Heartworm infection (Dirofilaria immitis) can cause kidney damage due to the presence of circulating microfilariae (mf) that contribute to the production and deposit of immune complexes. It has been shown that mf are a major source of Wolbachia antigen during active infection. Here the authors compared urine samples from 19 naturally infected dogs with (mf+) and 12 without (mf-) microfilariae for the presence of proteinuria and anti-Wolbachia Surface Protein (-WSP) IgG in ELISA. Kidneys from 6 mf+ and 3 mf- dogs were also examined by anti-WSP immuno-histochemistry. All infected dogs showed proteinuria, but mf+ dogs had significantly higher values compared to mf-dogs. Mf+ dogs had optical density values for anti-WSP IgG consistently higher than established cut-off values and were significantly higher than values for mf- dogs. Kidneys from mf+ dogs showed Wolbachia+ mf in glomerular capillaries. Results strongly suggest that Wolbachia associated with circulating mf may contribute to immune-mediated kidney disease in dogs with heartworm infection.