Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease.

cAMP stimulates cell proliferation and Cl(-)-dependent fluid secretion, promoting the progressive enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Intracellular cAMP levels are determined by the balance of cAMP synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Therefore, PDE isoform expression and activity strongly influence global and compartmentalized cAMP levels. We report here that PDE3 and PDE4 expression levels are lower in human ADPKD tissue and cells compared with those of normal human kidneys (NHKs), whereas PDE1 levels are not significantly different. Inhibition of PDE4 caused a greater increase in basal and vasopressin (AVP)-stimulated cAMP levels and Cl(-) secretion by ADPKD cells than inhibition of PDE1, and inhibition of PDE4 induced cyst-like dilations in cultured mouse Pkd1(-/-) embryonic kidneys. In contrast, inhibition of PDE1 caused greater stimulation of extracellular signal-regulated kinase (ERK) and proliferation of ADPKD cells than inhibition of PDE4, and inhibition of PDE1 enhanced AVP-induced ERK activation. Notably, inhibition of PDE1, the only family of Ca(2+)-regulated PDEs, also induced a mitogenic response to AVP in NHK cells, similar to the effect of restricting intracellular Ca(2+). PDE1 coimmunoprecipitated with B-Raf and A-kinase anchoring protein 79, and AVP increased this interaction in ADPKD but not NHK cells. These data suggest that whereas PDE4 is the major PDE isoform involved in the regulation of global intracellular cAMP and Cl(-) secretion, PDE1 specifically affects the cAMP signal to the B-Raf/MEK/ERK pathway and regulates AVP-induced proliferation of ADPKD cells.

Periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney disease.

In renal cystic diseases, sustained enlargement of fluid-filled cysts is associated with severe interstitial fibrosis and progressive loss of functioning nephrons. Periostin, a matricellular protein, is highly overexpressed in cyst-lining epithelial cells of autosomal-dominant polycystic disease kidneys (ADPKD) compared with normal tubule cells. Periostin accumulates in situ within the matrix subjacent to ADPKD cysts, binds to αVß3 and αVß5 integrins, and stimulates the integrin-linked kinase to promote cell proliferation. We knocked out periostin (Postn) in pcy/pcy mice, an orthologous model of nephronophthisis type 3, to determine whether periostin loss reduces PKD progression in a slowly progressive model of renal cystic disease. At 20 weeks of age, pcy/pcy:Postn(-/-) mice had a 34% reduction in kidney weight/body weight, a reduction in cyst number and total cystic area, a 69% reduction in phosphorylated S6, a downstream component of the mTOR pathway, and fewer proliferating cells in the kidneys compared with pcy/pcy:Postn(+/+) mice. The pcy/pcy Postin knockout mice also had less interstitial fibrosis with improved renal function at 20 weeks and significantly longer survival (51.4 compared with 38.0 weeks). Thus, periostin adversely modifies the progression of renal cystic disease by promoting cyst epithelial cell proliferation, cyst enlargement, and interstitial fibrosis, all contributing to the decline in renal function and premature death.

A PRoliferation-Inducing Ligand (APRIL) in the Pathogenesis of Immunoglobulin A Nephropathy: A Review of the Evidence.

A PRoliferation-Inducing Ligand (APRIL), the thirteenth member of the tumor necrosis factor superfamily, plays a key role in the regulation of activated B cells, the survival of long-lived plasma cells, and immunoglobulin (Ig) isotype class switching. Several lines of evidence have implicated APRIL in the pathogenesis of IgA nephropathy (IgAN). Globally, IgAN is the most common primary glomerulonephritis, and it can progress to end-stage kidney disease; yet, disease-modifying treatments for this condition have historically been lacking. The preliminary data in ongoing clinical trials indicate that APRIL inhibition can reduce proteinuria and slow the rate of kidney disease progression by acting at an upstream level in IgAN pathogenesis. In this review, we examine what is known about the physiologic roles of APRIL and evaluate the experimental and epidemiological evidence describing how these normal biologic processes are thought to be subverted in IgAN. The weight of the preclinical, clinical, and genetic data supporting a key role for APRIL in IgAN has galvanized pharmacologic research, and several anti-APRIL drug candidates have now entered clinical development for IgAN. Herein, we present an overview of the clinical results to date. Finally, we explore where more research and evidence are needed to transform potential therapies into clinical benefits for patients with IgAN.

Calmodulin-sensitive adenylyl cyclases mediate AVP-dependent cAMP production and Cl- secretion by human autosomal dominant polycystic kidney cells.

In autosomal dominant polycystic kidney disease (ADPKD), binding of AVP to the V2 receptor (V2R) increases cAMP and accelerates cyst growth by stimulating cell proliferation and Cl(-)-dependent fluid secretion. Basal cAMP is elevated in human ADPKD cells compared with normal human kidney (NHK) cells. V2R mRNA levels are elevated in ADPKD cells; however, AVP caused a greater increase in global cAMP in NHK cells, suggesting an intrinsic difference in cAMP regulation. Expression, regulatory properties, and receptor coupling of specific adenylyl cyclases (ACs) provide temporal and spatial regulation of the cAMP signal. ADPKD and NHK cells express mRNAs for all nine ACs. Ca(2+)-inhibited ACs 5 and 6 are increased in ADPKD cells, while Ca(2+)/CaM-stimulated ACs 1 and 3 are downregulated. ACs 1, 3, 5, and 6 were detected in cyst cells in situ, and codistribution with aquaporin-2 suggests that these cysts were derived from collecting ducts. To determine the contribution of CaM-sensitive ACs to AVP signaling, cells were treated with W-7, a CaM inhibitor. W-7 decreased AVP-induced cAMP production and Cl(-) secretion by ADPKD cells. CaMKII inhibition increased AVP-induced cAMP, suggesting that cAMP synthesis is mediated by AC3. In contrast, CaM and CaMKII inhibition in NHK cells did not affect AVP-induced cAMP production. Restriction of intracellular Ca(2+) switched the response in NHK cells, such that CaM inhibition decreased AVP-induced cAMP production. We suggest that a compensatory response to decreased Ca(2+) in ADPKD cells switches V2R coupling from Ca(2+)-inhibited ACs 5/6 to Ca(2+)/CaM-stimulated AC3, to mitigate high cAMP levels in response to continuous AVP stimulation.

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.

Complex changes in ecto-nucleoside 5'-triphosphate diphosphohydrolase expression in hypoxanthine phosphoribosyl transferase deficiency.

Lesch-Nyhan disease is caused by a deficiency of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT). The link between HPRT deficiency and the neuropsychiatric symptoms is unknown. In rat B103 neuroblastoma cell membranes and mouse Neuro2a neuroblastoma cell membranes, nucleoside 5'-triphosphatase (NTPase) activity is substantially reduced, whereas in fibroblast membranes from HPRT knock-out mice, NTPase activity is increased. Candidate genes for these NTPase activity changes are ecto-nucleoside 5'-triphosphate diphosphohydrolases (NTPDases). Therefore, we studied expression of NTPDases in B103 cells, Neuro2a cells and skin fibroblasts by reverse transcriptase polymerase chain reaction and restriction enzyme digestion of amplified cDNA fragments. In B103 cells, expression of NTPDases 1, 3 and 6 decreased, whereas expression of NTPDases 4 and 5 increased in HPRT deficiency. In Neuro2a cells, expression of NTPDases 3-6 increased in HPRT deficiency. In fibroblasts, NTPDase 3 expression decreased, and expression of NTPDases 4-6 increased in HPRT deficiency. Collectively, there are complex decreases and increases in NTPDase isoform expression in HPRT deficiency that depend on the specific cell type and species studied. These changes in NTPDase expression may reflect an (insufficient) attempt of cells to compensate for the changes in nucleotide metabolism caused by HPRT deficiency.

Decreased GTP-stimulated adenylyl cyclase activity in HPRT-deficient human and mouse fibroblast and rat B103 neuroblastoma cell membranes.

Defect of the purine salvage enzyme, hypoxanthine phosphoribosyl transferase (HPRT), results in Lesch-Nyhan disease (LND). It is unknown how the metabolic defect translates into the severe neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in GTP, UTP and CTP concentrations in HPRT-deficient cells. Moreover, GTP, ITP, XTP, UTP and CTP differentially support Gs-protein-mediated adenylyl cyclase (AC) activation. Based on these findings we hypothesized that abnormal AC regulation may constitute the missing link between HPRT deficiency and the neuropsychiatric symptoms in LND. To test this hypothesis, we studied AC activity in membranes from primary human skin and immortalized mouse skin fibroblasts, mouse Neuro-2a neuroblastoma cells and rat B103 neuroblastoma cells. In B103 control membranes, GTP, ITP, XTP and UTP exhibited profound stimulatory effects on basal AC activity that approached the effects of hydrolysis-resistant nucleotide analogs. In HPRT- membranes, the stimulatory effects of GTP, ITP, XTP and UTP were strongly reduced. Similarly, in human and mouse skin fibroblast membranes we also observed a decrease in GTP-stimulated AC activity in HPRT-deficient cells compared with the respective controls. In mouse Neuro-2a neuroblastoma membranes, AC activity in the presence of GTP was below the detection limit of the assay. We discuss several possibilities to explain the abnormalities in AC regulation in HPRT deficiency that encompass various species and cell types.

Altered membrane NTPase activity in Lesch-Nyhan disease fibroblasts: comparison with HPRT knockout mice and HPRT-deficient cell lines.

Lesch-Nyhan disease (LND) is a rare disorder caused by a defect of an enzyme in the purine salvage pathway, hypoxanthine phosphoribosyl transferase (HPRT). It is still unknown how the metabolic defect translates into the complex neuropsychiatric phenotype characterized by self-injurious behavior, dystonia and mental retardation. There are abnormalities in purine and pyrimidine nucleotide content in HPRT-deficient cells. We hypothesized that altered nucleotide concentrations in HPRT deficiency change G-protein-mediated signal transduction. Therefore, our original study aim was to examine the high-affinity GTPase activity of G-proteins in membranes from primary human skin and immortalized mouse skin fibroblasts, rat B103 neuroblastoma cells and mouse Neuro-2a neuroblastoma cells. Unexpectedly, in membranes from human fibroblasts, B103- and Neuro-2a cells, V(max) of low-affinity nucleoside 5'-triphosphatase (NTPase) activities was decreased up to 7-fold in HPRT deficiency. In contrast, in membranes from mouse fibroblasts, HPRT deficiency increased NTPase activity up to 4-fold. The various systems analyzed differed from each other in terms of K(m) values for NTPs, absolute V(max) values and K(i) values for nucleoside 5'-[beta,gamma-imido]triphosphates. Our data show that altered membrane NTPase activity is a biochemical hallmark of HPRT deficiency, but species and cell-type differences have to be considered. Thus, future studies on biochemical changes in LND should be conducted in parallel in several HPRT-deficient systems.