Abstract P006: Purinergic Receptor Activation Protects Glomerular Microvasculature From Increased Mechanical Stress In Angiotensin Ii-induced Hypertension: A Modeling Study

Angiotensin II (Ang II)-induced hypertension increases afferent and efferent arteriole resistances via the actions of Ang II on the AT1 receptor. In addition to the increased interstitial levels of Ang II, the increased arterial pressure increases interstitial ATP concentrations which act on the purinergic receptors P2X1 and P2X7, to constrict the AA, preventing increases in plasma flow and single nephron GFR (SNGFR). Blockade of the P2 receptors also mitigates the effects of Ang II, thus increasing blood flow and SNGFR, but the resulting increases in mechanical stresses (shear stress and circumferential hoop stress) on the glomerular microvasculature have not been quantified. A mathematical microvascular hemodynamic glomerular model was developed to simulate blood flow and plasma filtration at each of 320 capillary segments in an anatomically-accurate rat glomerular capillary network topology. Afferent and efferent arteriole resistances and network hydraulic conductivity were adjusted to match glomerular hemodynamic data for control, Ang II-induced hypertension and P2X1-blocked conditions (Franco, Martha, et al. Amer. J. Physiology-Renal 313.1 (2017): F9-F19). Ang II infusion increased both afferent and efferent resistances, reducing blood flow while slightly raising glomerular pressure. Blockade of the purinergic receptors reduced both afferent and efferent resistances, maintaining glomerular pressure at hypertensive levels but increasing blood flow significantly, increasing shear stress from 24.9 dynes/cm 2 in hypertensive conditions to 71.3 dynes/cm 2 after purinergic blockade. Because glomerular pressure was maintained, hoop stress barely changed from 69.5 kPa in hypertensive conditions to 70.9 kPa after purinergic blockade. Purinergic blockade also increased hydraulic conductivity and filtering surface area, increasing SNGFR. In hypertension, purinergic stimulation does not prevent the transmission of increased arterial pressure to the glomerular capillaries to reduce capillary hoop stress. However, activation of the purinergic system protects the glomerular microvasculature from increases in shear stress caused by a marked increase in blood flow that would occur in the absence of purinergic stimulation.

P0089KEAP1/NRF2 PATHWAY REGULATES GFR BY INCREASING THE GLOMERULAR EFFECTIVE AREA WITHOUT AFFECTING THE AFFERENT/EFFERENT ARTERIOLE RATIO

Background and Aims The Keap1/Nrf2 pathway regulates the expression of a series of cytoprotective, anti-inflammatory and antioxidant genes. The Nrf2 activator, bardoxolone methyl (BARD), has consistently increased estimated GFR (eGFR) in clinical studies in patients with chronic kidney disease. BARD demonstrated improvement of renal function assessed by inulin clearance, the clinical gold standard for measuring GFR, in diabetic kidney disease patients. These findings suggest the Keap1/Nrf2 system is deeply involved in the regulatory mechanisms of GFR. However, the precise mechanisms are not fully elucidated. We pharmacologically and genetically investigated the mechanisms of GFR regulation by Keap1/Nrf2 system using in vivo multiphoton microscope (MPM) imaging techniques. Method C57BL/6 (Cont), Nrf2 knockout (Nrf2-KO), and Nrf2-activated Keap1-knockdown mice (Keap1-KD) were used. The mice were treated the synthetic triterpenoid RTA dh404 (10 mg/kg/day by gavage) which is a Nrf2 activator for rodents, for a week. We successfully developed the technique to evaluate single-nephron GFR (SNGFR) using MPM (Circulation 2019). The glomerular hemodynamics, diameter of the afferent/efferent arterioles and glomerular permeability were also evaluated. The calcium influx into cells in response to ATP and angiotensin II stimulation and the effect on [Ca2+]i by RTAdh404 were evaluated using Fluo 4 and Fura red in cultured mesangial cells and podocytes. Production of reactive oxygen species and nitric oxide (NO) availability were assessed by fluorescent method using CellROX® Deep Red and diaminofluorescein-FM diacetate (DAF-FM DA) upon the exposure to these stimuli. Results SNGFR in Keap1-KD mice was significantly higher than in the control (9.13±0.55 vs 4.40±0.39 nl/min, Figure 1). RTA dh404 increased SNGFR in the control but not in the Nrf2-KO mice (6.00±0.40 vs 4.66±0.35 nl/min, Figure 1). There was no significant change in the ratio of the glomerular afferent/efferent arteriole diameter in all groups. RTA dh404 treatment increased glomerular volume but did not affect the glomerular permeability of albumin and 40kd-dextran. RTA dh404-treatment inhibited calcium influx into cultured podocytes and mesangial cells induced by angiotensin II or ATP, thereby affecting contractile responses. Oxidative stress and NO-bioavailablity were also ameliorated with RTA dh404. Conclusion The Keap1/Nrf2 pathway plays a pivotal role in controlling GFR and presumably underlies the effect of BARD on GFR in patients.

Proinsulin C-peptide reduces diabetes-induced glomerular hyperfiltration via efferent arteriole dilation and inhibition of tubular sodium reabsorption

C-peptide reduces diabetes-induced glomerular hyperfiltration in diabetic patients and experimental animal models. However, the mechanisms mediating the beneficial effect of C-peptide remain unclear. We investigated whether altered renal afferent-efferent arteriole tonus or alterations in tubular Na+ transport (TNa) in response to C-peptide administration mediate the reduction of diabetes-induced glomerular hyperfiltration. Glomerular filtration rate, filtration fraction, total and cortical renal blood flow, total kidney O2 consumption (Qo2), TNa, fractional Na+ and Li+ excretions, and tubular free-flow and stop-flow pressures were measured in anesthetized adult male normoglycemic and streptozotocin-diabetic Sprague-Dawley rats. The specific effect of C-peptide on transport-dependent Qo2 was investigated in vitro in freshly isolated proximal tubular cells. C-peptide reduced glomerular filtration rate (−24%), stop-flow pressure (−8%), and filtration fraction (−17%) exclusively in diabetic rats without altering renal blood flow. Diabetic rats had higher baseline TNa (+40%), which was reduced by C-peptide. Similarly, C-peptide increased fractional Na+ (+80%) and Li+ (+47%) excretions only in the diabetic rats. None of these parameters was affected by vehicle treatments in either group. Baseline Qo2 was 37% higher in proximal tubular cells from diabetic rats than controls and was normalized by C-peptide. C-peptide had no effect on ouabain-pretreated diabetic cells from diabetic rats. C-peptide reduced diabetes-induced hyperfiltration via a net dilation of the efferent arteriole and inhibition of tubular Na+ reabsorption, both potent regulators of the glomerular net filtration pressure. These findings provide new mechanistic insight into the beneficial effects of C-peptide on diabetic kidney function.

Determinants of renal microvascular response to ACh: afferent and efferent arteriolar actions of EDHF

The renal microvascular actions of ACh were investigated using the in vitro perfused hydronephrotic rat kidney. ACh reversed ANG II-induced vasoconstriction in the afferent and efferent arteriole by 106 ± 2 and 75 ± 5%, respectively. Inhibition of nitric oxide synthase [NOS; 100 μmol/l N G-nitro-l-arginine methyl ester (l-NAME)] and cyclooxygenase (COX; 10 μmol/l ibuprofen) prevented the sustained response of the afferent arteriole but did not reduce the magnitude of the initial dilation (97 ± 7%). However, NOS/COX inhibition abolished the response of the efferent arteriole. The underlying mechanisms mediating this endothelium-derived hyperpolarizing factor (EDHF)-like response were characterized using K channel blockers. Ba (100 μmol/l), tetraethylammonium (1 mmol/l), and ouabain (3 mmol/l) had no effect, arguing against a role of an inward rectifier K channel, large-conductance Ca-activated K channel, or Na,K-ATPase. Charybdotoxin (10 nmol/l) and apamin (1.0 μmol/l) attenuated the response when administered alone (63 ± 7% and 37 ± 5%, respectively) and abolished the response when coadministered (0.1 ± 1.0%). These findings indicate that, as in other vascular beds, the renal EDHF-like response to ACh involves K channels that are sensitive to a combination of apamin and charybdotoxin. Our finding that EDHF modulates preglomerular, but not postglomerular, tone is consistent with the evolving concept that vasomotor mechanisms in cortical efferent arterioles do not involve voltage-gated Ca entry.