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Background/Aim: Necrotizing crescentic glomerulonephritis (GN) associated with anti-neutrophil cytoplasmic antibodies (ANCA) against myeloperoxidase (MPO) is a devastating disease that quickly progresses to kidney failure. Current therapies are broadly immunosuppressive and associated with adverse effects. We wanted to set up a model that could be suitable for testing narrowly targeted therapies. Methods: The model was constructed in male Wistar Kyoto rats through injections of human MPO (hMPO) and pertussis toxin, followed by a sub-nephritogenic dose of sheep anti-rat glomerular basement membrane (GBM) serum to boost the disease. Rats were monitored for 35 days. Rats given hMPO alone, saline, or human serum albumin with or without anti-GBM serum were also studied. Results: Rats receiving hMPO developed circulating anti-hMPO and anti-rat MPO antibodies. Challenging hMPO-immunized rats with the anti-GBM serum led to more glomerular neutrophil infiltration and MPO release, and severe haematuria, heavy proteinuria, and higher blood urea nitrogen than hMPO alone. Pauci-immune GN developed with crescents, affecting 25% of glomeruli. The majority of crescents were fibrocellular. Necrotizing lesions and Bowman capsule ruptures were detected. Cells double positive for claudin-1 (a marker of parietal epithelial cells [PECs]) and neural cell adhesion molecule (NCAM; progenitor PECs) were present in crescents. Double staining for NCAM and Ki-67 established proliferative status of progenitor PECs. Podocyte damage was associated with endothelial and GBM changes by electron microscopy. Monocyte/macrophages and CD4+ and CD8+ T cells accumulated in glomeruli and the surrounding area and in the tubulointerstitium. Lung haemorrhage also manifested. Conclusion: This model reflects histological lesions of human ANCA-associated rapidly progressive GN and may be useful for investigating new therapies.

Background/Aims: Glomerular filtration rate (GFR) is the best index for evaluating renal function. We aimed to develop a simplified iohexol plasma clearance procedure for GFR measurement in rats without urine collection, animal catheterization or anesthesia, with limited sampling and requiring blood instead of plasma, to further reduce the sample volume and improve animal welfare. Methods: After iohexol injection (129.4 mg), samples were drawn according to 2-compartment kinetics and analyzed by high performance liquid chromatography. Healthy male Lewis rats were used to find a correction factor (CF) to obtain the ‘reference clearance' from the simplified 1-comparment model. This approach was validated using male or female (Lewis, Sprague-Dawley) rats and animals with renal mass reduction (RMR). In additional rats, different simplified approaches were evaluated. Results: Iohexol concentrations in blood and plasma strongly correlated (r = 0.9784, p < 0.0001). A CF of 0.90 enabled the calculation of the reference GFR. Validation results in male Lewis rats were 0.99 ± 0.27 for the reference GFR and 1.03 ± 0.29 ml/min/100 g for the simplified approach. Results in female Sprague-Dawley rats confirmed the suitability of the proposed method. In RMR rats, GFR was 0.14 ± 0.05 and 0.14 ± 0.04 ml/min/100 g for the reference and simplified model, respectively. Conclusion: The procedure we set up to measure GFR in conscious rats was proven to be reliable, required a small volume of blood at only 4 selected time points, without the need to collect urine or catheterize the animals, was applicable to rats from different strains and sexes, both healthy and with renal function impairment. Moreover, the procedure enables the monitoring of GFR changes over time in the same animal, thereby reducing the number of animals to be used.

Background/Aims: A critical involvement of the endocannabinoid/cannabinoid receptor system in diabetes and its complications has been recognized. Experimental evidence suggested that activation of the cannabinoid receptor type 2 (CB2), which is expressed in the kidney by podocytes and inflammatory cells, had a protective role in early streptozotocin-induced type 1 diabetes in mice. No experimental evidence is so far available on the effects of CB2 agonists in type 2 diabetes. In this study, we investigated the effects of a CB2 agonist given at a phase of overt disease on renal functional and structural changes in BTBR ob/ob mice, a model of type 2 diabetic nephropathy. Methods: BTBR ob/ob mice received, from 10 to 21 weeks of age, vehicle, the selective CB2 agonist HU910, or lisinopril used as standard therapy for comparison. BTBR wild-type mice served as controls. Results: Treatment with CB2 agonist reduced progressive albuminuria of BTBR ob/ob mice to a similar extent as ACE inhibitor. The antiproteinuric effect of CB2 agonist was associated with the amelioration of the defective nephrin expression in podocytes of diabetic mice. CB2 agonist limited mesangial matrix expansion, fibronectin accumulation and sclerosis. Glomerular infiltration of Mac-2-positive monocytes/machrophages was attenuated by CB2 agonist, at least in part due to the drug's ability to reduce MCP-1 chemotactic signals. Renoprotective effects of CB2 were similar to those achieved by ACE inhibitor. Conclusion: These results suggest that CB2 agonism is a potential option to be added to the available therapeutic armamentarium for type 2 diabetic nephropathy.