Rhea Bhargava

Circulating IL-6 mediates lung injury via CXCL1 production after acute kidney injury in mice

Serum IL-6 is increased in patients with acute kidney injury (AKI) and is associated with prolonged mechanical ventilation and increased mortality. Inhibition of IL-6 in mice with AKI reduces lung injury associated with a reduction in the chemokine CXCL1 and lung neutrophils. Whether circulating IL-6 or locally produced lung IL-6 mediates lung injury after AKI is unknown. We hypothesized that circulating IL-6 mediates lung injury after AKI by increasing lung endothelial CXCL1 production and subsequent neutrophil infiltration. To test the role of circulating IL-6 in AKI-mediated lung injury, recombinant murine IL-6 was administered to IL-6-deficient mice. To test the role of CXCL1 in AKI-mediated lung injury, CXCL1 was inhibited by use of CXCR2-deficient mice and anti-CXCL1 antibodies in mice with ischemic AKI or bilateral nephrectomy. Injection of recombinant IL-6 to IL-6-deficient mice with AKI increased lung CXCL1 and lung neutrophils. Lung endothelial CXCL1 was increased after AKI. CXCR2-deficient and CXCL1 antibody-treated mice with ischemic AKI or bilateral nephrectomy had reduced lung neutrophil content. In summary, we demonstrate for the first time that circulating IL-6 is a mediator of lung inflammation and injury after AKI. Since serum IL-6 is increased in patients with either AKI or acute lung injury and predicts prolonged mechanical ventilation and increased mortality in both conditions, our data suggest that serum IL-6 is not simply a biomarker of poor outcomes but a pathogenic mediator of lung injury.

Acute lung injury and acute kidney injury are established by four hours in experimental sepsis and are improved with pre, but not post, sepsis administration of TNF-α antibodies.

Introduction Acute kidney injury (AKI) and acute lung injury (ALI) are serious complications of sepsis. AKI is often viewed as a late complication of sepsis. Notably, the onset of AKI relative to ALI is unclear as routine measures of kidney function (BUN and creatinine) are insensitive and increase late. In this study, we hypothesized that AKI and ALI would occur simultaneously due to a shared pathophysiology (i.e., TNF-α mediated systemic inflammatory response syndrome [SIRS]), but that sensitive markers of kidney function would be required to identify AKI. Methods Sepsis was induced in adult male C57B/6 mice with 5 different one time doses of intraperitoneal (IP) endotoxin (LPS) (0.00001, 0.0001, 0.001, 0.01, or 0.25 mg) or cecal ligation and puncture (CLP). SIRS was assessed by serum proinflammatory cytokines (TNF-α, IL-1β, CXCL1, IL-6), ALI was assessed by lung inflammation (lung myeloperoxidase [MPO] activity), and AKI was assessed by serum creatinine, BUN, and glomerular filtration rate (GFR) (by FITC-labeled inulin clearance) at 4 hours. 20 µgs of TNF-α antibody (Ab) or vehicle were injected IP 2 hours before or 2 hours after IP LPS. Results Serum cytokines increased with all 5 doses of LPS; AKI and ALI were detected within 4 hours of IP LPS or CLP, using sensitive markers of GFR and lung inflammation, respectively. Notably, creatinine did not increase with any dose; BUN increased with 0.01 and 0.25 mg. Remarkably, GFR was reduced 50% in the 0.001 mg LPS dose, demonstrating that dramatic loss of kidney function can occur in sepsis without a change in BUN or creatinine. Prophylactic TNF-α Ab reduced serum cytokines, lung MPO activity, and BUN; however, post-sepsis administration had no effect. Conclusions ALI and AKI occur together early in the course of sepsis and TNF-α plays a role in the early pathogenesis of both.

Heparanase mediates renal dysfunction during early sepsis in mice

Heparanase, a heparan sulfate‐specific glucuronidase, mediates the onset of pulmonary neutrophil adhesion and inflammatory lung injury during early sepsis. We hypothesized that glomerular heparanase is similarly activated during sepsis and contributes to septic acute kidney injury (AKI). We induced polymicrobial sepsis in mice using cecal ligation and puncture (CLP) in the presence or absence of competitive heparanase inhibitors (heparin or nonanticoagulant N‐desulfated re‐N‐acetylated heparin [NAH]). Four hours after surgery, we collected serum and urine for measurement of renal function and systemic inflammation, invasively determined systemic hemodynamics, harvested kidneys for histology/protein/mRNA, and/or measured glomerular filtration by inulin clearance. CLP‐treated mice demonstrated early activation of glomerular heparanase with coincident loss of glomerular filtration, as indicated by a >twofold increase in blood urea nitrogen (BUN) and a >50% decrease in inulin clearance (P < 0.05) in comparison to sham mice. Administration of heparanase inhibitors 2 h prior to CLP attenuated sepsis‐induced loss of glomerular filtration rate, demonstrating that heparanase activation contributes to early septic renal dysfunction. Glomerular heparanase activation was not associated with renal neutrophil influx or altered vascular permeability, in marked contrast to previously described effects of pulmonary heparanase on neutrophilic lung injury during sepsis. CLP induction of renal inflammatory gene (IL‐6, TNF‐α, IL‐1β) expression was attenuated by NAH pretreatment. While serum inflammatory indices (KC, IL‐6, TNF‐α, IL‐1β) were not impacted by NAH pretreatment, heparanase inhibition attenuated the CLP‐induced increase in serum IL‐10. These findings demonstrate that glomerular heparanase is active during sepsis and contributes to septic renal dysfunction via mechanisms disparate from heparanase‐mediated lung injury.

Intratracheal IL-6 Protects against Lung Inflammation in Direct, but Not Indirect, Causes of Acute Lung Injury in Mice

Introduction Serum and bronchoalveolar fluid IL-6 are increased in patients with acute respiratory distress syndrome (ARDS) and predict prolonged mechanical ventilation and poor outcomes, although the role of intra-alveolar IL-6 in indirect lung injury is unknown. We investigated the role of endogenous and exogenous intra-alveolar IL-6 in AKI-mediated lung injury (indirect lung injury), intraperitoneal (IP) endotoxin administration (indirect lung injury) and, for comparison, intratracheal (IT) endotoxin administration (direct lung injury) with the hypothesis that IL-6 would exert a pro-inflammatory effect in these causes of acute lung inflammation. Methods Bronchoalveolar cytokines (IL-6, CXCL1, TNF-α, IL-1β, and IL-10), BAL fluid neutrophils, lung inflammation (lung cytokines, MPO activity [a biochemical marker of neutrophil infiltration]), and serum cytokines were determined in adult male C57Bl/6 mice with no intervention or 4 hours after ischemic AKI (22 minutes of renal pedicle clamping), IP endotoxin (10 µg), or IT endotoxin (80 µg) with and without intratracheal (IT) IL-6 (25 ng or 200 ng) treatment. Results Lung inflammation was similar after AKI, IP endotoxin, and IT endotoxin. BAL fluid IL-6 was markedly increased after IT endotoxin, and not increased after AKI or IP endotoxin. Unexpectedly, IT IL-6 exerted an anti-inflammatory effect in healthy mice characterized by reduced BAL fluid cytokines. IT IL-6 also exerted an anti-inflammatory effect in IT endotoxin characterized by reduced BAL fluid cytokines and lung inflammation; IT IL-6 had no effect on lung inflammation in AKI or IP endotoxin. Conclusion IL-6 exerts an anti-inflammatory effect in direct lung injury from IT endotoxin, yet has no role in the pathogenesis or treatment of indirect lung injury from AKI or IP endotoxin. Since intra-alveolar inflammation is important in the pathogenesis of direct, but not indirect, causes of lung inflammation, IT anti-inflammatory treatments may have a role in direct, but not indirect, causes of ARDS.

Cytokine production increases and cytokine clearance decreases in mice with bilateral nephrectomy

BACKGROUND Serum cytokines are increased in patients with acute kidney injury (AKI) and predict increased mortality. It is widely assumed that increased renal production of cytokines is the source of increased serum cytokines; the role of extra-renal cytokine production and impaired renal cytokine clearance is less well studied. We hypothesized that cytokine production in AKI was mononuclear phagocyte dependent, independent of production by the kidneys, and that serum cytokine clearance would be impaired in AKI. METHODS Bilateral nephrectomy was used as a model of AKI to assess cytokine production independent of kidney cytokine production. Mononuclear phagocytes were depleted utilizing intravenous (IV) administration of liposome-encapsulated clodronate (LEC). Twenty-three serum cytokines were determined utilizing a multiplex cytokine kit. Proteins for cytokines were determined in the spleen and liver by enzyme-linked immunosorbent assay. Recombinant cytokines were injected by IV into mice with bilateral nephrectomy to determine the effect of absent kidney function on serum cytokine clearance. RESULTS Serum interleukin (IL)-6, chemokine (C-X-C motif) ligand 1 (CXCL1), IL-10, IL-1β, monocyte chemotactic protein 1 (MCP-1), IL-5 and eotaxin were increased in the serum of mice after bilateral nephrectomy and were reduced with LEC. Serum IL-12p40 and regulated upon activation, normal T-cell expressed, and secreted (RANTES) were increased after bilateral nephrectomy and were further increased with LEC. Spleen IL-6, CXCL1, IL-10 and IL-1β and liver IL-6 and IL-10 were increased after bilateral nephrectomy. After IV injection, IL-6, CXCL1, IL-10 and IL-1β had a prolonged serum cytokine appearance in mice with bilateral nephrectomy versus sham operation. CONCLUSIONS Increased mononuclear phagocyte production and impaired renal clearance contribute to serum cytokine accumulation in AKI, independent of kidney injury. The effect of AKI on cytokine production and clearance may contribute to the increased mortality of patients with AKI.

Prolonged acute kidney injury exacerbates lung inflammation at 7 days post-acute kidney injury

Patients with acute kidney injury (AKI) have increased mortality; data suggest that the duration, not just severity, of AKI predicts increased mortality. Animal models suggest that AKI is a multisystem disease that deleteriously affects the lungs, heart, brain, intestine, and liver; notably, these effects have only been examined within 48 h, and longer term effects are unknown. In this study, we examined the longer term systemic effects of AKI, with a focus on lung injury. Mice were studied 7 days after an episode of ischemic AKI (22 min of renal pedicle clamping and then reperfusion) and numerous derangements were present including (1) lung inflammation; (2) increased serum proinflammatory cytokines; (3) liver injury; and (4) increased muscle catabolism. Since fluid overload may cause respiratory complications post‐AKI and fluid management is a critical component of post‐AKI care, we investigated various fluid administration strategies in the development of lung inflammation post‐AKI. Four different fluid strategies were tested – 100, 500, 1000, or 2000 μL of saline administered subcutaneously daily for 7 days. Interestingly, at 7 days post‐AKI, the 1000 and 2000 μL fluid groups had less severe AKI and less severe lung inflammation versus the 100 and 500 μL groups. In summary, our data demonstrate that appropriate fluid management after an episode of ischemic AKI led to both (1) faster recovery of kidney function and (2) significantly reduced lung inflammation, consistent with the notion that interventions to shorten AKI duration have the potential to reduce complications and improve patient outcomes.

CaMK4 compromises podocyte function in autoimmune and nonautoimmune kidney disease

Podocyte malfunction occurs in autoimmune and nonautoimmune kidney disease. Calcium signaling is essential for podocyte injury, but the role of Ca2+/calmodulin–dependent kinase (CaMK) signaling in podocytes has not been fully explored. We report that podocytes from patients with lupus nephritis and focal segmental glomerulosclerosis and lupus-prone and lipopolysaccharide- or adriamycin-treated mice display increased expression of CaMK IV (CaMK4), but not CaMK2. Mechanistically, CaMK4 modulated podocyte motility by altering the expression of the GTPases Rac1 and RhoA and suppressed the expression of nephrin, synaptopodin, and actin fibers in podocytes. In addition, it phosphorylated the scaffold protein 14-3-3&bgr;, which resulted in the release and degradation of synaptopodin. Targeted delivery of a CaMK4 inhibitor to podocytes preserved their ultrastructure, averted immune complex deposition and crescent formation, and suppressed proteinuria in lupus-prone mice and proteinuria in mice exposed to lipopolysaccharide-induced podocyte injury by preserving nephrin/synaptopodin expression. In animals exposed to adriamycin, podocyte-specific delivery of a CaMK4 inhibitor prevented and reversed podocyte injury and renal disease. We conclude that CaMK4 is pivotal in immune and nonimmune podocyte injury and that its targeted cell-specific inhibition preserves podocyte structure and function and should have therapeutic value in lupus nephritis and podocytopathies, including focal segmental glomerulosclerosis.

Early peritoneal dialysis reduces lung inflammation in mice with ischemic acute kidney injury

Although dialysis has been used in the care of patients with acute kidney injury (AKI) for over 50 years, very little is known about the potential benefits of uremic control on systemic complications of AKI. Since the mortality of AKI requiring renal replacement therapy (RRT) is greater than half in the intensive care unit, a better understanding of the potential of RRT to improve outcomes is urgently needed. Therefore, we sought to develop a technically feasible and reproducible model of RRT in a mouse model of AKI. Models of low- and high-dose peritoneal dialysis (PD) were developed and their effect on AKI, systemic inflammation, and lung injury after ischemic AKI was examined. High-dose PD had no effect on AKI, but effectively cleared serum IL-6, and dramatically reduced lung inflammation, while low-dose PD had no effect on any of these three outcomes. Both models of RRT using PD in AKI in mice reliably lowered urea in a dose-dependent fashion. Thus, use of these models of PD in mice with AKI has great potential to unravel the mechanisms by which RRT may improve the systemic complications that have led to increased mortality in AKI. In light of recent data demonstrating reduced serum IL-6 and improved outcomes with prophylactic PD in children, we believe that our results are highly clinically relevant.

Calcium/Calmodulin Kinase IV Controls the Function of Both T Cells and Kidney Resident Cells

Calcium calmodulin kinase IV (CaMK4) regulates multiple processes that significantly contribute to the lupus-related pathology by controlling the production of IL-2 and IL-17 by T cells, the proliferation of mesangial cells, and the function and structure of podocytes. CaMK4 is also upregulated in podocytes from patients with focal segmental glomerulosclerosis (FSGS). In both immune and non-immune podocytopathies, CaMK4 disrupts the structure and function of podocytes. In lupus-prone mice, targeted delivery of a CaMK4 inhibitor to CD4+ T cells suppresses both autoimmunity and the development of nephritis. Targeted delivery though to podocytes averts the deposition of immune complexes without affecting autoimmunity in lupus-prone mice and averts pathology induced by adriamycin in normal mice. Therefore, targeted delivery of a CaMK4 inhibitor to podocytes holds high therapeutic promise for both immune (lupus nephritis) and non-immune (FSGS) podocytopathies.

Rhabdomyolysis as the first manifestation of human immunodeficiency virus infection

Renal tubular acidosis (RTA) is a known complication of anti-retroviral medications. The presence of RTA in treatment naive-HIV patients is rare. A 49-year-old Caucasian woman presented with recurrent non-anion gap metabolic acidosis, AKI, rhabdomyolysis and hypokalemia on several occasions. Diagnosis of acquired distal RTA due to HIV was made given the history and laboratory data. To the best of our knowledge, this is the first case of HIV diagnosed with an initial presentation of rhabdomyolysis. We believe that acute renal failure was due to hypokalemia precipitating rhabdomyolysis caused by HIV-induced dRTA which was further exacerbated by amphetamine use.