Hyperproliferation of PKD1 cystic cells is induced by insulin-like growth factor-1 activation of the Ras/Raf signalling system.

Autosomal dominant polycystic kidney disease (ADPKD) largely results from mutations in the PKD1 gene leading to hyperproliferation of renal tubular epithelial cells and consequent cyst formation. Rodent models of PKD suggest that the multifunctional hormone insulin-like growth factor-1 (IGF-1) could play a pathogenic role in renal cyst formation. In order to test this possibility, conditionally immortalized renal epithelial cells were prepared from normal individuals and from ADPKD patients with known germline mutations in PKD1. All patient cell lines had a decreased or absence of polycystin-1 but not polycystin-2. These cells had an increased sensitivity to IGF-1 and to cyclic AMP, which required phosphatidylinositol-3 (PI3)-kinase and the mitogen-activated protein kinase, extracellular signal-regulated protein kinase (ERK) for enhanced growth. Inhibition of Ras or Raf abolished the stimulated cell proliferation. Our results suggest that haploinsufficiency of polycystin-1 lowers the activation threshold of the Ras/Raf signalling system leading to growth factor-induced hyperproliferation. Inhibition of Ras or Raf activity may be a therapeutic option for decreasing tubular cell proliferation in ADPKD.

The role of geranylgeranylated proteins in human mesangial cell proliferation.

The Rho family of guanine 5'-triphosphatases (GTPases) play a key role in regulating cell proliferation, tubulointerstitial fibrosis, and glomerular hemodynamics. The post-translational prenylation of RhoGTPases by the addition of a geranylgeranyl moiety is critical for cellular localization and signaling activity. This study investigates the effects of (i) inhibiting geranylgeranylation (GG) in human mesangial cell (HMC) proliferation and apoptosis, using GGTI 298, a specific inhibitor of GG and (ii) lovastatin, an HMG-coacetyl A-reductase inhibitor, which depletes the availability of prenylation substrates. HMC proliferation was assessed using an assay of viable cell number and measuring bromodeoxyuridine (BrdU) incorporation. Hoechst 33342 staining was used to determine apoptosis. Extracellular signal-regulated protein kinase (Erk)1/2 and Akt activation were analysed by Western blotting. Rho activation was determined using the Rhotekin pull-down assay. Immunocytochemistry was performed to study the effects on the actin cytoskeleton and RhoA localization. GGTI 298 (10-20 muM) and lovastatin (5-10 muM) potently inhibited platelet-derived growth factor and serum-stimulated HMC proliferation and induced apoptosis. These effects of lovastatin were attenuated by co-incubation with geranylgeranylpyrophosphate. C3 exoenzyme, a clostridial toxin that specifically targets Rho also inhibited BrdU incorporation and promoted apoptosis. GGTI 298 increased cytosolic expression of RhoA, prevented RhoA activation, and inhibited the activation of Erk1/2 and the survival protein Akt. GGTI 298, lovastatin, and C3 exoenzyme inhibit HMC proliferation and promote apoptosis. Inhibiting GG increases cytosolic RhoA expression, disrupts the actin cytoskeleton, and inhibits RhoA activation. These results suggest that targeting geranylgeranylated proteins with statins or GGTI 298 is a promising therapeutic strategy in human mesangioproliferative renal disease.

Evidence of a role for Ki-Ras in the stimulated proliferation of renal fibroblasts.

Progressive renal fibrosis is driven by a range of cytokines that act via membrane receptors and intracellular signaling cascades to evoke gene transcription events and related responses. The Ras family of GTPases has been implicated in many of these signaling cascades in model systems such as 3T3 fibroblasts. However, the roleof the specific Ras isoforms Ki, Ha, and N in the stimulation of renal fibroblasts has not been defined. In this study, Ras has been inhibited in primate renal fibroblasts (vero cells) using specific phosphorothioate oligodeoxynucleotides (oligos) targeting the three isoforms. Lipofectin transfection with 200 to 400 nM Ki-Ras oligo inhibited the epidermal growth factor- and fibroblast growth factor-stimulated proliferation of vero cells by 25 to 35% with a lesser effect on serum-stimulated growth. Oligos against Ha-Ras and N-Ras were inactive with respect to control oligo. Total cellular Ras protein (estimated by Western blotting) was reduced by 60 to 90% 24 h after transfection with Ki-Ras oligo. N-Ras, Ha-Ras, and control oligos were inactive. Total Ras synthesis over 4 h measured using [35S]-cys/met pulse chase was reduced by approximately 70% by Ki-Ras oligo and not altered by other oligos. The fractional prenylation of Ras was quantified from the discrete bands on polyacrylamide gel electrophoresis and was increased by the Ki-Ras oligo alone. These data demonstrate that these renal fibroblasts predominantly express the Ki isoform of Ras and that this GTPase plays a role in the stimulated proliferation of these cells. Ras GTPases may be a target for the inhibition of processes leading to renal fibrosis.