Development of genetic hypotheses in essential hypertension.

Essential hypertension illustrates the formidable task presented by the identification of genetic determinants of common disease. Making an initial genetic inference may prove difficult enough; the subsequent demonstration of functional significance at various levels of biological integration may be even more challenging. We review three instances in which an initial genetic inference has led to the development of testable hypotheses pursued at increasingly higher levels of biological organization. These include the adducin, the G protein beta3 subunit, and the angiotensinogen hypotheses.

Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: antizyme 3.

Previous studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase. The potential significance of antizyme 3 expression during spermatogenesis is discussed in this paper.

A conditionally immortalized cell line from murine proximal tubule.

We have developed a conditionally immortalized murine cell line with proximal tubule characteristics (tsMPT) and a background suitable for genetic manipulations. tsMPT was derived from the F1 progeny of crosses between: [1] a transgenic mouse harboring a gamma-interferon (IFN-gamma)-inducible, temperature sensitive SV40 large T antigen gene (tsA58) and [2] mice of the 129/SvEv strain, the background from which most embryonic stem (ES) cells are derived. Under permissive conditions (33 degrees C and in the presence of IFN-gamma), tsMPT cells grow rapidly as monolayers with a doubling time of 23 hours; the large T antigen can be detected by immunocytochemistry and by Western blotting. When transferred to non-permissive conditions (39 degrees C, without IFN-gamma), the cells undergo differentiation coinciding with the disappearance of the large T antigen. By electron microscopy, tsMPT cells are polarized and show microvilli at their apical surface. tsMPT cells express brush border enzymes gamma-glutamyl transpeptidase and carbonic anhydrase IV. They possess Na(+)-dependent transport systems for Pi, D-glucose and L-proline as well as an amiloride-insensitive Na(+)-H+ exchanger. Intracellular cAMP generation is stimulated by parathyroid hormone but not by arginine vasopressin. Angiotensinogen mRNA and protein are present in tsMPT with markedly higher levels at non-permissive conditions. tsMPT cells should be a useful model for investigation of the functional features of the proximal tubule epithelium in relation to cellular differentiation.

Functional analysis of a mutation occurring between the two in-frame AUG codons of human angiotensinogen.

Angiotensinogen (ANG) is the specific substrate of the renin-angiotensin system, a major participant in blood pressure control. We have identified a natural mutation at the -30 amino acid position of the angiotensinogen signal peptide, in which an arginine is replaced by a proline (R-30P). Heterozygous individuals with R-30P showed a tendency to lowered plasma angiotensinogen level (1563 ng of ANG I/ml (range 1129-1941)) compared with normal individuals in the family (1892 ng of ANG I/ml (range 1603-2072)). Human angiotensinogen mRNA has two in-phase translation initiation codons (AUG) starting upstream 39 and 66 nucleotides from the cap site. R-30P occurs in a cluster of basic residues adjacent to the first AUG codon that may affect intracellular sorting of the nascent protein. Pulse-chase experiments in transiently transfected cultured cells revealed that the R-30P mutation was associated with reduced amounts of both intra- and extracellular protein. In a cell-free system, we found that two forms of native angiotensinogen were generated by alternative initiation of translation at either AUG codon. Alteration of either the first or second AUG codons abolished the synthesis of the longer and the shorter form of native angiotensinogen, respectively. Furthermore, the rate of secretion of the shorter form was lower than that of the longer form. By transplanting angiotensinogen signal peptide onto green fluorescence protein, however, we found that both forms of the signal peptide could target green fluorescence protein, normally localized in the cytoplasm, to the secretory pathway. Although the R-30P mutation may not affect intracellular sorting of angiotensinogen in a qualitative manner, it leads to a quantitative reduction in the net secretion of mature angiotensinogen through decreased translocation or increased residence time in the endoplasmic reticulum.

A mutation of angiotensinogen in a patient with preeclampsia leads to altered kinetics of the renin-angiotensin system.

Angiotensinogen exhibits genetic linkage to and association with essential hypertension and preeclampsia, a common hypertensive disorder of pregnancy; however, the polymorphisms detected thus far provide no functional clues. In a preeclamptic patient, we have identified a mutation leading to the replacement of leucine by phenylalanine at position 10 of mature angiotensinogen (L10F), the site of renin cleavage. Kinetic analyses of the enzymes of the renin-angiotensin system, using either model peptides or full-length substrates, show that this mutation significantly alters the reactions with both renin and angiotensin-converting enzyme. For the renin reaction on a full-length substrate, this substitution leads to a 10-fold decrease in Km (from 1.1 to 0.09 microM) and a 5-fold decrease in kcat (from 1.0 to 0.22 s-1); as a result, catalytic efficiency (kcat/Km) is increased by a factor of 2 (1.1 versus 2.4 microM-1 s-1). In the reaction of angiotensin-converting enzyme on angiotensin decapeptides, the substitution has no effect on Km (38.0 versus 30.0 microM), but increases kcat and catalytic efficiency > 2-fold (kcat = 15.0 versus 37.0 s-1; kcat/Km = 0.41 versus 1.23). The renin-angiotensin system, challenged by the profound physiological adaptations of pregnancy, is perturbed in preeclampsia; consequently, the L10F mutation may promote this condition in carrier subjects.

Elements of a paracrine tubular renin-angiotensin system along the entire nephron.

The renin-angiotensin system is a major regulator of body sodium, predominantly through the actions of intrarenal angiotensin II of unclear origin. We show that polarized epithelium of the proximal tubule synthesizes and secretes angiotensinogen at its apical side and that the protein can be detected in urine as a function of dietary sodium. Furthermore, we demonstrate that renin is expressed and secreted in a restricted nephron segment, the connecting tubule, also in a sodium-dependent fashion. A paracrine renin-angiotensin system operating along the entire nephron may contribute to long-term arterial pressure regulation by integrating distant tubular sodium-reabsorbing functions.