Orpk Mouse Research Articles

Loss of cilia increases angiotensin II mediated apical angiotensin‐1‐receptor localization in collecting duct cells

Hypertension (HTN) is frequently present early on in Polycystic Kidney Disease (PKD) before a significant cystic burden has occurred. However, the mechanism for PKD‐induced HTN has not been fully elucidated. The renin‐angiotensin‐system (RAS) is one factor contributing to HTN in PKD, although there are no consistent changes in plasma renin/aldosterone levels in animals or humans with PKD. We hypothesized that intra‐renal‐RAS rather than the systemic RAS may be important in PKD. As an initial approach, we used immortalized collecting duct (CD) cell line derived from Orpk mice with a hypomorphic Ift88 gene resulting in stunted/absent cilia: cilia (−), and Ift88 rescued cells: cilia (+) with normal cilia. There was increased abundance of the angiotensin‐1‐receptor (AT1R) and in (pro) renin in cilia (−) compared to cilia (+) cells. Using biotinylation of apical membranes, 30 min treatment with Ang II (1nM) increased apical AT1R expression in cilia (−) cells but not in cilia (+) cells. Using a GFP‐AT1R construct transfected into these CD cells, we found that with Ang II administration cilia (−) but not cilia (+) cells had predominant luminal membrane localization of GFP‐AT1R. Finally, with addition of Ang II, phospho ERK was increased in cilia (−) compared to cilia (+). Thus, loss of cilia leads to increased abundance of AT1R and the targeting of AT1R to the apical membrane which may lead to increased RAS activity in the CD in PKD.

Gene therapy rescues cilia defects and restores olfactory function in a mammalian ciliopathy model

Cilia are evolutionarily conserved microtubule-based organelles that are crucial for diverse biological functions, including motility, cell signaling and sensory perception. In humans, alterations in the formation and function of cilia manifest clinically as ciliopathies, a growing class of pleiotropic genetic disorders. Despite the substantial progress that has been made in identifying genes that cause ciliopathies, therapies for these disorders are not yet available to patients. Although mice with a hypomorphic mutation in the intraflagellar transport protein IFT88 (Ift88Tg737Rpw mice, also known as ORPK mice)5 have been well studied, the relevance of IFT88 mutations to human pathology is unknown. We show that a mutation in IFT88 causes a hitherto unknown human ciliopathy. In vivo complementation assays in zebrafish and mIMCD3 cells show the pathogenicity of this newly discovered allele. We further show that ORPK mice are functionally anosmic as a result of the loss of cilia on their olfactory sensory neurons (OSNs). Notably, adenoviral-mediated expression of IFT88 in mature, fully differentiated OSNs of ORPK mice is sufficient to restore ciliary structures and rescue olfactory function. These studies are the first to use in vivo therapeutic treatment to reestablish cilia in a mammalian ciliopathy. More broadly, our studies indicate that gene therapy is a viable option for cellular and functional rescue of the complex ciliary organelle in established differentiated cells.

Transport, cilia, and PKD: must we in (cyst) on interrelationships? Focus on “Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease”

<i>Transport, cilia, and PKD: must we in (cyst) on interrelationships?</i> Focus on “Increased Na⁺/H⁺ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease”

Increased Na+/H+ exchanger activity on the apical surface of a cilium-deficient cortical collecting duct principal cell model of polycystic kidney disease

Pathophysiological anomalies in autosomal dominant and recessive forms of polycystic kidney disease (PKD) may derive from impaired function/formation of the apical central monocilium of ductal epithelia such as that seen in the Oak Ridge polycystic kidney or orpk (Ift88(Tg737Rpw)) mouse and its immortalized cell models for the renal collecting duct. According to a previous study, Na/H exchanger (NHE) activity may contribute to hyperabsorptive Na(+) movement in cilium-deficient ("mutant") cortical collecting duct principal cell monolayers derived from the orpk mice compared with cilium-competent ("rescued") monolayers. To examine NHE activity, we measured intracellular pH (pH(i)) by fluorescence imaging with the pH-sensitive dye BCECF, and used a custom-designed perfusion chamber to control the apical and basolateral solutions independently. Both mutant and rescued monolayers exhibited basolateral Na(+)-dependent acid-base transporter activity in the nominal absence of CO(2)/HCO(3)(-). However, only the mutant cells displayed appreciable apical Na(+)-induced pH(i) recoveries from NH(4)(+) prepulse-induced acid loads. Similar results were obtained with isolated, perfused collecting ducts from orpk vs. wild-type mice. The pH(i) dependence of basolateral cariporide/HOE-694-sensitive NHE activity under our experimental conditions was similar in both mutant and rescued cells, and 3.5- to 4.5-fold greater than apical HOE-sensitive NHE activity in the mutant cells (pH(i) 6.23-6.68). Increased apical NHE activity correlated with increased apical NHE1 expression in the mutant cells, and increased apical localization in collecting ducts of kidney sections from orpk vs. control mice. A kidney-specific conditional cilium-knockout mouse produced a more acidic urine compared with wild-type littermates and became alkalotic by 28 days of age. This study provides the first description of altered NHE activity, and an associated acid-base anomaly in any form of PKD.

Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.

Primary cilium is required for vasopressin mediated aquaporin‐2 trafficking

The collecting duct cell lines PCDNA (cilia (−)) and BAP2 (cilia (+)/rescue) derived from a PKD mouse model (orpk mouse) were used to determine the role of the primary cilium in vasopressin (AVP) mediated aquaporin 2 (AQP2) trafficking and fluid absorption. With apical AVP addition, cilia (−) cells grown on impermeable supports demonstrated an increased net fluid absorption as evident by the formation of epithelial domes, which were not seen in cilia (+) cells. Immunofluorescence studies showed AQP2 localization along the ciliary shaft and in subapical membrane compartment in cilia (+). Moreover, after apical AVP treatment, there was an increase in AQP2 expression at the apical membrane in cilia (−) but not in cilia (+) cells. This was confirmed by biotinylation and Western blot analysis, which demonstrated an AVP induced elevated glycosylated form of apically located AQP2 in cilia (−) vs. cilia (+) cells. Interestingly, in cilia (−) cells there was evidence for basolateral membrane insertion of AQP2. In the absence of cilia, there appears to be an AVP mediated targeting of the V2 receptor to the apical membrane and enhanced AQP2 localization at both apical and basolateral membranes. Thus, we propose AQP2 trafficking requires a normal cilium and when absent, the collecting duct cells becomes dedifferentiate leading to enhanced salt and water absorption which may contribute to the hypertension observed in PKD patients.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

Articular cartilage and growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737 orpk mice lacking the primary cilia protein polaris

Primary cilia are highly conserved organelles found on almost all eukaryotic cells. Tg737 orpk ( orpk) mice carry a hypomorphic mutation in the Tg737 gene resulting in the loss of polaris, a protein essential for ciliogenesis. Orpk mice have an array of skeletal patterning defects and show stunted growth after birth, suggesting defects in appositional and endochondral development. This study investigated the association between orpk tibial long bone growth and chondrocyte primary cilia expression using histomorphometric and immunohistochemical analysis. Wild-type chondrocytes throughout the developing epiphysis and growth plate expressed primary cilia, which showed a specific orientation away from the articular surface in the first 7–10 cell layers. In orpk mice, primary cilia were identified on very few cells and were significantly shorter. Orpk chondrocytes also showed significant increases in cytoplasmic tubulin, a likely result of failed ciliary assembly. The growth plates of orpk mice were significantly smaller in length and width, with marked changes in cellular organization in the presumptive articular cartilage, proliferative and hypertrophic zones. Cell density at the articular surface and in the hypertrophic zone was significantly altered, suggesting defects in both appositional and endochondral growth. In addition, orpk hypertrophic chondrocytes showed re-organization of the F-actin network into stress fibres and failed to fully undergo hypertrophy, while there was a marked reduction in type X collagen sequestration. These data suggest that failure to form a functional primary cilium affects chondrocyte differentiation and results in delayed chondrocyte hypertrophy within the orpk growth plate.

Mechanoregulation of intracellular Ca2+concentration is attenuated in collecting duct of monocilium-impairedorpkmice

Autosomal recessive polycystic kidney disease (ARPKD) is characterized by the progressive dilatation of collecting ducts, the nephron segments responsible for the final renal regulation of sodium, potassium, acid-base, and water balance. Murine models of ARPKD possess mutations in genes encoding cilia-associated proteins, including Tg737 in orpk mice. New findings implicate defects in structure/function of primary cilia as central to the development of polycystic kidney disease. Our group (Liu W, Xu S, Woda C, Kim P, Weinbaum S, and Satlin LM, Am J Physiol Renal Physiol 285: F998-F1012, 2003) recently reported that increases in luminal flow rate in rabbit collecting ducts increase intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells therein. We thus hypothesized that fluid shear acting on the apical membrane or hydrodynamic bending moments acting on the cilium increase renal epithelial [Ca(2+)](i). To further explore this, we tested whether flow-induced [Ca(2+)](i) transients in collecting ducts from mutant orpk mice, which possess structurally abnormal cilia, differ from those in controls. Isolated segments from 1- and 2-wk-old mice were microperfused in vitro and loaded with fura 2; [Ca(2+)](i) was measured by digital ratio fluorometry before and after the rate of luminal flow was increased. All collecting ducts responded to an increase in flow with an increase in [Ca(2+)](i), a response that appeared to be dependent on luminal Ca(2+) entry. However, the magnitude of the increase in [Ca(2+)](i) in 2- but not 1-wk-old mutant orpk animals was blunted. We speculate that this defect in mechano-induced Ca(2+) signaling in orpk mice leads to aberrant structure and function of the collecting duct in ARPKD.

Cyst fluid composition in human autosomal recessive polycystic kidney disease

Sirs, Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic renal disorder and frequently progresses to renal failure [1]. Because of its relative prevalence, it is the most well-characterized of the genetic cystic renal diseases. Histopathologically, the cysts are derived from all segments of the nephron, yet they “bud off” and separate from the tubule from which they are derived [1]. Cystic expansion is thought to be related to multiple biological processes including Na, Cl, and fluid secretion into cysts, processes that would tend to enlarge a discrete cyst and compress surrounding normal renal tissue [1]. Autosomal recessive polycystic kidney disease (ARPKD) is less common than ADPKD with an incidence of ~1 in 20,000 live births [1, 2]. Phenotypically, infants and children with ARPKD frequently present with hypertension, which generally appears well in advance of renal insufficiency [3]. Histologically, ARPKD is associated with ectasia and dilatation of renal collecting ducts and intra-hepatic bile ducts, and therefore does not retain the classic ADPKD “blind loop” sac-like cystic pattern [1]. We have previously examined the transepithelial Na transport properties of human ARPKD cystic epithelial cells in culture and reported that these cells absorb Na, as did normal age-matched counterparts. However, we showed, unexpectedly, that ARPKD cyst-lining cells reabsorbed Na at a rate ~50% greater than that of controls [4]. The Na absorptive flux in ARPKD cells was only modestly inhibited by high doses (100 mM) of amiloride, an inhibitor of the epithelial Na channel (ENaC) and Na-H antiporter [4]. Similar investigations of collecting duct epithelial cells derived from orpk mice, a murine model of ARPKD, suggested that these cells, like their human ARPKD counterparts, absorb Na at a higher rate than controls [5]. Based on these laboratory investigations, we were interested to determine if the in vitro studies could be validated in vivo. With institutional review board approval, we sought to characterize cyst fluid composition from ARPKD nephrectomy specimens at the time of renal transplantation. Cyst fluid (~0.1–0.3 ml samples, 8–11 samples per kidney) was collected in the operating room from freshly harvested pediatric ARPKD kidneys. Analysis of fluid aspirated from cysts within the renal parenchyma from three separate end-stage ARPKD kidney specimens revealed a low Na concentration and an osmolality similar to that of serum (Table 1). We speculate that the urine osmolality of ~300 mosmol/kg is due to the presence of urea, which was not measured. The cyst fluid R. Rohatgi Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA

Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization.

Polycystic kidney disease (PKD) includes a group of disorders that are characterized by the presence of cysts in the kidney and other organs, including the pancreas. Here we show that in orpk mice, a model system for PKD that harbors a mutation in the gene that encodes the polaris protein, pancreatic defects start to occur at the end of gestation, with an initial expansion of the developing pancreatic ducts. Ductal dilation continues rapidly after birth and results in the formation of large, interconnected cysts. Expansion of pancreatic ducts is accompanied by apoptosis of neighboring acinar cells, whereas endocrine cell differentiation and islet formation appears to be unaffected. Polaris has been shown to co-localize with primary cilia, and these structures have been implicated in the formation of renal cysts. In the orpk pancreas, cilia numbers are reduced and cilia length is decreased. Expression of polycystin-2, a protein involved in PKD, is mislocalized in orpk mice. Furthermore, the cellular localization of beta-catenin, a protein involved in cell adhesion and Wnt signaling, is altered. Thus, polaris and primary cilia function are required for the maturation and maintenance of proper tissue organization in the pancreas.

Polycystin-2 localizes to kidney cilia and the ciliary level is elevated in orpk mice with polycystic kidney disease

We thank Dr J. Lawrence and members of her laboratory for microscope time and assistance, Dr P. Furcinitti for assistance, and Drs Y. Cai and S. Somlo for the YCC2 antibody and for critically reading this manuscript. This work was supported by NIH GM60992 (GJP), GM30626 (GBW), GM14642 (JLR), and by the Robert W. Booth Fund at the Greater Worcester Community Foundation (GBW).

Phenotypic variations of orpk mutation and chromosomal localization of modifiers influencing kidney phenotype.

The Oak Ridge polycystic kidney (orpk) mutant mouse model resulted from a transgene insertion into the Tg737 gene and exhibits a pleiotropic syndrome with lesions in the kidney, liver, and pancreas. We found marked differences in the phenotypic expression of the orpk mutation when bred on different genetic backgrounds. In the FVB/N background, the phenotype is very severe for kidney, pancreas, and liver lesions. To evaluate better how genetic background might influence the expressivity of the orpk phenotype, we bred the transgene into the C3HeB/FeJLe (C3H) genetic background. We performed a genome-wide scan using backcross and intercross populations with more than 150 markers to map the chromosomal location of the modifier genes that differ in the FVB/N and C3H genetic backgrounds that affect the severity of kidney disease in the orpk mouse. Low-resolution interval mapping was performed using the Map Manager QTb program, with the interval explaining a significant portion of the variance being the distal end of chromosome 4.

Expression of the orpk disease gene during kidney development and maturation.

Further analysis of the orpk mouse model of human autosomal recessive polycystic kidney disease is providing more insight into the function of the Tg737 gene and the pathobiology of renal cystic disease. Here we have determined the temporal-spatial profile of Tg737 expression and ascertained the profile of disease pathology utilizing Tg737delta2-3betaGal/+ and Tg737delta2-3betaGal/ Tg737orpk compound heterozygotes from embryonic day 13.0 (E13.0) to postnatal day 270 (D270). This has allowed us to correlate disease progression and Tg737 expression in the context of the mutant orpk phenotype. These data reveal that Tg737 is dynamically regulated during kidney development and during postnatal kidney maturation in normal and in orpk mutants. This expression pattern correlates with the pathology of the disease, such that tubular segments with the highest expression levels are most protected from cystic disease. These data indicate that kidney tubules require a threshold level of Tg737 function for normal tubular development, structure, and function. In addition, these data demonstrate that the timing of cyst formation and severity of cyst progression is modulated differently in different regions of the nephron in this model.

New insights into the molecular pathophysiology of polycystic kidney disease

Polycystic kidney diseases are characterized by the progressive expansion of multiple cystic lesions, which compromise the function of normal parenchyma. Throughout the course of these diseases, renal tubular function and structure are altered, changing the tubular microenvironment and ultimately causing the formation and progressive expansion of cystic lesions. Renal tubules are predisposed to cystogenesis when a germ line mutation is inherited in either the human PKD1 or PKD2 genes in autosomal dominant polycystic kidney disease (ADPKD) or when a homozygous mutation in Tg737 is inherited in the orpk mouse model of autosomal recessive polycystic kidney disease (ARPKD). Recent information strongly suggests that the protein products of these disease genes may form a macromolecular signaling structure, the polycystin complex, which regulates fundamental aspects of renal epithelial development and cell biology. Here, we re-examine the cellular pathophysiology of renal cyst formation and enlargement in the context of our current understanding of the molecular genetics of ADPKD and ARPKD.

Cellular pathophysiology of cystic kidney disease: insight into future therapies.

Polycystic kidney disease (PKD) is a developmental kidney disorder which can be inherited as either an autosomal dominant trait, with an incidence of 1:50 to 1:1000, or as an autosomal recessive trait with an incidence of 1:6,000 to 1:40,000. Three different genes have now been cloned that are associated with mutations that cause PKD. Two of these are linked to the most common forms of the dominant disease while the third is associated with the orpk mouse model of recessive polycystic kidney disease. Advances in understanding the molecular genetics of PKD have been paralleled by new insights into the cellular pathophysiology of cyst formation and progressive enlargement. Current data suggest that a number of PKD proteins may interact in a complex, which when disrupted by mutations in PKD genes may lead to altered epithelial proliferative activity, secretion, and cell matrix biology. The identification of a unique cystic epithelial phenotype presents new opportunities for targeted therapies. These include targeted gene therapy, gene complementation, and specific immunological or pharmacological interruption of growth factor pathways.

Efficacy of taxol in the orpk mouse model of polycystic kidney disease.

Polycystic kidney disease (PKD) a is common disease in the human population that can lead to renal failure and death. Taxol has recently been reported to be of therapeutic benefit in the cpk mouse model of PKD. To determine whether these results also apply to other models of PKD, we studied the effects of taxol treatment on the development of renal cysts and biliary hyperplasia/dysplasia/fibrosis in the orpk mouse mutant, a unique murine model for human autosomal recessive PKD. We report no significant differences between the treatment and control groups with respect to weight gain, survival, urine to serum osmolality ratio, and serum concentration of liver enzymes. Moreover, renal cystic development was not affected by taxol treatment in the orpk mutant animals. This was confirmed by lectin staining and morphometric analysis of the renal cysts, which indicated no significant differences between treatment groups. Therefore, while taxol has a positive effect on the cystic kidney disease in cpk mutant mice, this effect is not applicable to all forms of PKD.