Metformin does not slow cyst growth in the PCK rat model of polycystic kidney disease.

Metformin (MET) has the potential to activate p-AMPK and block mTORC1-induced proliferation of tubular cells in PKD kidneys. The aim of this study was to determine the effects of MET on cyst growth, kidney function, AMPK and mTOR signaling, and lactate levels in male PCK rats, a Pkhd1 gene mutation model of human autosomal recessive polycystic kidney disease (ARPKD). MET 300 mg/kg/day IP from days 28 to 84 of age resulted in a mean serum metformin level that was 10 times the upper limit of therapeutic, no effect on cyst indices, nephrotoxicity, and increased serum lactate. MET 150 mg/kg resulted in a therapeutic serum metformin level but had no effect on kidney weight, cyst indices, kidney function, or mTOR and autophagy proteins. In summary, a standard dose of MET was ineffective in reducing PKD, did not activate p-AMPK or suppress mTOR and the higher dose resulted in increased lactate levels and nephrotoxicity. In conclusion, the study dampens enthusiasm for human studies of MET in PKD. Doubling the metformin dose resulted in a 10-fold increase in mean blood levels and toxicity suggesting that the dosage range between therapeutic and toxic is narrow.

IL-6 mediates the hepatic acute phase response after prerenal azotemia in a clinically defined murine model.

Prerenal azotemia (PRA) is a major cause of acute kidney injury and uncommonly studied in preclinical models. We sought to develop and characterize a novel model of PRA that meets the clinical definition: acute loss of glomerular filtration rate (GFR) that returns to baseline with resuscitation. Adult male C57BL/6J wild-type (WT) and IL-6-/- mice were studied. Intraperitoneal furosemide (4 mg) or vehicle was administered at time = 0 and 3 h to induce PRA from volume loss. Resuscitation began at 6 h with 1 mL intraperitoneal saline for four times for 36 h. Six hours after furosemide administration, measured glomerular filtration rate was 25% of baseline and returned to baseline after saline resuscitation at 48 h. After 6 h of PRA, plasma interleukin (IL)-6 was significantly increased, kidney and liver histology were normal, kidney and liver lactate were normal, and kidney injury molecule-1 immunofluorescence was negative. There were 327 differentially regulated genes upregulated in the liver, and the acute phase response was the most significantly upregulated pathway; 84 of the upregulated genes (25%) were suppressed in IL-6-/- mice, and the acute phase response was the most significantly suppressed pathway. Significantly upregulated genes and their proteins were also investigated and included serum amyloid A2, serum amyloid A1, lipocalin 2, chemokine (C-X-C motif) ligand 1, and haptoglobin; hepatic gene expression and plasma protein levels were all increased in wild-type PRA and were all reduced in IL-6-/- PRA. This work demonstrates previously unknown systemic effects of PRA that includes IL-6-mediated upregulation of the hepatic acute phase response.NEW & NOTEWORTHY Prerenal azotemia (PRA) accounts for a third of acute kidney injury (AKI) cases yet is rarely studied in preclinical models. We developed a clinically defined murine model of prerenal azotemia characterized by a 75% decrease in measured glomerular filtration rate (GFR), return of measured glomerular filtration rate to baseline with resuscitation, and absent tubular injury. Numerous systemic effects were observed, such as increased plasma interleukin-6 (IL-6) and upregulation of the hepatic acute phase response.

Immune checkpoint activity regulates polycystic kidney disease progression.

Innate and adaptive immune cells modulate the severity of autosomal dominant polycystic kidney disease (ADPKD), a common kidney disease with inadequate treatment options. ADPKD has parallels with cancer, in which immune checkpoint inhibitors have been shown to reactivate CD8+ T cells and slow tumor growth. We have previously shown that in PKD, CD8+ T cell loss worsens disease. This study used orthologous early-onset and adult-onset ADPKD models (Pkd1 p.R3277C) to evaluate the role of immune checkpoints in PKD. Flow cytometry of kidney cells showed increased levels of programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte associated protein 4 (CTLA-4) on T cells and programmed cell death ligand 1 (PD-L1)/CD80 on macrophages and epithelial cells in Pkd1RC/RC mice versus WT, paralleling disease severity. PD-L1/CD80 was also upregulated in ADPKD human cells and patient kidney tissue versus controls. Genetic PD-L1 loss or treatment with an anti-PD-1 antibody did not impact PKD severity in early-onset or adult-onset ADPKD models. However, treatment with anti-PD-1 plus anti-CTLA-4, blocking 2 immune checkpoints, improved PKD outcomes in adult-onset ADPKD mice; neither monotherapy altered PKD severity. Combination therapy resulted in increased kidney CD8+ T cell numbers/activation and decreased kidney regulatory T cell numbers correlative with PKD severity. Together, our data suggest that immune checkpoint activation is an important feature of and potential novel therapeutic target in ADPKD.

Putative Mode of Action of the Monoterpenoids Linalool, Methyl Eugenol, Estragole, and Citronellal on Ligand-Gated Ion Channels.

Essential oil has been used as sedatives, anticonvulsants, and local anesthetics in traditional medical remedies; as preservatives for food, fruit, vegetable, and grain storage; and as bio-pesticides for food production. Linalool (LL), along with a few other major components such as methyl eugenol (ME), estragole (EG), and citronellal, are the active chemicals in many essential oils such as basil oil. Basil oil and the aforementioned monoterpenoids are potent against insect pests. However, the molecular mechanism of action of these chemical constituents is not well understood. It is well-known that the γ-aminobutyric acid type A receptors (GABAARs) and nicotinic acetylcholine receptor (nAChR) are primary molecular targets of the synthetic insecticides used in the market today. Furthermore, the GABAAR-targeted therapeutics have been used in clinics for many decades, including barbiturates and benzodiazepines, to name just a few. In this research, we studied the electrophysiological effects of LL, ME, EG, and citronellal on GABAAR and nAChR to further understand their versatility as therapeutic agents in traditional remedies and as insecticides. Our results revealed that LL inhibits both GABAAR and nAChR, which may explain its insecticidal activity. LL is a concentration-dependent, non-competitive inhibitor on GABAAR, as the half-maximal effective concentration (EC50) values of γ-aminobutyric acid (GABA) for the rat α1ß3γ2L GABAAR were not affected by LL: (36.2 ± 7.9) µmol·L-1 and (36.1 ± 23.8) µmol·L-1 in the absence and presence of 5 mmol·L-1 LL, respectively. The half-maximal inhibitory concentration (IC50) of LL on GABAAR was approximately 3.2 mmol·L-1. Considering that multiple monoterpenoids are found within the same essential oil, it is likely that LL has a synergistic effect with ME, which has been previously characterized as both a GABAAR agonist and a positive allosteric modulator, and with other monoterpenoids, which offers a possible explanation for the sedative and anticonvulsant effects and the insecticidal activities of LL.