Abstract MP07: Long-term Suppression Of The Renin-angiotensin System Leads To Concentric Arteriolar Hypertrophy By Activation Of Renin Cells.

Hypertensive patients are frequently treated with inhibitors of the renin-angiotensin system (RASi). In renin knockout mice, cells programmed for the renin phenotype ( Renin null cells) stimulate the concentric hypertrophy of intrarenal arteries and arterioles. The Renin null cells invade the arteriolar walls and stimulate the concentric growth of smooth muscle cells (SMCs). EM exam showed disorganized glomerular arterioles, marked layering of SMCs and increased basement membrane, compared to a single organized SMC layer in WT mice. The hypertrophy leads to flow obstruction, ischemia, and renal failure.We hypothesize that Renin null cells or renin-expressing cells from animals treated with RASi possess a unique transcriptome that drives their own abnormal fate and the concentric accumulation of SMCs.To test this, we performed single-cell RNA-seq in WT and Renin null cells. We also tested genetically hypertensive mice and their normotensive controls treated with captopril for 6 months. Further, we examined renal biopsies from patients treated with RAS inhibitors for more than 5 years, age-matched controls without RAS inhibitors, and healthy control kidneys.The transcriptional profile of Renin null cells was markedly different from the profile of WT cells. Gene ontology indicated that Renin null cells possess a contractile rather than the endocrine phenotype of WT cells ( p <0.0001). The wall thickness of the afferent arterioles in both BPN/3 and BPH/2 mice treated with captopril was significantly increased when compared to the untreated controls (BPN/3: control; 5.54 ± 0.27 μm vs. captopril; 10.57 ± 0.61 μm, P <0.0001, BPH/2: control; 5.46 ± 0.26 μm vs. captopril; 10.44 ± 0.43 μm, P <0.0001), with arterioles positive for renin. Patients with long-term use of RASi had significantly thicker arterioles compared to the other groups (control; 6.55 ± 0.73 μm, without RAS; 8.54 ± 1.71 μm, vs. long-term RAS; 12.46 ± 1.86 μm, P <0.001). The renin-positive area was also increased in the kidneys with long-term use of RASi (control; 307.3 ± 74.7 μm 2 , without RAS; 677.8 ± 313.4 μm 2 , vs. long-term RAS; 1347 ± 529.9 μm 2 , P =0.003).In conclusion, renin cells stimulated by inhibition of RAS have specific molecular programs that contribute to arterial disease.

Genetically increased angiotensin I-converting enzyme alters peripheral and renal vascular reactivity to angiotensin II and bradykinin in mice

Angiotensin I-converting enzyme (ACE) levels in humans are under strong genetic influence. Genetic variation in ACE has been linked to risk for and progression of cardiovascular and renal diseases. Causality has been documented in genetically modified mice, but the mechanisms underlying causality are not completely elucidated. To further document the vascular and renal consequences of a moderate genetic increase in ACE synthesis, we studied genetically modified mice carrying three copies of the ACE gene (three-copy mice) and littermate wild-type animals (two-copy mice). We investigated peripheral and renal vascular reactivity to angiotensin II and bradykinin in vivo by measuring blood pressure and renal blood flow after intravenous administration and also reactivity of isolated glomerular arterioles by following intracellular Ca2+ mobilization. Carrying three copies of the ACE gene potentiated the systemic and renal vascular responses to angiotensin II over the whole range of peptide concentration tested. Consistently, the response of isolated glomerular afferent arterioles to angiotensin II was enhanced in three-copy mice. In these mice, signaling pathways triggered by endothelial activation by bradykinin or carbachol in glomerular arterioles were also altered. Although the nitric oxide (NO) synthase (NOS)/NO pathway was not functional in arterioles of two-copy mice, in muscular efferent arterioles of three-copy mice NOS3 gene expression was induced and NO mediated the effect of bradykinin or carbachol. These data document new and unexpected vascular consequences of a genetic increase in ACE synthesis. Enhanced vasoconstrictor effect of angiotensin II may contribute to the risk for cardiovascular and renal diseases linked to genetically high ACE levels. NEW & NOTEWORTHY A moderate genetic increase in angiotensin I-converting enzyme (ACE) in mice similar to the effect of the ACE gene D allele in humans unexpectedly potentiates the systemic and renal vasoconstrictor responses to angiotensin II. It also alters the endothelial signaling pathways triggered by bradykinin or carbachol in glomerular efferent arterioles.

Vascular smooth muscle function of renal glomerular and interlobar arteries predicts renal damage in rats

Previously, it was shown that individuals with good baseline (a priori) endothelial function in isolated (in vitro) renal arteries developed less renal damage after ⅚ nephrectomy (5/6Nx; Gschwend S, Buikema H, Navis G, Henning RH, de Zeeuw D, van Dokkum RP. J Am Soc Nephrol 13: 2909–2915, 2002). In this study, we investigated whether preexisting glomerular vascular integrity predicts subsequent renal damage after 5/6Nx, using in vivo intravital microscopy and in vitro myogenic constriction of small renal arteries. Moreover, we aimed to elucidate the role of renal ANG II type 1 receptor (AT1R) expression in this model. Anesthetized rats underwent intravital microscopy to visualize constriction to ANG II of glomerular afferent and efferent arterioles, with continuous measurement of blood pressure, heart rate, and renal blood flow. Thereafter, 5/6Nx was performed, interlobar arteries were isolated from the extirpated kidney, and myogenic constriction was assessed in a perfused vessel setup. Blood pressure and proteinuria were assessed weekly for 12 wk, and focal glomerulosclerosis (FGS) was determined at the end of study. Relative expression AT1R in the kidney cortex obtained at 5/6Nx was determined by PCR. Infusion of ANG II induced significant constriction of both afferent and efferent glomerular arterioles, which strongly positively correlated with proteinuria and FGS at 12 wk after 5/6Nx. Furthermore, in vitro measured myogenic constriction of small renal arteries negatively correlated with proteinuria 12 wk after 5/6Nx. Moreover, in vivo vascular reactivity negatively correlated with in vitro reactivity. Additionally, relative expression of AT1R positively correlated with responses of glomerular arterioles and with markers of renal damage. Both in vivo afferent and efferent responses to ANG II and in vitro myogenic constriction of small renal arteries in the healthy rat predict the severity of renal damage induced by 5/6Nx. This vascular responsiveness is highly dependent on AT1R expression. Intraorgan vascular integrity may provide a useful tool to guide the prevention and treatment of renal end-organ damage.

Voltage-gated divalent currents in descending vasa recta pericytes

Multiple voltage-gated Ca2+ channel (CaV) subtypes have been reported to participate in control of the juxtamedullary glomerular arterioles of the kidney. Using the patch-clamp technique, we examined whole cell CaV currents of pericytes that contract descending vasa recta (DVR). The dihydropyridine CaV agonist FPL64176 (FPL) stimulated inward Ca2+ and Ba2+ currents that activated with threshold depolarizations to −40 mV and maximized between −20 and −10 mV. These currents were blocked by nifedipine (1 μM) and Ni2+ (100 and 1,000 μM), exhibited slow inactivation, and conducted Ba2+ > Ca2+ at a ratio of 2.3:1, consistent with “long-lasting” L-type CaV. In FPL, with 1 mM Ca2+ as charge carrier, Boltzmann fits yielded half-maximal activation potential ( V1/2) and slope factors of −57.9 mV and 11.0 for inactivation and −33.3 mV and 4.4 for activation. In the absence of FPL stimulation, higher concentrations of divalent charge carriers were needed to measure basal currents. In 10 mM Ba2+, pericyte CaV currents activated with threshold depolarizations to −30 mV, were blocked by nifedipine, exhibited voltage-dependent block by diltiazem (10 μM), and conducted Ba2+ > Ca2+ at a ratio of ∼2:1. In Ca2+, Boltzmann fits to the data yielded V1/2 and slope factors of −39.6 mV and 10.0 for inactivation and 2.8 mV and 7.7 for activation. In Ba2+, V1/2 and slope factors were −29.2 mV and 9.2 for inactivation and −5.6 mV and 6.1 for activation. Neither calciseptine (10 nM), mibefradil (1 μM), nor ω-agatoxin IVA (20 and 100 nM) blocked basal Ba2+ currents. Calciseptine (10 nM) and mibefradil (1 μM) also failed to reverse ANG II-induced DVR vasoconstriction, although raising mibefradil concentration to 10 μM was partially effective. We conclude that DVR pericytes predominantly express voltage-gated divalent currents that are carried by L-type channels.

Renoprotective effect of calcium channel blockers

The advancing chronic renal failure is at most the consequence of secondary haemodynamic and metabolic factors as intraglomerular hypertension and glomerular hypertrophy. Although tight blood pressure control is the major preventive mechanism for progressive renal failure, ACE inhibitors and angiotensin receptor blockers have some other renoprotective mechanisms beyond the blood pressure control. That is why these two groups of antihypertensive drugs traditionally have advantages in treating renal patients especially those with proteinuria over 400-1000 mg/day. Even if earlier experimental studies have shown renoprotective effect of calcium channel blockers, later clinical studies did not prove that calcium channel blockers have any advantages in renal protection over ACE inhibitors given as monotherapy or in combination with ACE inhibitors. It was explained by action of calcium channel blockers on afferent but not on efferent glomerular arterioles; a well known mechanism that leads to intraglomerular hypertension. New generations of dihydropiridine calcium channel blockers can dilate even efferent arterioles not causing unfavorable haemodynamic disturbances. This finding was confirmed in clinical studies which showed that renoprotection established by calcium channel blockers was not inferior to that of ACE inhibitors and that calcium channel blockers and ACE inhibitors have additive effect on renoprotection. Newer generation of dihydropiridine calcium channel blockers seem to offer more therapeutic possibilities in renoprotection by their dual action on afferent and efferent glomerular arterioles and, possibly by other effects beyond the blood pressure control.

Expression of NTPDase1 and NTPDase2 in murine kidney: relevance to regulation of P2 receptor signaling

The regulation of renal function by extracellular nucleotides encompasses alterations in glomerular hemodynamics, microvascular function, tubuloglomerular feedback, tubular transport, cell growth or apoptosis, and transport of water and solutes in the medullary collecting duct. Nearly all cells can release ATP or other nucleotides that are then rapidly hydrolyzed in the extracellular milieu. However, little information is available on the cellular expression of ectoenzymes that hydrolyze extracellular nucleotides within the kidney. Nucleoside triphosphate diphosphohydrolases (NTPDases) are plasma membrane-bound ectonucleotidases. NTPDase1 has identity with CD39, a B lymphocyte activation marker, and hydrolyzes extracellular ATP and ADP to AMP within the vasculature, whereas NTPDase2/CD39L(ike)1 preferentially converts ATP to ADP outside of blood vessels. Using immunohistochemical and in situ hybridization approaches, we localized the protein and mRNA of NTPDase1 and 2 in murine renal tissues. In the renal cortex, NTPDase1 is expressed by vascular smooth muscle cells and endothelium in interlobular arteries, afferent glomerular arterioles, and peritubular capillaries. In the inner medulla, NTPDase1 is expressed in ascending thin limbs of Henle's loop, ducts of Bellini, and in the pelvic wall. In contrast, NTPDase2 is expressed in Bowman's capsule, glomerular arterioles, adventitia of blood vessels, and pelvic wall. Thus the distribution patterns of NTPDases have parallels to the known distribution of P2 receptors within the kidney. NTPDases may modulate regulatory effects of ATP and degradation products within the vasculature and other sites and thereby potentially influence physiological as well as multiple pathological events in the kidney.