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

Abstract

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.

Fig. 1.

IV administration of LEC reduces splenic mononuclear phagocytes. Sham operation (Sham) or bilateral nephrectomy (BNx) was performed with or without administration of LEC. Flow cytometry was performed to confirm mononuclear phagocyte depletion. IV LEC administration to before sham operation and bilateral nephrectomy resulted in a significant reduction in splenic mononuclear phagocytes defined as CD45-positive, F4/80-positive and Ly6G-negative cells (n= 4–7).

Fig. 2.

Serum IL-6, CXCL1, IL-10, IL-1β, IL-5, MCP-1 and eotaxin were increased after bilateral nephrectomy and reduced with LEC treatment. Sham operation (Sham) or bilateral nephrectomy (BNx) was performed with or without administration of LEC to deplete mononuclear phagocytes. Serum (A) IL-6, (B) CXCL1, (C) IL-10, (D) IL-1β, (E) IL-5, (F) MCP-1 and (G) eotaxin were all increased in mice with bilateral nephrectomy versus sham operation and reduced with LEC treatment. *P < 0.05 versus Sham,**P < 0.01 versus Sham, ^#P < 0.05 versus BNx, ^##P < 0.01 versus BNx (n= 4–7).

Fig. 3.

Serum IL-12 (p40) and RANTES were increased after bilateral nephrectomy and further increased with LEC treatment. Sham operation (Sham) or bilateral nephrectomy (BNx) was performed with or without administration of LEC to deplete mononuclear phagocytes. Serum (A) IL-12 (p40) and (B) RANTES were both increased in mice with bilateral nephrectomy versus sham operation and were further increased with LEC treatment. **P < 0.01 versus Sham, ^##P < 0.01 versus BNx (n= 4–7).

Fig. 4.

IL-6, CXCL1 and IL-10 protein are increased in the spleen 2 h after bilateral nephrectomy. Sham operation (Sham) or bilateral nephrectomy (BNx) was performed, and (A) IL-6, (B) IL-10, (C) CXCL1, (D) IL-1β and (E) TNF-α were measured in the spleen and liver by ELISA 2 h post-procedure. IL-6 and IL-10 were significantly increased in both organs. CXCL1 and IL-1β was significantly increased in the spleen but not the liver. TNF-α did not increase in either organ (n= 5–8).

Fig. 5.

IL-6 and CXCL1 are reduced in the spleen 2 h after bilateral nephrectomy and LEC. Bilateral nephrectomy (BNx) with LEC or Vehicle administration was performed, and (A) IL-6, (B) IL-10, (C) CXCL1, (D) IL-1β and (E) TNF-α were measured in the spleen and liver by ELISA 2 h post-procedure. IL-6 and CXCL1 were significantly decreased in the spleen after LEC treatment. IL-1β was significantly decreased in the liver after LEC treatment. IL-10 and TNF-α did not change in either organ (n= 5–8).

Fig. 6.

Serum clearance of IL-6, CXCL1, IL-10 and IL-1β is impaired in mice with bilateral nephrectomy. Vehicle (Veh) or 200 ng of (A) IL-6, (B) CXCL1, (C) IL-10 or (D) IL-1β were administered intravenously by tail vein injection to mice immediately after sham operation (Sham) or bilateral nephrectomy (BNx). Serum cytokine levels were determined 60 min post-injection. *P < 0.05 versus Sham + Veh; **P < 0.05 versus all other groups (n= 3–7).

Fig. 7.

Serum clearance of TNF-α is not affected by bilateral nephrectomy. Vehicle (Veh) or 200 ng of TNF-α was administered intravenously by tail vein injection to mice immediately after sham operation (Sham) or bilateral nephrectomy (BNx). Serum cytokine levels were determined 60 min post-injection. *P < 0.05 versus Sham + Veh; NS, not significant versus Sham + TNF-α (n= 4–10).