Additional comments on response of Jiang et al.

We appreciate the response to our concerns by Jiang et al. Some issues, however, were addressed inadequately or not at all.

(1) The authors have now provided a Western blot showing a 133 kDa band for EGFR-TRPC6. However, the strong bands at about 106 representing endogenous TRPC6 that are evident in the published paper are now hardly visible in the newly provided blot.

(2) The newly provided, uncropped blot for synaptopodin shows much reduced intensity of the 110 kDa band in the eGFP-transfected cells and a stronger band in the TRPC6-transfected cells. These data argue against the conclusion that synaptopodin expression is reduced by TRPC6. Furthermore, the uncropped Western blot shows no band at 44 kDa which is in contrast to the originally published data, where very strong bands at 44 kDA are shown in several figures.

(3) In contradiction to statements by Jiang et al. in their response, none of the cited references 10–14 used U73122 to inhibit OAG-induced TRPC6 activation. These studies used U73122 to inhibit Ca²⁺ responses to histamine or angiotensin II, which act upstream of PLC. Although the study by Estacion et al. suggested a potential direct effect of U73122 on TRPC6, the study by Ju et al. found that ‘U73122-induced inhibition of anti-PIP2 antibody-evoked TRPC6/C7 channel activity was overcome by co-application of 10 μM OAG, which rules out the possibility that U73122 was acting as a direct channel blocker’.

(4) Although cultured, differentiated, conditionally immortalized podocytes were described as arborized, process-bearing cells, these do not usually display such extremely enlarged processes as shown in the publication by Jiang et al. The significance of such morphological characteristics remains to be determined.