The role of TRPC6 calcium channels and P2 purinergic receptors in podocyte mechanical and metabolic sensing

Podocyte calcium (Ca2+) signaling plays important roles in the (patho)physiology of the glomerular filtration barrier. Overactivation of podocyte transient receptor potential canonical (TRPC) channels including TRPC6 and purinergic signaling via P2 receptors that are known mechanosensors can increase podocyte intracellular Ca2+ levels ([Ca2+]i) and cause cell injury, proteinuria and glomerular disease including in diabetes. However, important mechanistic details of the trigger and activation of these pathways in vivo in the intact glomerular environment are lacking. Here we show direct visual evidence that podocytes can sense mechanical overload (increased glomerular capillary pressure) and metabolic alterations (increased plasma glucose) via TRPC6 and purinergic receptors including P2Y2. Multiphoton microscopy of podocyte [Ca2+]i was performed in vivo using wild-type and TRPC6 or P2Y2 knockout (KO) mice expressing the calcium reporter GCaMP3/5 only in podocytes and in vitro using freshly dissected microperfused glomeruli. Single-nephron intra-glomerular capillary pressure elevations induced by obstructing the efferent arteriole lumen with laser-induced microthrombus in vivo and by a micropipette in vitro triggered >2-fold increases in podocyte [Ca2+]i. These responses were blocked in TRPC6 and P2Y2 KO mice. Acute elevations of plasma glucose caused >4-fold increases in podocyte [Ca2+]i that were abolished by pharmacological inhibition of TRPC6 or P2 receptors using SAR7334 or suramin treatment, respectively. This study established the role of Ca2+ signaling via TRPC6 channels and P2 receptors in mechanical and metabolic sensing of podocytes in vivo, which are promising therapeutic targets in conditions with high intra-glomerular capillary pressure and plasma glucose, such as diabetic and hypertensive nephropathy.

Impaired renal hemodynamics and glomerular hyperfiltration contribute to hypertension-induced renal injury

Recently, we reported a mutation in γ-adducin (ADD3) was associated with an impaired myogenic response of the afferent arteriole and hypertension-induced chronic kidney disease (CKD) in fawn hooded hypertensive (FHH) rats. However, the mechanisms by which altered renal blood flow (RBF) autoregulation promotes hypertension-induced renal injury remain to be determined. The present study compared the time course of changes in renal hemodynamics and the progression of CKD during the development of DOCA-salt hypertension in FHH 1BN congenic rats [wild-type (WT)] with an intact myogenic response versus FHH 1BN Add3KO ( Add3KO) rats, which have impaired myogenic response. RBF was well autoregulated in WT rats but not in Add3KO rats. Glomerular capillary pressure rose by 6 versus 14 mmHg in WT versus Add3KO rats when blood pressure increased from 100 to 150 mmHg. After 1 wk of hypertension, glomerular filtration rate increased by 38% and glomerular nephrin expression decreased by 20% in Add3KO rats. Neither were altered in WT rats. Proteinuria doubled in WT rats versus a sixfold increase in Add3KO rats. The degree of renal injury was greater in Add3KO than WT rats after 3 wk of hypertension. RBF, glomerular filtration rate, and glomerular capillary pressure were lower by 20%, 28%, and 19% in Add3KO rats than in WT rats, which was associated with glomerular matrix expansion and loss of capillary filtration area. The results indicated that impaired RBF autoregulation and eutrophic remodeling of preglomerular arterioles increase the transmission of pressure to glomeruli, which induces podocyte loss and accelerates the progression of CKD in hypertensive Add3KO rats.

Abstract MP46: Knockout Of Add3 Impairs Autoregulation Of Renal Blood Flow And Promotes Hypertension-induced Renal Injury By Increasing Glomerular Capillary Pressure And Podocyte Loss

Recently, we reported that a mutation in γ-Adducin (ADD3) alters the actin cytoskeleton and is associated with an impaired myogenic response of the afferent arteriole and enhanced hypertension-induced renal disease. However, it remains to be determined whether the loss of ADD3 function promotes renal injury by increasing glomerular capillary pressure (Pgc) and podocyte loss or other mechanisms. The present study compared the time course of changes in renal hemodynamics and the progression of renal injury during the development of DOCA-salt hypertension in FHH 1 BN rats (WT) with an intact myogenic response vs. FHH 1 BN Add3KO rats (Add3KO) in which the myogenic response is impaired. When transmural pressure rose from 40 to 100 mmHg, the inner diameter of the preglomerular artery constricted by 19% (47.7 ± 4.3 to 38.4 ± 3.4 μm, n = 5) in WT, but it dilated by 28% (53.0 ± 2.2 to 67.9 ± 4.3 μm, n = 7) in Add3KO. Pgc was similar (50.1 ± 0.4 vs. 51.2 ± 0.8 mmHg, n = 6) at 100 mmHg, but rose by 6 and 14 mmHg in WT vs. Add3KO when perfusion pressure rose to 150 mmHg. Mean arterial pressure increased similarly and reached 177.7 ± 3.5 vs. 182.6 ± 2.3 mmHg (n = 9) after 3 weeks of DOCA-salt hypertension in WT vs. Add3KO. After 1 week of DOCA-salt hypertension, glomerular filtration rate (GFR) increased by 38% (1.2 ± 0.1 to 1.6 ± 0.1 ml/min/kidney, n = 6) and glomerular nephrin expression decreased by 20% (165.4 ± 4.5 to 131.614 ± 5.2 RFU, n = 7) in Add3KO. Both were unaltered in WT. Proteinuria increased 2 folds in WT (56.9 ± 4.7 to 168.6 ± 26.7 mg/day, n = 12) in the first week of hypertension vs. a 6-fold increase in Add3KO (64.6 ± 4.1 to 446.0 ± 41.9 mg/day, n = 9). After 3 weeks of hypertension, the degree of glomerulosclerosis (3.4 ± 0.1 vs. 2.4 ± 0.1 glomerular injury score, n = 9~12), protein cast formation (9.0% ± 0.8% vs. 4.8% ± 0.4% of area, n = 6), epithelial-mesenchymal transition, interstitial fibrosis (17.9% ± 0.8% vs. 9.5% ± 0.3% of area, n = 6), and inflammation was significantly greater in Add3KO vs. WT. GFR and Pgc were 28% and 19% lower in Add3KO than WT. These results indicate that the impaired myogenic response increases the transmission of pressure to the glomerulus to induce the loss of podocytes, which accelerates the progression of renal injury during the development of hypertension in Add3KO.

Abstract 104: Dynamic Autoregulation of Glomerular Capillary Pressure

It is generally accepted that renal blood flow (RBF) autoregulation is mediated by myogenic and tubuloglomerular feedback responses acting on the pre-glomerular resistance. If this is so, then autoregulation of RBF and glomerular capillary pressure (PGC) should change in the same direction throughout an autoregulatory step response. We computed autoregulatory step responses from time series recordings of arterial blood pressure (BP) and RBF (Transonics) blood flow or tubular stop-flow pressure (micropuncture), which is a surrogate for PGC in Wistar-Froemter rats fed for one week on low or high salt diets (n=6-10 ). Autoregulatory step responses were generated from time series by an algorithm that treats BP as a leading indicator of RBF or PGC and uses the projection theorem to solve for the impulse response which is integrated to obtain the step response. Step responses shown in the figure represent the uncompensated changes in RBF and PGC (mean + SEM) following a 1 mmHg BP step. The data clearly reveal that the time courses of RBF and PGC differ such that changes in RBF cannot predict changes in PGC. This implies that the renal hemodynamic response to a blood pressure disturbance is not confined to the pre-glomerular resistance. Furthermore, the participation of post-glomerular resistance in the autoregulatory response is sensitive to dietary salt such that PGC is more sensitive to BP on low salt diet.

Regulation of vasomotor tone in the afferent and efferent arterioles

Pre-glomerular vessels are regulated by membrane potential alterations affecting the activity of L-type voltage-activated Ca2+ channels; whereas voltage-independent mechanisms regulate the efferent arteriole, notably influenced by angiotensin II and therefore by angiotensin converting enzyme inhibitors, and by non-steroidal anti-inflammatory drugs.These properties underlie the physiologic control of glomerular capillary pressure, for example, by prostaglandin E2, during conditions of reduced renal perfusion and the stabilization of glomerular capillary pressure when the kidney is exposed to pressure fluctuations. A wealth of hormones affects the tone of renal vessels. The chapter focuses on basic regulatory and signalling mechanisms, emphasizing the unique aspects of the renal vasculature and the underlying features that facilitate the independent regulation of pre-glomerular and post-glomerular tone.

Abstract 063: Impaired Myogenic Response of the Af-art Contributes to the Increased Susceptibility to Hypertension and Diabetic Induced Renal Disease in Milan Normotensive Rats

Previous studies have indicated that Milan normotensive (MNS) rats are more susceptible to the development of hypertension and diabetic induced renal injury than Milan hypertensive (MHS) rats, but the genes and pathways involved are unknown. MNS also develop proteinuria and chronic kidney disease (CKD) as they age, whereas hypertensive MHS do not. We compared the myogenic response of isolated perfused Af-Art and autoregulation of RBF and glomerular capillary pressure in 6-9 week old MNS and MHS rats. The diameter of Af-Art of MNS rats increased from 14.0 ± 0.5 to 14.2 ± 0.6 μm (n=6) when elevation in perfusion pressure from 60 to 120 mmHg. In contrast, the diameter of the Af-Art decreased significantly from 14.3 ± 0.5 to 11.5 ± 0.6 μm (n=6) in MHS rats. In vivo, RBF increased by 26% when RPP was increased from 100 to 140 mmHg in MNS rats but it remained unchanged in MHS rats. Glomerular capillary pressure rose by 11 mmHg in MNS following the elevation in RPP from 100 to 140 mm Hg but not in MHS rats. Protein excretion increased from 8.9 ± 0.7 to 158.2 ± 23.1 mg/day in MNS rats as the increased in age from 3 to 9 months of age but it did not increase in MHS rats. In com-parison to other strains susceptible and resistant to CKD, we noticed that both MNS and Fawn Hooded hypertensive (FHH) rats that do not autoregulate RBF also share the same sequence variant in the Adducin 3 gene. We performed a genetic complementation study to test whether this mutation might be responsible for the impaired myogenic response in MNS. The diameter of the Af-Art isolated from an F1 cross of MNS &FHH rats increased from 17.2 ± 0.9 to 18.5 ± 0.9 μM (n=5) in response to increase in perfusion pressure and RBF was not efficiently autoregulated in these animals. These data indicate a mutation in Adducin 3 which impairs myogenic response of the Af-Art and increased transmission of pressure to the glomerular capillaries may contribute to the development of CKD in MNS rats similar to what is seen in FHH rats.

Predominant postglomerular vascular resistance response to reflex renal sympathetic nerve activation during ANG II clamp in rabbits

We have shown previously that a moderate reflex increase in renal sympathetic nerve activity (RSNA) elevated glomerular capillary pressure, whereas a more severe increase in RSNA decreased glomerular capillary pressure. This suggested that the nerves innervating the glomerular afferent and efferent arterioles could be selectively activated, allowing differential control of glomerular capillary pressure. A caveat to this conclusion was that intrarenal actions of neurally stimulated ANG II might have contributed to the increase in postglomerular resistance. This has now been investigated. Anesthetized rabbits were prepared for renal micropuncture and RSNA recording. One group (ANG II clamp) received an infusion of an angiotensin-converting enzyme inhibitor (enalaprilat, 2 mg/kg bolus plus 2 mg·kg−1·h−1) plus ANG II (∼20 ng·kg−1·min−1), the other vehicle. Measurements were made before (room air) and during 14% O2. Renal blood flow decreased less during ANG II clamp compared with vehicle [9 ± 1% vs. 20 ± 4%, interaction term (PGT) < 0.05], despite a similar increase in RSNA in response to 14% O2 in the two groups. Arterial pressure and glomerular filtration rate were unaffected by 14% O2 in both groups. Glomerular capillary pressure increased from 33 ± 1 to 37 ± 1 mmHg during ANG II clamp and from 33 ± 2 to 35 ± 1 mmHg in the vehicle group before and during 14% O2, respectively (PGT < 0.05). During ANG II clamp, postglomerular vascular resistance was still increased in response to RSNA during 14% O2, demonstrating that the action of the renal nerves on the postglomerular vasculature was independent of the renin-angiotensin system. This further supports our hypothesis that increases in RSNA can selectively control pre- and postglomerular vascular resistance and therefore glomerular ultrafiltration.