mTORC1/2 and rapamycin in female Han:SPRD rats with polycystic kidney disease.

Rapamycin slows disease progression in the male Han:SPRD (Cy/+) rat with polycystic kidney disease (PKD). The aim of this study was to determine the effect of rapamycin on PKD and the relative contributions of the proproliferative mammalian target of rapamycin complexes 1 and 2 (mTORC1 and mTORC2) in female Cy/+ rats. Female Cy/+ rats were treated with rapamycin from 4 to 12 wk of age. In vehicle-treated Cy/+ rats, kidney volume increased by 40% and cyst volume density (CVD) was 19%. Phosphorylated S6 (p-S6) ribosomal protein, a marker of mTORC1 activity, was increased in Cy/+ rats compared with normal littermate controls (+/+) and decreased by rapamycin. Despite activation of mTORC1 in female Cy/+ rats, rapamycin had no effect on kidney size, CVD, number of PCNA-positive cystic tubular cells, caspase-3 activity, or the number of terminal deoxynucleotidyl transferase dUTP-mediated nick-end label-positive apoptotic cells. To determine a reason for the lack of effect of rapamycin, we studied the mTORC2 signaling pathway. On immunoblot of kidney, phosphorylated (Ser473) Akt (p-Akt), a marker of mTORC2 activity, was increased in female Cy/+ rats treated with rapamycin. Phosphorylated (Ser657) PKCα, a substrate of mTORC2, was unaffected by rapamycin in females. In contrast, in male rats, where rapamycin significantly decreases PKD, p-Akt (Ser473) was decreased by rapamcyin. PKCα (Ser657) was increased in male Cy/+ rats but was unaffected by rapamycin. In summary, in female Cy/+ rats, rapamycin had no effect on PKD and proproliferative p-Akt (Ser473) activity was increased by rapamycin. There were differential effects of rapamycin on mTORC2 signaling in female vs. male Cy/+ rats.

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD).

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.

Sirolimus attenuates disease progression in an orthologous mouse model of human autosomal dominant polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD), abnormal proliferation of tubular cells drives cyst development and growth. Sirolimus, an inhibitor of the protein kinase mammalian target of rapamycin (mTOR) and a potent anti-proliferative agent, decreases cyst growth in several genetically distinct rodent models of polycystic kidney disease (PKD). We determined here the effect of sirolimus on renal cyst growth in Pkd2WS25/- mice; an ortholog of human ADPKD involving mutation of the Pkd2 gene. In Pkd2WS25/- mice treated with sirolimus, both the two kidney/total body weight (2K/TBW) ratio and the cyst volume density (CVD) were significantly decreased by over half compared with untreated mice suffering with PKD. However, there was no effect on the increased blood urea nitrogen (BUN) levels as an index of kidney function. There are two distinct complexes containing mTOR depending on its binding partners: mTORC1 and mTORC2. Western blot analysis of whole kidney lysates and immunohistochemistry of the cysts found that phospho-S6 ribosomal protein, a marker of mTORC1 activity, was increased in Pkd2WS25/- mice and its phosphorylation was decreased by sirolimus treatment. Phospho-Akt at serine 473, a marker associated with mTORC2 activity, was not different between Pkd2WS25/- mice and normal littermate controls. Hence, our study found that inhibition of mTORC1 by sirolimus correlated with decreased renal cyst growth in this model of human ADPKD but had no effect on the decline in renal function.

Long-term rapamycin therapy in the Han:SPRD rat model of polycystic kidney disease (PKD).

BACKGROUND: Short-term studies have demonstrated that rapamycin or everolimus treatment decreases cyst formation and improves renal function in animal models of polycystic kidney disease (PKD). Autosomal dominant polycystic kidney disease (ADPKD) patients would likely require life-long treatment with rapamycin. METHODS: Male Han:SPRD rats with PKD (Cy/+) were treated with rapamycin (0.2 mg/kg/day IP) or vehicle from 1 to 12 months of age. Mean trough levels of rapamycin (ng/mL) were 6.6 +/- 0.1 at 8 weeks of age. Twelve-month-old littermates (+/+) were used as normal controls. RESULTS: Twelve-month-old male Cy/+ rats treated with the vehicle had a more than doubling of kidney volume, severe chronic renal failure, severe hypertension and increased heart weight compared to normal littermate controls (+/+). After rapamycin treatment, 12-month-old Cy/+ rats had markedly improved kidney volume, renal function, blood pressure and heart weight not statistically different from controls. Rapamycin reduced the cyst volume density (CVD) by 72%. Mammalian target of rapamycin (mTOR) activation in the heart, as evidenced by a marked increase in the phospho-S6 protein that was inhibited by rapamycin, was demonstrated in 12-month-old Cy/+ rats. CONCLUSION: In conclusion, long-term rapamycin treatment in Cy/+ rats results in a normalization of kidney volume, renal function, blood pressure and heart weight. The novel finding that rapamycin decreases hypertension, heart enlargement and mTOR signalling in the heart in PKD rats is reported. The only side effect of rapamycin treatment was an 11% decrease in body weight.

Metastatic Renal Cancer: What Role for Everolimus?

Metastatic renal cell carcinoma is uncommon (only 3% of cancers worldwide) but of poor prognosis. Renal cell carcinoma has traditionally been treated with cytokines (interferon-α or interleukin-2). More recently, a more clear understanding of the molecular and cellular mechanisms of the disease, involving the VEGF receptor and mTOR, has led to the discovery of novel therapies. Therapeutic options in patients with advanced RCC include the VEGF receptor inhibitors Sunitinib and Sorafenib, the anti-VEGF monoclonal antibody Bevacizumab and the mTORC1 inhibitors Temsirolimus and Everolimus. In 2009, Everolimus was FDA-approved for the treatment of patients with advanced clear cell RCC which had progressed within 6 months of stopping treatment with Sunitinib or sorafenib, or both drugs. Everolimus resulted in a 70% reduction in the risk of disease recurrence or death. The purpose of this review is to update on the current knowledge of the role of Everolimus in metastatic renal cell carcinoma.

Novel targets for the treatment of autosomal dominant polycystic kidney disease.

IMPORTANCE OF THE FIELD: Autosomal dominant (AD) polycystic kidney disease (PKD) is the most common life-threatening hereditary disorder. There is currently no therapy that slows or prevents cyst formation and kidney enlargement in humans. An increasing number of animal studies have advanced our understanding of molecular and cellular targets of PKD. AREAS COVERED IN THE REVIEW: The purpose of this review is to summarize the molecular and cellular targets involved in cystogenesis and to update on the promising therapies that are being developed and tested based on knowledge of these molecular and cellular targets. WHAT THE READER WILL GAIN: Insight into the pathogenesis of PKD and how a better understanding of the pathogenesis of PKD has led to the development of potential therapies to inhibit cyst formation and/or growth and improve kidney function. TAKE HOME MESSAGE: The results of animal studies in PKD have led to the development of clinical trials testing potential new therapies to reduce cyst formation and/or growth. A vasopressin V2 receptor antagonist, mTOR inhibitors, blockade of the renin-angiotensin system and statins that reduce cyst formation and improve renal function in animal models of PKD are being tested in interventional studies in humans.

The effect of caffeine on renal epithelial cells from patients with autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder characterized by the progressive enlargement of cysts derived from tubules. Tubule cell proliferation and chloride-dependent fluid accumulation, mechanisms underlying cyst expansion, are accelerated by adenosine 3':5'-cyclic monophosphate (cAMP). This study examined the extent to which caffeine may stimulate the production of cAMP by cyst epithelial cells, thereby adversely increasing proliferation and fluid secretion. Mural epithelial cells from ADPKD cysts and normal human kidney cortex cells (HKC) were cultured, and cAMP levels were determined in response to caffeine and receptor-mediated agonists linked to adenylyl cyclase. Caffeine, a methylxanthine, slightly increased basal levels of cAMP, as did other nonselective phosphodiesterase (PDE) inhibitors, 1-methyl-3- isobutyl xanthine and theophylline and rolipram, a specific PDE IV inhibitor. More importantly, clinically relevant concentrations of caffeine (10 to 50 micro M) potentiated the effects of desmopressin (DDAVP), prostaglandin E(2) (PGE(2)), and isoproterenol to increase cAMP levels in both ADPKD and HKC cells. By contrast, at concentrations that augmented the DDAVP response, caffeine attenuated cAMP accumulation by adenosine, implicating an action apart from the inhibition of PDE. Caffeine enhanced the effect of DDAVP to stimulate transepithelial short-circuit current of polarized ADPKD monolayers, reflecting an increase in chloride secretion. Caffeine potentiated the effect of DDAVP and PGE(2) to increase the levels of phosphorylated extracellular signal-regulated kinase (P-ERK). By contrast, P-ERK levels in HKC cells were not raised by increased intracellular concentrations of cAMP. It is concluded that PDE inhibition by caffeine increases the accumulation of cAMP, and through this mechanism activates the ERK pathway to cellular proliferation and increases transepithelial fluid secretion in ADPKD cystic epithelium. Caffeine is, therefore, a risk factor for the promotion of cyst enlargement in patients with ADPKD.

Electrolyte and fluid secretion by cultured human inner medullary collecting duct cells.

Inner medullary collecting ducts (IMCD) are the final nephron segments through which urine flows. To investigate epithelial ion transport in human IMCD, we established primary cell cultures from initial (hIMCD(i)) and terminal (hIMCD(t)) inner medullary regions of human kidneys. AVP, PGE(2), and forskolin increased cAMP in both hIMCD(i) and hIMCD(t) cells. The effects of AVP and PGE2 were greatest in hIMCD(i); however, forskolin increased cAMP to the same extent in hIMCD(i) and hIMCD(t). Basal short-circuit current (I(SC)) of hIMCD(i) monolayers was 1.4 +/- 0.5 microA/cm2 and was inhibited by benzamil, a Na+ channel blocker. 8-Bromo-cAMP, AVP, PGE(2), and forskolin increased I(SC); the current was reduced by blocking PKA, apical Cl- channels, basolateral NKCC1 (a Na+ - K+ - 2Cl- cotransporter), and basolateral Cl-/HCO(3)(-) exchangers. In fluid transport studies, hIMCD(i) monolayers absorbed fluid in the basal state and forskolin reversed net fluid transport to secretion. In hIMCD(t) monolayers, basal current was not different from zero and cAMP had no effect on I(SC). We conclude that AVP and PGE2 stimulate cAMP-dependent Cl- secretion by hIMCD(i) cells, but not hIMCD(t) cells, in vitro. We suggest that salt secretion at specialized sites along human collecting ducts may be important in the formation of the final urine.

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys.

BACKGROUND: The proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3', 5' cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells. METHODS: Primary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot. RESULTS: 8-Br-cAMP increased phosphorylated ERK (2.7- +/- 0.6-fold, N = 7), and B-Raf kinase activity (3.6- +/- 1.1-fold, N = 5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK. CONCLUSION: cAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts.

Cyclic AMP promotes growth and secretion in human polycystic kidney epithelial cells.

BACKGROUND: Progressive cyst enlargement, the hallmark of autosomal-dominant polycystic kidney disease (ADPKD) and autosomal-recessive (ARPKD) polycystic kidney disease, precedes the eventual decline of function in these conditions. The expansion of individual cysts in ADPKD is determined to a major extent by mural epithelial cell proliferation and transepithelial fluid secretion. This study determined if common receptor-mediated agonists and an anonymous lipid stimulate the production of 3' 5'-cyclic monophosphate (cAMP) in mural epithelial cells from the two major types of human cystic diseases. METHODS: cAMP responses to maximally effective concentrations of renal agonists were determined together with measurements of transepithelial anion current and cellular proliferation and extracellular signal-related kinase (ERK 1/2) expression in primary cultures of epithelial cells from human ADPKD and ARPKD cysts. RESULTS: The rank orders of responses to ligands for ADPKD and ARPKD cells were identical: epinephrine > desmopressin (DDAVP) approximately arginine vasopressin (AVP) > adenosine > prostaglandin E(2) (PGE(2)) > parathyroid hormone (PTH). cAMP concentrations elevated by epinephrine, DDAVP, adenosine, and PGE(2) were diminished by receptor-specific inhibitors. Pools of cyst fluid collected individually from 16 of 19 ADPKD kidneys increased, to varying degrees, cAMP levels in ADPKD and ARPKD cells. PGE(2), beta-adrenergic and AVP antagonists partially inhibited cAMP accumulation in response to fluids from three kidneys, but a large portion of the endogenous activity was attributed to yet-to-be identified bioactive lipid, designated cyst activating factor (CAF). CAF stimulated cAMP production in ADPKD and ARPKD cells, activated ERK(1/2), and increased cellular proliferation in ADPKD cells. CAF increased positive short circuit current (I(SC)) in polarized ADPKD and T-84 monolayers, indicating stimulation of net anion secretion. CONCLUSION: Endogenous adenylyl cyclase agonists promote cell proliferation and electrolyte secretion of human ADPKD and ARPKD cells in vitro. We suggest that increased levels of cAMP may accelerate cyst growth and overall renal enlargement in patients with PKD.