Rapamycin ameliorates PKD resulting from conditional inactivation of Pkd1

Abstract

Aberrant activation of the mammalian target of rapamycin (mTOR) pathway occurs in polycystic kidney disease (PKD). mTOR inhibitors, such as rapamycin, are highly effective in several rodent models of PKD, but these models result from mutations in genes other than Pkd1 and Pkd2, which are the primary genes responsible for human autosomal dominant PKD. To address this limitation, we tested the efficacy of rapamycin in a mouse model that results from conditional inactivation of Pkd1. Mosaic deletion of Pkd1 resulted in PKD and replicated characteristic features of human PKD including aberrant mTOR activation, epithelial proliferation and apoptosis, and progressive fibrosis. Treatment with rapamycin was highly effective: It reduced cyst growth, preserved renal function, inhibited epithelial cell proliferation, increased apoptosis of cyst-lining cells, and inhibited fibrosis. These data provide in vivo evidence that rapamycin is effective in a human-orthologous mouse model of PKD.

Figure 1.

Rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice significantly improves the renal cystic phenotype. All mice were treated daily with vehicle or 5 mg/kg rapamycin starting at day 28 and ending at day 49. (A) Representative low-power hematoxylin and eosin (H&E) renal sections from 49-day-old nontreated Pkd1^cond/cond:Nestin^cre mice (middle) showing numerous cysts compared with nontreated wild-type mice (left). Treatment of Pkd1^cond/cond:Nestin^cre mice with rapamycin (right) significantly improves the renal cystic phenotype. (B) Representative high-power H&E renal sections as in A. Bar = 100μm for all images. (C through F) The significant increases in the two-kidney/total body weight (2K/TBW) ratios (C), two-kidney weights (D), and BUN levels (F) observed in nontreated Pkd1^cond/cond:Nestin^cre mice are significantly decreased in rapamycin-treated Pkd1^cond/cond:Nestin^cre mice (ANOVA, Newman-Keuls posttest), whereas final body weight is not significantly different between treatment groups (E). (G) The cystic index is significantly decreased in rapamycin-treated Pkd1^cond/cond:Nestin^cre mice (n = 4) compared with nontreated P28 through P33 (n = 7) and P49 (n = 4) Pkd1^cond/cond:Nestin^cre mice (ANOVA, Newman-Keuls posttest).

Figure 2.

Renal fibrosis is reduced in rapamycin-treated Pkd1^cond/cond:Nestin^cre mice. All mice were treated daily with vehicle or 5 mg/kg rapamycin starting at day 28 and ending at day 49. (A) Representative Masson trichrome–stained renal sections from indicated animal genotypes. Note the extensive deposition of collagen (blue) in Pkd1^cond/cond:Nestin^cre mice (middle), which is significantly reduced in rapamycin-treated Pkd1^cond/cond:Nestin^cre mice (right). Bar = 100 μm. (B and C) Representative SMA-stained renal sections from indicated animal genotypes. Rapamycin treatment significantly reduces SMA-positive interstitial cells surrounding morphologically normal tubules (B and C, right) versus nontreated (B and C, middle) Pkd1^cond/cond:Nestin^cre mice. Bars = 100 and 50 μm in B and C, respectively.

Figure 3.

Renal cysts in Pkd1^cond/cond:Nestin^cre mice are derived from distal tubules. Renal sections derived from 49-day-old Pkd1^cond/cond (A) and Pkd1^cond/cond:Nestin^cre (B through D) mice and 28-d-old Pkd1^cond/cond:Nestin^cre mice (E and F) were subjected to immunofluorescence staining with DBA (red, distal tubules), LTL (green, proximal tubules), and DAPI (blue). Note the predominantly collecting duct/distal tubule origin of cysts in Pkd1^cond/cond:Nestin^cre mice. Bar = 100 μm.

Figure 4.

Rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice decreases renal cell proliferation. All mice were treated daily with vehicle or 5 mg/kg rapamycin starting at day 28 and ending at day 49. (A through C) Representative low-power fields of renal sections from nontreated Pkd1^cond/cond:Nestin^cre (A), rapamycin-treated Pkd1^cond/cond:Nestin^cre (B) and nontreated wild-type (C) mice immunostained with a Ki-67 antibody. (D) Quantification of Ki-67 immunostaining (n = 3 for all treatment groups). Note the decrease in proliferation of cystic and morphologically normal epithelial cells in response to rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice. Bar = 50 μm.

Figure 5.

Rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice increases apoptosis of cyst-lining epithelial cells. All mice were treated daily with vehicle or 5 mg/kg rapamycin starting at day 28 and ending at day 49. (A through C) Representative low-power fields of renal sections from nontreated Pkd1^cond/cond:Nestin^cre (A), rapamycin-treated Pkd1^cond/cond:Nestin^cre (B), and nontreated wild-type (C) mice processed for TUNEL staining. (D) Quantification of TUNEL-positive nuclei (n = 3 for all treatment groups). Note the increase in apoptosis of cystic epithelial cells in response to rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice. Bar = 50 μm.

Figure 6.

Rapamycin treatment of Pkd1^cond/cond:Nestin^cre mice reduces mTOR pathway activity. All mice were treated daily with vehicle or 5 mg/kg rapamycin starting at day 28 and ending at day 49. (A) Representative Western blots of kidney tissue lysates demonstrating that phosphorylated ribosomal protein S6 (P-rpS6) increases in Pkd1^cond/cond:Nestin^cre mice and is decreased by rapamycin treatment. Total levels of rpS6 and β-tubulin remain unchanged. Total levels of Akt and phosphorylated Akt (P-Akt) are increased and decreased in nontreated and rapamycin-treated cystic animals, respectively. (B and C) Immunohistochemistry showing intense P-rpS6 staining of cyst-lining and immediately adjacent epithelial cells in Pkd1^cond/cond:Nestin^cre mice (B) and undetectable staining in rapamycin-treated Pkd1^cond/cond:Nestin^cre mice (C). Bar = 50 μm.