Objective To compare the infection efficiency of podocytes in the kidney following recombinant adeno-associated virus (rAAV) delivery by three routes: tail vein injection, renal parenchymal (multiple-site) injection, and renal pelvis injection via the renal parenchyma.

Methods An rAAV vector carrying a podocyte-specific promoter and a fluorescent reporter was constructed. A total of 24 four-week-old specific-pathogen-free (SPF) male C57BL/6 mice were randomly assigned according to the infection method to a control group, a tail vein injection group, a renal parenchymal injection group, or a renal pelvis injection group (6 mice per group). Four weeks after rAAV infection, samples were collected from all mice. Biosafety was assessed by measuring serum biochemical indices and the urine albumin-to-creatinine ratio (UACR), and by hematoxylin and eosin (HE) staining of tissue sections. Renal expression of rAAV was analyzed by quantitative real-time PCR (qPCR) and Western blotting. Fluorescence staining of kidney tissue was used to determine the localization of rAAV expression.

Results During the experiment, one mouse in the renal parenchymal injection group and three mice in the renal pelvic injection group died; the remaining mice survived. Four weeks after injection, AST (P＝0.036) and UACR levels (P＝0.028) differed significantly among the four groups; however, UACR in the renal parenchymal injection group was not significantly higher than that in the control group. HE staining revealed no significant pathological damage in the heart, liver, spleen, lungs, or kidneys. Western blotting and qPCR showed that expression of relative green fluorescent protein (GFP) and the EGFP target gene were higher in the renal parenchymal injection group than in the other three groups (P＜0.01). Quantitative analysis of fluorescence intensity in kidney tissue showed that GFP intensity was higher in the renal parenchymal injection group than in the control group (P＜0.0001), tail vein injection group (P＜0.0001), and renal pelvis injection group (P＜0.001).

Conclusions None of the three injection methods caused evident tissue toxicity. Among them, renal parenchymal injection of rAAV carrying a podocyte-specific promoter achieved the highest infection efficiency.