The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease

Abstract

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.

Fig. 1.

The N-terminal cytoplasmic domain of PC1 (NTM-PC1) colocalizes with mTOR and colocalizes and interacts with tuberin. (A–D) Expression of NTM-PC1 was induced with DOX for 16 h before fixation and immunostaining with antibodies against the indicated proteins. Noninduced NTM-PC1 cells served as controls (C and D, −DOX). Grp94 and GM130 are resident proteins of the endoplasmic reticulum and Golgi apparatus, respectively. (E) FLM-PC1 and CTM-PC1 cells were cultured in the absence or presence of DOX for 16 h, the PC1 fusion proteins were immunoprecipitated by using an antibody against CD16, and binding proteins were analyzed by Western blot with the indicated antibodies. The starting material (MDCK lysate) serves as a control. [Scale bars, 3 μm (A–D).]

Fig. 2.

Phospho-mTOR, -S6K, and -S6 are specifically induced in cyst-lining epithelial cells in ADPKD and mouse models. (A–D) Renal tissue sections isolated from human ADPKD patients (A and C) or normal patients (B and D) were analyzed by immunohistochemistry using antibodies against phospho-mTOR (Ser 2448) (A and B) or phospho-S6K (Thr 389) (C and D). Note specific staining of cystic epithelial cells (A and C, arrows) but not surrounding normal epithelium (A and C, arrowheads). (E–I) Renal tissue sections derived from Pkd1^cond/cond:TgN(balancer1)2Cgn (E) and MAL-overexpressing (F) mice, nontreated (G) or rapamycin-treated (H) (5 mg/kg of body weight) oprk-rescue (Tg737^orpk/orpk;TgRsq) mutant mice, and nontreated orpk-heterozygous-rescue (Tg737^orpk/+;TgRsq) (I) control mice were immunostained by using an antibody against phospho-S6 ribosomal protein (Ser 235/236). Note specific cytoplasmic staining of cystic-epithelial cells (E–G, arrows) but not surrounding normal epithelium (E and F, arrowheads; and I). The cyst-specific cytoplasmic immunosignal is abolished after rapamycin treatment (H, arrows). (Scale bars, 20 μm.)

Fig. 3.

Rapamycin treatment significantly improves renal cystogenesis in the orpk-rescue mouse model. Day-150 postpartum orpk-rescue (Tg737^orpk/orpk;TgRsq) mutant mice and orpk-heterozygous-rescue (Tg737^orpk/+;TgRsq) control mice were given daily i.p. injections of rapamycin (5 mg/kg of body weight) or vehicle for 28 days before isolation, fixation, and sectioning of whole kidneys. (A–F) Representative whole kidney (A, C, and E) and H&E-stained sections (B, D, and F) derived from non-rapamycin-treated (A and B) or rapamycin-treated (C and D) (5 mg/kg of body weight) orpk-rescue mutant mice and orpk-heterozygous-rescue (E and F) control mice. Note the nonuniform appearance of the nontreated mutant kidney (A, arrow) compared with the smooth surface of the treated mutant kidney (C). (G) Cystic indices were calculated based on representative renal sections derived from non-rapamycin-treated and rapamycin-treated orpk-rescue mutant mice as described in Materials and Methods. (H) Kidney volumes were measured by MRI scanning of live animals at the beginning (day 150 postpartum), middle (day 164 postpartum), and end (day 178 postpartum) of treatment with rapamycin or vehicle. Each individual line represents a separate kidney for each respective genotype, and the averages for each group are indicated with horizontal lines. [Scale bars, 3 mm (A, C, and E) and 200 μm (B, D, and F).] ∗∗∗, P < 0.001.

Fig. 4.

Rapamycin treatment significantly improves the renal cystic phenotype and kidney function in the bpk mouse model. Mutant (bpk/bpk) and control (bpk/+) mice received daily i.p. injections of rapamycin (5 or 1.67 mg/kg of body weight) starting at day 7 for a period of 14 days. (A–C) Representative low-power (Left) and high-power (Right) H&E-stained sections derived from non-rapamycin-treated (A) or rapamycin-treated (B) (5 mg/kg of body weight) bpk/bpk mutant and bpk/+ (C) animals. (D) Kidney weight expressed as a percentage of total body weight. (E) Cystic indices were calculated based on representative renal sections derived from non-rapamycin-treated and rapamycin-treated bpk/bpk mutant mice (n = 4 kidneys) as described in Materials and Methods. (F) Blood drawn from anesthetized animals was assayed for blood urea nitrogen (BUN) according to standard procedures. [Scale bars, 3 mm (A–C, whole kidney H&E) and 200 μm (A–C, histology H&E).] ∗∗∗, P < 0.001; ∗∗, P < 0.01.

Fig. 5.

Rapamycin treatment induces increased apoptosis and luminal shedding of cystic epithelial cells. Orpk-rescue (Tg737^orpk/orpk;TgRsq) mutant and orpk-heterozygous-rescue (Tg737^orpk/+;TgRsq) control mice were treated with rapamycin or vehicle as indicated. Renal tissue sections were processed and analyzed for the presence of apoptotic cells by TUNEL. (A) TUNEL-positive, cyst-lining epithelial cells were observed in sections of nontreated Tg737^orpk/orpk;TgRsq mutant mice, whereas a significant increase in TUNEL-positive, cyst-lining and cyst-luminal epithelial cells were observed in sections of rapamycin-treated Tg737^orpk/orpk;TgRsq mutant mice. (B) Quantification of TUNEL-positive cells in renal sections derived from vehicle or rapamycin-treated orpk-rescue mutant animals expressed as an average of TUNEL-positive cells per cyst (n = 4 kidneys). ∗∗∗, P < 0.001.