Philip R. Mayeux

PATHOBIOLOGY OF LIPOPOLYSACCHARIDE

Lipopolysaccharide is a component of the gram-negative bacterial cell wall that is responsible for 25,000-50,000 deaths in the United States each year. The sequelae of gram-negative infection and septicemia leading to death include fever, hypotension with inadequate tissue perfusion, and disseminated intravascular coagulation. It is clear that different cell types respond differently to lipopolysaccharide. Furthermore, various autacoids and cytokines are released that can affect cellular function even in cell types that do not recognize lipopolysaccharide. Despite advances made in the etiology of septic shock and organ failure, therapy is still for the most part supportive and largely ineffective. The aim of this review is to summarize the current understanding of the role of lipopolysaccharide in the development of septicemia by examining signal transduction and therapeutic approaches.

Resveratrol improves renal microcirculation, protects the tubular epithelium, and prolongs survival in a mouse model of sepsis-induced acute kidney injury.

The mortality rate of patients who develop acute kidney injury during sepsis nearly doubles. The effectiveness of therapy is hampered because it is usually initiated only after the onset of symptoms. As renal microvascular failure during sepsis is correlated with the generation of reactive nitrogen species, the therapeutic potential of resveratrol, a polyphenol vasodilator that is also capable of scavenging reactive nitrogen species, was investigated using the cecal ligation and puncture (CLP) murine model of sepsis-induced acute kidney injury. Resveratrol when given at 5.5 h following CLP reversed the decline in cortical capillary perfusion, assessed by intravital microscopy, at 6 h in a dose-dependent manner. Resveratrol produced the greatest improvement in capillary perfusion and increased renal blood flow and the glomerular filtration rate without raising systemic pressure. A single dose at 6 h after CLP was unable to improve renal microcirculation assessed at 18 h; however, a second dose at 12 h significantly improved microcirculation and decreased the levels of reactive nitrogen species in tubules, while improving renal function. Moreover, resveratrol given at 6, 12, and 18 h significantly improved survival. Hence, resveratrol may have a dual mechanism of action to restore the renal microcirculation and scavenge reactive nitrogen species, thus protecting the tubular epithelium even when administered after the onset of sepsis.

Evidence for the Role of Reactive Nitrogen Species in Polymicrobial Sepsis-Induced Renal Peritubular Capillary Dysfunction and Tubular Injury

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.

Development of Oxidative Stress in the Peritubular Capillary Microenvironment Mediates Sepsis-Induced Renal Microcirculatory Failure and Acute Kidney Injury

Acute kidney injury is a frequent and serious complication of sepsis. To better understand the development of sepsis-induced acute kidney injury, we performed the first time-dependent studies to document changes in renal hemodynamics and oxidant generation in the peritubular microenvironment using the murine cecal ligation and puncture (CLP) model of sepsis. CLP caused an increase in renal capillary permeability at 2 hours, followed by decreases in mean arterial pressure, renal blood flow (RBF), and renal capillary perfusion at 4 hours, which were sustained through 18 hours. The decline in hemodynamic parameters was associated with hypoxia and oxidant generation in the peritubular microenvironment and a decrease in glomerular filtration rate. The role of oxidants was assessed using the superoxide dismutase mimetic/peroxynitrite scavenger MnTMPyP [Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin]. At 10 mg/kg administered 6 hours after CLP, MnTMPyP did not alter blood pressure, but blocked superoxide and peroxynitrite generation, reversed the decline in RBF, capillary perfusion, and glomerular filtration rate, preserved tubular architecture, and increased 48-hour survival. However, MnTMPyP administered at CLP did not prevent capillary permeability or the decrease in RBF and capillary perfusion, which suggests that these early events are not mediated by oxidants. These data demonstrate that renal hemodynamic changes occur early after sepsis and that targeting the later oxidant generation can break the cycle of injury and enable the microcirculation and renal function to recover.

Role of nitric oxide in lipopolysaccharide-induced oxidant stress in the rat kidney

Lipopolysaccharide (LPS)-induced renal oxidant injury and the role of nitric oxide (NO) were evaluated using the inducible nitric oxide synthase (iNOS) inhibitor L-iminoethyl-lysine (L-NIL). One group of male rats received LPS (Salmonella minnesota; 2 mg/kg, i.v.). A second group received LPS plus L-NIL (3 mg/kg, i.p.). A third group received saline i.v. At 6 hr, iNOS protein was induced in the kidney cortex, and plasma nitrate/nitrite levels were increased from 4 +/- 2 nmol/mL in the Saline group to 431 +/- 23 nmol/mL in the LPS group. The value for the LPS + L-NIL group was reduced significantly to 42 +/- 9 nmol/mL. LPS increased blood urea nitrogen levels from 13 +/- 1 to 47 +/- 3 mg/dL. LPS + L-NIL reduced these levels significantly to 29 +/- 2 mg/dL. Plasma creatinine levels were unchanged in all groups. Tissue lipid peroxidation products in the kidney were increased from 0.16 +/- 0.01 nmol/mg in the Saline group to 0.30 +/- 0.03 nmol/mg in the LPS group. LPS + L-NIL reduced the values significantly to 0.22 +/- 0.02 nmol/mg. Intracellular glutathione levels were decreased in the kidneys from 1.32 +/- 0.1 nmol/mg in the Saline group to 0.66 +/- 0.08 nmol/mg in the LPS group. LPS + L-NIL increased the levels significantly to 0.99 +/- 0.13 nmol/mg. LPS increased the 3-nitrotyrosine-protein adducts in renal tubules as detected by immunohistochemistry, indicating the generation of peroxynitrite. L-NIL decreased adduct formation. These data indicated that LPS-induced NO generation resulted in peroxynitrite formation and oxidant stress in the kidney and that inhibitors of iNOS may offer protection against LPS-induced renal toxicity.

Inactivation of renal mitochondrial respiratory complexes and manganese superoxide dismutase during sepsis: mitochondria-targeted antioxidant mitigates injury.

Acute kidney injury (AKI) is a complication of sepsis and leads to a high mortality rate. Human and animal studies suggest that mitochondrial dysfunction plays an important role in sepsis-induced multi-organ failure; however, the specific mitochondrial targets damaged during sepsis remain elusive. We used a clinically relevant cecal ligation and puncture (CLP) murine model of sepsis and assessed renal mitochondrial function using high-resolution respirometry, renal microcirculation using intravital microscopy, and renal function. CLP caused a time-dependent decrease in mitochondrial complex I and II/III respiration and reduced ATP. By 4 h after CLP, activity of manganese superoxide dismutase (MnSOD) was decreased by 50% and inhibition was sustained through 36 h. These events were associated with increased mitochondrial superoxide generation. We then evaluated whether the mitochondria-targeted antioxidant Mito-TEMPO could reverse renal mitochondrial dysfunction and attenuate sepsis-induced AKI. Mito-TEMPO (10 mg/kg) given at 6 h post-CLP decreased mitochondrial superoxide levels, protected complex I and II/III respiration, and restored MnSOD activity by 18 h. Mito-TEMPO also improved renal microcirculation and glomerular filtration rate. Importantly, even delayed therapy with a single dose of Mito-TEMPO significantly increased 96-h survival rate from 40% in untreated septic mice to 80%. Thus, sepsis causes sustained inactivation of three mitochondrial targets that can lead to increased mitochondrial superoxide. Importantly, even delayed therapy with Mito-TEMPO alleviated kidney injury, suggesting that it may be a promising approach to treat septic AKI.

The endothelium in sepsis

ABSTRACT Sepsis affects practically all aspects of endothelial cell (EC) function and is thought to be the key factor in the progression from sepsis to organ failure. Endothelial functions affected by sepsis include vasoregulation, barrier function, inflammation, and hemostasis. These are among other mechanisms often mediated by glycocalyx shedding, such as abnormal nitric oxide metabolism, up-regulation of reactive oxygen species generation due to down-regulation of endothelial-associated antioxidant defenses, transcellular communication, proteases, exposure of adhesion molecules, and activation of tissue factor. This review covers current insight in EC-associated hemostatic responses to sepsis and the EC response to inflammation. The endothelial cell lining is highly heterogeneous between different organ systems and consequently also in its response to sepsis. In this context, we discuss the response of the endothelial cell lining to sepsis in the kidney, liver, and lung. Finally, we discuss evidence as to whether the EC response to sepsis is adaptive or maladaptive. This study is a result of an Acute Dialysis Quality Initiative XIV Sepsis Workgroup meeting held in Bogota, Columbia, between October 12 and 15, 2014.

Molecular Cloning and Functional Characterization of a Vasotocin Receptor Subtype That Is Expressed in the Shell Gland and Brain of the Domestic Chicken

Abstract In chickens, oviposition is correlated with increased plasma levels of the neurohypophysial hormone vasotocin, and vasotocin stimulates contraction of uterine strips in vitro. A gene encoding a vasotocin receptor subtype that we have designated the VT1 receptor was cloned from the domestic chicken. The open reading frame encodes a 370-amino acid polypeptide that displays seven segments of hydrophobic amino acids, typical of guanine nucleotide-protein-coupled receptors. Other structural features of the VT1 receptor include two potential N-linked glycosylation sites in the extracellular N-terminal region, a conserved aspartic acid in transmembrane domain 2 that is found in nearly all guanine nucleotide-protein-coupled receptors, and two potential protein kinase C phosphorylation sites in the third intracellular loop and C-terminal tail. Expressed VT1 receptors in COS7 cells bind neurohypophysial hormones with the following rank order of potency: vasotocin ≅ vasopressin > oxytocin ≅ mesotocin > isotocin. In addition, the expressed VT1 receptor mediates vasotocin-induced phosphatidylinositol turnover and Ca2+ mobilization. In the chicken, expression of VT1 receptor gene transcripts is limited to the shell gland (uterus) and the brain. Thus, the VT1 receptor that we have cloned may mediate contractions of the shell gland during oviposition and activate reproductive behaviors known to be stimulated by vasotocin in lower vertebrates.

Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin alpha are capable of generating biologically active IL-1beta from its precursor pro-IL-1beta. Amino-acid sequencing analysis reveals that meprin A and meprin alpha cleave pro-IL-1beta at the His(115)-Asp(116) bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin beta site. The biological activity of the pro-IL-1beta cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1beta product produced by meprin beta or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1beta, meprin inhibitor actinonin significantly reduces levels of serum IL-1beta. Meprin A and meprin alpha may therefore play a critical role in the production of active IL-1beta during inflammation and tissue injury.

Hemodynamic changes in the kidney in a pediatric rat model of sepsis-induced acute kidney injury

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17-18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min(-1)·g(-1)), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.