS (A). Data are means ± SEM (n = 4 to 6). *P < 0.05 versus all other groups. Rhodamine fluorescence was determined in the same fields of view that were used in Figure 6 to determine capillary flow. L-NIL treatment significantly reduced the rise in rhodamine fluorescence, suggesting that iNOS-derived RNS was the major source for DHR oxidation. Data are means ± SEM (n = 4 to 6). *P < 0.05 versus all other groups.
Figure 8 shows representative images that were captured fromthe kidney of mice after sham surgery (Figure 8, A and D), 16h after CLP (Figure 8, B and E), and 16 h after CLP and treatmentwith L-NIL (Figure 8, C and F). Images of perfusion (Figure 8,A through C) and rhodamine fluorescence (Figure 8, D throughF) are from the same fields of view, respectively. Arrows indicatecapillaries with no perfusion. These images provide additionalevidence to suggest a link between iNOS-derived NO and RNS generationand the increased generation of RNS by tubules in areas thatwere bordered by capillaries with reduced perfusion.
Figure 8. Representative images of the effects of L-NIL treatment on peritubular capillary perfusion and tubular ROS/RNS generation. Shown in the top row are representative video images that were used to assess capillary flow in sham (A), CLP 16 h (B), and CLP 16 h + L-NIL (C) groups. Shown in the bottom row are representative video images of rhodamine fluorescence (D through F) captured from the same fields of view that are shown in A through C. Arrows in A through C indicate capillaries with no perfusion.
L-NIL Protects Against Renal Injury
Serum creatinine concentration and morphologic scoring wereused to assess the effects of L-NIL treatment on renal injury.Mice that were treated with CLP + L-NIL had significantly lowerserum creatinine than the CLP group, indicating that L-NIL treatmentpreserved renal function (Figure 9A). In addition, L-NIL showedprotection as based on tubular injury score (Figure 9B).
Figure 9. Effects of L-NIL treatment on renal injury. Serum creatinine levels were measured 16 h after CLP in sham, sham + L-NIL, CLP, and CLP + L-NIL groups (A). Treatment with L-NIL significantly reduced the rise in serum creatinine level. Tissue injury scoring also supported a protective effect of L-NIL (B). Data are means ± SEM (n = 4 to 6). SE values for sham and CLP + L-NIL groups were 0 because all mice within each group earned the same score. *P < 0.05 versus the corresponding sham group.
Microvascular dysfunction is believed to be a key feature ofsepsis that contributes to end-organ failure (21). These studiesestablish that peritubular capillary perfusion is dramaticallydecreased during the course of CLP-induced sepsis. Furthermore,these studies are the first to demonstrate real-time generationof RNS by renal tubules and link decreased peritubular capillaryperfusion to the generation of RNS and tubular injury. Moreover,studies with the iNOS inhibitor L-NIL suggest that iNOS-derivedNO/RNS is critical to the development of capillary dysfunctionand AKI after CLP.
It is recognized that endothelial injury can have detrimentaleffects on peritubular capillary flow and permeability barrier(22). Although several reports indicate that endothelial cellsare targets of bacterial toxins (6) and RNS (23), the role ofthe peritubular capillary in the development of renal injuryafter sepsis is suspected but not fully elucidated. Studiesby Yasuda et al. (7) were the first to suggest the involvementof the peritubular capillary in CLP-induced AKI. Peritubularcapillary leakage, as assessed by Evans blue dye leakage, occursas early as 6 h after CLP. Moreover, evidence of hypoxia (increasein pimonidazole-protein adducts) observed at 24 h after CLPmight have resulted from decreased capillary flow (7). The strikingdecrease in peritubular capillary perfusion revealed by IVVMsupports the notion that regional hypoxia may contribute tomitochondrial dysfunction and oxidant generation.
Decreased capillary perfusion coupled to capillary leak (7)suggests the occurrence of direct injury to the renal microvasculatureafter CLP. A unique feature of IVVM is the ability to monitorboth capillary perfusion and tubular RNS generation in realtime. These studies are the first to examine specifically therenal peritubular microenvironment and implicate RNS as a possiblemediator of peritubular capillary dysfunction and tubular injurythat are associated with CLP-induced sepsis. Pathophysiologiceffects of NO are observed when excessive amounts are generated.For example, during iNOS induction, NO reacts with oxygen orsuperoxide to produce RNS that can initiate protein oxidation,lipid oxidation, and DNA damage (24,25). One RNS, peroxynitrite(ONOO–), is formed from the chemical reaction betweenNO and superoxide (26) and can damage not only membranes butalso nitrate protein tyrosine and change the activities of anumber of important enzymes (27,28). Moreover, decreased capillaryperfusion can lead to parenchymal hypoxia (29) and superoxidegeneration to fuel RNS synthesis (30). Therefore, it is likelythat increased NO generation (as a result of iNOS induction)in the setting of a pro-oxidant, hypoxic microenvironment causedby decreased perfusion, exacerbates renal injury (31).
In models of AKI associated with iNOS induction, the contributionof RNS has drawn recent attention, particularly in ischemia/reperfusioninjury and LPS-induced injury (32–35). These studies arethe first to examine the potential therapeutic benefits of pharmacologicinhibition of iNOS in CLP-induced AKI. In other models of CLP-inducedsepsis, pharmacologic inhibition of iNOS prevents the fall inBP and other hemodynamic changes in the rat (36), and transgenicmice that are deficient in iNOS maintain arteriolar responsivenessto catecholamines and show improved survival (37). The appearanceof nitrated proteins and oxidation products in the kidney duringLPS-induced renal failure in rats and the ability of iNOS inhibitorsto reduce these products and preserve renal function (14,15)suggest that iNOS-derived RNS are important mediators of renalinjury. In these studies, L-NIL treatment ameliorated CLP-inducedcapillary dysfunction, morphologic injury, and renal dysfunction,suggesting a role for iNOS. Nevertheless, pharmacologic studieswith iNOS inhibitors must be interpreted with caution becausethere are no absolutely selective iNOS inhibitors, and constitutiveNOS isoforms may be induced during sepsis as well (38). L-NILhas been used extensively as an iNOS inhibitor and displaysa 14-fold selectivity for iNOS compared with endothelial NOS(39). The dosage used in these studies was chosen because itdid not significantly reduce basal serum NOx levels, suggestinga minimal effect on constitutive isoforms. Still, additionalstudies with more selective inhibitors such as 1400W (39) areneeded to establish iNOS fully as an important therapeutic target.
Although induction of iNOS in inflammatory cells occurs duringsepsis, sepsis-induced AKI is not associated with a significantinflux of activated inflammatory cells to the kidney (10). Rather,kidney epithelial cells seem to be the major source of iNOS-derivedNO in the kidney during sepsis (40). These studies extend thesefinding to suggest that renal tubular epithelial cells are perhapsthe major source of NO-derived oxidants. Two independent methods(IVVM and immunohistochemistry) for detecting the generationof RNS both indicated the generation of RNS by renal tubulesafter CLP. Furthermore, both IVVM and immunohistochemistry revealedRNS generation in tubular vacuoles. The potential clinical relevanceof these findings is supported by the detection of nitratedplasma proteins in patients with sepsis and renal failure (13).
Although these studies do not establish cause–effect,it is reasonable to speculate that RNS generation contributedto tubular injury. Additional support for the role of oxidantscomes from the findings that the anti-inflammatory antioxidantethyl pyruvate is protective against CLP-induced AKI (10). Itmust be acknowledged that oxidation of DHR to rhodamine is notsolely dependent on RNS. However, the ability of L-NIL to blockcompletely the rise in rhodamine fluorescence in CLP-treatedmice compared with sham mice suggests that RNS were the majoroxidants generated after CLP. It is interesting that sham miceshowed a basal DHR oxidation localized to tubules that werebordered by capillaries with reduced flow that was not inhibitedby L-NIL. The absence of nitrotyrosine staining in sham micesuggests that generation of ROS rather than RNS was responsiblefor DHR oxidation in the absence of iNOS induction.
A rapid decline in peritubular capillary perfusion accompaniedby tubular injury is also a feature of LPS-induced AKI (8,17).However, in the CLP model, the decline in capillary perfusionseems to be greater than in the LPS model, and tubular morphologicchanges are more severe. Still, studies in both types of sepsismodels show that a change in the peritubular microenvironmentis a common feature. Sepsis-induced AKI has an extremely complexetiology. While mediators of the early response by the kidneymay be different in the two models (9), iNOS-mediated peritubularcapillary dysfunction and tubular injury occur in both modelsof sepsis-induced AKI (17). This suggests that common pathwaysare activated in the microenvironment after inflammatory stimulithat mediate tubular injury. From a clinical perspective, therapeuticagents that target later events might be the most beneficialbecause downstream mediators may be relatively independent ofupstream events and because therapy is generally started afterinitiation of sepsis (3).
Our findings have implications beyond sepsis-induced AKI. Itis becoming increasingly clear that endothelial injury and peritubularcapillary dysfunction may initiate and extend the pathogenesisof both acute and chronic renal injury (41–44) by compromisingrenal vascular responsiveness and tubule function (41,45,46).In addition, reductions in peritubular capillary perfusion causeslocalized hypoxia, which over time may lead to tubulointerstitialinjury (47). We found that even in the absence of disease, localizeddecreases in peritubular capillary perfusion are associatedwith localized areas of oxidant generation. Therefore, eventransient localized oxidant generation may sensitize the kidneyto injury.
We have uncovered a previously unrecognized role for capillarydysfunction and tubular RNS generation in CLP-induced AKI. Theclose association between capillary dysfunction, RNS generation,and tubular damage suggests that the peritubular capillary/tubularmicroenvironment plays a critical role in sepsis-induced AKI.Furthermore, studies with the iNOS inhibitor L-NIL suggest thatpharmacologic inhibition of iNOS and/or RNS should be consideredas potential therapeutic approaches for the prevention of sepsis-inducedAKI.
This research was supported by a Pilot Study Grant (P.R.M.)and the Graduate Student Research Fund (L.W.) from the Universityof Arkansas for Medical Sciences.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
