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.

Endothelial prostaglandin D2 opposes angiotensin II contractions in mouse isolated perfused intracerebral microarterioles

Hypothesis: A lack of contraction of cerebral microarterioles to Ang II (“resilience”) depends on cyclooxygenase (COX) and lipocalin type prostaglandin D sythase L-PGDS producing PGD2 that activates prostaglandin D type 1 receptors (DP1Rs) and nitric oxide synthase (NOS). Materials & Methods: Contractions were assessed in isolated, perfused vessels and NO by fluorescence microscopy. Results: The mRNAs of penetrating intraparenchymal cerebral microarterioles versus renal afferent arterioles were >3000-fold greater for L-PGDS and DP1R and 5-fold for NOS III and COX 2. Larger cerebral arteries contracted with Ang II. However, cerebral microarterioles were entirely unresponsive but contracted with endothelin 1 and perfusion pressure. Ang II contractions were evoked in cerebral microarterioles from COX1 –/– mice or after blockade of COX2, L-PGDS or NOS and in deendothelialized vessels but effects of deendothelialization were lost during COX blockade. NO generation with Ang II depended on COX and also was increased by DP1R activation. Conclusion: The resilience of cerebral arterioles to Ang II contractions is specific for intraparenchymal microarterioles and depends on endothelial COX1 and two products that are metabolized by L-PGDS to generate PGD2 that signals via DP1Rs and NO.

Abstract P004: Endothelial Function In Rat Juxtamedullary Afferent Arterioles After Renal Ischemia-reperfusion Injury

Renal ischemia-reperfusion (IR) induced kidney injury exhibits reduced renal blood flow (RBF) and glomerular filtration rate (GFR). Both RBF and GFR are regulated by renal microvascular function. Our previous study showed that IR caused impaired afferent arteriole autoregulatory capability via excess ROS accumulation and inflammation in renal microvessels. Other mechanisms contributing to renal microvascular dysfunction in IR have not been fully defined. In this study, we conducted acetylcholine (ACh) concentration-response experiments (n=5-7/each group) to assess endothelial function using the in vitro blood-perfused juxtamedullary nephron preparation. IR was induced by 60 minutes of bilateral renal artery occlusion followed by 24 hours or 7 days of reperfusion. Renal microvessels (RMV) were collected at either 24 hours or 7 days post-surgery for mRNA analysis. Systolic blood pressure was normal in IR rats vs. sham ranging from 126-136 mmHg (>0.05) over 7 days. IR markedly increased plasma creatinine concentration (Cr) to 3.9±0.2 mg/dL at 24 hours post-IR ( P <0.05 vs. 0.9±0.1 mg/dL in sham). Plasma Cr remained elevated in IR rats by day 7 of reperfusion (1.5±0.2 vs. 1.0±0.0 mg/dL in sham, P <0.05). Baseline afferent arteriole diameter was smaller in IR rats 24 hours post-IR and averaged 11.1±0.7 μm (P<0.05 vs. 14.2±0.7 μm in sham). Superfusion of ACh (10 -8 to 10 -4 M) caused concentration-dependent vasodilation in sham rats, increasing diameter to 23.5±2.6 μm (169±14% of the control, P <0.05) at 10 -4 M ACh. The ACh-induced vasodilation was not altered by IR insult and was indistinguishable from the sham responses. IR diameter reached a maximum of 19.7±0.9 μm (179±8% of the control) at 10 -5 M ACh. The ACh responses were also similar between sham and IR rats on day 7 post-IR. mRNA expression of endothelial markers, iCAM and vCAM, were not statistically different in RMV collected at 24 hours post-surgery between two groups, but was markedly elevated in IR RMV on day 7 post-IR (4-fold in iCAM and 2-fold in vCAM, P<0.05 vs. sham). In conclusion, upregulation of iCAM and vCAM mRNA expression in RMV 7 days post-IR suggests renal microvascular inflammation, but rat juxtamedullary afferent arterioles still maintain normal endothelium-dependent reactivity to ACh.

Purinergic P2X1 receptor, purinergic P2X7 receptor, and angiotensin II type 1 receptor interactions in the regulation of renal afferent arterioles in angiotensin II-dependent hypertension

In ANG II-dependent hypertension, ANG II activates ANG II type 1 receptors (AT1Rs), elevating blood pressure and increasing renal afferent arteriolar resistance (AAR). The increased arterial pressure augments interstitial ATP concentrations activating purinergic P2X receptors (P2XRs) also increasing AAR. Interestingly, P2X1R and P2X7R inhibition reduces AAR to the normal range, raising the conundrum regarding the apparent disappearance of AT1R influence. To evaluate the interactions between P2XRs and AT1Rs in mediating the increased AAR elicited by chronic ANG II infusions, experiments using the isolated blood perfused juxtamedullary nephron preparation allowed visualization of afferent arteriolar diameters (AAD). Normotensive and ANG II-infused hypertensive rats showed AAD responses to increases in renal perfusion pressure from 100 to 140 mmHg by decreasing AAD by 26 ± 10% and 19 ± 4%. Superfusion with the inhibitor P2X1Ri (NF4490; 1 μM) increased AAD. In normotensive kidneys, superfusion with ANG II (1 nM) decreased AAD by 16 ± 4% and decreased further by 19 ± 5% with an increase in renal perfusion pressure. Treatment with P2X1Ri increased AAD by 30 ± 6% to values higher than those at 100 mmHg plus ANG II. In hypertensive kidneys, the inhibitor AT1Ri (SML1394; 1 μM) increased AAD by 10 ± 7%. In contrast, treatment with P2X1Ri increased AAD by 21 ± 14%; combination with P2X1Ri plus P2X7Ri (A438079; 1 μM) increased AAD further by 25 ± 8%. The results indicate that P2X1R, P2X7R, and AT1R actions converge at receptor or postreceptor signaling pathways, but P2XR exerts a dominant influence abrogating the actions of AT1Rs on AAR in ANG II-dependent hypertension.

Differential effects of inotropes and inodilators on renal function in acute cardiac care

Pathological interplay between the heart and kidneys is widely encountered in heart failure (HF) and is linked to worse prognosis and quality of life. Inotropes, along with diuretics and vasodilators, are a core medical response to HF but decompensated patients who need inotropic support often present with an acute worsening of renal function. The impact of inotropes on renal function is thus potentially an important influence on the choice of therapy. There is currently relatively little objective data available to guide the selection of inotrope therapy but recent direct observations on the effects of levosimendan and milrinone on glomerular filtration favour levosimendan. Other lines of evidence indicate that in acute decompensated HF levosimendan has an immediate renoprotective effect by increasing renal blood flow through preferential vasodilation of the renal afferent arterioles and increases in glomerular filtration rate: potential for renal medullary ischaemia is avoided by an offsetting increase in renal oxygen delivery. These indications of a putative reno-protective action of levosimendan support the view that this calcium-sensitizing inodilator may be preferable to dobutamine or other adrenergic inotropes in some settings by virtue of its renal effects. Additional large studies will be required, however, to clarify the renal effects of levosimendan in this and other relevant clinical situations, such as cardiac surgery.

Influence of connexin45 on renal autoregulation

Renal autoregulation is mediated by the myogenic response and tubuloglomerular feedback (TGF) working in concert to maintain renal blood flow and glomerular filtration rate despite fluctuations in renal perfusion pressure. Intercellular communication through gap junctions may play a role in renal autoregulation. We examine if one of the building blocks in gap junctions, connexin45 (Cx45), which is expressed in vascular smooth muscle cells, has an influence on renal autoregulatory efficiency. The isolated perfused juxtamedullary nephron preparation was used to measure afferent arteriolar diameter changes in response to acute changes in renal perfusion pressure. In segmental arteries, pressure myography was used to study diameter changes in response to pressure changes. Wire myography was used to study vasoconstrictor and vasodilator responses. A mathematical model of the vascular wall was applied to interpret experimental data. We found a significant reduction in the afferent arteriolar constriction in response to acute pressure increases in Cx45 knockout (KO) mice compared with wild-type (WT) mice. Abolition of TGF caused a parallel upward shift in the autoregulation curve of WT animals but had no effect in KO animals, which is compatible with TGF providing a basal tonic contribution in afferent arterioles whereas Cx45 KO animals were functionally papillectomized. Analysis showed a shift toward lower stress sensitivity in afferent arterioles from Cx45 KO animals, indicating that the absence of Cx45 may also affect myogenic properties. Finally, loss of Cx45 in vascular smooth muscle cells appeared to associate with a change in both structure and passive properties of the vascular wall.

Rho kinase inhibitors reduce voltage-dependent Ca2+ channel signaling in aortic and renal microvascular smooth muscle cells

Voltage-dependent L-type Ca2+ channels (L-VDCCs) and the RhoA/Rho kinase pathway are two predominant intracellular signaling pathways that regulate renal microvascular reactivity. Traditionally, these two pathways have been thought to act independently; however, recent evidence suggests that these pathways could be convergent. We hypothesized that Rho kinase inhibitors can influence L-VDCC signaling. The effects of Rho kinase inhibitors Y-27632 or RKI-1447 on KCl-induced depolarization or the L-VDCC agonist Bay K8644 were assessed in afferent arterioles using an in vitro blood-perfused rat juxtamedullary nephron preparation. Superfusion of KCl (30–90 mM) led to concentration-dependent vasoconstriction of afferent arterioles. Administration of Y-27632 (1, 5, and 10 µM) or RKI-1447 (0.1, 1, and 10 µM) significantly increased the starting diameter by 16–65%. KCl-induced vasoconstriction was markedly attenuated with 5 and 10 µM Y-27632 and with 10 µM RKI-1447 ( P < 0.05 vs. KCl alone). Y-27632 (5 µM) also significantly attenuated Bay K8644-induced vasoconstriction ( P < 0.05). Changes in intracellular Ca2+ concentration ([Ca2+]i) were estimated by fura-2 fluorescence during KCl-induced depolarization in cultured A7r5 cells and in freshly isolated preglomerular microvascular smooth muscle cells. Administration of 90 mM KCl significantly increased fura-2 fluorescence in both cell types. KCl-mediated elevation of [Ca2+]i in A7r5 cells was suppressed by 1–10 µM Y-27632 ( P < 0.05), but 10 µM Y-27632 was required to suppress Ca2+ responses in preglomerular microvascular smooth muscle cells. RKI-1447, however, significantly attenuated KCl-mediated elevation of [Ca2+]i. Y-27632 markedly inhibited Bay K8644-induced elevation of [Ca2+]i in both cell types. The results of the present study indicate that the Rho kinase inhibitors Y-27632 and RKI-1447 can partially inhibit L-VDCC function and participate in L-VDCC signaling.

The Key Role of Epithelial to Mesenchymal Transition (EMT) in Hypertensive Kidney Disease

Accumulating evidence indicates that epithelial-to-mesenchymal transition (EMT), originally described as a key process for organ development and metastasis budding in cancer, plays a key role in the development of renal fibrosis in several diseases, including hypertensive nephroangiosclerosis. We herein reviewed the concept of EMT and its role in renal diseases, with particular focus on hypertensive kidney disease, the second leading cause of end-stage renal disease after diabetes mellitus. After discussing the pathophysiology of hypertensive nephropathy, the ‘classic’ view of hypertensive nephrosclerosis entailing hyalinization, and sclerosis of interlobular and afferent arterioles, we examined the changes occurring in the glomerulus and tubulo-interstitium and the studies that investigated the role of EMT and its molecular mechanisms in hypertensive kidney disease. Finally, we examined the reasons why some studies failed to provide solid evidence for renal EMT in hypertension.