Jasper J. Saris

Identification and characterization of the tuberous sclerosis gene on chromosome 16

Tuberous sclerosis (TSC) is an autosomal dominant multisystem disorder with loci assigned to chromosomes 9 and 16. Using pulsed-field gel electrophoresis (PFGE), we identified five TSC-associated deletions at 16p13.3. These were mapped to a 120 kb region that was cloned in cosmids and from which four genes were isolated. One gene, designated TSC2, was interrupted by all five PFGE deletions, and closer examination revealed several intragenic mutations, including one de novo deletion. In this case, Northern blot analysis identified a shortened transcript, while reduced expression was observed in another TSC family, confirming TSC2 as the chromosome 16 TSC gene. The 5.5 kb TSC2 transcript is widely expressed, and its protein product, tuberin, has a region of homology to the GTPase-activating protein GAP3.Tuberous sclerosis (TSC) is an autosomal dominant multisystem disorder with loci assigned to chromosomes 9 and 16. Using pulsed-field gel electrophoresis (PFGE), we identified five TSC-associated deletions at 16p 13.3. These were mapped to a 120 kb region that was cloned in cosmids and from which four genes were isolated. One gene, designated TSC2, was interrupted by all five PFGE deletions, and closer examination revealed several intragenic mutations, including one de novo deletion. In this case, Northern blot analysis identified a shortened transcript, while reduced expression was observed in another TSC family, confirming TSC2 as the chromosome 16 TSC gene. The 5.5 kb TSC2 transcript is widely expressed, and its protein product, tuberin, has a region of homology to the GTPaseactivating protein GAP3.

PKD2, a Gene for Polycystic Kidney Disease That Encodes an Integral Membrane Protein

A second gene for autosomal dominant polycystic kidney disease was identified by positional cloning. Nonsense mutations in this gene (PKD2) segregated with the disease in three PKD2 families. The predicted 968-amino acid sequence of the PKD2 gene product has six transmembrane spans with intracellular amino- and carboxyl-termini. The PKD2 protein has amino acid similarity with PKD1, the Caenorhabditis elegans homolog of PKD1, and the family of voltage-activated calcium (and sodium) channels, and it contains a potential calcium-binding domain.

Prorenin Induces Intracellular Signaling in Cardiomyocytes Independently of Angiotensin II

Tissue accumulation of circulating prorenin results in angiotensin generation, but could also, through binding to the recently cloned (pro)renin receptor, lead to angiotensin-independent effects, like p42/p44 mitogen-activated protein kinase (MAPK) activation and plasminogen-activator inhibitor (PAI)-1 release. Here we investigated whether prorenin exerts angiotensin-independent effects in neonatal rat cardiomyocytes. Polyclonal antibodies detected the (pro)renin receptor in these cells. Prorenin affected neither p42/p44 MAPK nor PAI-1. PAI-1 release did occur during coincubation with angiotensinogen, suggesting that this effect is angiotensin mediated. Prorenin concentration-dependently activated p38 MAPK and simultaneously phosphorylated HSP27. The latter phosphorylation was blocked by the p38 MAPK inhibitor SB203580. Rat microarray gene (n=4800) transcription profiling of myocytes stimulated with prorenin detected 260 regulated genes (P<0.001 versus control), among which genes downstream of p38 MAPK and HSP27 involved in actin filament dynamics and (cis-)regulated genes confined in blood pressure and diabetes QTL regions, like Syntaxin-7, were overrepresented. Quantitative real-time RT-PCR of 7 selected genes (Opg, Timp1, Best5, Hsp27, pro-Anp, Col3a1, and Hk2) revealed temporal regulation, with peak levels occurring after 4 hours of prorenin exposure. This regulation was not altered in the presence of the renin inhibitor aliskiren or the angiotensin II type 1 receptor antagonist eprosartan. Finally, pilot 2D proteomic differential display experiments revealed actin cytoskeleton changes in cardiomyocytes after 48 hours of prorenin stimulation. In conclusion, prorenin exerts angiotensin-independent effects in cardiomyocytes. Prorenin-induced stimulation of the p38 MAPK/HSP27 pathway, resulting in alterations in actin filament dynamics, may underlie the severe cardiac hypertrophy that has been described previously in rats with hepatic prorenin overexpression.

A spectrum of mutations in the second gene for autosomal dominant polycystic kidney disease (PKD2).

Recently the second gene for autosomal dominant polycystic kidney disease (ADPKD), located on chromosome 4q21-q22, has been cloned and characterized. The gene encodes an integral membrane protein, polycystin-2, that shows amino acid similarity to the PKD1 gene product and to the family of voltage-activated calcium (and sodium) channels. We have systematically screened the gene for mutations by single-strand conformation-polymorphism analysis in 35 families with the second type of ADPKD and have identified 20 mutations. So far, most mutations found seem to be unique and occur throughout the gene, without any evidence of clustering. In addition to small deletions, insertions, and substitutions leading to premature translation stops, one amino acid substitution and five possible splice-site mutations have been found. These findings suggest that the first step toward cyst formation in PKD2 patients is the loss of one functional copy of polycystin-2.

Prorenin accumulation and activation in human endothelial cells: importance of mannose 6-phosphate receptors

Abstract—ACE inhibitors improve endothelial dysfunction, possibly by blocking endothelial angiotensin production. Prorenin, through its binding and activation by endothelial mannose 6-phosphate (M6P) receptors, may contribute to this production. Here, we investigated this possibility as well as prorenin activation kinetics, the nature of the prorenin-activating enzyme, and M6P receptor–independent prorenin binding. Human umbilical vein endothelial cells (HUVECs) were incubated with wild-type prorenin, K/A-2 prorenin (in which Lys42 is mutated to Ala, thereby preventing cleavage by known proteases), M6P-free prorenin, and nonglycosylated prorenin, with or without M6P, protease inhibitors, or angiotensinogen. HUVECs bound only M6P-containing prorenin (Kd 0.9±0.1 nmol/L, maximum number of binding sites [Bmax] 1010±50 receptors/cell). At 37°C, because of M6P receptor recycling, the amount of prorenin internalized via M6P receptors was >25 times Bmax. Inside the cells, wild-type and K/A-2 prorenin were proteolytically activated to renin. Renin was subsequently degraded. Protease inhibitors interfered with the latter but not with prorenin activation, thereby indicating that the activating enzyme is different from any of the known prorenin-activating enzymes. Incubation with angiotensinogen did not lead to endothelial angiotensin generation, inasmuch as HUVECs were unable to internalize angiotensinogen. Most likely, therefore, in the absence of angiotensinogen synthesis or endocytosis, M6P receptor–mediated prorenin internalization by endothelial cells represents prorenin clearance.

Cultured neonatal rat cardiac myocytes and fibroblasts do not synthesize renin or angiotensinogen : evidence for stretch-induced cardiomyocyte hypertrophy independent of angiotensin II

OBJECTIVE The hypertrophic response of cardiomyocytes exposed to mechanical stretch is assumed to depend on the release of angiotensin (Ang) II from these cells. Here we studied the synthesis of renin-angiotensin system (RAS) components by cardiac cells under basal conditions and after stretch. METHODS Myocytes and fibroblasts were isolated by enzymatic dissociation from hearts of 1-3-day-old Wistar rat strain pups, grown for 1 day in serum-supplemented medium and then cultured in a chemically defined, serum-free medium. Medium and cell lysate were collected 5 days later or after exposure of the cells to cyclic stretch for 24 h. Prorenin, renin and angiotensinogen were measured by enzyme-kinetic assay; Ang I and Ang II were measured by radioimmunoassay after SepPak extraction and HPLC separation. RESULTS Prorenin, but none of the other RAS components, could be detected in the medium of both cell types. However, its levels were low and the Ang I-generating activity corresponding with these low prorenin levels could not be inhibited by the specific rat renin inhibitor CH-732, suggesting that it was most likely due to bovine and/or horse prorenin sequestered from the serum-containing medium to which the cells had been exposed prior to the serum-free period. When incubated with Ang I, both myocytes and fibroblasts generated Ang II in a captopril-inhibitable manner. Myocyte and fibroblast cell lysates did not contain prorenin, renin, angiotensinogen, Ang I or Ang II in detectable quantities. Stretch increased myocyte protein synthesis by 20%, but was not accompanied by Ang II release into the medium. CONCLUSION Cardiac myocytes and fibroblasts do not synthesize renin, prorenin or angiotensinogen in concentrations that are detectable or, it not detectable, high enough to result in Ang II concentrations of physiological relevance. These cells do synthesize ACE, thereby allowing the synthesis of Ang II at cardiac tissue sites when renin and angiotensinogen are provided via the circulation. Ang II is not a prerequisite to observe a hypertrophic response of cardiomyocytes following stretch.

Cellular localization and tissue distribution of polycystin-1

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid‐filled cysts in both kidneys, in addition to a variety of extra‐renal manifestations. The PKD1 gene product, polycystin‐1, encodes a novel protein with a putative role in cell–cell/cell–matrix interactions. The present study we focused on the (sub)cellular localization of polycystin‐1 in cultured cells, and on its tissue distribution in various organs. In Madin Darby canine kidney (MDCK) cells, several polyclonal antibodies showed intense staining at the sites of interaction between adjacent cells, which remained after Triton extraction. Weak cytoplasmic staining was observed. No signal was detected at the free borders of cell aggregates, supporting a role for polycystin‐1 in cell–cell interactions. At the tissue level, polycystin‐1 expression was observed in specific cell types in tissues with known manifestations of the disease, but also in tissues of organs which have not been reported to be affected in ADPKD. Expression was frequently seen in epithelia, but also in endocrine cells (pancreatic islets, parathyroid‐producing cells, clusters in the adenohypophysis, clusters in the adrenal gland, and Leydig cells in the testis). In addition, expression was observed in myocardium and more weakly in myocytes of cardiac valves, of the cerebral arteries, and of skeletal muscles. Copyright

Is there a local renin—angiotensin system in the heart?

The existence of a local renin-angiotensin system in the heart is still a controversial issue. This review discusses the evidence, obtained from studies in cardiac cells, in isolated perfused hearts and in intact animals and humans, both under normal and pathological conditions, for local production of prorenin, renin, angiotensinogen, angiotensin-converting enzyme, angiotensin I and angiotensin II at cardiac tissue sites. In addition, the role of alternative angiotensin-generating enzymes (cathepsin, chymase) and the possibility of (pro)renin uptake from the circulation is evaluated.

Improved early diagnosis of adult polycystic kidney disease with flanking DNA markers.

A new polymorphic DNA marker for the diagnosis of autosomal dominant adult polycystic kidney disease (APKD) has been identified. The new marker, 24-1, flanks the APKD gene on the side opposite to the alpha globin on the short arm of chromosome 16. When both DNA polymorphisms bracketing the gene are informative the reliability of prenatal and presymptom diagnosis of polycystic kidney disease in non-recombinants (92% of cases) is more than 99%.

Cardiomyocytes bind and activate native human prorenin : role of soluble mannose 6-phosphate receptors

Cardiomyocytes bind, internalize, and activate recombinant human prorenin through mannose 6-phosphate/insulin-like growth factor II (M6P/IGFII) receptors. To investigate whether this also applies to native human prorenin, neonatal rat myocytes were incubated for 4 hours at 37°C with various prorenin-containing human body fluids. Uptake and activation by M6P/IGFII receptors were observed for plasma prorenin from subjects with renal artery stenosis and/or hypertension and for follicular fluid prorenin. The total amount of cellular renin and prorenin (expressed as percentage of the levels of renin and prorenin in the medium) after 4 hours of incubation was 4 to 10 times lower than after incubation with recombinant human prorenin. Although plasma contains alkaline phosphatases capable of inactivating the M6P label as well as soluble M6P/IGFII receptors that block prorenin binding in a competitive manner and proteins (eg, insulin, IGFII) that increase the number of cell-surface M6P/IGFII receptors, these factors were not responsible for the modest uptake of native human prorenin. Uptake did not occur during incubation of myocytes with plasma prorenin from anephric subjects or with amniotic fluid prorenin, and this was not due to the presence of excessively high levels of M6P/IGFII receptors and/or phosphatase activity in these fluids. In conclusion, myocytes are capable of binding, internalizing, and activating native human prorenin of renal and ovarian origin through M6P/IGFII receptors. Differences in prorenin glycosylation and/or phosphorylation as well as the concentration of soluble M6P/IGFII receptors and growth factors affecting cell-surface M6P/IGFII receptor density determine the amount of prorenin entering the heart and thus cardiac angiotensin II production.