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

COMMENTARY ON:A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation. Fedeles SV, Tian X, Gallagher AR, Mitobe M, Nishio S, Lee SH, Cai Y, Geng L, Crews CM, Somlo S. Nat Genet. 2011;43(7):639–47. Copyright (2011). Reprinted by permission from Macmillan Publishers Ltd.http://www.ncbi.nlm.nih.gov/pubmed/21685914Abstract: Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIβ and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIβ 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 IIβ and that treatment with a proteasome inhibitor reduces cystic disease in orthologous gene models of human autosomal dominant polycystic liver.The cilium, an antenna-like organelle on the surface of most cells, currently arises much interest and an emerging number of diseases with a wide phenotypic spectrum have been shown to be related to ciliary dysfunction, collectively termed “ciliopathies” (Fig. 1A) [[1]Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar]. Literally all organs can be affected, however, it is becoming increasingly evident that most ciliopathies have a cystogenic, especially renal cystogenic component making kidney cyst formation a hallmark feature of ciliopathies. Cystic and polycystic kidney diseases (PKD) are clinically and genetically heterogeneous. Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common Mendelian genetic disorders with a prevalence of 1:500–1:1000 and 10–15 million affected individuals worldwide [[2]Harris P.C. Torres V.E. Polycystic kidney disease.Annu Rev Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar]. Most ADPKD patients carry a germline mutation in the PKD1 gene encoding polycystin-1, the remaining 20% of patients a mutation in PKD2 encoding polycystin-2. While usually an adult-onset disease, a minor subset of ADPKD patients show an early and severe manifestation that is clinically indistinguishable from patients with “infantile” autosomal recessive polycystic kidney disease (ARPKD) and mutations in the Polycystic Kidney and Hepatic Disease 1 (PKHD1) gene. As a potential explanation for this clinical overlap, we could recently show that some severely affected patients carry multiple PKD alleles in trans and/or de novo in addition to the familial germline mutation, suggesting a dosage effect and complex network for PKD [[3]Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar].While the kidneys are usually the main organ involved, ADPKD and ARPKD display profound extrarenal disease burden especially in the liver (Fig. 1B). About 75% of ADPKD patients display liver cysts in their sixties. Polycystic liver disease (ADPLD) can also occur isolated as a distinct autosomal dominant trait and a mutation in either the non-catalytic β subunit of glucosidase II, also termed Protein Kinase C Substrate, Heavy Chain (PRKCSH), or the SEC63 gene encoding a protein involved in post-translational translocation of proteins into the endoplasmic reticulum (ER). Liver cysts in ADPLD are thought to arise from disconnected intralobular bile ductules and ductal plate malformation during embryogenesis [[4]Strazzabosco M. Somlo S. Polycystic liver diseases: congenital disorders of cholangiocyte signaling.Gastroenterology. 2011; 140: 1855-1859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Usually, ADPLD is a benign disorder with preserved organ function, characterized by the formation and progressive enlargement of multiple cysts scattered throughout the liver parenchyma which may lead to gross liver enlargement and displacement of other abdominal organs. Rare complications are bleeding or cyst infection. In clinical practice, it can be challenging to decide with certainty if a patient has ADPKD or ADPLD, particularly because some ADPLD patients also harbor some kidney cysts. Morphologically, cysts in ADPLD are indistinguishable from those found in ADPKD. While factually all proteins disturbed in PKD and other ciliopathies have their main site of action in primary cilia and related organelles, it has always been puzzling that the two proteins altered in ADPLD are non-ciliary with roles in protein translocation, folding and quality control in the ER, the largest controllable intracellular Ca2+ store in non-excitable cells. Thus, Ca2+ homeostasis, known to be of relevance in PKD, is largely regulated by the ER. Moreover, the largest pool of polycystin-2, a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable cation channels, is located in the ER.The recent study by Fedeles and colleagues [[5]Fedeles S.V. Tian X. Gallagher A.R. Mitobe M. Nishio S. Lee S.H. et al.A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation.Nat Genet. 2011; 43: 639-647Crossref PubMed Scopus (169) Google Scholar] nicely demonstrates that ciliopathies and related disorders not only depend on proper cilia function and structure, but that also other mechanisms such as trafficking and quality control processes within the cell play a major role (Fig. 1C). Almost every third protein encoded by the human genome passes through the translocation and quality control machinery in the ER. This especially holds true for secretory proteins and transmembrane proteins, as all PKD genes are. Thus, the authors hypothesized an interrelationship through which PKD and PLD overlap not only clinically, but also genetically and functionally. To confirm this, they performed a series of different experiments using both genetically engineered mouse models and biochemical analyses.In mice, they showed that tissue-selective loss of the ADPLD genes/proteins is sufficient to cause cyst formation in both the liver and kidney. While the first is not unexpected, the latter was surprising and prompted the authors to hypothesize that renal cyst formation and progression was related to a reduced dosage of the ADPKD proteins. Indeed, the homozygous Prkcsh and Sec63 floxed Ksp-Cre PKD mice compounded with either a Pkd1+/− or Pkd2+/− background displayed a more severe phenotype especially with Pkd1. In contrast to published data with overexpression of either polycystin-1 or polycystin-2 in mice leading to PKD [6Thivierge C. Kurbegovic A. Couillard M. Guillaume R. Coté O. Trudel M. Overexpression of PKD1 causes polycystic kidney disease.Mol Cell Biol. 2006; 26: 1538-1548Crossref PubMed Scopus (121) Google Scholar, 7Burtey S. Riera M. Ribe E. Pennekamp P. Passage E. Rance R. et al.Overexpression of PKD2 in the mouse is associated with renal tubulopathy.Nephrol Dial Transplant. 2008; 23: 1157-1165Crossref PubMed Scopus (32) Google Scholar], Fedeles and colleagues found that expressing more polycystin-1 abrogated the kidney and liver cystic phenotype in both tissue-selective ADPLD mouse models, whereas this was not the case for Pkd2. In this experimental setup, some functional polycystin-2 was necessary for the rescuing function of Pkd1 in line with the finding that both proteins form an active polycystin-complex [1Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar, 2Harris P.C. Torres V.E. Polycystic kidney disease.Annu Rev Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar]. Based on these data and known interrelationships, impaired biogenesis following loss of either ADPLD gene product could be expected for both polycystins and other integral membrane proteins. However, only polycystin-1 trafficking to cilia, and not polycystin-2, was considerably reduced. Overall, cyst formation in both ADPLD mouse models was dynamically determined by the levels of functional polycystin-1 dosage. Therefore, the authors speculated that a potential novel therapeutic target for ADPLD might be proteasome inhibition with stabilization of polycystin-1, and indeed this hypothesis was substantiated in a final experiment in which it was demonstrated that the proteasome inhibitors MG132 and carfilzomib increased polycystin-1 levels resulting in reduced cystic disease in ADPLD mice.In a further series of experiments, the interrelationship between the above genes and the ARPKD gene Pkhd1 was examined for good reasons: first, the ARPKD protein fibrocystin/polyductin is another ER-passing transmembrane protein, and second, there is increasing evidence for genetic interaction between ADPKD and ARPKD. Thus, aggravation of the phenotype when crossing Sec63flox/flox; Ksp-Cre and Pkhd1del4/del4 mice was not surprising; however, the phenotype was not as severe as when crossed with Pkd1+/− mice. In line with our results in PKD patients [[3]Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar] and other published data [[8]Garcia-Gonzalez M.A. Menezes L.F. Piontek K.B. Kaimori J. Huso D.L. Watnick T. et al.Genetic interaction studies link autosomal dominant and recessive polycystic kidney disease in a common pathway.Hum Mol Genet. 2007; 16: 1940-1950Crossref PubMed Scopus (87) Google Scholar], reduction of the Pkd1 dosage led to a more severe phenotype in Pkhd1 mice. Intriguingly and in contrast to other studies [3Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar, 9Kim I. Fu Y. C. et renal formation by polycystin-2 expression and Am Soc Nephrol. 2008; PubMed Scopus Google Scholar] the reduced dosage of polycystin-2 effect on the of study is for the of polycystic liver and kidney disease and increased in genetic and the of affected of the mechanisms that explain disease expression is one of the major in We have from different individuals from PKD and/or PLD that there is clinical in the member might be affected, whereas another one only clinical are to explain these and of mutations in the of remaining a major for disease [1Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar, N. and human genetic Genet. 26: Full Text Full Text PDF PubMed Scopus Google study by Fedeles and colleagues with of a network for polycystic liver and kidney disease may a for the of disease expression and could have for a of other In this an effect and to and more severe disease a a recessive disease has been for ADPKD and ADPLD Watnick L.F. The of cyst formation in human autosomal dominant polycystic kidney disease Full Text Full Text PDF PubMed Scopus Google Scholar, E. R. J. et mutations might cyst formation in patients with autosomal dominant polycystic liver 2011; Full Text Full Text PDF PubMed Scopus Google Scholar]. to of mutation and resulting loss of has been as the cyst formation. However, there is increasing evidence that this a and a germline mutation to one and a mutation to the other one explain The most of evidence for this is that in individuals with germline on both alleles by not every renal cell or to a cyst as to be expected in the Y. N. Watnick T. et disease and in autosomal dominant polycystic kidney J Genet. Full Text Full Text PDF PubMed Scopus Google Scholar]. Therefore, in line with a more complex the mutation to the other disease may as a that the of cyst and/or cyst progression cyst can be hypothesized that in addition to the and data recently some of the patients with liver and kidney cysts carry mutations in more one gene of the with the by Fedeles et Therefore, multiple genes may have to be to be in a patient it is to all those genes in of this is and genetic can be in a and with a with and of a of different disease genes is currently to be and all such as in when in However, the in this of this may and or genome may to the of the of an patient can be expected to be the first on the to a and increased also with to therapeutic and study of authors that they not have to or of interest with to this from the and and the PKD is by the and the COMMENTARY genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation. Fedeles SV, Tian X, Gallagher AR, Mitobe M, Nishio S, Lee SH, Cai Y, Geng L, Crews CM, Somlo S. Nat Genet. 2011;43(7):639–47. Copyright (2011). Reprinted by permission from Macmillan Publishers Autosomal dominant polycystic liver disease results from mutations in PRKCSH or SEC63. The respective gene products, glucosidase IIβ and SEC63p, function in protein translocation and quality control pathways in the endoplasmic reticulum. Here we show that glucosidase IIβ 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 IIβ and that treatment with a proteasome inhibitor reduces cystic disease in orthologous gene models of human autosomal dominant polycystic The cilium, an antenna-like organelle on the surface of most cells, currently arises much interest and an emerging number of diseases with a wide phenotypic spectrum have been shown to be related to ciliary dysfunction, collectively termed “ciliopathies” (Fig. 1A) [[1]Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar]. Literally all organs can be affected, however, it is becoming increasingly evident that most ciliopathies have a cystogenic, especially renal cystogenic component making kidney cyst formation a hallmark feature of ciliopathies. Cystic and polycystic kidney diseases (PKD) are clinically and genetically heterogeneous. Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common Mendelian genetic disorders with a prevalence of 1:500–1:1000 and 10–15 million affected individuals worldwide [[2]Harris P.C. Torres V.E. Polycystic kidney disease.Annu Rev Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar]. Most ADPKD patients carry a germline mutation in the PKD1 gene encoding polycystin-1, the remaining 20% of patients a mutation in PKD2 encoding polycystin-2. While usually an adult-onset disease, a minor subset of ADPKD patients show an early and severe manifestation that is clinically indistinguishable from patients with “infantile” autosomal recessive polycystic kidney disease (ARPKD) and mutations in the Polycystic Kidney and Hepatic Disease 1 (PKHD1) gene. As a potential explanation for this clinical overlap, we could recently show that some severely affected patients carry multiple PKD alleles in trans and/or de novo in addition to the familial germline mutation, suggesting a dosage effect and complex network for PKD [[3]Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar]. While the kidneys are usually the main organ involved, ADPKD and ARPKD display profound extrarenal disease burden especially in the liver (Fig. 1B). About 75% of ADPKD patients display liver cysts in their sixties. Polycystic liver disease (ADPLD) can also occur isolated as a distinct autosomal dominant trait and a mutation in either the non-catalytic β subunit of glucosidase II, also termed Protein Kinase C Substrate, Heavy Chain (PRKCSH), or the SEC63 gene encoding a protein involved in post-translational translocation of proteins into the endoplasmic reticulum (ER). Liver cysts in ADPLD are thought to arise from disconnected intralobular bile ductules and ductal plate malformation during embryogenesis [[4]Strazzabosco M. Somlo S. Polycystic liver diseases: congenital disorders of cholangiocyte signaling.Gastroenterology. 2011; 140: 1855-1859Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar]. Usually, ADPLD is a benign disorder with preserved organ function, characterized by the formation and progressive enlargement of multiple cysts scattered throughout the liver parenchyma which may lead to gross liver enlargement and displacement of other abdominal organs. Rare complications are bleeding or cyst infection. In clinical practice, it can be challenging to decide with certainty if a patient has ADPKD or ADPLD, particularly because some ADPLD patients also harbor some kidney cysts. Morphologically, cysts in ADPLD are indistinguishable from those found in ADPKD. While factually all proteins disturbed in PKD and other ciliopathies have their main site of action in primary cilia and related organelles, it has always been puzzling that the two proteins altered in ADPLD are non-ciliary with roles in protein translocation, folding and quality control in the ER, the largest controllable intracellular Ca2+ store in non-excitable cells. Thus, Ca2+ homeostasis, known to be of relevance in PKD, is largely regulated by the ER. Moreover, the largest pool of polycystin-2, a member of the transient receptor potential (TRP) superfamily of Ca2+-permeable cation channels, is located in the ER. The recent study by Fedeles and colleagues [[5]Fedeles S.V. Tian X. Gallagher A.R. Mitobe M. Nishio S. Lee S.H. et al.A genetic interaction network of five genes for human polycystic kidney and liver diseases defines polycystin-1 as the central determinant of cyst formation.Nat Genet. 2011; 43: 639-647Crossref PubMed Scopus (169) Google Scholar] nicely demonstrates that ciliopathies and related disorders not only depend on proper cilia function and structure, but that also other mechanisms such as trafficking and quality control processes within the cell play a major role (Fig. 1C). Almost every third protein encoded by the human genome passes through the translocation and quality control machinery in the ER. This especially holds true for secretory proteins and transmembrane proteins, as all PKD genes are. Thus, the authors hypothesized an interrelationship through which PKD and PLD overlap not only clinically, but also genetically and functionally. To confirm this, they performed a series of different experiments using both genetically engineered mouse models and biochemical In mice, they showed that tissue-selective loss of the ADPLD genes/proteins is sufficient to cause cyst formation in both the liver and kidney. While the first is not unexpected, the latter was surprising and prompted the authors to hypothesize that renal cyst formation and progression was related to a reduced dosage of the ADPKD proteins. Indeed, the homozygous Prkcsh and Sec63 floxed Ksp-Cre PKD mice compounded with either a Pkd1+/− or Pkd2+/− background displayed a more severe phenotype especially with Pkd1. In contrast to published data with overexpression of either polycystin-1 or polycystin-2 in mice leading to PKD [6Thivierge C. Kurbegovic A. Couillard M. Guillaume R. Coté O. Trudel M. Overexpression of PKD1 causes polycystic kidney disease.Mol Cell Biol. 2006; 26: 1538-1548Crossref PubMed Scopus (121) Google Scholar, 7Burtey S. Riera M. Ribe E. Pennekamp P. Passage E. Rance R. et al.Overexpression of PKD2 in the mouse is associated with renal tubulopathy.Nephrol Dial Transplant. 2008; 23: 1157-1165Crossref PubMed Scopus (32) Google Scholar], Fedeles and colleagues found that expressing more polycystin-1 abrogated the kidney and liver cystic phenotype in both tissue-selective ADPLD mouse models, whereas this was not the case for Pkd2. In this experimental setup, some functional polycystin-2 was necessary for the rescuing function of Pkd1 in line with the finding that both proteins form an active polycystin-complex [1Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar, 2Harris P.C. Torres V.E. Polycystic kidney disease.Annu Rev Med. 2009; 60: 321-337Crossref PubMed Scopus (581) Google Scholar]. Based on these data and known interrelationships, impaired biogenesis following loss of either ADPLD gene product could be expected for both polycystins and other integral membrane proteins. However, only polycystin-1 trafficking to cilia, and not polycystin-2, was considerably reduced. Overall, cyst formation in both ADPLD mouse models was dynamically determined by the levels of functional polycystin-1 dosage. Therefore, the authors speculated that a potential novel therapeutic target for ADPLD might be proteasome inhibition with stabilization of polycystin-1, and indeed this hypothesis was substantiated in a final experiment in which it was demonstrated that the proteasome inhibitors MG132 and carfilzomib increased polycystin-1 levels resulting in reduced cystic disease in ADPLD mice. In a further series of experiments, the interrelationship between the above genes and the ARPKD gene Pkhd1 was examined for good reasons: first, the ARPKD protein fibrocystin/polyductin is another ER-passing transmembrane protein, and second, there is increasing evidence for genetic interaction between ADPKD and ARPKD. Thus, aggravation of the phenotype when crossing Sec63flox/flox; Ksp-Cre and Pkhd1del4/del4 mice was not surprising; however, the phenotype was not as severe as when crossed with Pkd1+/− mice. In line with our results in PKD patients [[3]Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar] and other published data [[8]Garcia-Gonzalez M.A. Menezes L.F. Piontek K.B. Kaimori J. Huso D.L. Watnick T. et al.Genetic interaction studies link autosomal dominant and recessive polycystic kidney disease in a common pathway.Hum Mol Genet. 2007; 16: 1940-1950Crossref PubMed Scopus (87) Google Scholar], reduction of the Pkd1 dosage led to a more severe phenotype in Pkhd1 mice. Intriguingly and in contrast to other studies [3Bergmann C. von Bothmer J. Bruchle N. Venghaus A. Frank V. Fehrenbach H. et al.Mutations in multiple PKD genes may explain early and severe polycystic kidney disease.J Am Soc Nephrol. 2011; 22: 2047-2056Crossref PubMed Scopus (167) Google Scholar, 9Kim I. Fu Y. C. et renal formation by polycystin-2 expression and Am Soc Nephrol. 2008; PubMed Scopus Google Scholar] the reduced dosage of polycystin-2 effect on the of ARPKD. The study is for the of polycystic liver and kidney disease and increased in genetic and the of affected of the mechanisms that explain disease expression is one of the major in We have from different individuals from PKD and/or PLD that there is clinical in the member might be affected, whereas another one only clinical are to explain these and of mutations in the of remaining a major for disease [1Bergmann C. Ciliopathies. Eur J Pediatr, in press. doi:10.1007/s00431-011-1553-z. [Epub Sep 7, 2011].Google Scholar, N. and human genetic Genet. 26: Full Text Full Text PDF PubMed Scopus Google Scholar]. The study by Fedeles and colleagues with of a network for polycystic liver and kidney disease may a for the of disease expression and could have for a of other In this an effect and to and more severe disease a a recessive disease has been for ADPKD and ADPLD Watnick L.F. The of cyst formation in human autosomal dominant polycystic kidney disease Full Text Full Text PDF PubMed Scopus Google Scholar, E. R. J. et mutations might cyst formation in patients with autosomal dominant polycystic liver 2011; Full Text Full Text PDF PubMed Scopus Google Scholar]. to of mutation and resulting loss of has been as the cyst formation. However, there is increasing evidence that this a and a germline mutation to one and a mutation to the other one explain The most of evidence for this is that in individuals with germline on both alleles by not every renal cell or to a cyst as to be expected in the Y. N. Watnick T. et disease and in autosomal dominant polycystic kidney J Genet. Full Text Full Text PDF PubMed Scopus Google Scholar]. Therefore, in line with a more complex the mutation to the other disease may as a that the of cyst and/or cyst progression cyst can be hypothesized that in addition to the and data recently some of the patients with liver and kidney cysts carry mutations in more one gene of the with the by Fedeles et Therefore, multiple genes may have to be to be in a patient it is to all those genes in of this is and genetic can be in a and with a with and of a of different disease genes is currently to be and all such as in when in However, the in this of this may and or genome may to the of the of an patient can be expected to be the first on the to a and increased also with to therapeutic and study of authors that they not have to or of interest with to this The authors that they not have to or of interest with to this from the and and the PKD is by the and the from the and and the PKD is by the and the