Marcel E. Meima

Angiotensin II induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter independent of aldosterone

We studied here the independent roles of angiotensin II and aldosterone in regulating the sodium chloride cotransporter (NCC) of the distal convoluted tubule. We adrenalectomized three experimental and one control group of rats. Following surgery, the experimental groups were treated with either a high physiological dose of aldosterone, a non-pressor, or a pressor dose of angiotensin II for 8 days. Aldosterone and both doses of angiotensin II lowered sodium excretion and significantly increased the abundance of NCC in the plasma membrane compared with the control. Only the pressor dose of angiotensin II caused hypertension. Thiazides inhibited the sodium retention induced by the angiotensin II non-pressor dose. Both aldosterone and the non-pressor dose of angiotensin II significantly increased phosphorylation of NCC at threonine-53 and also increased the intracellular abundance of STE20/SPS1-related, proline alanine-rich kinase (SPAK). No differences were found in other modulators of NCC activity such as oxidative stress responsive protein type 1 or with-no-lysine kinase 4. Thus, our in vivo study shows that aldosterone and angiotensin II independently increase the abundance and phosphorylation of NCC in the setting of adrenalectomy; effects are likely mediated by SPAK. These results may explain, in part, the hormonal control of renal sodium excretion and the pathophysiology of several forms of hypertension.

Aldosterone does not require angiotensin II to activate NCC through a WNK4–SPAK–dependent pathway

We and others have recently shown that angiotensin II can activate the sodium chloride cotransporter (NCC) through a WNK4–SPAK-dependent pathway. Because WNK4 was previously shown to be a negative regulator of NCC, it has been postulated that angiotensin II converts WNK4 to a positive regulator. Here, we ask whether aldosterone requires angiotensin II to activate NCC and if their effects are additive. To do so, we infused vehicle or aldosterone in adrenalectomized rats that also received the angiotensin receptor blocker losartan. In the presence of losartan, aldosterone was still capable of increasing total and phosphorylated NCC twofold to threefold. The kinases WNK4 and SPAK also increased with aldosterone and losartan. A dose-dependent relationship between aldosterone and NCC, SPAK, and WNK4 was identified, suggesting that these are aldosterone-sensitive proteins. As more functional evidence of increased NCC activity, we showed that rats receiving aldosterone and losartan had a significantly greater natriuretic response to hydrochlorothiazide than rats receiving losartan only. To study whether angiotensin II could have an additive effect, rats receiving aldosterone with losartan were compared with rats receiving aldosterone only. Rats receiving aldosterone only retained more sodium and had twofold to fourfold increase in phosphorylated NCC. Together, our results demonstrate that aldosterone does not require angiotensin II to activate NCC and that WNK4 appears to act as a positive regulator in this pathway. The additive effect of angiotensin II may favor electroneutral sodium reabsorption during hypovolemia and may contribute to hypertension in diseases with an activated renin–angiotensin–aldosterone system.

K+-induced natriuresis is preserved during Na+ depletion and accompanied by inhibition of the Na+-Cl− cotransporter

During hypovolemia and hyperkalemia, the kidneys defend homeostasis by Na(+) retention and K(+) secretion, respectively. Aldosterone mediates both effects, but it is unclear how the same hormone can evoke such different responses. To address this, we mimicked hypovolemia and hyperkalemia in four groups of rats with a control diet, low-Na(+) diet, high-K(+) diet, or combined diet. The low-Na(+) and combined diets increased plasma and kidney ANG II. The low-Na(+) and high-K(+) diets increased plasma aldosterone to a similar degree (3-fold), whereas the combined diet increased aldosterone to a greater extent (10-fold). Despite similar Na(+) intake and higher aldosterone, the high-K(+) and combined diets caused a greater natriuresis than the control and low-Na(+) diets, respectively (P < 0.001 for both). This K(+)-induced natriuresis was accompanied by a decreased abundance but not phosphorylation of the Na(+)-Cl(-) cotransporter (NCC). In contrast, the epithelial Na(+) channel (ENaC) increased in parallel with aldosterone, showing the highest expression with the combined diet. The high-K(+) and combined diets also increased WNK4 but decreased Nedd4-2 in the kidney. Total and phosphorylated Ste-20-related kinase were also increased but were retained in the cytoplasm of distal convoluted tubule cells. In summary, high dietary K(+) overrides the effects of ANG II and aldosterone on NCC to deliver sufficient Na(+) to ENaC for K(+) secretion. K(+) may inhibit NCC through WNK4 and help activate ENaC through Nedd4-2.

The (pro)renin receptor. A decade of research: what have we learned?

The discovery of a (pro)renin receptor ((P)RR) in 2002 provided a long-sought explanation for tissue renin–angiotensin system (RAS) activity and a function for circulating prorenin, the inactive precursor of renin, in end-organ damage. Binding of renin and prorenin (referred to as (pro)renin) to the (P)RR increases angiotensin I formation and induces intracellular signalling, resulting in the production of profibrotic factors. However, the (pro)renin concentrations required for intracellular signalling in vitro are several orders of magnitude above (patho)physiological plasma levels. Moreover, the phenotype of prorenin-overexpressing animals could be completely attributed to angiotensin generation, possibly even without the need for a receptor. The efficacy of the only available putative (pro)renin receptor blocker handle region peptide remains doubtful, leading to inconclusive results. The fact that, in contrast to other RAS components, (P)RR knock-outs, even tissue-specific, are lethal, points to an important, (pro)renin-independent, function of the (P)RR. Indeed, recent research has highlighted ancillary functions of the (P)RR as an essential accessory protein of the vacuolar-type H+-ATPase (V-ATPase), and in this role, it acts as an intermediate in Wnt signalling independent of (pro)renin. In conclusion, (pro)renin-dependent signalling is unlikely in non-(pro)renin synthesizing organs, and the (P)RR role in V-ATPase integrity and Wnt signalling may explain some, if not all of the phenotypes previously associated with (pro)renin-(P)RR interaction.

(Pro)renin receptor is required for prorenin-dependent and -independent regulation of vacuolar H⁺-ATPase activity in MDCK.C11 collecting duct cells.

Prorenin binding to the prorenin receptor [(P)RR] results in nonproteolytic activation of prorenin but also directly (i.e., independent of angiotensin generation) activates signal transduction cascades that can lead to the upregulation of profibrotic factors. The (P)RR is an accessory protein of vacuolar-type H⁺-ATPase (V-ATPase) and is required for V-ATPase integrity. In addition, in collecting duct cells, prorenin-induced activation of Erk depends on V-ATPase activity. However, whether prorenin binding to the (P)RR directly regulates V-ATPase activity is as yet unknown. Here, we studied the effect of prorenin on plasma membrane V-ATPase activity in Madin-Darby canine kidney clone 11 (MDCK.C11) cells, which resemble intercalated cells of the collecting duct. Prorenin increased V-ATPase activity at low nanomolar concentrations, and the V-ATPase inhibitor bafilomycin A1, but not the angiotensin II type 1 and 2 receptor blockers irbesartan and PD-123319, prevented this. Increased, but not basal, V-ATPase activity was abolished by small interfering RNA depletion of the (P)RR. Unexpectedly, the putative peptidic (P)RR blocker handle region peptide also increased V-ATPase activity in a (P)RR-dependent manner. Finally, [Arg⁸]-vasopressin-stimulated V-ATPase activity and cAMP production were also abolished by (P)RR depletion. Our results show that in MDCK.C11 cells, the (P)RR is required for prorenin-dependent and -independent regulation of V-ATPase activity.

Phosphodiesterase 1 regulation is a key mechanism in vascular aging.

Reduced nitric oxide (NO)/cGMP signalling is observed in age-related vascular disease. We hypothesize that this disturbed signalling involves effects of genomic instability, a primary causal factor in aging, on vascular smooth muscle cells (VSMCs) and that the underlying mechanism plays a role in human age-related vascular disease. To test our hypothesis, we combined experiments in mice with genomic instability resulting from the defective nucleotide excision repair gene ERCC1 (Ercc1(d/-) mice), human VSMC cultures and population genome-wide association studies (GWAS). Aortic rings of Ercc1(d/-) mice showed 43% reduced responses to the soluble guanylate cyclase (sGC) stimulator sodium nitroprusside (SNP). Inhibition of phosphodiesterase (PDE) 1 and 5 normalized SNP-relaxing effects in Ercc1(d/-) to wild-type (WT) levels. PDE1C levels were increased in lung and aorta. cGMP hydrolysis by PDE in lungs was higher in Ercc1(d/-) mice. No differences in activity or levels of cGMP-dependent protein kinase 1 or sGC were observed in Ercc1(d/-) mice compared with WT. Senescent human VSMC showed elevated PDE1A and PDE1C and PDE5 mRNA levels (11.6-, 9- and 2.3-fold respectively), which associated with markers of cellular senescence. Conversely, PDE1 inhibition lowered expression of these markers. Human genetic studies revealed significant associations of PDE1A single nucleotide polymorphisms with diastolic blood pressure (DBP; β=0.28, P=2.47×10(-5)) and carotid intima-media thickness (cIMT; β=-0.0061, P=2.89×10(-5)). In summary, these results show that genomic instability and cellular senescence in VSMCs increase PDE1 expression. This might play a role in aging-related loss of vasodilator function, VSMC senescence, increased blood pressure and vascular hypertrophy.

Identification of the (Pro)renin Receptor as a Novel Regulator of Low-Density Lipoprotein Metabolism

RATIONALE The (pro)renin receptor ([P]RR) interacts with (pro)renin at concentrations that are >1000× higher than observed under (patho)physiological conditions. Recent studies have identified renin-angiotensin system-independent functions for (P)RR related to its association with the vacuolar H(+)-ATPase. OBJECTIVE To uncover renin-angiotensin system-independent functions of the (P)RR. METHODS AND RESULTS We used a proteomics-based approach to purify and identify (P)RR-interacting proteins. This resulted in identification of sortilin-1 (SORT1) as a high-confidence (P)RR-interacting protein, a finding which was confirmed by coimmunoprecipitation of endogenous (P)RR and SORT1. Functionally, silencing (P)RR expression in hepatocytes decreased SORT1 and low-density lipoprotein (LDL) receptor protein abundance and, as a consequence, resulted in severely attenuated cellular LDL uptake. In contrast to LDL, endocytosis of epidermal growth factor or transferrin remained unaffected by silencing of the (P)RR. Importantly, reduction of LDL receptor and SORT1 protein abundance occurred in the absence of changes in their corresponding transcript level. Consistent with a post-transcriptional event, degradation of the LDL receptor induced by (P)RR silencing could be reversed by lysosomotropic agents, such as bafilomycin A1. CONCLUSIONS Our study identifies a renin-angiotensin system-independent function for the (P)RR in the regulation of LDL metabolism by controlling the levels of SORT1 and LDL receptor.

Colocalization of the (Pro)renin Receptor/Atp6ap2 with H+-ATPases in Mouse Kidney but Prorenin Does Not Acutely Regulate Intercalated Cell H+-ATPase Activity

The (Pro)renin receptor (P)RR/Atp6ap2 is a cell surface protein capable of binding and non-proteolytically activate prorenin. Additionally, (P)RR is associated with H+-ATPases and alternative functions in H+-ATPase regulation as well as in Wnt signalling have been reported. Kidneys express very high levels of H+-ATPases which are involved in multiple functions such as endocytosis, membrane protein recycling as well as urinary acidification, bicarbonate reabsorption, and salt absorption. Here, we wanted to localize the (P)RR/Atp6ap2 along the murine nephron, exmaine whether the (P)RR/Atp6ap2 is coregulated with other H+-ATPase subunits, and whether acute stimulation of the (P)RR/Atp6ap2 with prorenin regulates H+-ATPase activity in intercalated cells in freshly isolated collecting ducts. We localized (P)PR/Atp6ap2 along the murine nephron by qPCR and immunohistochemistry. (P)RR/Atp6ap2 mRNA was detected in all nephron segments with highest levels in the collecting system coinciding with H+-ATPases. Further experiments demonstrated expression at the brush border membrane of proximal tubules and in all types of intercalated cells colocalizing with H+-ATPases. In mice treated with NH4Cl, NaHCO3, KHCO3, NaCl, or the mineralocorticoid DOCA for 7 days, (P)RR/Atp6ap2 and H+-ATPase subunits were regulated but not co-regulated at protein and mRNA levels. Immunolocalization in kidneys from control, NH4Cl or NaHCO3 treated mice demonstrated always colocalization of PRR/Atp6ap2 with H+-ATPase subunits at the brush border membrane of proximal tubules, the apical pole of type A intercalated cells, and at basolateral and/or apical membranes of non-type A intercalated cells. Microperfusion of isolated cortical collecting ducts and luminal application of prorenin did not acutely stimulate H+-ATPase activity. However, incubation of isolated collecting ducts with prorenin non-significantly increased ERK1/2 phosphorylation. Our results suggest that the PRR/Atp6ap2 may form a complex with H+-ATPases in proximal tubule and intercalated cells but that prorenin has no acute effect on H+-ATPase activity in intercalated cells.

Hemodynamic effects of intravenous nicardipine in severely pre-eclamptic women with a hypertensive crisis

Nicardipine permits rapid control of blood pressure in women with severe pre‐eclampsia (PE) and hypertensive crisis. Our objective was to investigate its maternal and fetal hemodynamic effects.

Functional Analysis of Novel Genetic Variation in the Thyroid Hormone Activating Type 2 Deiodinase

CONTEXT Thyroid hormones (TH) are important for normal brain development and abnormal TH regulation in the brain results in neurocognitive impairments. The type 2 deiodinase (D2) is important for local TH control in the brain by generating the active hormone T3 from its precursor T4. Dysfunction of D2 likely results in a neurocognitive phenotype. No mutations in D2 have been reported yet. OBJECTIVE The objective of the study was to identify D2 mutations in patients with intellectual disability and to test their functional consequences. DESIGN, SETTING, AND PATIENTS The patients were selected from the multicenter Thyroid Origin of Psychomotor Retardation study, which is a cohort of 946 subjects with unexplained intellectual disability. Based on characteristic serum TH values, the coding region of the DIO2 gene was sequenced in 387 patients. Functional consequences were assessed by in vitro D2 assays or intact cell metabolism studies using cells transfected with wild-type or mutant D2. RESULTS Sequence analysis revealed two heterozygous mutations: c.11T>A (p.L4H) in three subjects and c.305C>T (p.T102I) in one subject. Sequence analysis of family members revealed several carriers, but no segregation was observed with thyroid parameters or neurocognitive phenotype. Extensive tests with different in vitro D2 assays did not show differences between wild-type and mutant D2. CONCLUSION This study describes the identification and functional consequences of novel genetic variation in TH activating enzyme D2. Family studies and functional tests suggest that these variants do not underlie the neurocognitive impairment. Altogether our data provide evidence of the existence of rare but apparently harmless genetic variants of D2.