Autosomal-dominante polyzystische Nierenerkrankung

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic renal disease, and it is even one of the most common hereditary diseases in man. In 85% of the cases the disease is caused by mutations in the PKD1 gene, which encodes for the ciliary protein polycystin-1. A milder variant of the disease is caused by mutations in the PKD2 gene, which encodes for the calcium channel-related protein polycystin-2. The disease is characterized by the progressive development of innumerable cysts in both kidneys, which gradually replace the normal kidney tissue. In ADPKD patients the inexorable cyst growth leads to a progressive deterioration of renal function over decades, which ultimately can only be treated by renal replacement therapy or renal transplantation. Until now a causal treatment was not available, and treatment options were limited to regular clinical controls and treatment of complications (hypertension, cyst infections). Several promising clinical studies are currently examining new or even existing drugs as therapeutic options to retard cyst growth in ADPKD (vasopressin receptor-2 antagonists [V2RA], mammalian target of rapamycin [mTOR] inhibitors, somatostatin). It can be anticipated that ADPKD patients will benefit from these new treatment options in the near future.     

Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.     

Regulation of ryanodine receptor-dependent calcium signaling by polycystin-2

Mutations in polycystin-2 (PC2) cause autosomal dominant polycystic kidney disease. A function for PC2 in the heart has not been described. Here, we show that PC2 coimmunoprecipitates with the cardiac ryanodine receptor (RyR2) from mouse heart. Biochemical assays showed that the N terminus of PC2 binds the RyR2, whereas the C terminus only binds to RyR2 in its open state. Lipid bilayer electrophysiological experiments indicated that the C terminus of PC2 functionally inhibited RyR2 channel activity in the presence of calcium (Ca(2+)). Pkd2(-/-) cardiomyocytes had a higher frequency of spontaneous Ca(2+) oscillations, reduced Ca(2+) release from the sarcoplasmic reticulum stores, and reduced Ca(2+) content compared with Pkd2(+/+) cardiomyocytes. In the presence of caffeine, Pkd2(-/-) cardiomyocytes exhibited decreased peak fluorescence, a slower rate of rise, and a longer duration of Ca(2+) transients compared with Pkd2(+/+). These data suggest that PC2 is important for regulation of RyR2 function and that loss of this regulation of RyR2, as occurs when PC2 is mutated, results in altered Ca(2+) signaling in the heart.     

Autosomal dominant polycystic kidney disease is associated with an increased prevalence of radiographic bronchiectasis

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is a common disease with several known extrarenal manifestations, although no known pulmonary features. The formation of renal cysts in ADPKD has been attributed to dysfunction of primary cilia and the primary cilia-related proteins polycystin-1 (in 85% of cases) and polycystin-2 in renal epithelial cells. The goals of this study were to characterize the normal expression of polycystin-1 in the motile cilia of airway epithelial cells and to evaluate lung structure in ADPKD patients. METHODS: Airway epithelium from non-ADPKD patients was immunostained to localize polycystin-1 expression, and lung tissue from ADPKD patients was examined for pathologic changes. CT scans from ADPKD patients (n = 95) and a control group of non-ADPKD chronic kidney disease patients (n = 95) were retrospectively reviewed for the presence of bronchiectasis using defined criteria. RESULTS: Immunostaining revealed polycystin-1 expression in the motile cilia of non-ADPKD airway epithelial cells. Lung tissue from one of five available ADPKD patient autopsies revealed histologic changes of bronchiectasis. Review of CT scans revealed a threefold-increased prevalence of bronchiectasis in the ADPKD group compared to the control group (37% vs 13%, p = 0.002). CONCLUSIONS: ADPKD patients demonstrate an increased prevalence of radiographic bronchiectasis, a previously unrecognized manifestation of the disease. This association suggests that patients with primary cilia-associated diseases may be at risk for airway disease.     

G-protein signaling modulator 1 deficiency accelerates cystic disease in an orthologous mouse model of autosomal dominant polycystic kidney disease

Polycystic kidney diseases are the most common genetic diseases that affect the kidney. There remains a paucity of information regarding mechanisms by which G proteins are regulated in the context of polycystic kidney disease to promote abnormal epithelial cell expansion and cystogenesis. In this study, we describe a functional role for the accessory protein, G-protein signaling modulator 1 (GPSM1), also known as activator of G-protein signaling 3, to act as a modulator of cyst progression in an orthologous mouse model of autosomal dominant polycystic kidney disease (ADPKD). A complete loss of Gpsm1 in the Pkd1^V/V mouse model of ADPKD, which displays a hypomorphic phenotype of polycystin-1, demonstrated increased cyst progression and reduced renal function compared with age-matched cystic Gpsm1^+/+ and Gpsm1^+/− mice. Electrophysiological studies identified a role by which GPSM1 increased heteromeric polycystin-1/polycystin-2 ion channel activity via Gβγ subunits. In summary, the present study demonstrates an important role for GPSM1 in controlling the dynamics of cyst progression in an orthologous mouse model of ADPKD and presents a therapeutic target for drug development in the treatment of this costly disease.     

Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments

Quantitative analysis of Ca(2+) fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca(2+)-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca(2+) binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca(2+) release kinetics with an off-rate estimated at around 700 s(-1) and appropriate Ca(2+) binding affinity, likely attributable to local Ca(2+)-induced conformational changes around the designed Ca(2+) binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca(2+) dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca(2+) in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca(2+) release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca(2+) homeostasis.     

The mTOR pathway is regulated by polycystin-1, and its inhibition reverses renal cystogenesis in polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is a common genetic disorder that frequently leads to renal failure. Mutations in polycystin-1 (PC1) underlie most cases of ADPKD, but the function of PC1 has remained poorly understood. No preventive treatment for this disease is available. Here, we show that the cytoplasmic tail of PC1 interacts with tuberin, and the mTOR pathway is inappropriately activated in cyst-lining epithelial cells in human ADPKD patients and mouse models. Rapamycin, an inhibitor of mTOR, is highly effective in reducing renal cystogenesis in two independent mouse models of PKD. Treatment of human ADPKD transplant-recipient patients with rapamycin results in a significant reduction in native polycystic kidney size. These results indicate that PC1 has an important function in the regulation of the mTOR pathway and that this pathway provides a target for medical therapy of ADPKD.     

Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation in the kidney, liver, and pancreas and is associated often with cardiovascular abnormalities such as hypertension, mitral valve prolapse, and intracranial aneurysms. It is caused by mutations in PKD1 or PKD2, encoding polycystin-1 and -2, which together form a cell surface nonselective cation ion channel. Pkd2-/- mice have cysts in the kidney and pancreas and defects in cardiac septation, whereas Pkd1(del34) -/- and Pkd1(L) -/- mice have cysts but no cardiac abnormalities, although vascular fragility was reported in the latter. Here we describe mice carrying a targeted mutation in Pkd1 (Pkd1(del17-21betageo)), which defines its expression pattern by using a lacZ reporter gene and may identify novel functions for polycystin-1. Although Pkd1(del17-21betageo) +/- adult mice develop renal and hepatic cysts, Pkd1(del17-21betageo) -/- embryos die at embryonic days 13.5-14.5 from a primary cardiovascular defect that includes double outflow right ventricle, disorganized myocardium, and abnormal atrio-ventricular septation. Skeletal development is also severely compromised. These abnormalities correlate with the major sites of Pkd1 expression. During nephrogenesis, Pkd1 is expressed in maturing tubular epithelial cells from embryonic day 15.5. This expression coincides with the onset of cyst formation in Pkd1(del34) -/-, Pkd1(L) -/-, and Pkd2-/- mice, supporting the hypothesis that polycystin-1 and polycystin-2 interact in vivo and that their failure to do so leads to abnormalities in tubule morphology and function.     

Altered store operated calcium entry increases cyclic 3',5'-adenosine monophosphate production and extracellular signal-regulated kinases 1 and 2 phosphorylation in polycystin-2-defective cholangiocytes

Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER?; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6). CONCLUSION: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production.     

Heterozygosity loss and somatic mutations in type I and II dominant autosomal renal polycystic kidney disease: evidence of a recessive mechanism at a cell level in cystogenesis

Autosomal dominant polycystic kidney disease (ADPKD) is a systemic disorder mainly characterized by renal cyst formation. Cysts in ADPKD are focal in nature, since only a small fraction of nephrons become cystic. The hypothesis that a second hit may be required for cyst formation has been proposed. This hypothesis suggests that inactivation of the inherited wild-type allele by a somatic mutation triggers cyst formation. In some cases, this second hit eliminates the normal allele and the affected cells remain with a single allele, which is the inherited mutated copy, and we only visualize one allele after the amplification by polymerase chain reaction; this is called loss of heterozygosity (LOH). In this study we have analysed the DNA isolated from epitehlial cells from 164 cysts of 8 kidneys affected by ADPKD type I and 30 cysts form a kidney affected by ADPKD type II. We have demonstrated the presence of LOH in 20.1% of PKD1 cysts and in 10% of PKD2 cysts. We have also found eight other different mutations in PKD2 cysts without LOH; so the percentage of somatic mutations in the PKD2 kidney reaches 36.6% of cysts. In conclusion, our data suggest that a recessive mechanism at the cellular level is implicated in cyst formation in the PKD1 and the PKD2 disease. The loss of both copies of the gene triggers the proliferation of a single cell, resulting in the cyst formation.     

It's not all in the cilium, but on the road to it: genetic interaction network in polycystic kidney and liver diseases and how trafficking and quality control matter

Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIb and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIb and Sec63p are required in mice for adequate expression of a functional complex of the polycystic kidney disease gene products, polycystin-1 and polycystin-2. We find that polycystin-1 is the rate-limiting component of this complex and that there is a dose–response relationship between cystic dilation and levels of functional polycystin-1 following mutation of Prkcsh or Sec63.Reduced expression of polycystin-1 also serves to sensitize the kidney to cyst formation resulting from mutations in Pkhd1, the recessive polycystic kidney disease gene. Finally, we show that proteasome inhibition increases steady-state levels of polycystin-1 in cells lacking glucosidase IIb and that treatment with a proteasome inhibit or reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver.     

Follicle-stimulating hormone-induced Galphah/phospholipase C-delta1 signaling mediating a noncapacitative Ca2+ influx through T-type Ca2+ channels in rat sertoli cells

Our previous study demonstrated that FSH-induced immediate Ca(2+) influx in rat Sertoli cells (SCs) is mediated by the Galphah/phospholipase C-delta1 (PLC-delta1) signaling pathway. As to which Ca(2+) channel is responsible for such Ca(2+) influx was not understood. In this study, thapsigargin triggered an in-store calcium release and evoked a 1.5-fold elevation of intracellular Ca(2+) in Ca(2+)-free media, whereas FSH exhibited no effect. The readdition of CaCl(2) (2.5 mm) to FSH-pretreated or thapsigargin-sensitized SCs in Ca(2+)-free media immediately elicited a rapid Ca(2+) influx or a 2-fold increase of second intracellular Ca(2+) elevation, respectively. The addition of Ca(2+) chelator EGTA (0.2 mm) reduced the FSH-induced elevation of intracellular Ca(2+) in SCs incubated with CaCl(2). However, pretreatment with dantrolene (25 microM), which inhibits in-store calcium release, did not affect the FSH-induced elevation of intracellular Ca(2+). NiCl(2) (10 microM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca(2+) influx. Furthermore, mibefradil (10 and 100 microm), another specific blocker for T-type Ca(2+) channels, dose-dependently suppressed the FSH-induced Ca(2+) influx. In contrast, nifedipine (10 and 50 microm) or omega-conotoxin GVIA (100 and 500 nm), blocker of L- or N-type Ca(2+) channels, respectively, did not affect the FSH-induced SC Ca(2+) influx. On the other hand, FSH-induced Ca(2+) influx was significantly reduced by pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 microm) of PLC-delta1 fragment TIPWNSLKQGYRHVHLL but not affected by 2',5'-dideoxyadenosine (3 and 15 microm), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced Galphah/PLC-delta1 pathway-dependent Ca(2+) influx of rat SCs is mediated by T-type Ca(2+) channels and independent of in-store calcium release.     

Mutant polycystin-2 induces proliferation in primary rat tubular epithelial cells in a STAT-1/p21-independent fashion accompanied instead by alterations in expression of p57KIP2 and Cdk2

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.

Methods

Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.

Results

Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57^KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.

Conclusion

Our results indicate the probable involvement of p57^KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.     

Activation of angiotensin II type I receptor promotes protein kinase C translocation and cell proliferation in human cultured breast epithelial cells

The effect of angiotensin II (Ang II) on Ca(2+) signalling in human primary cultured breast epithelial cells was investigated by using fura-2 as the Ca(2+) probe. Ang II (0.1-1000 nM) induced an intracellular free calcium ([Ca(2+)](i)) transient peak which was unchanged by external Ca(2+ )removal. In Ca(2+)-free medium pretreatment with thapsigargin abolished Ang II-induced Ca(2+ )release. Suppression of 1,4,5-inositol trisphosphate formation by U73122, a phospholipase C inhibitor, blocked the Ang II-induced Ca(2+) response. Losartan (DuP753), an inhibitor of Ang II type I receptor (AT1), decreased the [Ca(2+)](i) increase evoked by Ang II, while CGP4221A, an inhibitor of Ang II type II receptor (AT2) did not. AT1 desensitisation was demonstrated with respect to the Ca(2+) response after subsequent exposure of cells to Ang II and also after pretreatment for 25 min with 1000 nM phorbol 12-myristate 13-acetate. Staurosporine, an inhibitor of protein kinases C (PKC), inhibited the AT1 desensitisation. Epithelial breast cells expressed PKC-alpha, -beta1, -delta and -zeta isozymes, and Ang II provoked translocation from the cytosol to the membranes of PKC-alpha, -beta1, and -delta (but not -zeta). Ang II was also able to stimulate cell proliferation in a dose-dependent manner; this effect was blocked by G? 6976, a specific inhibitor of PKC-alpha and -beta1, the Ca(2+)-dependent isozymes. The main conclusion of this study is that the the Ang II signalling mechanism in breast epithelial cells is based on the elevation of [Ca(2+)](i )released from intracellular stores through AT1 activation. In addition, Ang II stimulates cell proliferation by the activation of PKC isozymes.     

Ca2+-dependent modulation of single human cardiac L-type calcium channels by the calcineurin inhibitor cyclosporine

OBJECTIVE: Activity of single L-type calcium channels (LTCC) is enhanced in human failing myocardium (Circulation 98 (1998) 969.), most likely due to impaired dephosphorylation. Protein phosphatase 2B (calcineurin) has recently been shown to be involved in heart failure pathophysiology. We now focus on the regulation of single LTCC by calcineurin that were prevented by Ca(2+)-free experimental conditions in our previous study. METHODS: Single LTCC currents were recorded in myocytes from human atrium and ventricle. Charge carriers were 70 mM Ba(2+), or a mixture of 30 mM Ca(2+) and 60 mM Ba(2+) to facilitate Ca(2+) permeation through recorded channels. The calcineurin inhibitor cyclosporine (10 microM) was used to reveal a putative role for calcineurin in regulation of LTCC. RESULTS: A mixture of Ca(2+) and Ba(2+) as charge carriers allowed for Ca(2+) permeation through recombinant human embryonic kidney cells and native (atrial and ventricular) human cardiac LTCC. With only Ba(2+) as the charge carrier, activities of both ventricular and atrial LTCC were strongly decreased by cyclosporine. In contrast, channel activity remained constant when Ca(2+) permeation was provided. In the presence of thapsigargin and (S)-BayK 8644, cyclosporine here even increased channel activity. CONCLUSIONS: We propose a dual cyclosporine effect on human cardiac LTCC. A non-specific inhibitory effect prevails with Ba(2+) permeation but can be compensated or overcome by a specific Ca(2+)-dependent stimulation with Ca(2+) permeation. More complete restoration of physiological Ca(2+) movements (e.g., Ca(2+) release from sarcoplasmic reticulum) will help to define even more precisely the involvement of calcineurin in regulation of human cardiac LTCC.     

R93W mutation in Orai1 causes impaired calcium influx in platelets

The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses.     

Impaired insulin secretion and glucose intolerance in synaptotagmin-7 null mutant mice

Vertebrates express at least 15 different synaptotagmins with the same domain structure but diverse localizations and tissue distributions. Synaptotagmin-1,-2, and -9 act as calcium sensors for the fast phrase of neurotransmitter release, and synaptotagmin-12 acts as a calcium-independent modulator of release. The exact functions of the remaining 11 synaptotagmins, however, have not been established. By analogy to the role of synaptotagmin-1, -2, and -9 in neurotransmission, these other synaptotagmins may serve as Ca(2+) transducers regulating other Ca(2+)-dependent membrane processes, such as insulin secretion in pancreatic beta-cells. Of these other synaptotagmins, synaptotagmin-7 is one of the most abundant and is present in pancreatic beta-cells. To determine whether synaptotagmin-7 regulates Ca(2+)-dependent insulin secretion, we analyzed synaptotagmin-7 null mutant mice for glucose tolerance and insulin release. Here, we show that synaptotagmin-7 is required for the maintenance of systemic glucose tolerance and glucose-stimulated insulin secretion. Mutant mice have normal insulin sensitivity, insulin production, islet architecture and ultrastructural organization, and metabolic and calcium responses but exhibit impaired glucose-induced insulin secretion, indicating a calcium-sensing defect during insulin-containing secretory granule exocytosis. Taken together, our findings show that synaptotagmin-7 functions as a positive regulator of insulin secretion and may serve as a calcium sensor controlling insulin secretion in pancreatic beta cells.