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• W2095432889 abstract "Genetic loss-of-function defects of connexin 40 in renal juxtaglomerular cells are associated with renin-dependent hypertension. The dysregulation of renin secretion results from an intrarenal displacement of renin cells and an interruption of the negative feedback control of renin secretion by blood pressure. It is unknown whether this phenotype is secondary to developmental defects of juxtaglomerular renin cells due to connexin 40 malfunction, or whether acute functional defects of connexin 40 in the normal adult kidney can also lead to a similar dysregulation of renin secretion and hypertension. To address this question, we generated mice with an inducible deletion of connexin 40 in the adult kidney by crossing connexin 40–floxed mice with mice harboring a ubiquitously expressed tamoxifen-inducible Cre recombinase. Tamoxifen treatment in these mice strongly reduced connexin 40 mRNA and protein expression in the kidneys. These mice displayed persistent hypertension with renin expression shifted from the media layer of afferent arterioles to juxtaglomerular periglomerular cells. Control of renin secretion by the perfusion pressure was abolished in vitro, whereas in vivo plasma renin concentrations were increased. Thus, interruption of the connexin 40 gene in the adult kidney produced very similar changes in the renin system as had embryonic deletion. Hence, impairments of connexin 40 function in the normal adult kidney can cause renin-dependent hypertension. Genetic loss-of-function defects of connexin 40 in renal juxtaglomerular cells are associated with renin-dependent hypertension. The dysregulation of renin secretion results from an intrarenal displacement of renin cells and an interruption of the negative feedback control of renin secretion by blood pressure. It is unknown whether this phenotype is secondary to developmental defects of juxtaglomerular renin cells due to connexin 40 malfunction, or whether acute functional defects of connexin 40 in the normal adult kidney can also lead to a similar dysregulation of renin secretion and hypertension. To address this question, we generated mice with an inducible deletion of connexin 40 in the adult kidney by crossing connexin 40–floxed mice with mice harboring a ubiquitously expressed tamoxifen-inducible Cre recombinase. Tamoxifen treatment in these mice strongly reduced connexin 40 mRNA and protein expression in the kidneys. These mice displayed persistent hypertension with renin expression shifted from the media layer of afferent arterioles to juxtaglomerular periglomerular cells. Control of renin secretion by the perfusion pressure was abolished in vitro, whereas in vivo plasma renin concentrations were increased. Thus, interruption of the connexin 40 gene in the adult kidney produced very similar changes in the renin system as had embryonic deletion. Hence, impairments of connexin 40 function in the normal adult kidney can cause renin-dependent hypertension. The protease renin is the key regulator of the activity of the renin–angiotensin–aldosterone system involved in body fluid and blood pressure homeostasis. Its synthesis and secretion is controlled by a number of systemic and intrarenal parameters, which mainly act in the sense of negative feedback loops. One important negative feedback loop in this context is the influence of the systemic blood pressure and the intrarenal perfusion pressure on renin secretion. As activation of the renin–angiotensin–aldosterone system elevates blood pressure, high blood pressure suppresses renin secretion and vice versa, resulting in a characteristic inverse relationship between blood pressure and plasma renin concentrations.1.Davis J.O. Freeman R.H. Mechanisms regulating renin release.Physiol Rev. 1976; 56: 1-56PubMed Google Scholar There is common agreement that a defective feedback regulation of renin secretion by the blood pressure can be a major reason for hypertension.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar, 3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4.Lubkemeier I. Machura K. Kurtz L. et al.The connexin 40 A96S mutation causes renin-dependent hypertension.J Am Soc Nephrol. 2011; 22: 1031-1040Crossref PubMed Scopus (38) Google Scholar The mechanisms along which the blood or renal perfusion pressure control renin secretion from the juxtaglomerular cells of the kidney were rather speculative, until the discovery that genetic defects of the gap-junction protein connexin 40 (Cx40) interrupts the feedback control of renin secretion by blood pressure2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar,3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,5.Krattinger N. Capponi A. Mazzolai L. et al.Connexin40 regulates renin production and blood pressure.Kidney Int. 2007; 72: 814-822Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar and as a consequence they are associated with high blood pressure.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar, 4.Lubkemeier I. Machura K. Kurtz L. et al.The connexin 40 A96S mutation causes renin-dependent hypertension.J Am Soc Nephrol. 2011; 22: 1031-1040Crossref PubMed Scopus (38) Google Scholar, 5.Krattinger N. Capponi A. Mazzolai L. et al.Connexin40 regulates renin production and blood pressure.Kidney Int. 2007; 72: 814-822Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 6.de Wit C. Roos F. Bolz S.S. et al.Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion.Physiol Genomics. 2003; 13: 169-177Crossref PubMed Scopus (176) Google Scholar In addition, those defects are also associated with a translocation of renin expression from juxtaglomerular cells of afferent arterioles to the periglomerular interstitium7.Kurtz L. Schweda F. de Wit C. et al.Lack of connexin 40 causes displacement of renin-producing cells from afferent arterioles to the extraglomerular mesangium.J Am Soc Nephrol. 2007; 18: 1103-1111Crossref PubMed Scopus (98) Google Scholar,8.Kurtz L. Gerl M. Kriz W. et al.Replacement of connexin 40 by connexin 45 causes ectopic localization of renin-producing cells in the kidney but maintains in vivo control of renin gene expression.Am J Physiol Renal Physiol. 2009; 297: F403-F409Crossref PubMed Scopus (16) Google Scholar and with an abolition of the so-called ‘calcium paradox’ of renin secretion.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar,3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar The ‘calcium paradox’ circumscribes the phenomenon that lowering of the extracellular concentration of calcium stimulates renin secretion from juxtaglomerular cells,9.Schweda F. Kurtz A. Cellular mechanism of renin release.Acta Physiol Scand. 2004; 181: 383-390Crossref PubMed Scopus (59) Google Scholar, 10.Scholz H. Hamann M. Gotz K.H. et al.Role of calcium ions in the pressure control of renin secretion from the kidneys.Pflugers Arch. 1994; 428: 173-178Crossref PubMed Scopus (38) Google Scholar, 11.Ortiz-Capisano M.C. Ortiz P.A. Harding P. et al.Adenylyl cyclase isoform v mediates renin release from juxtaglomerular cells.Hypertension. 2007; 49: 618-624Crossref PubMed Scopus (53) Google Scholar which is unexpected, as calcium is in general considered a positive trigger for secretory events.12.Trikha S. Lee E.C. Jeremic A.M. Cell secretion: current structural and biochemical insights.ScientificWorldJournal. 2010; 10: 2054-2069Crossref PubMed Scopus (6) Google Scholar The abundant expression of Cx40 in preglomerular endothelial cells and renin-producing cells raises the question in which cell type the Cx40 expression is required to enable negative feedback of blood pressure on renin secretion and positioning of renin-producing cells in the juxtaglomerular area. Selective deletion of Cx40 in renin-producing cells exactly mimics the phenotype of global Cx40 deletion, whereas endothelial deletion of Cx40 has no effect on renin secretion or blood pressure.3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar It is unclear so far whether these profound changes of the function of renin-expressing cells induced by genetically determined malfunctions of Cx40 is a consequence of defective differentiation or maturation of renin-producing cells, or whether acutely occurring defects of Cx40 function in the normal adult kidney can produce similar changes in the function of renin-producing cells in the kidney. Proper function of Cx40 is dependent on the synthesis of a correct protein on one hand and of correct insertion into the plasma membrane. Although little is known yet about the intracellular control of Cx40 synthesis and trafficking,13.Segretain D. Falk M.M. Regulation of connexin biosynthesis, assembly, gap junction formation, and removal.Biochim Biophys Acta. 2004; 1662: 3-21Crossref PubMed Scopus (248) Google Scholar,14.Brink P.R. Gap junctions in vascular smooth muscle.Acta Physiol Scand. 1998; 164: 349-356Crossref PubMed Scopus (58) Google Scholar changes of Cx40 expression have already been described for clinically relevant situations such as hypertension, sepsis/inflammation, diabetes, or hypercholesteremia.15.Barriga M. Cal R. Cabello N. et al.Low density lipoproteins promote unstable calcium handling accompanied by reduced SERCA2 and connexin-40 expression in cardiomyocytes.PLoS One. 2013; 8: e58128Crossref PubMed Scopus (11) Google Scholar, 16.Rignault S. Haefliger J.A. Gasser D. et al.Sepsis up-regulates the expression of connexin 40 in rat aortic endothelium.Crit Care Med. 2005; 33: 1302-1310Crossref PubMed Scopus (22) Google Scholar, 17.Rignault S. Haefliger J.A. Waeber B. et al.Acute inflammation decreases the expression of connexin 40 in mouse lung.Shock. 2007; 28: 78-85Crossref PubMed Scopus (38) Google Scholar, 18.Kansui Y. Fujii K. Nakamura K. et al.Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats.Am J Physiol Heart Circ Physiol. 2004; 287: H216-H224Crossref PubMed Scopus (73) Google Scholar, 19.Zhang J.H. Kawashima S. Yokoyama M. et al.Increased eNOS accounts for changes in connexin expression in renal arterioles during diabetes.Anat Rec A Discov Mol Cell Evol Biol. 2006; 288: 1000-1008Crossref PubMed Scopus (20) Google Scholar Therefore, it appeared of interest for us to find out whether changes of Cx40 function in the normal adult kidney can also induce fundamental changes of the function of renin-producing cells of the kidney and thus provide a basis for renin-dependent hypertension. To address this aim, we have generated and characterized mice with an inducible deletion of Cx40 in the adult animals. To find the best treatment protocol for tamoxifen-induced Cre recombinase activation and secondary Cx40 deletion, adult Cx40flflCreER mice and Cx40flfl controls were either injected with tamoxifen (40mg/kg/day) every other day for 10 days or were fed a tamoxifen-containing chow (40mg/kg/day) over different time periods. Most efficient and reproducible deletion of Cx40 in the kidneys was achieved with feeding the tamoxifen-containing chow for 2 weeks. Animals were then analyzed 2 and 14 weeks after stop of tamoxifen feeding. Already 2 weeks after completing the tamoxifen treatment, renal Cx40 mRNA abundance and Cx40 protein levels had dropped to 20 and to 10% of the respective values of Cx40flfl control mice (Figure 1a). The reduction of Cx40 expression by tamoxifen treatment of Cx40flflCreER mice was also clearly visible by immunohistochemical analysis of the kidneys. In kidneys of Cx40flfl control mice, Cx40 immunoreactivity was clearly detectable in intraglomerular (presumably mesangial) cells, in juxtaglomerular renin-producing cells, and in the endothelial cell layer of afferent arterioles (Figure 1b). In kidneys of Cx40flflCreER mice, Cx40 immunoreactivity was occasionally seen as a residual faint intraglomerular staining, but renin-producing cells were frequently free of Cx40 immunoreactivity (Figure 1c). Determination of the renal mRNA abundance of the other vascular connexin isoforms Cx37, Cx43, and Cx45 revealed no obvious differences between the two genotypes (Table 1).Table 1Measurements of renal mRNA abundance of connexin 37, connexin 43, and connexin 45 in Cx40flfl and Cx40flflCreER miceCx40flflCx40flflCreERCx37 mRNA1.12±0.181.19±0.07Cx43 mRNA0.98±0.091.13±0.03Cx45 mRNA1.07±0.071.26±0.11Abbreviation: Cx40, connexin 40.Renin mRNA quantification was performed using glyceraldehyde 3-phosphate dehydrogenase as a control. Data are means±s.e.m. (n=15–20 mice). Open table in a new tab Abbreviation: Cx40, connexin 40. Renin mRNA quantification was performed using glyceraldehyde 3-phosphate dehydrogenase as a control. Data are means±s.e.m. (n=15–20 mice). Already 2 weeks after the completion of tamoxifen treatment, Cx40flflCreER mice had developed robust hypertension, which sustained throughout the time of observation, whereas tamoxifen-treated Cx40flfl control mice remained normotensive (Figure 2c; Table 2). Treatment with enalapril (10mg/kg/day), an angiotensin I–converting enzyme inhibitor, for a time span of 12 days reduced the blood pressure of Cx40flfl mice significantly and decreased blood pressure in Cx40flflCreER mice to the levels observed in control littermates (Table 2). Plasma renin concentrations were approximately doubled in Cx40flflCreER mice relative to Cx40flfl control mice throughout the experimental period (Figure 2b), whereas renin mRNA abundance in the kidneys was not different between the two genotypes (Figure 2a).Table 2Measurements of systolic blood pressure in Cx40flfl and Cx40flflCreER mice under control conditions and during enalapril treatmentExperimental groupSystolic blood pressure (mmHg)Cx40flfl122±3.7Cx40flfl+enalapril110±3.1*Cx40flflCreER156±4.9*Cx40flflCreER+enalapril125±3.4§+Abbreviation: Cx40, connexin 40.*P<0.05 versus Cx40flfl control; §P<0.05 versus Cx40flflCreER control; +P<0.05 versus Cx40flfl+enalapril. Data are means±s.e.m. (n=5 mice). Open table in a new tab Abbreviation: Cx40, connexin 40. *P<0.05 versus Cx40flfl control; §P<0.05 versus Cx40flflCreER control; +P<0.05 versus Cx40flfl+enalapril. Data are means±s.e.m. (n=5 mice). Hypertension in combination with elevated plasma renin concentrations suggests abnormalities in the negative feedback control of renin secretion by the blood pressure. We therefore analyzed the pressure control of renin secretion from isolated kidneys of tamoxifen-treated Cx40flflCreER mice and from tamoxifen-treated Cx40flfl control mice. In kidneys from Cx40flfl control mice, renin secretion was inversely related to the perfusion pressure as normal, whereas in kidneys from Cx40flflCreER mice renin secretion showed no dependency on the perfusion pressure (Figure 3a). As the normal pressure control of renin secretion is a calcium-sensitive process,10.Scholz H. Hamann M. Gotz K.H. et al.Role of calcium ions in the pressure control of renin secretion from the kidneys.Pflugers Arch. 1994; 428: 173-178Crossref PubMed Scopus (38) Google Scholar we next considered the calcium sensitivity of renin secretion in isolated kidneys of tamoxifen-treated Cx40flfl control mice and of tamoxifen-treated Cx40flflCreER mice. In kidneys of Cx40flfl control mice, renin secretion is stimulated by the β-adrenoreceptor activator isoproterenol, and this stimulation of renin secretion is strongly enhanced by lowering the extracellular concentration of calcium owing to the addition of the calcium chelator ethylene glycol-bis (aminoethyl ether) tetraacetic acid (EGTA) to the perfusate. In kidneys of Cx40flflCreER mice, isoproterenol induced a similar stimulation of renin secretion as in kidneys of Cx40flfl control mice, but further addition of EGTA to the perfusate produced no additional effect on renin secretion (Figure 3b). The abnormalities of renin secretion in tamoxifen-treated Cx40flflCreER mice prompted us to study the morphology of renin-secreting cells in more detail. In kidneys of tamoxifen-treated Cx40flfl control mice, renin immunoreactive cells were restricted to the media layer of afferent arterioles at the juxtaglomerular junction sites of afferent arterioles and glomerular capillaries as normal (Figure 1b). In kidneys of tamoxifen-treated Cx40fl/flCreER mice, a more variable localization pattern of renin immunoreactive cells emerged, which could be described as a gradual shift of renin expression from the media layer of afferent arterioles into the extraglomerular mesangial (lacis) cell field accompanied with a local increase in the number of ectopic renin immunoreactive cells (Figures 1c and 4). The development of such larger ectopic renin cell fields increased with the age of the animals. In the older tamoxifen-pretreated Cx40flflCreER mice (24 weeks), about 15% of glomeruli showed extended ectopic renin cell fields, as illustrated in Figure 4b and c, whereas 30% of glomeruli displayed no renin immunoreactive cells. A number of previous studies have shown that genetically fixed loss of functions of Cx40 in renin-secreting cells of the kidneys cause major functional and structural alterations of the cells.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar, 3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4.Lubkemeier I. Machura K. Kurtz L. et al.The connexin 40 A96S mutation causes renin-dependent hypertension.J Am Soc Nephrol. 2011; 22: 1031-1040Crossref PubMed Scopus (38) Google Scholar, 7.Kurtz L. Schweda F. de Wit C. et al.Lack of connexin 40 causes displacement of renin-producing cells from afferent arterioles to the extraglomerular mesangium.J Am Soc Nephrol. 2007; 18: 1103-1111Crossref PubMed Scopus (98) Google Scholar, 20.Schweda F. Kurtz L. de Wit C. et al.Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension.Kidney Int. 2009; 75: 482-489Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Most prominent is an interruption of the renal baroreceptor control of renin secretion that mediates a negative feedback of blood pressure on renin secretion.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar,3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar As a consequence of the interruption of the baroreceptor control, the mice experience a relative hypersecretion of renin leading to hypertension.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar, 3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4.Lubkemeier I. Machura K. Kurtz L. et al.The connexin 40 A96S mutation causes renin-dependent hypertension.J Am Soc Nephrol. 2011; 22: 1031-1040Crossref PubMed Scopus (38) Google Scholar, 5.Krattinger N. Capponi A. Mazzolai L. et al.Connexin40 regulates renin production and blood pressure.Kidney Int. 2007; 72: 814-822Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar A second striking alteration of renin cells with defective Cx40 function is a shift of renin expression from the typical juxtaglomerular cells of afferent arterioles into the extraglomerular mesangium and periglomerular interstitium.7.Kurtz L. Schweda F. de Wit C. et al.Lack of connexin 40 causes displacement of renin-producing cells from afferent arterioles to the extraglomerular mesangium.J Am Soc Nephrol. 2007; 18: 1103-1111Crossref PubMed Scopus (98) Google Scholar This study aimed to determine which of those earlier-mentioned changes of renin cells depend on dysfunction of Cx40 during development or can be induced anytime, that is, also in the normal adult kidney. We found that inducible Cre activation can lead to a strong downregulation of Cx40 expression without alterations of the expression of other vascular connexins in the kidney. In global Cx40 knockout mice, slightly divergent results about renal mRNA levels of Cx37, Cx43, and Cx45 have been reported by several groups.5.Krattinger N. Capponi A. Mazzolai L. et al.Connexin40 regulates renin production and blood pressure.Kidney Int. 2007; 72: 814-822Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar,20.Schweda F. Kurtz L. de Wit C. et al.Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension.Kidney Int. 2009; 75: 482-489Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar The results of this study strongly suggest that interruption of the baroreceptor control of renin secretion and the translocation of renin expression by defective Cx40 function are not developmentally fixed but can be induced anytime even in the adult kidney. Thus, induced deletion of Cx40 in the adult organism produces very similar phenotypic changes as global or conditional knockouts of Cx40 do. In detail, induced deletion of Cx40 in the adult mouse led to hypertension, increased plasma renin concentrations, abrogation of the renal baroreceptor control of renin secretion, desensitization of renin secretion toward changes of the extracellular calcium concentration, and to translocation of renin expression, similar to embryonic deletion of Cx40.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar, 3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 4.Lubkemeier I. Machura K. Kurtz L. et al.The connexin 40 A96S mutation causes renin-dependent hypertension.J Am Soc Nephrol. 2011; 22: 1031-1040Crossref PubMed Scopus (38) Google Scholar, 7.Kurtz L. Schweda F. de Wit C. et al.Lack of connexin 40 causes displacement of renin-producing cells from afferent arterioles to the extraglomerular mesangium.J Am Soc Nephrol. 2007; 18: 1103-1111Crossref PubMed Scopus (98) Google Scholar, 20.Schweda F. Kurtz L. de Wit C. et al.Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension.Kidney Int. 2009; 75: 482-489Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar There is evidence that global deletion of Cx40 variably attenuates macula densa–triggered tubuloglomerular feedback response.21.Just A. Kurtz L. de Wit C. et al.Connexin 40 mediates the tubuloglomerular feedback contribution to renal blood flow autoregulation.J Am Soc Nephrol. 2009; 20: 1577-1585Crossref PubMed Scopus (49) Google Scholar,22.Oppermann M. Carota I. Schiessl I. et al.Direct assessment of tubuloglomerular feedback responsiveness in connexin 40-deficient mice.Am J Physiol Renal Physiol. 2013; 304: F1181-F1186Crossref PubMed Scopus (11) Google Scholar We have therefore also examined in as far altered macula densa signaling might contribute to the interruption of the baroreceptor control of renin secretion. For this aim, we have performed experiments with isolated perfused kidneys with transient ureteral ligation. This maneuver, however, exerted no effect on renin secretion in wild-type mice and was therefore not further pursued (data not shown). Our data obtained with ACE inhibition, moreover, support the assumption that the hypertension of Cx40-deficient mice is significantly triggered by the renin–angiotensin system. It is noteworthy, however, that inhibition of the renin–angiotensin system did not abolish the blood pressure difference between wild-type and Cx40-deficient mice. Similar observations have already been reported by several groups.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar,5.Krattinger N. Capponi A. Mazzolai L. et al.Connexin40 regulates renin production and blood pressure.Kidney Int. 2007; 72: 814-822Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar,6.de Wit C. Roos F. Bolz S.S. et al.Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion.Physiol Genomics. 2003; 13: 169-177Crossref PubMed Scopus (176) Google Scholar,20.Schweda F. Kurtz L. de Wit C. et al.Substitution of connexin40 with connexin45 prevents hyperreninemia and attenuates hypertension.Kidney Int. 2009; 75: 482-489Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar In line, renin cell–specific re-expression of Cx40 in mice with global Cx40 knockout attenuates, but does not abrogate, hypertension.23.Le Gal L. Alonso F. Wagner C. et al.Restoration of connexin 40 (cx40) in Renin-producing cells reduces the hypertension of cx40 null mice.Hypertension. 2014; 63: 1198-1204Crossref PubMed Scopus (28) Google Scholar These findings suggest that also factors independent of the renin–angiotensin system may contribute to hypertension in Cx40-defective mice. A possible factor contributing to renin-independent elevation of blood pressure in mice with induced Cx40 deletion could be a disturbed endothelium-dependent spread of dilator responses.6.de Wit C. Roos F. Bolz S.S. et al.Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion.Physiol Genomics. 2003; 13: 169-177Crossref PubMed Scopus (176) Google Scholar It has been shown that the endothelium of arterioles is abundantly coupled by Cx37 and Cx40 in many species24.Hakim C.H. Jackson W.F. Segal S.S. Connexin isoform expression in smooth muscle cells and endothelial cells of hamster cheek pouch arterioles and retractor feed arteries.Microcirculation. 2008; 15: 503-514Crossref PubMed Scopus (44) Google Scholar, 25.Wolfle S.E. Schmidt V.J. Hoepfl B. et al.Connexin45 cannot replace the function of connexin40 in conducting endothelium-dependent dilations along arterioles.Circ Res. 2007; 101: 1292-1299Crossref PubMed Scopus (79) Google Scholar, 26.Hill C.E. Phillips J.K. Sandow S.L. Heterogeneous control of blood flow amongst different vascular beds.Med Res Rev. 2001; 21: 1-60Crossref PubMed Scopus (148) Google Scholar and that Cx40 deficiency within the endothelial layer causes a deficient spreading of vasodilator stimuli along the vessel wall.6.de Wit C. Roos F. Bolz S.S. et al.Lack of vascular connexin 40 is associated with hypertension and irregular arteriolar vasomotion.Physiol Genomics. 2003; 13: 169-177Crossref PubMed Scopus (176) Google Scholar Deletion or functional impairment of Cx40 in the endothelium, however, does not cause hypertension.3.Wagner C. Jobs A. Schweda F. et al.Selective deletion of Connexin 40 in renin-producing cells impairs renal baroreceptor function and is associated with arterial hypertension.Kidney Int. 2010; 78: 762-768Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar,27.Jobs A. Schmidt K. Schmidt V.J. et al.Defective Cx40 maintains Cx37 expression but intact Cx40 is crucial for conducted dilations irrespective of hypertension.Hypertension. 2012; 60: 1422-1429Crossref PubMed Scopus (45) Google Scholar There are several conclusions that arise from these findings. First, the functional alterations of renin-producing cells induced by defective Cx40 function as seen in previous studies are probably directly related to Cx40 gap junction or hemi channel functions, rather than to indirect effects resulting from misdirected development. This conclusion is supported by the observation of two previous studies that demonstrate that chemicals known to block gap junctions affect renin secretion in vivo in the normal adult kidney and also in vitro in the isolated perfused kidney.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar,28.Takenaka T. Inoue T. Kanno Y. et al.Expression and role of connexins in the rat renal vasculature.Kidney Int. 2008; 73: 415-422Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Second, the translocation of renin expression from juxtaglomerular cells of afferent arterioles into the lacis cell field and periglomerular interstitium is not genetically programmed but can be induced anytime by loss of Cx40 function. This conclusion fits with the high degree of plasticity of renin expression, such as the development of juxtaglomerular hypertrophy in states of a chronically challenged renin–angiotensin–aldosterone system.29.Kurt B. Karger C. Wagner C. et al.Control of renin secretion from kidneys with renin cell hyperplasia.Am J Physiol Renal Physiol. 2014; 306: F327-F332Crossref PubMed Scopus (6) Google Scholar It will be of interest to know what comes first, hypertension or translocation of the cells, and to know whether the prevention of hypertension prevents the translocation of the renin cells. Third, possibly any form of developing malfunction of Cx40 in renin cells of the adult kidney can lead to dysfunction and in consequence to renin-induced hypertension. This issue appears important, as there is growing information about changes of Cx40 expression/function in clinically relevant disease states such as hypertension, inflammation, diabetes, hyperlipidemia, and others.15.Barriga M. Cal R. Cabello N. et al.Low density lipoproteins promote unstable calcium handling accompanied by reduced SERCA2 and connexin-40 expression in cardiomyocytes.PLoS One. 2013; 8: e58128Crossref PubMed Scopus (11) Google Scholar, 16.Rignault S. Haefliger J.A. Gasser D. et al.Sepsis up-regulates the expression of connexin 40 in rat aortic endothelium.Crit Care Med. 2005; 33: 1302-1310Crossref PubMed Scopus (22) Google Scholar, 17.Rignault S. Haefliger J.A. Waeber B. et al.Acute inflammation decreases the expression of connexin 40 in mouse lung.Shock. 2007; 28: 78-85Crossref PubMed Scopus (38) Google Scholar, 18.Kansui Y. Fujii K. Nakamura K. et al.Angiotensin II receptor blockade corrects altered expression of gap junctions in vascular endothelial cells from hypertensive rats.Am J Physiol Heart Circ Physiol. 2004; 287: H216-H224Crossref PubMed Scopus (73) Google Scholar, 19.Zhang J.H. Kawashima S. Yokoyama M. et al.Increased eNOS accounts for changes in connexin expression in renal arterioles during diabetes.Anat Rec A Discov Mol Cell Evol Biol. 2006; 288: 1000-1008Crossref PubMed Scopus (20) Google Scholar Two main questions arising from this study await future clarification. First, how is Cx40 directly involved in the control of renin secretion by pressure and by external calcium, and how does Cx40 prevent the spreading of renin expression into the periglomerular interstitium? Second, which clinical disorders or diseases are associated with altered expression of Cx40 in renin cells of the kidney? Animal experiments were conducted according to the National Institutes of Health guidelines for the care and use of animals in research and were approved by the local ethics committee. All laboratory mice in experiments were at the age of 12 to 24 weeks. Mice with a floxed Cx40 gene (Cx40flfl) generated by TAB van Veen, University Medical Center Utrecht, and mice with tamoxifen-inducible Cre recombinase (CreER) generated by R Feil,30.Feil R. Wagner J. Metzger D. et al.Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (731) Google Scholar and provided by A Ludwig, University of Erlangen, were mated to generate mice that were homozygous for the floxed Cx40 gene (Cx40flfl) carrying one allele of CreER. Corresponding littermates exhibiting no Cre recombinase Cx40flfl served as controls. Mice were genotyped by PCR from tail-tip biopsies using primers as described.30.Feil R. Wagner J. Metzger D. et al.Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.Biochem Biophys Res Commun. 1997; 237: 752-757Crossref PubMed Scopus (731) Google Scholar,31.Chadjichristos C.E. Scheckenbach K.E. van Veen T.A. et al.Endothelial-specific deletion of connexin40 promotes atherosclerosis by increasing CD73-dependent leukocyte adhesion.Circulation. 2010; 121: 123-131Crossref PubMed Scopus (113) Google Scholar Cx40 gene ablation in Cx40flflCreER mice was induced by tamoxifen administration via intraperitoneal injection every other day for 10 days (40mg/kg/day) or via tamoxifen-containing chow (40mg/kg/day) at the age of 8 weeks. Tamoxifen chow was applied as tamoxifen citrate (400mg/kg diet, 66% tamoxifen, Harlan Laboratories, Rossdorf, Germany) for different time periods (2–4 weeks). After tamoxifen treatment, mice were set back to normal rodent chow and were used in experiments at the age of 12 and 24 weeks, respectively. Blood samples were taken from tail vein to measure the plasma renin concentration. The animals were deeply anesthetized with sevoflurane and killed by cervical dislocation. For RNA analysis, the kidneys were removed quickly and frozen in liquid nitrogen. In addition, the kidneys of both genotypes were perfusion-fixed with 4% paraformaldehyde for immunohistology. Measurements of systolic blood pressure were performed noninvasively by tail cuff manometry (TSE Systems, Frankfurt, Germany). Therefore, the blood pressure of conscious mice, before being accustomed to the experimental setup for 5–7 days, was measured on a 30°C warm platform. Data from 10 sequential days at a certain time of the day were analyzed to determine the values. In a separate group of 20 mice (10 Cx40flfl, 10 Cx40flflCreER), systolic blood pressure was measured on 10 subsequent days under control conditions or under treatment with the angiotensin I–converting enzyme inhibitor enalapril (10mg/kg/day) for 12 days through drinking water. Primary antibodies used for immunohistochemistry were goat anti-Cx40 (Santa Cruz Biotechnology, Santa Cruz, CA), chicken anti-renin (Davids Biotechnology, Regensburg, Germany), and anti-α-smooth muscle actin (anti-α-SMA; Beckman Coulter Immunotech, Krefeld, Germany). Cy2-, tetramethyl-rhodamineisothiocyanate (TRITC)-, and Cy5-conjugated secondary antibodies were purchased from Dianova (Hamburg, Germany). Primary antibodies for immunoblotting were anti-β-actin (Sigma Aldrich, Taufkirchen, Germany) and Cx40 (Santa Cruz Biotechnology). The secondary antibodies were purchased from Santa Cruz Biotechnology. Kidneys from Cx40flflCreER mice and their controls were perfusion fixed with 4% paraformaldeyhde, dehydrated, and embedded in paraffin. Immunolabeling was performed on 5-μm paraffin sections. For Cx40 immunostaining, an antigen retrieval with Tris-EDTA (pH 9.0) for 45min at 97°C was necessary, followed by a blocking step with 5% horse serum in phosphate-buffered saline with Tween 20 (PBST; 0.3% Triton X-100 in PBS buffer). The blocking reagent was also used for diluting the primary, as well as the secondary, antibodies. Incubation with the primary antibody mix was performed overnight at 4°C. The following antibody dilutions were used: Cx40 diluted to 1:100, renin diluted to 1:400, and α-SMA diluted to 1:100. After washing the sections in PBS, the tissue was incubated for 90min at room temperature with the fluorescent-linked secondary antibodies each diluted to a ratio of 1:400. All sections for fluorescent imaging were mounted with glycergel (DakoCytomation, Hamburg, Germany) and viewed with an Zeiss Axiovert Microscope (Jena, Germany). Total RNA was isolated from kidneys as described by Chomczynski and Sacchi32.Chomczynski P. Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63147) Google Scholar and was quantified using a photometer. The complementary DNA was synthesized by Moloney murine leukemia virus room temperature (Superscript, Invitrogen, Darmstadt, Germany) and amplified with primers, as described previously.33.Kurtz L. Janssen-Bienhold U. Kurtz A. et al.Connexin expression in renin-producing cells.J Am Soc Nephrol. 2009; 20: 506-512Crossref PubMed Scopus (48) Google Scholar,34.Sauter A. Machura K. Neubauer B. et al.Development of renin expression in the mouse kidney.Kidney Int. 2008; 73: 43-51Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar For quantification of mRNA expression, real-time PCR was performed using a Light Cycler Instrument and the LightCycler 480 SYBR Green I Master kit (Roche Diagnostics, Mannheim, Germany), and glyceraldehyde 3-phosphate dehydrogenase was used as a control. Fifty micrograms of membrane protein was loaded per lane, separated by a 10% sodium dodecyl sulfate–polyacrylamide gel, and transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), which was blocked for 90min in 5% nonfat dry milk diluted in Tris-buffered saline with 0.1% Tween-20, and then incubated overnight at 4°C with primary antibodies β-actin (1:5000) and Cx40 (1:500). After being washed, the membrane was incubated for 2h with the secondary antibodies (1:2000) and subjected to a chemiluminescence detection system (ECL, Amersham, Braunschweig, Germany). For quantitative analysis, the band intensities were quantified densitometrically (Image lab 5.0, Bio-Rad). For the determination of plasma renin concentration, the blood samples taken from the tail vein were centrifuged and the plasma was incubated for 1.5h at 37°C, with plasma from bilaterally nephrectomized male rats as the renin substrate.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar The generated angiotensin I (ng/ml/h) was determined using radioimmunoassay (Byk&DiaSorin Diagnostics, Dietzenbach, Germany). Induced Cx40flflCreER mice and age-matched Cx40flfl mice of either sex were used as kidney donors. The isolated perfused mouse kidney model has been described in detail previously.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar Briefly, the animals were anesthetized with an intraperitoneal injection of 12mg/kg xylazine (Rompun, Bayer, Leverkusen, Germany) and 80mg/kg ketamine-HCl (WDT, Garbsen, Germany), the abdominal aorta was cannulated, and the right kidney was excised, placed in a thermostated moistening chamber, and perfused at constant pressure (90mmHg). Using an electronic feedback control, perfusion pressure could be changed and held constant in a pressure range between 40 and 140mmHg. Finally, the renal vein was cannulated and the venous effluent was collected for the determination of renin activity and venous blood flow. The basic perfusion medium consisted of a modified Krebs–Henseleit solution supplemented with 6g/100ml bovine serum albumin and with freshly washed human red blood cells (10% hematocrit). Stock solutions of isoproterenol were dissolved in freshly prepared perfusate. Drugs were infused into the arterial limb of the perfusion circuit. For lowering the extracellular calcium concentration into the submicromolar range, we added the calcium chelator EGTA 3.12mmol/l to the perfusate. For the determination of perfusate renin concentration, three samples of the venous effluent were taken in intervals of 2min during each experimental period. Renin activity in the venous effluent was determined by radioimmunoassay (Byk&DiaSorin Diagnostics), as described previously.2.Wagner C. de Wit C. Kurtz L. et al.Connexin40 is essential for the pressure control of renin synthesis and secretion.Circ Res. 2007; 100: 556-563Crossref PubMed Scopus (171) Google Scholar Values are given as means±s.e.m. Differences between groups were analyzed using analysis of variance and Bonferroni adjustment for multiple comparisons. P<0.05 was considered statistically significant. We thank Professor Dr A. Ludwig, Institute of Pharmacology and Toxicology, University of Erlangen for providing us CreER mice. We are very grateful to Gerda Treuner, Anna M’Bangui, Ramona Mogge, and Marcela DeLoza for helpful technical assistance. The authors’ work was financially supported by Deutsche Forschungsgemeinschaft Grant (WA-2137/2-2)." @default.
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