Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

Hypertension induces glomerulosclerosis in phospholipase C-ε1 deficiency.

Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.

A case of tumor lysis syndrome and acute renal failure associated with elotuzumab treatment in multiple myeloma.

Renal dysfunction is a common comorbidity of multiple myeloma. However, tumor lysis syndrome is a rare cause of renal dysfunction in multiple myeloma. Elotuzumab is a newly US FDA-approved monoclonal antibody used in the treatment of refractory multiple myeloma. To our knowledge, elotuzumab has not been associated with a case of tumor lysis syndrome. We present the case of a patient who developed clinical tumor lysis syndrome 1 week after treatment with elotuzumab accompanied by renal failure with hyperphosphatemia, hyperkalemia, and profound hyperuricemia. His course was further complicated by significant epistaxis from the accumulation of dabigatran in acute renal failure. In spite of treatment with rasburicase and hemodiafiltration, the patient decompensated and eventually died. Risk factors for the development of tumor lysis syndrome in multiple myeloma are discussed.

Recruited renin-containing renal microvascular cells demonstrate the calcium paradox regulatory phenotype.

Renin is the critical regulatory enzyme for production of angiotensin (Ang)-II, a potent vasoconstrictor involved in regulating blood pressure and in the pathogenesis of hypertension. Chronic sodium deprivation enhances renin secretion from the kidney, due to recruitment of additional cells from the afferent renal microvasculature to become renin-producing rather than just increasing release from existing juxtaglomerular (JG) cells. JG cells secrete renin inversely proportional to extra- and intracellular calcium, a unique phenomenon characteristic of the JG regulatory phenotype known as the "calcium paradox." It is not known if renin secreted from recruited renin-containing cells is regulated similarly to native JG cells, and therefore acquires this JG cell phenotype. We hypothesized that non-JG cells in renal microvessels recruited to produce renin in response to chronic dietary sodium restriction would demonstrate the calcium paradox, characteristic of the JG cell phenotype. Histology showed recruitment of upstream arteriolar renin in response to sodium restriction compared to normal-diet rats. Renin fluorescence intensity increased 53% in cortices of sodium-restricted rats (P<0.001). We measured renin release from rat afferent microvessels, isolated using iron oxide nanopowder and incubated in either normal or low-calcium media. Basal renin release from normal sodium-diet rat microvessels in normal calcium media was 298.1±44.6 ng AngI/mL/hour/mg protein, and in low-calcium media increased 39% to 415.9±71.4 ng AngI/mL/hour/mg protein (P<0.025). Renin released from sodium-restricted rat microvessels increased 50% compared to samples from normal-diet rats (P<0.04). Renin release in normal calcium media was 447.0±54.3 ng AngI/mL/hour/mg protein, and in low-calcium media increased 36% to 607.6±96.1 ng AngI/mL/hour/mg protein (P<0.05). Thus, renin-containing cells recruited in the afferent microvasculature not only express and secrete renin but demonstrate the calcium paradox, suggesting renin secretion from recruited renin-containing cells share the JG phenotype for regulating renin secretion.

Vitamin D increases plasma renin activity independently of plasma Ca2+ via hypovolemia and ß-adrenergic activity.

1, 25-Dihydroxycholechalciferol (calcitriol) and 19-nor-1, 25-dihydroxyvitamin D2 (paricalcitol) are vitamin D receptor (VDR) agonists. Previous data suggest VDR agonists may actually increase renin-angiotensin activity, and this has always been assumed to be mediated by hypercalcemia. We hypothesized that calcitriol and paricalcitol would increase plasma renin activity (PRA) independently of plasma Ca(2+) via hypercalciuria-mediated polyuria, hypovolemia, and subsequent increased ß-adrenergic sympathetic activity. We found that both calcitriol and paricalcitol increased PRA threefold (P < 0.01). Calcitriol caused hypercalcemia, but paricalcitol did not. Both calcitriol and paricalcitol caused hypercalciuria (9- and 7-fold vs. control, P < 0.01) and polyuria (increasing 2.6- and 2.2-fold vs. control, P < 0.01). Paricalcitol increased renal calcium-sensing receptor (CaSR) expression, suggesting a potential cause of paricalcitol-mediated hypercalciuria and polyuria. Volume replacement completely normalized calcitriol-stimulated PRA and lowered plasma epinephrine by 43% (P < 0.05). ß-Adrenergic blockade also normalized calcitriol-stimulated PRA. Cyclooxygenase-2 inhibition had no effect on calcitriol-stimulated PRA. Our data demonstrate that vitamin D increases PRA independently of plasma Ca(2+) via hypercalciuria, polyuria, hypovolemia, and increased ß-adrenergic activity.

Adenosine inhibits renin release from juxtaglomerular cells via an A1 receptor-TRPC-mediated pathway.

Renin is synthesized and released from juxtaglomerular (JG) cells. Adenosine inhibits renin release via an adenosine A1 receptor (A1R) calcium-mediated pathway. How this occurs is unknown. In cardiomyocytes, adenosine increases intracellular calcium via transient receptor potential canonical (TRPC) channels. We hypothesized that adenosine inhibits renin release via A1R activation, opening TRPC channels. However, higher concentrations of adenosine may stimulate renin release through A2R activation. Using primary cultures of isolated mouse JG cells, immunolabeling demonstrated renin and A1R in JG cells, but not A2R subtypes, although RT-PCR indicated the presence of mRNA of both A2AR and A2BR. Incubating JG cells with increasing concentrations of adenosine decreased renin release. Different concentrations of the adenosine receptor agonist N-ethylcarboxamide adenosine (NECA) did not change renin. Activating A1R with 0.5 µM N6-cyclohexyladenosine (CHA) decreased basal renin release from 0.22 ± 0.05 to 0.14 ± 0.03 µg of angiotensin I generated per milliliter of sample per hour of incubation (AngI/ml/mg prot) (P < 0.03), and higher concentrations also inhibited renin. Reducing extracellular calcium with EGTA increased renin release (0.35 ± 0.08 µg AngI/ml/mg prot; P < 0.01), and blocked renin inhibition by CHA (0.28 ± 0.06 µg AngI/ml/mg prot; P < 0. 005 vs. CHA alone). The intracellular calcium chelator BAPTA-AM increased renin release by 55%, and blocked the inhibitory effect of CHA. Repeating these experiments in JG cells from A1R knockout mice using CHA or NECA demonstrated no effect on renin release. However, RT-PCR showed mRNA from TRPC isoforms 3 and 6 in isolated JG cells. Adding the TRPC blocker SKF-96365 reversed CHA-mediated inhibition of renin release. Thus A1R activation results in a calcium-dependent inhibition of renin release via TRPC-mediated calcium entry, but A2 receptors do not regulate renin release.

The influence of extracellular and intracellular calcium on the secretion of renin.

Changes in plasma, extracellular, and intracellular calcium can affect renin secretion from the renal juxtaglomerular (JG) cells. Elevated intracellular calcium directly inhibits renin release from JG cells by decreasing the dominant second messenger intracellular cyclic adenosine monophosphate (cAMP) via actions on calcium-inhibitable adenylyl cyclases and calcium-activated phosphodiesterases. Increased extracellular calcium also directly inhibits renin release by stimulating the calcium-sensing receptor (CaSR) on JG cells, resulting in parallel changes in the intracellular environment and decreasing intracellular cAMP. In vivo, acutely elevated plasma calcium inhibits plasma renin activity (PRA) via parathyroid hormone-mediated elevations in renal cortical interstitial calcium that stimulate the JG cell CaSR. However, chronically elevated plasma calcium or CaSR activation may actually stimulate PRA. This elevation in PRA may be a compensatory mechanism resulting from calcium-mediated polyuria. Thus, changing the extracellular calcium in vitro or in vivo results in inversely related acute changes in cAMP, and therefore renin release, but chronic changes in calcium may result in more complex interactions dependent upon the duration of changes and the integration of the body's response to these changes.

Anandamide inhibits transport-related oxygen consumption in the loop of Henle by activating CB1 receptors.

The energy required for active Na chloride reabsorption in the thick ascending limb (TAL) depends on oxygen consumption and oxidative phosphorylation (OXP). In other cells, Na transport is inhibited by the endogenous cannabinoid anandamide through the activation of the cannabinoid receptors (CB) type 1 and 2. However, it is unclear whether anandamide alters TAL transport and the mechanisms that could be involved. We hypothesized that anandamide inhibits TAL transport via activation of CB1 receptors and NO. For this, we measured oxygen consumption (Q(O(2))) in TAL suspensions to monitor the anandamide effects on transport and OXP. Anandamide reduced Q(O(2)) in a concentration-dependent manner. During Na-K-2Cl cotransport and Na/H exchange inhibition, anandamide did not inhibit TAL Q(O(2)). To test the role of the cannabinoid receptors, we used specific agonists and antagonists of CB1 and CB2 receptors. The CB1-selective agonist WIN55212-2 reduced Q(O(2)) in a concentration-dependent manner. Also, the CB1 receptor antagonist rimonabant blocked the effect of anandamide on Q(O(2)). In contrast, the CB2-selective agonist JHW-133 had no effect on Q(O(2)), while the CB2 receptor antagonist AM-630 failed to block the anandamide effects on Q(O(2)). To confirm these results, we measured CB1 and CB2 receptor expression and only CB1 expression was detected. Because CB1 receptors are strong nitric oxide synthase (NOS) stimulators and NO inhibits transport in TALs, we evaluated the role of NO. Anandamide stimulated NO production and the NOS inhibitor N(G)-nitro-L-arginine methyl ester blocked the anandamide effects on Q(O(2)). We conclude that anandamide inhibits TAL Na transport-related Q(O(2)) via activation of CB1 receptor and NOS.

Parathyroid hormone stimulates juxtaglomerular cell cAMP accumulation without stimulating renin release.

Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log(10) means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P < 0.001), it did not affect renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P < 0.001), again without effect on renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P < 0.01) and from 3.29 ± 0.18 to 3.63 ± 0.22 fmol/mg protein (P < 0.05), respectively, with no effect on renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P < 0.001) without affecting renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P < 0.01) and renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I·mg prot(-1)·h(-1) (P < 0.01). Thus PTH increases JG cell cAMP via non-calcium-sensitive adenylate cyclases without affecting renin release. These data suggest compartmentalization of cAMP signaling in JG cells.

Parathyroid hormone-related protein stimulates plasma renin activity via its anorexic effects on sodium chloride intake.

Parathyroid hormone-related protein (PTHrP) increases renin release from isolated perfused kidneys and may act as an autacoid regulator of renin secretion, but its effects on renin in vivo are unknown. In vivo, PTHrP causes hypercalcemia and anorexia, which may affect renin. We hypothesized that chronically elevated PTHrP would increase plasma renin activity (PRA) indirectly via its anorexic effects, reducing sodium chloride (NaCl) intake and causing NaCl restriction. We infused male Sprague-Dawley rats with the vehicle (control) or 125 µg PTHrP/day (PTHrP) via subcutaneous osmotic minipumps for 5 days. To replenish NaCl consumption, a third group of PTHrP-infused rats received 0.3% NaCl (PTHrP + NaCl) in their drinking water. PTHrP increased PRA from a median control value of 3.68 to 18.4 ng Ang I·ml(-1)·h(-1) (P < 0.05), whereas the median PTHrP + NaCl PRA value was normal (7.82 ng Ang I·ml(-1)·h(-1), P < 0.05 vs. PTHrP). Plasma Ca(2+) (median control: 10.2 mg/dl; PTHrP: 13.7 mg/dl; PTHrP + NaCl: 14.1 mg/dl; P < 0.05) and PTHrP (median control: 0.03 ng/ml; PTHrP: 0.12 ng/ml; PTHrP + NaCl: 0.15 ng/ml; P < 0.05) were elevated in PTHrP- and PTHrP + NaCl-treated rats. Body weights and caloric consumption were lower in PTHrP- and PTHrP + NaCl-treated rats. NaCl consumption was lower in PTHrP-treated rats (mean Na(+): 28.5 ± 4.1 mg/day; mean Cl(-): 47.8 mg/day) compared with controls (Na(+): 67.3 ± 2.7 mg/day; Cl(-): 112.8 ± 4.6 mg/day; P < 0.05). NaCl consumption was comparable with control in the PTHrP + NaCl group; 0.3% NaCl in the drinking water had no effect on PRA in normal rats. Thus, our data support the hypothesis that PTHrP increases PRA via its anorexic effects, reducing NaCl intake and causing NaCl restriction.

Hypercalcemia reduces plasma renin via parathyroid hormone, renal interstitial calcium, and the calcium-sensing receptor.

Acute hypercalcemia inhibits plasma renin activity (PRA). How this occurs is unknown. We hypothesized that acute hypercalcemia inhibits PRA via the calcium-sensing receptor because of parathyroid hormone-mediated increases in renal cortical interstitial calcium via TRPV5. To test our hypothesis, acute in vivo protocols were run in sodium-restricted, anesthetized rats. TRPV5 messenger RNA expression was measured with real-time quantitative RT-PCR. Acute hypercalcemia significantly decreased PRA by 37% from 32.0±3.3 to 20.3±2.6 ng of angiotensin I per milliliter per hour (P<0.001). Acute hypercalcemia also significantly increased renal cortical interstitial calcium by 38% (1.73±0.06 mmol/L) compared with control values (1.25±0.05 mmol/L; P<0.001). PRA did not decrease in hypercalcemia in the presence of a calcium-sensing receptor antagonist, Ronacaleret (22.8±4.3 versus 21.6±3.6 ng of angiotensin I per milliliter per hour). Increasing plasma calcium did not decrease PRA in parathyroidectomized rats (22.5±2.6 versus 22.0±3.0 ng of angiotensin I per milliliter per hour). Parathyroidectomized rats were unable to increase their renal cortical interstitial calcium in response to hypercalcemia (1.01±0.11 mmol/L). Acutely replacing plasma parathyroid hormone levels did not modify the hypercalcemic inhibition of PRA in parathyroid-intact rats (39.1±10.9 versus 16.3±3.2 ng of angiotensin I per milliliter per hour; P<0.05). Renal cortical TRPV5 messenger RNA expression decreased by 67% in parathyroidectomized (P<0.001) compared with intact rats. Our data suggest that acute hypercalcemia inhibits PRA via the calcium-sensing receptor because of parathyroid hormone-mediated increases in renal cortical interstitial calcium via TRPV5.

Acute activation of the calcium-sensing receptor inhibits plasma renin activity in vivo.

In vitro, the renin-secreting juxtaglomerular cells express the calcium-sensing receptor, and its activation with the calcimimetic cinacalcet inhibits renin release. To test whether the activation of calcium-sensing receptor similarly inhibits plasma renin activity (PRA) in vivo, we hypothesized that the calcium-sensing receptor is expressed in juxtaglomerular cells in vivo, and acutely administered cinacalcet would inhibit renin activity in anesthetized rats. Since cinacalcet inhibits parathyroid hormone, which may stimulate renin activity, we sought to determine whether cinacalcet inhibits renin activity by decreasing parathyroid hormone. Lastly, we hypothesized that chronically administered cinacalcet would inhibit basal and stimulated renin in conscious rats. Calcium-sensing receptors and renin were localized in the same juxtaglomerular cells using immunofluorescence in rat cortical slices fixed in vivo. Cinacalcet was administered acutely via intravenous bolus in anesthetized rats and chronically in conscious rats by oral gavage. Acute administration of cinacalcet decreased basal renin activity from 13.6 ± 2.4 to 6.1 ± 1.1 ng ANG I·ml(-1)·h(-1) (P < 0.001). Likewise, cinacalcet decreased furosemide-stimulated renin from 30.6 ± 2.3 to 21.3 ± 2.3 ng ANG I·ml(-1)·h(-1) (P < 0.001). In parathyroidectomized rats, cinacalcet decreased renin activity from 9.3 ± 1.3 to 5.2 ± 0.5 ng ANG I·ml(-1)·h(-1) (P < 0.05) similar to sham-operated controls (13.5 ± 2.2 to 6.6 ± 0.8 ng ANG I·ml(-1)·h(-1), P < 0.05). Chronic administration of cinacalcet over 7 days had no significant effect on PRA under basal or stimulated conditions. In conclusion, calcium-sensing receptors are expressed in juxtaglomerular cells in vivo, and acute activation of these receptors with cinacalcet inhibits PRA in anesthetized rats, independent of parathyroid hormone.