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-1ß, 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-1ß and liver IL-6 and IL-10 were increased after bilateral nephrectomy. After IV injection, IL-6, CXCL1, IL-10 and IL-1ß 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.

Increased macrophage infiltration and fractalkine expression in cisplatin-induced acute renal failure in mice.

Inflammatory mechanisms contribute to cisplatin-induced acute renal failure (CisARF). Our first aim was to determine renal macrophage infiltration in CisARF. A more than 2-fold increase in CD11b-positive macrophages in the kidney on day 2 preceded the increase in blood urea nitrogen (BUN) and serum creatinine (SCr). Our next aim was to determine the chemoattractant for macrophage infiltration in CisARF. Fractalkine (CX(3)CL1) is expressed on activated endothelial cells and is a potent chemoattractant for macrophages that express its receptor (CX(3)CR1). Immunoblotting showed that whole-kidney CX(3)CL1 expression on days 1, 2, and 3 after cisplatin administration was increased. On immunofluorescence, the intensity of renal endothelial staining of CX(3)CL1 in blood vessels was significantly increased on day 2. Circulating von Willebrand factor (vWF), a measure of systemic endothelial injury, was increased on day 2. Next we determined whether macrophages played an injurious role in CisARF. Macrophages were depleted with injections of liposome-encapsulated clodronate (LEC). LEC resulted in a decrease in renal CD11b-positive macrophages on day 3. However, LEC-treated mice were not protected from CisARF on day 3. To determine the role of CX(3)CR1, both a specific anti-CX(3) CR1 antibody and CX(3) CR1(-/-) mice were used. Administration of the CX(3)CR1 antibody and CX(3) CR1(-/-) mice was not protected against CisARF. In summary, in CisARF, macrophage infiltration in the kidney, CX(3)CL1 expression in whole kidney and blood vessels, and the increase in circulating vWF precede BUN and SCr increase. However, inhibition of macrophage infiltration in the kidney or CX(3)CR1 blockade is not sufficient to prevent CisARF.

Fractalkine receptor (CX3CR1) inhibition is protective against ischemic acute renal failure in mice.

Fractalkine (CX3CL1) is expressed on injured endothelial cells and is a potent chemoattractant and adhesion molecule for macrophages carrying the fractalkine receptor (CX3CR1). The aim of this study was to investigate the role of CX3CL1, and its ligand CX3CR1, in ischemic acute renal failure (ARF) in mice. On immunoblotting, CX3CL1 protein expression in the kidney increased markedly in ischemic ARF. On immunofluorescence staining, the intensity of CX3CL1 staining in blood vessels was significantly more prominent in ischemic ARF compared with controls. A specific anti-CX3CR1 antibody (25 microg i.p. 1 h before induction of ischemia) was functionally and histologically protective against ischemic ARF. CX3CR1 is predominantly expressed on macrophages. Macrophage infiltration in the kidney in ischemic ARF was significantly decreased after anti-CX3CR1 antibody treatment. To determine the role of macrophages in ischemic ARF, macrophages in the kidney were depleted using liposomal-encapsulated clodronate (LEC). LEC resulted in significant functional and histological protection against ischemic ARF. In summary, in ischemic ARF, 1) there is upregulation of CX3CL1 protein in the kidney, specifically in blood vessels; 2) CX3CR1 inhibition using a specific antibody is partially protective and is associated with reduced macrophage infiltration in the kidney; and 3) macrophage depletion in the kidney is protective.

Interleukin-18 binding protein transgenic mice are protected against ischemic acute kidney injury.

IL-18 function is neutralized in IL-18 binding protein transgenic (IL-18BP Tg) mice. First, we determined whether IL-18BP Tg mice are protected against ischemic acute kidney injury (AKI). Ischemic AKI was induced by bilateral renal pedicle clamping. IL-18BP Tg mice were functionally and histologically protected against ischemic AKI as determined by blood urea nitrogen, serum creatinine, and acute tubular necrosis score. We have demonstrated that the injurious effect of IL-18 in the kidney is independent of neutrophils and lymphocytes. Thus the effect of IL-18 inhibition on renal macrophage infiltration was determined. The number of macrophages was significantly reduced in IL-18BP Tg compared with wild-type kidneys. To determine the cytokines and chemokines that are dependent on IL-18, we performed flow cytometry based assays. Multiple chemokines/cytokines, IL-3, IL-6, IL-15, IL-18, leukemia inhibitory factor, macrophage colony-stimulating factor, macrophage inflammatory protein-2, granulocyte-macrophage colony-stimulating factor, and monocyte chemotactic protein-1 were significantly increased in AKI vs. sham kidneys. Only CXCL1 (also known as KC or IL-8) was significantly increased in AKI vs. sham kidneys and significantly reduced in IL-18BP Tg AKI vs. wild-type AKI kidneys. To determine whether macrophages are the source of CXCL1 in the kidney, we depleted macrophages with liposomal encapsulated clodronate. CXCL1 was significantly decreased in macrophage-depleted vs. control AKI mice. In summary, in ischemic AKI in mice, 1) IL-18BP Tg mice are functionally and histologically protected, 2) macrophage infiltration in the kidney and CXCL1 are significantly reduced in IL-18BP Tg mice, and 3) macrophage depletion significantly reduces CXCL1 in the kidney. In conclusion, protection against ischemic AKI in IL-18BP Tg mice is associated with less macrophage infiltration and less production of CXCL1 in the kidney.

Proximal tubules from caspase-1-deficient mice are protected against hypoxia-induced membrane injury.

BACKGROUND: Caspase-1 is a proinflammatory caspase via activation of the cytokine IL-18. We have recently demonstrated that the caspase-1-mediated production of IL-18 plays a deleterious role in ischaemic acute renal failure (ARF) which is independent of neutrophils and CD4+ T cells. The role of caspase-1 in hypoxia-induced membrane injury of proximal tubules (PT) in vitro is unknown. METHODS: Freshly isolated mouse PT exposed to 25 min of hypoxia were used to study the role of caspases, caspase-1 and IL-18 in hypoxia-induced membrane injury. Lactate dehydrogenase (LDH) release into the PT medium was used as a biochemical parameter of cell membrane damage. IL-18 was determined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: PT pre-incubated with the novel pancaspase inhibitor IDN-8050 were protected; LDH release (%) was 35+/-3 in vehicle-treated hypoxic PT and 21+/-2 in IDN-8050-treated hypoxic PT (P<0.01, n=6). To investigate the mechanism of protection and examine the role of caspase-1 specifically, PT were isolated in parallel from wild-type and caspase-1- deficient (-/-) mice. PT from caspase-1-/-mice demonstrated less hypoxia-induced membrane injury. LDH release was 37+/-2 in wild-type hypoxic PT and 28+/-2 in caspase-1-/-hypoxic PT (P<0.01, n=12). IL-18 was detected in PT by immunoblotting and ELISA. PT pre-incubated with IL-18 binding protein, an inhibitor of IL-18, were not protected. CONCLUSIONS: These studies demonstrate a deleterious effect of the proinflammatory caspase, caspase-1, on PT in vitro in the absence of inflammatory cells and vascular effects.

Cisplatin-induced acute renal failure is associated with an increase in the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil infiltration in the kidney.

We have demonstrated that caspase-1-deficient (caspase-1(-/-)) mice are functionally and histologically protected against cisplatin-induced acute renal failure (ARF). Caspase-1 exerts proinflammatory effects via the cytokines interleukin (IL)-1beta, IL-18, IL-6, and neutrophil recruitment. We sought to determine the role of the cytokines IL-1beta, IL-18, and IL-6 and neutrophil recruitment in cisplatin-induced ARF. We first examined IL-1beta; renal IL-1beta increased nearly 2-fold in cisplatin-induced ARF and was reduced in the caspase-1(-/-) mice. However, inhibition with IL-1 receptor antagonist (IL-1Ra) did not attenuate cisplatin-induced ARF. Renal IL-18 increased 2.5-fold; however, methods to inhibit IL-18 using IL-18 antiserum and transgenic mice that overproduce IL-18-binding protein (a natural inhibitor of IL-18) did not protect. Renal IL-6 increased 3-fold; however, IL-6-deficient (IL-6(-/-)) mice still developed cisplatin-induced ARF. We next examined neutrophils; blood neutrophils increased dramatically after cisplatin injection; however, prevention of peripheral neutrophilia and renal neutrophil infiltration with the neutrophil-depleting antibody RB6-8C5 did not protect against cisplatin-induced ARF. In summary, our data demonstrated that cisplatin-induced ARF is associated with increases in the cytokines IL-1beta, IL-18, and IL-6 and neutrophil infiltration in the kidney. However, inhibition of IL-1beta, IL-18, and IL-6 or neutrophil infiltration in the kidney is not sufficient to prevent cisplatin-induced ARF.

Acute renal failure after bilateral nephrectomy is associated with cytokine-mediated pulmonary injury.

Clinical studies demonstrate that acute renal failure (ARF) is associated with increased mortality, which may be due to pulmonary complications. ARF may affect the lung via increased renal production or impaired clearance of mediators of lung injury, such as proinflammatory cytokines. Bilateral nephrectomy is a method to examine directly the deleterious systemic effects of absent renal clearance in ARF without the confounding effects that are associated with ischemia-reperfusion injury (e.g., ischemic ARF) or systemic toxicity (e.g., cisplatin-induced ARF). This study contrasts the effects of ischemic ARF and bilateral nephrectomy on serum cytokines and lung injury. It demonstrates that the acute absence of kidney function after both ischemic ARF and bilateral nephrectomy is associated with an increase in multiple serum cytokines, including IL-6 and IL-1beta, and that the cytokine profiles were distinct. Lung injury after ischemic ARF and bilateral nephrectomy was similar and was characterized by pulmonary vascular congestion and neutrophil infiltration. For investigation of the role of proinflammatory cytokines in pulmonary injury after ARF, the anti-inflammatory cytokine IL-10 was administered before bilateral nephrectomy. IL-10 treatment improved pulmonary architecture and was associated with a reduction in inflammatory markers, including bronchoalveolar lavage fluid total protein, pulmonary myeloperoxidase activity (a biochemical marker of neutrophils), and the chemokine macrophage inflammatory protein 2. These data demonstrate for the first time that the acute absence of kidney function results in pulmonary injury independent of renal ischemia and highlight the critical role of the kidney in the maintenance of serum cytokine balance and pulmonary homeostasis.