Pathophysiology of Renal Disease and Progression

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
JASN June 2007, 18 (6) 1807-1815; DOI: https://doi.org/10.1681/ASN.2006121402

Abstract

Acute kidney injury (AKI) remains a frequent and serious complication of 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 sepsis was studied using intravital video microscopy (IVVM) of the kidney. IVVM with FITC-dextran was used to determine the percentage of capillaries with continuous, intermittent or no flow at 0 (sham), 10, 16, and 22 h after CLP. There was a dramatic fall in capillary perfusion as early as 10 h after CLP that persisted through 22 h. The percentage of vessels with continuous flow at 16 h decreased from 73 ± 2% in shams to 16 ± 2% (P < 0.05), whereas the percentage of vessels with no flow increased from 4 ± 1% in shams to 42 ± 2% (P < 0.05). The capillary perfusion defect preceded the rise in serum creatinine. IVVM with dihydrorhodamine-123 was used to quantify in real time reactive nitrogen species (RNS) generation by renal tubules, and the inducible nitric oxide synthase inhibitor l-iminoethyl-lysine (mg/kg) was used to examine the role of inducible nitric oxide synthase inhibitor on capillary dysfunction and RNS generation. Tubular generation of RNS was significantly elevated at 10 h after CLP and was associated with tubules that were bordered by capillaries with reduced perfusion. l-Iminoethyl-lysine significantly reversed the capillary perfusion defect, blocked RNS generation, and reduced AKI. These data show that capillary dysfunction and RNS generation contribute to tubular injury and suggest that RNS should be considered a potential therapeutic target in the treatment of sepsis-induced AKI.

Acute kidney injury (AKI) remains a frequent and serious complication of 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 failure approaches 70% (1,2). Sepsis-induced AKI is most common in the elderly patient in the intensive care unit, for whom current therapy is, for the most part, still primarily supportive (3). Therefore, there is a critical need to uncover new therapeutic approaches because the incidence of sepsis-induced AKI is predicted to increase as the population ages (4).

The timing of therapy in the patient with sepsis clearly affects outcome (3,5), particularly the development of AKI (1). A major hindrance to the advance of new therapeutic approaches is a lack of understanding regarding the temporal relationships between the evolution of the inflammatory response, mediator signaling, and end-organ failure. This is especially important because treatment in the patient with sepsis is generally begun only after the onset of symptoms (e.g., systemic inflammatory response syndrome, hypotension) (3).

Endothelial cells are a well-established target for bacterial toxins (6), and although endothelial injury and microvascular failure are thought to play important roles in the development of organ failure during sepsis, direct evidence of renal capillary dysfunction during sepsis has only recently begun to emerge (7). We recently reported that peritubular capillary perfusion is dramatically reduced before the development of renal injury in a murine model of lipopolysaccharide (LPS)-induced AKI (8). These findings suggest a possible relationship between capillary dysfunction and the development of renal injury during sepsis. However, LPS models of sepsis-induced AKI typically do not to replicate the hyperdynamic changes that are observed in human sepsis. Therefore, it is important to examine peritubular capillary perfusion in more clinically relevant models of sepsis-induced AKI. The cecal ligation and puncture (CLP) murine model of polymicrobial sepsis is generally accepted to exhibit more key pathogenic features that are observed in humans than does LPS administration (9), including a hyperdynamic phase that is initiated by fluid resuscitation (10). In this model, older rather than younger adult mice are used because sepsis-induced renal failure is more commonly a disease of the elderly, and antibiotics and fluids are administered to mimic standard care for the patient with sepsis (7,11). An understanding of the development of renal microvascular injury after sepsis and its relationship to tubular injury 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 hemodynamic failure that is associated with sepsis, but its role in CLP-induced AKI has never been examined. It has also been proposed that reactive nitrogen species (RNS) derived from iNOS may contribute to the development of sepsis-induced AKI. This notion is supported by the appearance of nitrated plasma proteins in the patient with sepsis and kidney injury (13) and the appearance of nitrated proteins and oxidation products in the kidney during LPS-induced renal injury in rats (14,15). Although the generation of RNS in the kidney after CLP has never been examined, the protective effects of anti-inflammatory antioxidants in CLP suggest that oxidants may play a role in CLP-induced AKI (10,16). Accordingly, these studies were undertaken to monitor, in real time, peritubular capillary perfusion using intravital videomicroscopy (IVVM) and to establish whether RNS generation occurs in the peritubular capillary/tubular microenvironment during the development of CLP-induced AKI.

Materials and Methods

FITC-dextran 500,000-Da conjugate was purchased from Sigma-Aldrich (St. Louis, MO). Dihydrorhodamine-123 (DHR) was purchased from Invitrogen (Eugene, OR). Rabbit polyclonal anti-nitrotyrosine and anti-iNOS antibodies were purchased from Upstate Cell Signaling Solutions (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 by Miyaji et al. (10). All animals were housed and killed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee. Male C57/BL6 mice (Harlan, Indianapolis, IN) at 39 to 40 wk of age were acclimated for 1 wk with free access to food and water. At the time of surgery, the mice were anesthetized using isoflurane inhalation. After laparotomy, a 4-0 silk ligature was placed 1.5 cm from the cecal tip. The cecum was punctured twice with a 21-G needle and gently squeezed to express approximately a 1-mm column of fecal material. In sham-operated mice, the cecum was located but neither ligated nor punctured. The abdominal incision was closed in two layers with 4-0 silk sutures. After surgery, 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 on a warming pad.

IVVM

At 10, 16, or 22 h after CLP or 16 h after sham surgery, mice were anesthetized with isoflurane and underwent laparotomy to expose the left kidney. The kidney was positioned on a glass stage above an inverted fluorescence microscope (Zeiss Axiovert 200; Jena, Germany) equipped with a digitizing camera (Hamamatsu, Bridgewater, NJ) and kept moist with saline and covered. The entire IVVM procedure, including surgery, was completed within 30 min. During this time, core temperature was monitored and maintained at 36 to 37°C using an infrared heat lamp. At the conclusion of IVVM, blood was collected by cardiac puncture and the mouse was killed by cervical dislocation. Kidneys were fixed in 10% phosphate-buffered formalin. Serum creatinine concentration was determined using a Roche Cobas Mira Clinical Analyzer (Roche Diagnostic Systems, Branchburg, NJ) using the picric acid-based colorimetric 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 via tail vein in a volume of 70 μl. FITC-dextran was used to visualize the renal intravascular space and red blood cell (RBC) movement at an excitation of 470 nm and emission of 520 nm. For each mouse, five videos of 10 s each were captured at approximately 15 frames per second from five randomly selected fields of view (×200). Capillary perfusion was analyzed as described previously (17). Briefly, approximately 150 randomly selected vessels per mouse were classified into three categories of blood perfusion: “Continuous flow,” whereby RBC movement in the vessel was not interrupted during the video; “intermittent flow,” whereby RBC movement stopped or reversed any time during the video; and “no flow,” whereby no RBC movement was detected. Data were expressed as the percentage of vessels in each of the three categories.

Under the same fields of view that were used to assess capillary perfusion, oxidation of DHR to rhodamine was visualized at 535 nm excitation and 590 nm emission. For minimization of photo bleaching, a <3-s exposure was used to capture videos of the same five randomly selected fields of view per mouse that were used to assess RBC movement. Gain and other contrast enhancement settings were identical for all fields of view. Fluorescence intensity was quantified densitometrically using AxioVision Imaging Software (Zeiss). Data were expressed as arbitrary units per μm2.

Dosing Protocol for L-NIL

The iNOS inhibitor L-NIL (18) was administered as two doses of 3 mg/kg (intraperitoneally) each. The first dose was administered at the end of surgery, and the second dose was administered at 6 h.

Assessment of Kidney Morphology

Paraffin-embedded sections (3 μm) were prepared from kidneys that were fixed in 10% phosphate-buffered formalin. The periodic acid-Schiff (PAS) stain was used for the analysis of morphology with light microscopy (Nikon E800; Melville, NY) by a blinded observer. A semiquantitative score for tubular injury and acute tubular 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 tubule dilation were scored as follows: 0, none; 1, <10%; 2, 11 to 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 nonspecific protein binding were blocked using reagents that were supplied in 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 incubated with the second antibody supplied by the kit at room temperature for 30 min. Sections were then incubated with streptavidin peroxidase for 30 min, then with chromagen solution as described by the manufacturer. Gill's hematoxylin II was used as a counterstain. The negative control for detection of nitrotyrosine was preincubation of the antibody with 10 mM nitrotyrosine for 1 h before use.

Serum Nitrate/Nitrite Levels

Serum nitrate/nitrite (NOx) levels were determined using a Kamiya Biomedical assay kit (Seattle, WA) as directed by the manufacturer. Briefly, nitrate in serum samples was first converted to nitrite using nitrate reductase. Data were expressed as serum NOx concentration in μM.

Statistical Analyses

Data were analyzed with Prism 4.0 software for Mac (GraphPad Software, San Diego, CA). Data are expressed as means ± SEM. A one-way ANOVA followed by the Student-Newman-Keuls test was used to compare groups. For analysis of tissue injury scores, a one-way ANOVA followed by the Kruskal-Wallis test was used to compare groups. P < 0.05 was considered significant. Each n represents data from one mouse.

Results

Time Course of Kidney Injury

Representative images of PAS-stained sections from sham-treated mice and mice at 10, 16, and 22 h after CLP are shown in Figure 1, A through D, respectively. Morphologic changes including loss of brush border, tubular cell sloughing, tubular dilation, and tubule vacuolization increased in severity over time. Serum creatinine concentration increased over time to a significant level at 16 h and remained elevated through 22 h (Figure 1E), indicating the development of functional renal injury after CLP.

Figure 1.
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, ×400.

Time Course of Disruption of Capillary Perfusion

Mice were subjected to IVVM with FITC-dextran to assess cortical peritubular capillary perfusion at 10, 16, or 22 h after CLP. These data are presented in Figure 2. Sham controls are indicated as 0 h. The percentage of vessels with continuous flow decreased from 73 ± 2% in sham controls to 49 ± 8% at 10 h (P < 0.05 compared with sham) and decreased further to 16 ± 2% at 16 h (P < 0.05 compared with 10 h). The decrease in the percentage of vessels with continuous flow was accompanied by a significant increase in the percentage of vessels with no flow. At 16 h after CLP, the percentage of vessels with intermittent flow was significantly elevated also. The decrease in cortical peritubular capillary perfusion persisted through 22 h.

Figure 2.
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 used as an indicator of reactive oxygen species (ROS)/RNS generation. Rhodamine fluorescence was visualized in real time and localized using IVVM. Representative images of FITC-dextran and rhodamine fluorescence from the same field of view from the kidney of a mouse 16 h after CLP are shown in Figure 3. Capillaries with no flow were defined as having no detectable RBC movement during the 10-s video capture. In single-frame images, as in Figure 3, these capillaries may appear white (because of the lack of RBC movement), collapsed, or to contain stacked RBC. Single-frame images of capillaries with continuous perfusion generally appear as dark because of the movement of RBC that obscure FITC-dextran fluorescence. Rhodamine fluorescence was increased in tubules that were bordered by capillaries with compromised perfusion. Moreover, rhodamine fluorescence seemed to be most intense in discrete areas within the tubular epithelium. For further examination of this, the same fields of view from the kidneys of mice 16 h after CLP were subjected to IVVM to detect rhodamine fluorescence and epi-illumination to reveal surface contour. Shown in Figure 4 are representative images from the same field of view of rhodamine fluorescence (Figure 4A) and epi-illumination (Figure 4B) at 16 h after CLP. Increased rhodamine fluorescence was observed in what seemed to be surface tubule vacuoles. Figure 4C shows a representative PAS-stained kidney section from a mouse 16 h after CLP. The circle encloses to vacuoles in the tubular epithelium.

Figure 3.
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.
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, ×400.

Time Course of Systemic NO Generation and Tubular ROS/RNS Generation

The time course of changes in serum NOx concentration and tubular rhodamine fluorescence are presented in Figure 5. Serum NOx concentration, an indicator of systemic NO synthesis, was significantly elevated at 10 h after CLP and remained elevated through 22 h (Figure 5A). Tubular rhodamine fluorescence was also significantly elevated at 10 h after CLP, paralleling systemic NO generation (Figure 5B). Values for mice that were subjected to sham surgery at each time point are presented to rule out the effects of surgery trauma on these parameters. Immunohistochemistry was performed to detect iNOS protein and nitrotyrosine-protein adducts, a marker of peroxynitrite generation, in kidney section from sham mice and mice at 16 h after CLP. Specific staining for iNOS and nitrotyrosine was very weak in sham mice (Figure 5, C and E, respectively). In contrast, iNOS and nitrotyrosine staining were observed in the kidneys from mice at 16 h after CLP (Figure 5, D and F, respectively) in both glomeruli and tubules. Sections from the kidneys of mice at 16 h after CLP did not show staining for iNOS in the absence of primary antibody or staining for nitrotyrosine when the anti-nitrotyrosine antibody was preincubated with 10 mM nitrotyrosine before use (data not shown). Both IVVM and immunohistochemistry indicated that proximal tubules were the major site of RNS generation. Although our IVVM studies were not capable of imaging murine cortical glomeruli because they reside too deep within the cortex, immunohistochemistry revealed some staining for nitrotyrosine in the glomeruli and very weak staining in some distal tubules of the cortex.

Figure 5.
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, ×200.

L-NIL Protects Against Capillary Dysfunction and Tubular ROS/RNS Generation

To assess the effects of pharmacologic inhibition of iNOS on capillary perfusion and ROS/RNS generation after CLP, mice were treated with L-NIL (3 mg/kg, intraperitoneally) at the time of surgery and received a second dose at 6 h. Four groups of mice (sham, sham + L-NIL, CLP 16 h, and CLP 16 h + L-NIL) underwent IVVM with FITC-dextran to assess peritubular capillary perfusion and DHR to assess ROS/RNS generation. The effects of L-NIL on peritubular capillary flow at 16 h after CLP is presented in Figure 6. The percentage of vessels with continuous flow in the CLP + L-NIL group was significantly higher compared with the CLP group (42 ± 8 versus 18 ± 3; P < 0.05). L-NIL treatment also significantly reduced the percentage of vessels with no flow compared with the CLP group (21 ± 4 versus 40 ± 1; P < 0.05) but did not affect the percentage of vessels with intermittent flow. L-NIL treatment did not affect the distribution of flow in sham mice.

Figure 6.
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 inhibited NO synthesis, serum NOx levels were measured. At 16 h after CLP, L-NIL significantly blocked the rise in serum NOx levels (Figure 7A), indicating induction of iNOS activity and the effectiveness of L-NIL treatment. The effects of L-NIL on rhodamine fluorescence are presented in Figure 7B. L-NIL treatment blocked the increase in rhodamine fluorescence that was observed in CLP-treated mice. These data suggest that after CLP, DHR oxidation (rhodamine fluorescence) is predominantly due to iNOS-derived RNS.

Figure 7.
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 from the kidney of mice after sham surgery (Figure 8, A and D), 16 h after CLP (Figure 8, B and E), and 16 h after CLP and treatment with L-NIL (Figure 8, C and F). Images of perfusion (Figure 8, A through C) and rhodamine fluorescence (Figure 8, D through F) are from the same fields of view, respectively. Arrows indicate capillaries with no perfusion. These images provide additional evidence to suggest a link between iNOS-derived NO and RNS generation and the increased generation of RNS by tubules in areas that were bordered by capillaries with reduced perfusion.

Figure 8.
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 were used to assess the effects of L-NIL treatment on renal injury. Mice that were treated with CLP + L-NIL had significantly lower serum creatinine than the CLP group, indicating that L-NIL treatment preserved renal function (Figure 9A). In addition, L-NIL showed protection as based on tubular injury score (Figure 9B).

Figure 9.
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.

Discussion

Microvascular dysfunction is believed to be a key feature of sepsis that contributes to end-organ failure (21). These studies establish that peritubular capillary perfusion is dramatically decreased during the course of CLP-induced sepsis. Furthermore, these studies are the first to demonstrate real-time generation of RNS by renal tubules and link decreased peritubular capillary perfusion to the generation of RNS and tubular injury. Moreover, studies with the iNOS inhibitor L-NIL suggest that iNOS-derived NO/RNS is critical to the development of capillary dysfunction and AKI after CLP.

It is recognized that endothelial injury can have detrimental effects on peritubular capillary flow and permeability barrier (22). Although several reports indicate that endothelial cells are targets of bacterial toxins (6) and RNS (23), the role of the peritubular capillary in the development of renal injury after sepsis is suspected but not fully elucidated. Studies by Yasuda et al. (7) were the first to suggest the involvement of the peritubular capillary in CLP-induced AKI. Peritubular capillary leakage, as assessed by Evans blue dye leakage, occurs as early as 6 h after CLP. Moreover, evidence of hypoxia (increase in pimonidazole-protein adducts) observed at 24 h after CLP might have resulted from decreased capillary flow (7). The striking decrease in peritubular capillary perfusion revealed by IVVM supports the notion that regional hypoxia may contribute to mitochondrial dysfunction and oxidant generation.

Decreased capillary perfusion coupled to capillary leak (7) suggests the occurrence of direct injury to the renal microvasculature after CLP. A unique feature of IVVM is the ability to monitor both capillary perfusion and tubular RNS generation in real time. These studies are the first to examine specifically the renal peritubular microenvironment and implicate RNS as a possible mediator of peritubular capillary dysfunction and tubular injury that are associated with CLP-induced sepsis. Pathophysiologic effects of NO are observed when excessive amounts are generated. For example, during iNOS induction, NO reacts with oxygen or superoxide 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 between NO and superoxide (26) and can damage not only membranes but also nitrate protein tyrosine and change the activities of a number of important enzymes (27,28). Moreover, decreased capillary perfusion can lead to parenchymal hypoxia (29) and superoxide generation to fuel RNS synthesis (30). Therefore, it is likely that increased NO generation (as a result of iNOS induction) in the setting of a pro-oxidant, hypoxic microenvironment caused by decreased perfusion, exacerbates renal injury (31).

In models of AKI associated with iNOS induction, the contribution of RNS has drawn recent attention, particularly in ischemia/reperfusion injury and LPS-induced injury (32–35). These studies are the first to examine the potential therapeutic benefits of pharmacologic inhibition of iNOS in CLP-induced AKI. In other models of CLP-induced sepsis, pharmacologic inhibition of iNOS prevents the fall in BP and other hemodynamic changes in the rat (36), and transgenic mice that are deficient in iNOS maintain arteriolar responsiveness to catecholamines and show improved survival (37). The appearance of nitrated proteins and oxidation products in the kidney during LPS-induced renal failure in rats and the ability of iNOS inhibitors to reduce these products and preserve renal function (14,15) suggest that iNOS-derived RNS are important mediators of renal injury. In these studies, L-NIL treatment ameliorated CLP-induced capillary dysfunction, morphologic injury, and renal dysfunction, suggesting a role for iNOS. Nevertheless, pharmacologic studies with iNOS inhibitors must be interpreted with caution because there are no absolutely selective iNOS inhibitors, and constitutive NOS isoforms may be induced during sepsis as well (38). L-NIL has been used extensively as an iNOS inhibitor and displays a 14-fold selectivity for iNOS compared with endothelial NOS (39). The dosage used in these studies was chosen because it did not significantly reduce basal serum NOx levels, suggesting a minimal effect on constitutive isoforms. Still, additional studies with more selective inhibitors such as 1400W (39) are needed to establish iNOS fully as an important therapeutic target.

Although induction of iNOS in inflammatory cells occurs during sepsis, sepsis-induced AKI is not associated with a significant influx of activated inflammatory cells to the kidney (10). Rather, kidney epithelial cells seem to be the major source of iNOS-derived NO in the kidney during sepsis (40). These studies extend these finding to suggest that renal tubular epithelial cells are perhaps the major source of NO-derived oxidants. Two independent methods (IVVM and immunohistochemistry) for detecting the generation of RNS both indicated the generation of RNS by renal tubules after CLP. Furthermore, both IVVM and immunohistochemistry revealed RNS generation in tubular vacuoles. The potential clinical relevance of these findings is supported by the detection of nitrated plasma 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 contributed to tubular injury. Additional support for the role of oxidants comes from the findings that the anti-inflammatory antioxidant ethyl pyruvate is protective against CLP-induced AKI (10). It must be acknowledged that oxidation of DHR to rhodamine is not solely dependent on RNS. However, the ability of L-NIL to block completely the rise in rhodamine fluorescence in CLP-treated mice compared with sham mice suggests that RNS were the major oxidants generated after CLP. It is interesting that sham mice showed a basal DHR oxidation localized to tubules that were bordered by capillaries with reduced flow that was not inhibited by L-NIL. The absence of nitrotyrosine staining in sham mice suggests that generation of ROS rather than RNS was responsible for DHR oxidation in the absence of iNOS induction.

A rapid decline in peritubular capillary perfusion accompanied by tubular injury is also a feature of LPS-induced AKI (8,17). However, in the CLP model, the decline in capillary perfusion seems to be greater than in the LPS model, and tubular morphologic changes are more severe. Still, studies in both types of sepsis models show that a change in the peritubular microenvironment is a common feature. Sepsis-induced AKI has an extremely complex etiology. While mediators of the early response by the kidney may be different in the two models (9), iNOS-mediated peritubular capillary dysfunction and tubular injury occur in both models of sepsis-induced AKI (17). This suggests that common pathways are activated in the microenvironment after inflammatory stimuli that mediate tubular injury. From a clinical perspective, therapeutic agents that target later events might be the most beneficial because downstream mediators may be relatively independent of upstream events and because therapy is generally started after initiation of sepsis (3).

Our findings have implications beyond sepsis-induced AKI. It is becoming increasingly clear that endothelial injury and peritubular capillary dysfunction may initiate and extend the pathogenesis of both acute and chronic renal injury (41–44) by compromising renal vascular responsiveness and tubule function (41,45,46). In addition, reductions in peritubular capillary perfusion causes localized hypoxia, which over time may lead to tubulointerstitial injury (47). We found that even in the absence of disease, localized decreases in peritubular capillary perfusion are associated with localized areas of oxidant generation. Therefore, even transient localized oxidant generation may sensitize the kidney to injury.

Conclusion

We have uncovered a previously unrecognized role for capillary dysfunction and tubular RNS generation in CLP-induced AKI. The close association between capillary dysfunction, RNS generation, and tubular damage suggests that the peritubular capillary/tubular microenvironment plays a critical role in sepsis-induced AKI. Furthermore, studies with the iNOS inhibitor L-NIL suggest that pharmacologic inhibition of iNOS and/or RNS should be considered as potential therapeutic approaches for the prevention of sepsis-induced AKI.

Disclosures

None.

Acknowledgments

This research was supported by a Pilot Study Grant (P.R.M.) and the Graduate Student Research Fund (L.W.) from the University of Arkansas for Medical Sciences.

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

References

  1. ↵
    Schrier RW, Wang W: Acute renal failure and sepsis. N Engl J Med 351 : 159 –169, 2004
    OpenUrlCrossRefPubMed
  2. ↵
    Wan L, Bellomo R, Di Giantomasso D, Ronco C: The pathogenesis of septic acute renal failure. Curr Opin Crit Care 9 : 496 –502, 2003
    OpenUrlCrossRefPubMed
  3. ↵
    Nguyen HB, Rivers EP, Abrahamian FM, Moran GJ, Abraham E, Trzeciak S, Huang DT, Osborn T, Stevens D, Talan DA: Severe sepsis and septic shock: Review of the literature and emergency department management guidelines. Ann Emerg Med 48 : 28 –54, 2006
    OpenUrlCrossRefPubMed
  4. ↵
    Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR: Epidemiology of severe sepsis in the United States: Analysis of incidence, outcome, and associated costs of care. Crit Care Med 29 : 1303 –1310, 2001
    OpenUrlCrossRefPubMed
  5. ↵
    Annane D, Bellissant E, Cavaillon JM: Septic shock. Lancet 365 : 63 –78, 2005
    OpenUrlCrossRefPubMed
  6. ↵
    Dauphinee SM, Karsan A: Lipopolysaccharide signaling in endothelial cells. Lab Invest 86 : 9 –22, 2006
    OpenUrlCrossRefPubMed
  7. ↵
    Yasuda H, Yuen PS, Hu X, Zhou H, Star RA: Simvastatin improves sepsis-induced mortality and acute kidney injury via renal vascular effects. Kidney Int 69 : 1535 –1542, 2006
    OpenUrlCrossRefPubMed
  8. ↵
    Wu L, Tiwari MM, Messer KJ, Holthoff JH, Gokden N, Brock RW, Mayeux PR: Peritubular capillary dysfunction and renal tubular epithelial cell stress following lipopolysaccharide administration in mice. Am J Physiol Renal Physiol 292 : F261 –F268, 2007
    OpenUrlCrossRefPubMed
  9. ↵
    Remick DG, Ward PA: Evaluation of endotoxin models for the study of sepsis. Shock 24[Suppl 1] : 7 –11, 2005
    OpenUrl
  10. ↵
    Miyaji T, Hu X, Yuen PS, Muramatsu Y, Iyer S, Hewitt SM, Star RA: Ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice. Kidney Int 64 : 1620 –1631, 2003
    OpenUrlCrossRefPubMed
  11. ↵
    Dear JW, Yasuda H, Hu X, Hieny S, Yuen PS, Hewitt SM, Sher A, Star RA: Sepsis-induced organ failure is mediated by different pathways in the kidney and liver: Acute renal failure is dependent on MyD88 but not renal cell apoptosis. Kidney Int 69 : 832 –836, 2006
    OpenUrlCrossRefPubMed
  12. ↵
    Holly MK, Dear JW, Hu X, Schechter AN, Gladwin MT, Hewitt SM, Yuen PS, Star RA: Biomarker and drug-target discovery using proteomics in a new rat model of sepsis-induced acute renal failure. Kidney Int 70 : 496 –506, 2006
    OpenUrlPubMed
  13. ↵
    Fukuyama N, Takebayashi Y, Hida M, Isida H, Ichimori K, Nakazawa H: Clinical evidence of peroxynitrite formation in chronic renal failure patients with septic shock. Free Radic Biol Med 22 : 771 –774, 1997
    OpenUrlCrossRefPubMed
  14. ↵
    Bian K, Davis K, Kuret J, Binder L, Murad F: Nitrotyrosine formation with endotoxin-induced kidney injury detected by immunohistochemistry. Am J Physiol 277 : F33 –F40, 1999
    OpenUrlPubMed
  15. ↵
    Zhang C, Walker LM, Mayeux PR: Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney. Biochem Pharmacol 59 : 203 –209, 2000
    OpenUrlCrossRefPubMed
  16. ↵
    Wu F, Wilson JX, Tyml K: Ascorbate protects against impaired arteriolar constriction in sepsis by inhibiting inducible nitric oxide synthase expression. Free Radic Biol Med 37 : 1282 –1289, 2004
    OpenUrlCrossRefPubMed
  17. ↵
    Tiwari MM, Brock RW, Kaushal GP, Mayeux PR: Disruption of renal peritubular blood flow in lipopolysaccharide-induced renal failure: Role of nitric oxide and caspases. Am J Physiol Renal Physiol 289 : F1324 –F1332, 2005
    OpenUrlCrossRefPubMed
  18. ↵
    Moore WM, Webber RK, Jerome GM, Tjoeng FS, Misko TP, Currie MG: L-N6-(1-Iminoethly)lysine: A selective inhibitor of inducible nitric oxide synthase. J Med Chem 37 : 3886 –3888, 1994
    OpenUrlCrossRefPubMed
  19. ↵
    Wang W, Faubel S, Ljubanovic D, Mitra A, Kim J, Tao Y, Soloviev A, Reznikov LL, Dinarello CA, Schrier RW, Edelstein CL: Endotoxemic acute renal failure (ARF) is attenuated in caspase-1 deficient mice. Am J Physiol Renal Physiol 288 : F997 –F1004, 2005
    OpenUrlCrossRefPubMed
  20. ↵
    Halliwell B, Whiteman M: Measuring reactive species and oxidative damage in vivo and in cell culture: How should you do it and what do the results mean? Br J Pharmacol 142 : 231 –255, 2004
    OpenUrlCrossRefPubMed
  21. ↵
    Ince C: The microcirculation is the motor of sepsis. Crit Care 9[Suppl 4] : S13 –S19, 2005
    OpenUrl
  22. ↵
    Sutton TA, Mang HE, Campos SB, Sandoval RM, Yoder MC, Molitoris BA: Injury of the renal microvascular endothelium alters barrier function after ischemia. Am J Physiol Renal Physiol 285 : F191 –F198, 2003
    OpenUrlCrossRefPubMed
  23. ↵
    Wolin MS: Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol 20 : 1430 –1442, 2000
    OpenUrlAbstract/FREE Full Text
  24. ↵
    Beckman JS, Beckman TW, Chen J, Marshall PA, Freeman BA: Apparent hydroxyl radical production by peroxynitrite: Implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci U S A 87 : 1620 –1624, 1990
    OpenUrlAbstract/FREE Full Text
  25. ↵
    Brune B, Zhou J, von Knethen A: Nitric oxide, oxidative stress, and apoptosis. Kidney Int Suppl 63 : S22 –S24, 2003
    OpenUrl
  26. ↵
    Pryor WA, Squadrito GL: The chemistry of peroxynitrite: A product from the reaction of nitric oxide with superoxide. Am J Physiol 268 : L699 –L722, 1995
    OpenUrlPubMed
  27. ↵
    MacMillan-Crow LA, Crow JP, Kerby JK, Beckman JS: Nitration and inactivation of manganese superoxide dismutase in chronic rejection of human renal allografts. Proc Natl Acad Sci U S A 93 : 11853 –11858, 1996
    OpenUrlAbstract/FREE Full Text
  28. ↵
    Neumann P, Gertzberg N, Vaughan E, Weisbrot J, Woodburn R, Lambert W, Johnson A: Peroxynitrite mediates TNF-alpha-induced endothelial barrier dysfunction and nitration of actin. Am J Physiol Lung Cell Mol Physiol 290 : L674 –L684, 2006
    OpenUrlCrossRefPubMed
  29. ↵
    Paxian M, Keller SA, Cross B, Huynh TT, Clemens MG: High-resolution visualization of oxygen distribution in the liver in vivo. Am J Physiol Gastrointest Liver Physiol 286 : G37 –G44, 2004
    OpenUrlCrossRefPubMed
  30. ↵
    Wolin MS, Ahmad M, Gupte SA: The sources of oxidative stress in the vessel wall. Kidney Int 67 : 1659 –1661, 2005
    OpenUrlCrossRefPubMed
  31. ↵
    Xie Y-W, Wolin MS: Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration: Involvement in response to hypoxia/reoxygenation. Circulation 94 : 2580 –2586, 1996
    OpenUrlAbstract/FREE Full Text
  32. ↵
    Yaqoob M, Edelstein CL, Wieder ED, Alkhunaizi AM, Gengaro PE, Nemenoff RA, Schrier RW: Nitric oxide kinetics during hypoxia in proximal tubules: Effects of acidosis and glycine. Kidney Int 49 : 1314 –1319, 1996
    OpenUrlCrossRefPubMed
  33. Noiri E, Peresleni T, Miller F, Goligorsky MS: In vivo targeting of inducible NO synthase with oligodeoxynucleotides protects rat kidney against ischemia. J Clin Invest 97 : 2377 –2383, 1996
    OpenUrlCrossRefPubMed
  34. Chiao H, Kohda Y, McLeroy P, Craig L, Housini I, Star RA: alpha-Melanocyte-stimulating hormone protects against renal injury after ischemia in mice and rats. J Clin Invest 99 : 1165 –1172, 1997
    OpenUrlCrossRefPubMed
  35. ↵
    Walker LM, Walker PD, Imam SZ, Ali SF, Mayeux PR: Evidence for peroxynitrite formation in renal ischemia-reperfusion injury: Studies with the inducible nitric oxide synthase inhibitor L-N6-(1-iminoethyl)-lysine. J Pharmacol Exp Ther 295 : 417 –422, 2000
    OpenUrlAbstract/FREE Full Text
  36. ↵
    Kadoi Y, Goto F: Selective inducible nitric oxide inhibition can restore hemodynamics, but does not improve neurological dysfunction in experimentally-induced septic shock in rats. Anesth Analg 99 : 212 –220, 2004
    OpenUrlCrossRefPubMed
  37. ↵
    Hollenberg SM, Broussard M, Osman J, Parrillo JE: Increased microvascular reactivity and improved mortality in septic mice lacking inducible nitric oxide synthase. Cir Res 86 : 774 –778, 2000
    OpenUrlAbstract/FREE Full Text
  38. ↵
    Mayeux PR, Garner HR, Gibson JD, Beanum VC: Effect of lipopolysaccharide on nitric oxide synthase activity in rat proximal tubules. Biochem Pharmacol 49 : 115 –118, 1995
    OpenUrlCrossRefPubMed
  39. ↵
    Boer R, Ulrich WR, Klein T, Mirau B, Haas S, Baur I: The inhibitory potency and selectivity of arginine substrate site nitric-oxide synthase inhibitors is solely determined by their affinity toward the different isoenzymes. Mol Pharmacol 58 : 1026 –1034, 2000
    OpenUrlAbstract/FREE Full Text
  40. ↵
    Bultinck J, Sips P, Vakaet L, Brouckaert P, Cauwels A: Systemic NO production during (septic) shock depends on parenchymal and not on hematopoietic cells: In vivo iNOS expression pattern in (septic) shock. FASEB J 20 : 2363 –2365, 2006
    OpenUrlCrossRefPubMed
  41. ↵
    O'Riordan E, Chen J, Brodsky SV, Smirnova I, Li H, Goligorsky MS: Endothelial cell dysfunction: The syndrome in making. Kidney Int 67 : 1654 –1658, 2005
    OpenUrlCrossRefPubMed
  42. Yuan HT, Li XZ, Pitera JE, Long DA, Woolf AS: Peritubular capillary loss after mouse acute nephrotoxicity correlates with down-regulation of vascular endothelial growth factor-A and hypoxia-inducible factor-1 alpha. Am J Pathol 163 : 2289 –2301, 2003
    OpenUrlCrossRefPubMed
  43. Molitoris BA, Sutton TA: Endothelial injury and dysfunction: Role in the extension phase of acute renal failure. Kidney Int 66 : 496 –499, 2004
    OpenUrlCrossRefPubMed
  44. ↵
    Brodsky SV, Yamamoto T, Tada T, Kim B, Chen J, Kajiya F, Goligorsky MS: Endothelial dysfunction in ischemic acute renal failure: Rescue by transplanted endothelial cells. Am J Physiol Renal Physiol 282 : F1140 –F1149, 2002
    OpenUrlCrossRefPubMed
  45. ↵
    Piepot HA, Groeneveld AB, van Lambalgen AA, Sipkema P: Endotoxin impairs endothelium-dependent vasodilation more in the coronary and renal arteries than in other arteries of the rat. J Surg Res 110 : 413 –418, 2003
    OpenUrlCrossRefPubMed
  46. ↵
    Linas SL, Repine JE: Endothelial cells regulate proximal tubule epithelial cell sodium transport. Kidney Int 55 : 1251 –1258, 1999
    OpenUrlCrossRefPubMed
  47. ↵
    Nangaku M: Chronic hypoxia and tubulointerstitial injury: A final common pathway to end-stage renal failure. J Am Soc Nephrol 17 : 17 –25, 2006
    OpenUrlAbstract/FREE Full Text
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Evidence for the Role of Reactive Nitrogen Species in Polymicrobial Sepsis-Induced Renal Peritubular Capillary Dysfunction and Tubular Injury
Liping Wu, Neriman Gokden, Philip R. Mayeux
JASN Jun 2007, 18 (6) 1807-1815; DOI: 10.1681/ASN.2006121402
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