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Recently, the use of overexpression of telomerase reverse transcriptase (TERT) has led to the generation of immortalized human cell lines. However, this cell immortalization approach has not been reported in well-differentiated mouse cells, such as renal epithelial cells. We sought to establish and then characterize a mouse collecting duct cell line, using ectopic expression of mTERT. Isolated primary cortical collecting duct (CCD) cell lines were transduced with mouse (m)TERT, using a lentiviral vector. mTERT-negative cells did not survive blasticidin selection, whereas mTERT-immortalized cells proliferated in selection media for over 40 subpassages, mTERT messenger RNA and telomerase activity was elevated in these cells, compared with an SV40-immortalized cell line. Flow cytometry with Dolichos biflorus agglutinin was used to select the CCD principal cells, and we designated this cell line mTERT-CCD. Cells were well differentiated and exhibited morphological characteristics typically found in renal epithelial cells, such as tight junction formation, microvilli, and primary cilia. Further characterization using standard immunofluorescence revealed abundant expression of aquaporin-2 and the vasopressin type 2 receptor. mTERT-CCD cells exhibited cAMP-stimulated/benzamil-inhibited whole cell currents. Whole cell patch-clamp currents were also enhanced after a 6-day treatment with aldosterone. In conclusion, we have successfully used mTERT to immortalize mouse collecting duct cells that retain the basic in vivo phenotypic characteristics of collecting duct cells. This technique should be valuable in generating cell lines from genetically engineered mouse models. epithelial; polycystic doi: 10.1152/ajprenal.00183.2010

Mutations in PKD1 and TRPP2 account for nearly all cases of autosomal dominant polycystic kidney disease (ADPKD). These 2 proteins form a receptor/ion channel complex on the cell surface. Using a combination of biochemistry, crystallography, and a single-molecule method to determine the subunit composition of proteins in the plasma membrane of live cells, we find that this complex contains 3 TRPP2 and 1 PKD1. A newly identified coiled-coil domain in the C terminus of TRPP2 is critical for the formation of this complex. This coiled-coil domain forms a homotrimer, in both solution and crystal structure, and binds to a single coiled-coil domain in the C terminus of PKD1. Mutations that disrupt the TRPP2 coiled-coil domain trimer abolish the assembly of both the full-length TRPP2 trimer and the TRPP2/PKD1 complex and diminish the surface expression of both proteins. These results have significant implications for the assembly, regulation, and function of the TRPP2/PKD1 complex and the pathogenic mechanism of some ADPKD-producing mutations. autosomal dominant polycystic kidney disease | single-molecule imaging | stoichiometry | transient receptor potential channel | X-ray crystallography

Autosomal dominant polycystic kidney disease (ADPKD) is a highly prevalent genetic disease that results in cyst formation in kidney and liver. Cytokines and growth factors secreted by the cyst-lining epithelia are positioned to initiate autocrine/paracrine signaling and promote cyst growth. Comparative analyses of human kidney and liver cyst fluids revealed disparate cytokine/growth factor profiles. CXCR2 agonists, including IL-8, epithelial neutrophil-activating peptide (ENA-78), growth-related oncogene-[alpha] (GRO-[alpha]), are potent proliferative agents that were found at high levels in liver but not kidney cyst fluids. Liver cysts are lined by epithelial cells derived from the intrahepatic bile duct (i.e., cholangiocytes). In polarized pkd2(WS25/-) mouse liver cyst epithelial monolayers, CXCR2 agonists were released both apically and basally, indicating that they may act both on the endothelial and epithelial cells within or lining the cyst wall. IL-8 and human liver cyst fluid induced cell proliferation of HMEC-1 cells, a human microvascular endothelial cell line, and Mz-ChA1 cells, a human cholangiocyte cell model. IL-8 expression can be regulated by specific stresses. Hypoxia and mechanical stretch, two likely stressors acting on the liver cyst epithelia, significantly increased IL-8 secretion and promoter activity. AP-1, c/EBP, and NF-[kappa]B were required but not sufficient to drive the stress-induced increase in IL-8 transcription. An upstream element between -272 and -1,481 bp allowed for the stress-induced increase in IL-8 transcription. These studies support the hypothesis that CXCR2 signaling promotes ADPKD liver cyst growth. interleukin-8; cholangiocytes; endothelium; cytokines; growth factors; autosomal dominant polycystic kidney disease

Polycystin-1 (PC1) has an essential function in renal tubular morphogenesis and disruption of its function causes cystogenesis in human autosomal dominant polycystic kidney disease. We have previously shown that recombinant human PC1 is cis-autoproteolytically cleaved at the G protein-coupled receptor proteolytic site domain. To investigate the role of cleavage in vivo, we generated by gene targeting a Pkd1 knockin mouse ([Pkd1.sup.V/V]) that expresses noncleavable PC1. The [Pkd1.sup.V/V] mice show a hypomorphic phenotype, characterized by a delayed onset and distal nephron segment involvement of cystogenesis at postnatal maturation stage. We show that PC1 is ubiquitously and incompletely cleaved in wild-type mice, so that uncleaved and cleaved PC1 molecules coexist. Our study establishes a critical but restricted role of cleavage for PC1 function and suggests a differential function of the two types of PC1 molecules in vivo. autosomal dominant polycystic kidney disease | cis-autoproteoiytic cleavage | GPS | knockin mouse | tubulogenesis

The root cause for most cases of autosomal-dominant polycystic kidney disease (ADPKD) is mutations in the polycystin-1 (PC1) gene. While PC1 has been implicated in a perplexing variety of protein interactions and signaling pathways, what its normal function is and why its disruption leads to the proliferation of renal epithelial cells are unknown. Recent results suggest that PC1 is involved in mechanotransduction by primary cilia measuring the degree of luminal fluid flow. PC1 has also recently been shown to regulate the mTOR and signal transducers and activators of transcription (STAT) 6 pathways. These two pathways are normally dormant in the healthy kidney but are activated in response to injury and appear to drive a proliferative repair response. This review develops the idea that a critical function of PC1 and primary cilia in the adult kidney may be to sense renal injury by detecting changes in luminal fluid flow and to trigger proliferation. Constitutive activation of these pathways in ADPKD would lead to the futile attempt to repair a nonexisting injury, resulting in cyst growth. The existence of many known cellular and molecular similarities between renal repair and ADPKD supports this model. mTOR; STAT6; rapamycin; kidney injury and repair; ischemia-reperfusion injury

The genetics and physiology of polycystic kidney disease are reviewed. Topics include autosomal dominant and autosomal recessive polycystic kidney disease, familial nephronophthisis, medullary cystic kidney disease, cell biology, molecular biology, developmental regulation and programming, and prospects for prognosis and therapy. Polycystic kidney disease is caused by cysts in the kidneys that can cause kidney failure.

Autosomal dominant polycystic kidney disease (AD-PKD) is the result of mutations in one allele of the PKD1 or PKD2 genes, followed by 'second hit' somatic mutations of the other allele in renal tubule cells. Continued proliferation of clonal cells originating from different nephron segments leads to cyst formation. In vitro studies of the mechanisms of cyst formation have been hampered by the scarcity of nephrectomy specimens and the limited life span of cyst-derived cells in primary culture. We describe the development of a series of immortalized epithelial cell lines from over 30 individual renal cysts obtained from 11 patients with AD-PKD. The cells were immortalized with either wild-type (WT) or temperature-sensitive (TS) recombinant adeno-simian virus (SV)40 viruses. SV40 DNA integration into the cell genome was verified by PCR analysis. The cells have been passaged over 50 times with no apparent phenotypic change. By light microscopy, the cells appear pleomorphic but mostly polygonal and resemble the primary cultures. Transmission electron microscopy shows polarized epithelia with tight junctions. The SV40 large T antigen was detected by immunocytochemistry and by Western blot analysis at 37[degrees]C in the WT cell lines and at 33[degrees]C in the TS cell lines. It disappeared in TS cells 72 h following transfer to 39[degrees]C. The majority (29) of the cell lines show binding of Dolichos biflorus lectin, suggesting distal tubule origin. Three cell lines show binding of Lotus tetragonolobus lectin or express aminopeptidase N, suggesting proximal tubule origin. Three cell lines were derived from a mixture of cysts and express features of both tubules. The PKD1 and PKD2 mRNA and protein were detected in all cells by RT-PCR and by immunocytochemistry. The majority of the cells tested also express the epidermal growth factor receptor, cystic fibrosis transmembrane conductance regulator, epithelial sodium channel, and renin. These new series of cyst-derived cell lines represent useful and readily available in vitro models for studying the cellular and molecular biology of ADPKD. polycystic kidney disease; SV40

Research demonstrates that Polycystin-2 carboxyl terminal domain interacts with the cardiac troponin I with a 1:1 binding stoichiometry. Troponin I is an inhibitory subunit of the troponin complex and functions as an inhibitor of angiogenesis in the autosomal dominant polycystic kidney disease condition.

Polycystin-1 plays an essential role in renal tubular morphogenesis, and disruption of its function causes cystogenesis in human autosomal-dominant polycystic kidney disease (ADPKD). We demonstrated that polycystin-1 undergoes cleavage at G protein coupled receptor proteolytic site in a process that requires the receptor for egg jelly domain. Most of the N-terminal fragment remains tethered at the cell surface, although a small amount is secreted. PKD1-associated mutations in the receptor for egg jelly domain disrupt cleavage, abolish the ability of polycystin-1 to activate signal transducer and activator of transcription-1, and induce tubulogenesis in vitro. We conclude that the cleavage of polycystin-1 is likely essential for its biologic activity.

ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic [Ca.sup.2+] concentration and secretory [Cl.sup.-] transport show P2Y and P2X purinergic receptor-mediated stimulation of [Cl.sup.-] secretion via cytosolic [Ca.sup.2+]-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression. polycysfic kidney disease; adenosine 5'-triphosphate; autocrine; epithelia; purinergic receptors

Cilia are organelles that play diverse roles, from fluid movement to sensory reception. Polaris, a protein associated with cystic kidney disease in [Tg737.sup.orpk] mice, functions in a ciliogenic pathway. Here, we explore the role of polaris in primary cilia on Madin-Darby canine kidney cells. The results indicate that polaris localization and solubility change dramatically during cilia formation. These changes correlate with the formation of basal bodies and large protein rafts at the apical surface of the epithelia. A cortical collecting duct cell line has been derived from mice with a mutation in the Tg737 gene. These cells do not develop normal cilia, which can be corrected by reexpression of the wild-type Tg737 gene. These data suggest that the primary cilia are important for normal renal function and/or development and that the ciliary defect may be a contributing factor to the cystic disease in [Tg737.sup.orpk] mice. Further characterization of these cells will be important in elucidating the physiological role of renal cilia and in determining their relationship to cystic disease. Tg737; ciliogenesis; cell line; polycystic kidney disease; Madin-Darby canine kidney ceils; Oak Ridge Polycystic Kidney

Polycystins are a distinct family of integral membrane proteins, related by sequence and structural homology. These proteins play important roles in organ morphogenesis and function, and mutations in two of the five known members cause a common renal cystic disease. Recent data on the function of polycystins have revealed them to be [Ca.sup.2+]-permeable cation channels and putative ion-channel regulators. Their roles in the regulation of intracellular [Ca.sup.2+] concentration and in polycystic kidney disease are discussed.

PKD2 encodes a protein of unknown function that is mutated in 15% of autosomal dominant polycystic kidney disease (ADPKD) families. Polyclonal antisera were employed against PKD2 to study the pattern of Pkd2 expression in staged mouse embryos. Staining for Pkd2 was documented beginning on the 6th embryonic day in the embryonic ectoderm and endoderm. The greatest intensity of staining was seen in the fetal but not adult adrenal cortex and in red blood cell precursors. Expression was also seen in multiple endocrine organs, in cardiac, skeletal and smooth muscle, and in multiple mesenchymal tissues. The diffuse distribution and early expression of Pkd2 indicate a fundamental developmental role.

High-resolution, nonradioactive in situ hybridization was employed to investigate cell differentiation and cell polarity during cyst formation in the rat model of ADPKD. Results show that alkaline phosphatase, aquaporin-1 and sodium potassium adenosine triphosphatase were normally expressed during the early stages of cyst development. Change in cell polarity was not observed at any stage. Findings indicate that cell differentiation is late in the course of cyst formation.

The function, expression and regulation of anion exchangers were analyzed in normal intrahepatic biliary epithelium (IBE) and liver cyst derived epithelial (LCDE) cell lines. Analysis of altered cyst fluid secretion in autosomal dominant polycystic kidney disease (ADPKD) cells indicated the presence of reduced Cl-/HCO- anion exchanger activity in LCDE cells compared to normal IBE cells. The decreased function of the anion exchanger in LCDE cells was also caused by reduced levels of plasmalemmal AE2 polypeptides.

Aquaporins (AQP) are membrane water channels in the renal collecting duct (CD) which are involved in water transport. Expression of two AQPs, AQP1 and AQP2, was investigated during pathogenesis of autosomal dominant polycystic kidney disease. During 12-wk. gestation, AQP1 was detected in developing proximal tubule epithelia while AQP2 was detected in the apical cell membranes of the ureteric bud and the CD system. AQP1 was later seen in descending thin limbs of Henle and AQP2 was restricted to CD cells.

The pH regulatory pathways in epithelial cells derived from liver cysts (LCDE) of autosomal dominant polycystic kidney disease cell lines are the same as those in normal intrahepatic biliary epithelium. The LCDE cells are unable to produce HCO3- in response to secretin due to a defect in the Cl- substitution dependent alkalinization. The appearance of the LCDE cells is typically that of the biliary epithelium. The cells show a positive response to biliary specific markers and a negative response to hepatocyte markers.

Programmed cell death may be related to the loss of kidney function in polycystic kidney disease. Programmed cell death is known as apoptosis. Polycystic kidney disease involves the growth of cysts in extremely enlarged kidneys that gradually lose their function. Researchers analyzed the DNA in the kidneys from 16 patients with the disease, 12 normal patients, and diseased and normal mice. DNA associated with apoptosis was found in all but one of the diseased human kidneys, but not in the normal kidneys. All of the diseased mice had apoptotic DNA, but the normal mice did not. Apoptotic DNA was also detected in cells cultured from diseased kidneys and in diseased kidney tissue stained directly. In tissue staining, apoptotic DNA was found in the glomeruli, the noncystic tubular epithelial cells, and cells lining the cysts. Apoptotic DNA was not found in normal kidney cells or tissue, or in tissue from kidneys from patients with other kidney diseases that were analyzed later.

Autosomal dominant polycystic kidney disease is characterized by enlarged kidneys and the development of numerous cysts on the kidneys. The term autosomal dominant refers to the pattern of genetic transmission of the disease. Other manifestations of the disease include the development of cysts in the spleen, pancreas, liver, ovaries, gastrointestinal tract and central nervous system. Patients who are less than 16 years old rarely develop cysts outside the kidneys. However, the chance of cysts occurring in areas other than the kidneys increases with age. Diagnosis is usually made using ultrasonography, computed tomography or magnetic resonance imaging. Diagnosis usually requires that at least three cysts are present on the kidneys and that there is a family history of the disease. Many patients have a mild form of the disease, but others can have severe pain and many complications, including kidney failure.

The cysts present in autosomal dominant polycystic kidney disease seem capable of secreting fluid. This secretion can be increased by as yet unidentified substances in the cyst fluid. Polycystic kidney disease is characterized by enlarged kidneys and the presence of cysts. The term autosomal dominant refers to the genetic mode of disease transmission. Eighteen cysts were removed from three kidneys taken from women with polycystic kidney disease. Tests were performed to determine secretion rates of both natural cyst fluid and a laboratory-made fluid inserted into the cysts. Cysts containing cyst fluid secreted more fluid than cysts containing artificial fluid. Cyst fluid also stimulated fluid secretion in canine and human kidney cells. Drug therapy may eventually provide a means of controlling the size of cysts.

Polycystic kidney disease is characterized by the development of many cysts, sac-like structures that contain fluid or solid material, in the kidney. Hypertension (high blood pressure) is found in 50 to 75 percent of patients with this disorder and has been putatively linked to inadequate blood flow (ischemia) to the kidney caused by compression of the renal vessels by cysts. This study investigates the possibility that hypertension in polycystic kidney disease is produced by pathologic alterations of the renin-angiotensin-aldosterone system. The active kidney enzyme renin is produced from an inactive form, prorenin, that transforms the liver protein angiotensinogen to angiotensin I, which is then converted by angiotensin-converting enzyme to angiotensin II. This factor, angiotensin II, constricts blood vessels and activates the production and release of the adrenal hormone aldosterone, which regulates sodium, potassium, and chloride metabolism. Through a prospective study of polycystic kidney disease, patients' blood and urine levels of these hormones and other factors have been measured and related to blood pressure readings. Various drugs that block specific metabolic steps in this hormonal system were used to identify elements of the system that affected hypertension. The renin-angiotensin-aldosterone system is found to be significantly stimulated in hypertensive polycystic kidney disease patients. Increased renin release may be related to kidney ischemia. (Consumer Summary produced by Reliance Medical Information, Inc.)

Results show that seven of the 12 transmembrane domains of polycystin function as membrane-spanning domains with an additional four added on on their topogenic potential to be membrane-spanning domains as shown by a combination of glycosylation, protease protection, and carbonate extraction methods.

Developmental control of cell proliferation is crucial, and abnormal principal cell proliferation may contribute to cystogenesis in polycystic kidney disease. This study investigates roles of cAMP and its primary effector, cAMP-dependent protein kinase (protein kinase A; PKA), in control of cell proliferation in filter-grown noncystic (NC) and cystic (CY)-derived principal cell cultures. These cultures had similar cAMP pathway characteristics upstream of PKA subunit distribution but differed in predicted PKA subtype distribution. Functionally, cultures were proliferative before polarization, with constitutively higher proliferation in CY cultures. NC cultures achieved levels similar to those of CY cultures on pharmacological manipulation of cAMP production or PKA activation or inhibition of PKA subtype I activity. Inhibition of overall PKA activity, or of PKA subtype II anchoring, diminished cAMP/PKA-mediated proliferation in NC cultures but had no effect on CY cultures. Polarized CY monolayers remained proliferative, but NC monolayers lost responsiveness. No large proliferation changes resulted from treatments of polarized cultures; however, polarized NC and CY cultures differed in poststimulation handling of PKA catalytic and type II regulatory subunits. Our results support PKA subtype regulation of prepolarization proliferation in NC principal cells and altered regulation of PKA in CY cells and suggest that differences at or downstream of PKA can contribute to altered proliferation in a developmental renal disease. epidermal growth factor receptor; polycystic kidney disease; protein kinase subtypes; immunolocalization

The cysts in autosomal dominant polycystic kidney disease (ADPKD) may cause kidney failure by damaging normal tissue, rather than by simply crowding out the local tissue. Patients with ADPKD develop cysts in their kidney tubules and about 50% of them eventually experience kidney failure. The inherited gene mutation that leads to cyst growth in some forms of the disease has been identified. The cysts may damage other kidney tissue by triggering the release of cytokines and other chemicals that lead to the growth of scar tissue. A 1995 study showed that programmed cell death, known as apoptosis may be a characteristic of ADPKD. This study suggests that the genes responsible for ADPKD may also cause apoptosis. Through apoptosis, the cysts may cause destruction of kidney tissue.

Two articles in the October 18, 1990 issue of The New England Journal of Medicine are concerned with the measurement of various elements of the renin-angiotensin-aldosterone system. This system is important in the maintenance of blood pressure, and involves the secretion of preprorenin and conversion of this compound to prorenin by a group of endocrine cells, the juxtaglomerular cells, located in the cortex of the kidney. The active kidney enzyme renin is produced from an inactive form, prorenin, that transforms the liver protein angiotensinogen to angiotensin I, which is then converted by angiotensin-converting enzyme to angiotensin II. This factor, angiotensin II, constricts blood vessels and activates the production and release of the adrenal hormone aldosterone, which regulates sodium, potassium, and chloride metabolism. One of these articles deals with the identification of diabetic children and adolescents who are at increased risk of retinal and kidney pathologies when their levels of prorenin are found to be elevated. While the results of this work do not show a causal relationship between blood prorenin and complications of diabetes, the data do establish the value of prorenin as a marker of incipient pathologic difficulties. The second study investigates the risk of microcirculatory abnormalities (small vessel disease) in the kidney as a result of a congenital form of polycystic disease, suggesting that the renin-angiotensin-aldosterone system is an important underlying causative factor in the condition. This study establishes that measurements of plasmal prorenin may be useful in the identification of patients at risk, thus allowing more intensive clinical intervention. (Consumer Summary produced by Reliance Medical Information, Inc.)