Darren P. Wallace

Calcium Restores a Normal Proliferation Phenotype in Human Polycystic Kidney Disease Epithelial Cells

Polycystic kidney disease (PKD) is a lethal disorder characterized by progressive expansion of renal cysts. Genetic mutations associated with PKD are thought to disrupt intracellular Ca2+ regulation, leading to abnormal proliferation of tubule epithelial cells. cAMP stimulates the B-Raf/MEK/extracellular signal-regulated kinase (B-Raf/MEK/ERK) pathway and accelerates the proliferation of cells that are cultured from PKD cysts. By contrast, cAMP inhibits the proliferation of cells from normal human kidneys (NHK) and M-1 mouse collecting duct cells. Previously, it was found that a sustained reduction of intracellular Ca2+ levels in NHK and M-1 cells that were treated with Ca2+ entry blockers allowed cAMP activation of the B-Raf/MEK/ERK pathway, switching the cells to a cAMP-growth stimulated phenotype. In this study, primary cultures of cyst epithelial cells from autosomal dominant (ADPKD) and recessive (ARPKD) PKD kidneys were used to determine whether controlled addition of Ca2+ could reverse the aberrant mitogenic response to cAMP. Steady-state intracellular Ca2+ levels were found to be 20 nM lower in cyst-derived ADPKD cells (57 +/- 2 nM) compared with NHK cells (77 +/- 2 nM). Treatment of ADPKD cells or ARPKD cells with either Bay K8644, a Ca2+ channel activator, or A23187, a Ca2+ ionophore, caused sustained increases in intracellular Ca2+ levels and completely reversed the mitogenic response to cAMP. Elevation of intracellular Ca2+ levels in ADPKD cells increased Akt activity and blocked cAMP-dependent B-Raf and ERK activation. Thus, increases in [Ca2+]i are able to restore the normal anti-mitogenic response to cAMP in cells that are derived from two genetically distinct forms of PKD.

Increased Water Intake Decreases Progression of Polycystic Kidney Disease in the PCK Rat

Renal enlargement in polycystic kidney disease (PKD) is caused by the proliferation of mural epithelial cells and transepithelial fluid secretion into the cavities of innumerable cysts. Arginine vasopressin (AVP) stimulates the proliferation of human PKD cells in vitro via cAMP-dependent activation of the B-Raf/MEK (MAPK/ERK kinase/extracellular signal-regulated kinase (ERK) pathway. ERK activity is elevated in cells that line the cysts in animals with PKD, and AVP receptor antagonists reduce ERK activity and halt disease progression. For suppression of the effect of AVP physiologically, water intake was increased in PCK rats, a model of PKD, and the effect on renal morphology, cellular mechanism, and function was determined. The addition of 5% glucose in the drinking water increased fluid intake approximately 3.5-fold compared with rats that received tap water. In PCK rats, increased water intake for 10 wk reduced urinary AVP excretion (68.3%), and urine osmolality fell below 290 mOsmol/kg. High water intake was associated with reduced renal expression of AVP V2 receptors (41.0%), B-Raf (15.4%), phosphorylated ERK (38.1%), and proliferating cell nuclear antigen-positive renal cells (61.7%). High water intake reduced the kidney/body weight ratio 28.0% and improved renal function. Taken together, these data demonstrate that water intake that is sufficient to cause persistent water diuresis suppresses B-Raf/MEK/ERK activity and decreases cyst and renal volumes in PCK rats. It is suggested that limiting serum AVP levels by increased water intake may be beneficial to some patients with PKD.

A tumor necrosis factor-α-mediated pathway promoting autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is caused by heterozygous mutations in either PKD1 or PKD2, genes that encode polycystin-1 and polycystin-2, respectively. We show here that tumor necrosis factor-α (TNF-α), an inflammatory cytokine present in the cystic fluid of humans with ADPKD, disrupts the localization of polycystin-2 to the plasma membrane and primary cilia through a scaffold protein, FIP2, which is induced by TNF-α. Treatment of mouse embryonic kidney organ cultures with TNF-α resulted in formation of cysts, and this effect was exacerbated in the Pkd2+/− kidneys. TNF-α also stimulated cyst formation in vivo in Pkd2+/− mice. In contrast, treatment of Pkd2+/− mice with the TNF-α inhibitor etanercept prevented cyst formation. These data reveal a pathway connecting TNF-α signaling, polycystins and cystogenesis, the activation of which may reduce functional polycystin-2 below a critical threshold, precipitating the ADPKD cellular phenotype.

Early embryonic renal tubules of wild-type and polycystic kidney disease kidneys respond to cAMP stimulation with cystic fibrosis transmembrane conductance regulator/Na(+),K(+),2Cl(-) Co-transporter-dependent cystic dilation.

Metanephric organ culture has been used to determine whether embryonic kidney tubules can be stimulated by cAMP to form cysts. Under basal culture conditions, wild-type kidneys from embryonic day 13.5 to 15.5 mice grow in size and continue ureteric bud branching and tubule formation over a 4- to 5-d period. Treatment of these kidneys with 8-Br-cAMP or the cAMP agonist forskolin induced the formation of dilated tubules within 1 h, which enlarged over several days and resulted in dramatically expanded cyst-like structures of proximal tubule and collecting duct origin. Tubule dilation was reversible upon withdrawal of 8-Br-cAMP and was inhibited by the cAMP-dependent protein kinase inhibitor H89 and the cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor CFTR(inh)172. For further testing of the role of CFTR, metanephric cultures were prepared from mice with a targeted mutation of the Cftr gene. In contrast to kidneys from wild-type mice, those from Cftr -/- mice showed no evidence of tubular dilation in response to 8-Br-cAMP, indicating that CFTR Cl(-) channels are functional in embryonic kidneys and are required for cAMP-driven tubule expansion. A requirement for transepithelial Cl(-) transport was demonstrated by inhibiting the basolateral Na(+),K(+),2Cl(-) co-transporter with bumetanide, which effectively blocked all cAMP-stimulated tubular dilation. For determination of whether cystic dilation occurs to a greater extent in PKD kidneys in response to cAMP, Pkd1(m1Bei) -/- embryonic kidneys were treated with 8-Br-cAMP and were found to form rapidly CFTR- and Na(+),K(+),2Cl(-) co-transporter-dependent cysts that were three- to six-fold larger than those of wild-type kidneys. These results suggest that cAMP can stimulate fluid secretion early in renal tubule development during the time when renal cysts first appear in PKD kidneys and that PKD-deficient renal tubules are predisposed to abnormally increased cyst expansion in response to elevated levels of cAMP.

Urinary Excretion of Monocyte Chemoattractant Protein-1 in Autosomal Dominant Polycystic Kidney Disease

Autosomal dominant polycystic kidney disease (ADPKD) progresses to renal insufficiency in >50% of patients and is characterized by interstitial inflammation and fibrosis in the end stage. In a rat model of ADPKD, monocytes accumulate within the renal interstitium in association with increased levels of monocyte chemoattractant protein-1 (MCP-1) in cyst mural cells and increased excretion of this chemokine into the urine. For determining the extent to which this chemokine is abnormally expressed in patients with ADPKD, a cross-section study was performed of MCP-1 in urine, serum, and cyst fluid and MCP-1 production by mural epithelial cells cultured from the cysts of human patients with ADPKD. Upper boundaries for urinary MCP-1 excretion (>263 pg/mg creatinine) and serum creatinine concentration (>1.5 mg/dl) determined in 19 normal individuals were used to sort 55 ADPKD patients into three groups. In group 1 (n = 13), urine MCP-1 excretion (136 +/- 14 pg/mg creatinine) was not different from normal volunteers (152 +/- 16 pg/mg); serum creatinine levels and urine total protein excretion were normal as well. In group 2 (n = 27), urine MCP-1 excretion was increased (525 +/- 39 pg/mg creatinine), but serum creatinine levels and urine protein excretion were not different from normal. In group 3 (n = 15), urine MCP-1 excretion increased further (1221 +/- 171 pg/mg), serum creatinine levels increased to 4.3 +/- 0.8 mg/dl, and urine protein excretion rose to 0.64 +/- 0.28 mg/mg creatinine. Serum MCP-1 levels of ADPKD patients (84 +/- 9.9 pg/ml; n = 15) did not differ from normal. Levels of MCP-1 much higher than in serum or urine were found in cyst fluids obtained from nephrectomy specimens (range, 767 to 40,860 pg/ml; mean, 6434 +/- 841 pg/ml; n = 73). Polarized, confluent cultures of ADPKD cyst epithelial cells secreted MCP-1 into the apical fluid to levels eightfold greater than in the basolateral medium. Similar results were obtained with tubule epithelial cells cultured from normal human renal cortex. On the basis of these results, it is concluded that urinary excretion of MCP-1 is increased in the majority of adult patients with ADPKD and that the source of some of this chemokine may be the mural epithelium of cysts. Furthermore, it seemed that urinary MCP-1 excretion may have increased in these ADPKD patients before appreciable increases in serum creatinine concentration or urine protein excretion were detected. It is reasonable to include urine MCP-1 excretion among candidate surrogate markers in controlled, longitudinal studies of ADPKD.

Cyclic AMP-Mediated Cyst Expansion

In polycystic kidney disease (PKD), intracellular cAMP promotes cyst enlargement by stimulating mural epithelial cell proliferation and transepithelial fluid secretion. The proliferative effect of cAMP in PKD is unique in that cAMP is anti-mitogenic in normal renal epithelial cells. This phenotypic difference in the proliferative response to cAMP appears to involve cross-talk between cAMP and Ca(2+) signaling to B-Raf, a kinase upstream of the MEK/ERK pathway. In normal cells, B-Raf is repressed by Akt (protein kinase B), a Ca(2+)-dependent kinase, preventing cAMP activation of ERK and cell proliferation. In PKD cells, disruption of intracellular Ca(2+) homeostasis due to mutations in the PKD genes relieves Akt inhibition of B-Raf, allowing cAMP stimulation of B-Raf, ERK and cell proliferation. Fluid secretion by cystic cells is driven by cAMP-dependent transepithelial Cl(-) secretion involving apical cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels. This review summarizes the current knowledge of cAMP-dependent cyst expansion, focusing on cell proliferation and Cl(-)-dependent fluid secretion, and discusses potential therapeutic approaches to inhibit renal cAMP production and its downstream effects on cyst enlargement. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

Tolvaptan inhibits ERK-dependent cell proliferation, Cl− secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ∼10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.

Macrophages promote polycystic kidney disease progression.

Renal M2-like macrophages have critical roles in tissue repair stimulating tubule cell proliferation and, if they remain, fibrosis. M2-like macrophages have also been implicated in promoting cyst expansion in mouse models of autosomal dominant polycystic kidney disease (ADPKD). While renal macrophages have been documented in human ADPKD, there are no studies in autosomal recessive polycystic kidney disease (ARPKD). Here we evaluated the specific phenotype of renal macrophages and their disease-impacting effects on cystic epithelial cells. We found an abundance of M2-like macrophages in the kidneys of patients with either ADPKD or ARPKD and in the cystic kidneys of cpk mice, a model of ARPKD. Renal epithelial cells from either human ADPKD cysts or non-cystic human kidneys promote differentiation of naive macrophages to a distinct M2-like phenotype in culture. Reciprocally, these immune cells stimulate the proliferation of renal tubule cells and microcyst formation in vitro. Further, depletion of macrophages from cpk mice indicated that macrophages contribute to PKD progression regardless of the genetic etiology. Thus M2-like macrophages are two-pronged progression factors in PKD promoting cyst cell proliferation, cyst growth, and fibrosis. Agents that block the emergence of these cells or their effects in the cystic kidney may be effective therapies for slowing PKD progression.

Prospects for mTOR Inhibitor Use in Patients with Polycystic Kidney Disease and Hamartomatous Diseases

Mammalian target of rapamycin (mTOR) is the core component of two complexes, mTORC1 and mTORC2. mTORC1 is inhibited by rapamycin and analogues. mTORC2 is impeded only in some cell types by prolonged exposure to these compounds. mTOR activation is linked to tubular cell proliferation in animal models and human autosomal dominant polycystic kidney disease (ADPKD). mTOR inhibitors impede cell proliferation and cyst growth in polycystic kidney disease (PKD) models. After renal transplantation, two small retrospective studies suggested that mTOR was more effective than calcineurin inhibitor-based immunosuppression in limiting kidney and/or liver enlargement. By inhibiting vascular remodeling, angiogenesis, and fibrogenesis, mTOR inhibitors may attenuate nephroangiosclerosis, cyst growth, and interstitial fibrosis. Thus, they may benefit ADPKD at multiple levels. However, mTOR inhibition is not without risks and side effects, mostly dose-dependent. Under certain conditions, mTOR inhibition interferes with adaptive increases in renal proliferation necessary for recovery from injury. They restrict Akt activation, nitric oxide synthesis, and endothelial cell survival (downstream from mTORC2) and potentially increase the risk for glomerular and peritubular capillary loss, vasospasm, and hypertension. They impair podocyte integrity pathways and may predispose to glomerular injury. Administration of mTOR inhibitors is discontinued because of side effects in up to 40% of transplant recipients. Currently, treatment with mTOR inhibitors should not be recommended to treat ADPKD. Results of ongoing studies must be awaited and patients informed accordingly. If effective, lower dosages than those used to prevent rejection would minimize side effects. Combination therapy with other effective drugs could improve tolerability and results.