FIGURE 1.

Cystin deficiency in cpk kidneys leads to FPC loss, with partial rescue of FPC following cystin–GFP expression (r‐cpk), but FPC loss (Del34, Del67 and Del367) does not alter cystin expression in mouse kidneys. (A) WB experiments demonstrate significantly lower FPC levels in cpk, compared to wt cells, with partial rescue of FPC levels in r‐cpk kidneys. Whole kidney lysates (N = 2, n = 6, male mice) from wt (C57BL/6), cpk and r‐cpk mouse kidneys were analyzed for FPC (anti‐FPC, C‐terminal, rat monoclonal antibody, top), β‐actin (loading control, middle) and cystin (anti‐cystin polyclonal, rabbit antibody, bottom). In wt samples, cystin is present (arrow). In r‐cpk samples, a cystin–GFP fusion protein (cystin‐GFP, arrow) and multiple unspecific bands are also present (brackets). No endogenous cystin is present in cpk and r‐cpk kidneys. (B) Densitometry confirms higher FPC levels in r‐cpk kidneys compared to cpk. Wt, cpk, and r‐cpk kidney lysates were tested for FPC levels are plotted relative to β‐actin (****: n = 6, p < .0001; ***p < .0005, unpaired t‐test). (C) No reduction in relative Pkhd1 mRNA levels in cpk kidneys compared to wt suggest FPC loss at the protein level. Relative Pkhd1 mRNA levels in wt and cpk kidneys were measured by real‐time RT‐PCR using Ppia mRNA as internal reference. Pkhd1 mRNA levels were higher, not lower in cpk kidneys compared to wt (n = 6, ***p < .001, unpaired t‐test). (D) Cystin levels are not reduced in Pkhd1 mutant (Del3‐4, Del67 and Del3‐67) kidneys. Whole kidney lysates from Pkhd1 mutant kidneys (Del3‐4, Del67, and Del3‐67) were tested for cystin and FPC expression and compared to age and gender matched wt kidney lysates (n = 3, male mice). Cystin expression was similar in wt and mutant (Del3‐4, Del67 and Del3‐67) kidney lysates (top). β‐actin was analyzed as loading control (middle). FPC levels were compared in the same lysates (bottom). We observed the loss of full‐length FPC in all Pkhd1 mutant kidneys tested (Del3‐4, Del67, and Del3‐67).