Background/Aims: Toll-like receptor 4 (TLR4) and its adaptor protein MyD88 play an important role in ischemia/reperfusion (I/R) injury in the kidney, and pituitary adenylate cyclase-activating polypeptide (PACAP) could ameliorate renal I/R injury. Methods: Primary cultures of proximal tubule epithelial cells (PTEC) were prepared from wild-type and MyD88^–/– mice, and subjected to hypoxia in vitro. Acute kidney injury (AKI) was induced by I/R in vivo in wild-type mice only. Results: Hypoxia resulted in significant increases in cytokine production and apoptosis/necrosis in wild-type PTEC, but these responses were markedly blunted in MyD88^–/– PTEC. Treatment with PACAP38 before or after hypoxia further suppressed the hypoxia-induced cytokine responses and apoptosis in both MyD88^+/+ and MyD88^–/– PTEC cultures. PACAP38 significantly inhibited TLR4/MyD88/TRAF6 as well as TRIF and IRF3 expression in mouse kidney and PTEC, and inhibited the secretion and mRNA expression of cytokines in kidneys from mice after I/R, paralleling the cytokine responses in vitro. Moreover, treatment with PACAP38 protected mice from renal failure, histological damage, neutrophil influx and tubule cell apoptosis after I/R. Conclusion: Our data reveal that the TLR4-mediated cytokine responses to hypoxia are primarily dependent on MyD88 signaling and highlight the pivotal role of MyD88-dependent mechanisms in the coordination of the innate immune responses to ischemic/hypoxic acute renal tubular injury. The renoprotective effect of PACAP in AKI involves both MyD88-dependent and -independent pathways.

We determined whether pituitary adenylate cyclase-activating polypeptide 38 (PACAP38) prevents contrast-induced nephropathy using human renal proximal tubule epithelial (HK-2) cells and homozygous endothelial nitric oxide synthase-deficient (eNOS−/−) mice as a novel in vivo model. Cultured HK-2 cells were pretreated with 10−9–10−6 mol/L PACAP or vasoactive intestinal peptide (VIP) for 1 h, and then exposed to ionic (Urografin) or nonionic (iohexol) contrast media at 50 mg iodine/mL for 24 h. Male eNOS−/− mice received Urografin (1.85 g iodine/kg) intravenously after water deprivation for 24 h, and PACAP38 (10 μg) intraperitoneally 1 h before and 12 h after Urografin injection. Urografin and iohexol increased lactate dehydrogenase and kidney injury molecule 1 in the culture medium, induced apoptosis, and inhibited cell proliferation in HK-2 cell cultures. PACAP38 and VIP reduced these changes in a dose-dependent manner. PACAP38 was more potent than VIP. In eNOS−/− mice, Urografin raised serum creatinine and cystatin C levels, caused renal tubule damage, induced apoptosis, and promoted neutrophil influx. Urografin also increased kidney protein levels of proinflammatory cytokines, and kidney mRNA levels of proinflammatory cytokines, kidney injury biomarkers, and enzymes responsible for reactive oxygen and nitrogen species. PACAP38 significantly reduced these Urografin-induced changes in eNOS−/− mice. This study shows that both Urografin and iohexol are toxic to HK-2 cells, but Urografin is more toxic than iohexol. Urografin causes acute kidney injury in eNOS−/− mice. PACAP38 protects HK-2 cells and mouse kidneys from contrast media and is a potential therapeutic agent for contrast-induced nephropathy.

Pituitary adenylate cyclase-activating polypeptide 38 ameliorated renal tubular injury, reduced oxidative injury, and inhibited the expression of TGF-β1 in CsA-exposed murine kidneys. Pituitary adenylate cyclase-activating polypeptide could be a novel renoprotective and antifibrotic agent for CsA nephrotoxicity.

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We investigated the effects of human light chain (LC) protein-overload in mice kidney to gain further insights into the molecular mechanisms involved in the pathogenesis of myeloma kidney. Intact male C57BL/6, 10- to 12-week-old mice were given daily intraperitoneal (i.p.) injections of 1 ml of human ĸ-LCs (1.5 mg/ml, low dose), or (100 mg/ml, high dose) to uninephrectomized mice for 2 weeks. Intact, sham-operated or uninephrectomized control animals were given the same volume (1 ml/day) of saline, human serum albumin (10 mg/ml) or bovine serum albumin (100 mg/ml) i.p. for 2 weeks in place of LCs. The low-dose LC-treated mice had human LCs in their urine and a significant increase in monocyte chemoattractant protein-1 (MCP-1) mRNA in the kidneys. Uninephrectomized mice treated with high-dose ĸ-LCs showed tubule casts, and foci of intracytoplasmic rhomboid crystals within the proximal tubules, along with cytoskeletal disruptions and alterations in the brush-border membrane, and high concentrations of human ĸ-LC were present in their sera. High-dose LC treatment also led to increases in serum creatinine and tumor necrosis factor-α levels, and marked increases in interleukin-6 and MCP-1 expression as well as cellular apoptosis in the kidneys. These studies demonstrate that myeloma LC overload over a range of LC concentrations in mice causes significant functional and morphological kidney injury. The model should be helpful in investigating pathophysiologic mechanisms and exploring therapeutic interventions for myeloma kidney and other LC-associated renal disorders.