Glomerular Blood Flow Research Articles

Glomerular pressure and tubular oxygen supply: a critical dual target for renal protection.

The primary treatment goal of chronic kidney disease (CKD) is preserving renal function and preventing its progression to end-stage renal disease. Glomerular hypertension and tubular hypoxia are critical risk factors in CKD progression. However, the renal hemodynamics make it difficult to avoid both factors due to the existence of peritubular capillaries that supply oxygen to the renal tubules downstream from the glomerulus through the efferent arteriole. In the treatment strategies for balancing glomerular pressure and tubular oxygen supply, afferent and efferent arterioles of the glomerulus determine glomerular filtration rate and blood flow to the peritubular capillaries. Therefore, sodium-glucose cotransporter 2 inhibitors and angiotensin receptor-neprilysin inhibitors as well as classical renin-angiotensin system inhibitors, which can change the diameter of afferent and/or efferent arterioles, are promising options for balancing this dual target and achieving renal protection. This review focuses on the clinical importance of glomerular pressure and tubular oxygen supply and proposes an effective treatment modality for this dual target.

Novel in vivo optogenetic tools reveal autonomous pacemaker control of renal hemodynamics by macula densa cells

Macula densa (MD) cells, the tubular component of the juxtaglomerular apparatus (JGA) are generally considered as sensor cells detecting tubular salt (NaCl) concentration and based on this information provide feedback control of glomerular hemodynamics (tubuloglomerular feedback, TGF). However, the neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the paradigm-shifting hypothesis that rather than being simply the sensory arm of TGF, MD cells function as the nephron’s autonomous pacemaker and the primary driver and oscillator of glomerular hemodynamics. Single cell MD Ca2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-GT mice that selectively express the ratiometric Ca2+ reporter GCaMP6f/tdTomato in MD cells. Optogenetic MD stimulation was induced in vivo by blue light (470nm) exposure of MD-Ai27 mouse kidneys that express the depolarizing channelrhodopsin ChR2 specifically in MD cells. Conversely, MD inhibition was induced by yellow light (560nm) exposure of MD-Ai39 mouse kidneys that selectively express the hyperpolarizing halorhodopsin NpHR in MD cells. Cell membrane expression of ChR2 (in MD-Ai27 mice) and NpHR (in MD-Ai39 mice) only in the MD among all renal cell types was confirmed using immunohistochemistry. Pacemaker-like regular Ca2+ oscillations (4-fold elevations in baseline Ca2+ and average frequency of 0.03 Hz) and their cell-to-cell propagation within the MD plaque via long cell processes were observed in MD-GT mice. These Ca2+ oscillations were MD cell autonomous, as they were preserved in freshly dissected single MD cells and in the in vitro dissected and microperfused JGA, and were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high MD expression of genes related to pacemaker function, cell membrane depolarization, voltage-dependent ion channels, Ca2+ signaling and synaptic transmission that was validated by immunohistochemistry. Blue light exposure of single glomeruli/JGAs using ROI scan in MD-Ai27 mouse kidneys that previously underwent superficial corticotomy (open loop nephrons) acutely and reversibly increased capillary and afferent (AA)/efferent arteriole (EA) blood flow by approx. 50% in the light-exposed glomerulus but not in adjacent glomeruli. In contrast, yellow light exposure of single glomeruli/JGAs in MD-Ai39 mouse kidneys acutely and reversibly decreased blood flow but increased glomerular capillary diameter (suggesting preferential EA vs AA vasoconstriction) in the exposed but not in adjacent single nephrons. In addition, yellow light stimulation of single glomeruli in MD-Ai39 mouse kidneys augmented the spontaneous glomerular blood flow oscillations in these open-loop nephrons (frequency of 0.03 Hz), but not in adjacent control nephrons. Altogether, these studies identified MD cells as the nephron’s central pacemaker and oscillator of single nephron blood flow, and emphasized the importance of MD cell membrane potential and Ca2+-dependent vasodilator release as a major driver of nephron function. DK064234 DK123564 DK135290. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modeling the interaction between tubuloglomerular feedback and myogenic mechanisms in the control of glomerular mechanics.

Introduction: Mechanical stresses and strains exerted on the glomerular cells have emerged as potentially influential factors in the progression of glomerular disease. Renal autoregulation, the feedback process by which the afferent arteriole changes in diameter in response to changes in blood pressure, is assumed to control glomerular mechanical stresses exerted on the glomerular capillaries. However, it is unclear how the two major mechanisms of renal autoregulation, the afferent arteriole myogenic mechanism and tubuloglomerular feedback (TGF), each contribute to the maintenance of glomerular mechanical homeostasis. Methods: In this study, we made a mathematical model of renal autoregulation and combined this model with an anatomically accurate model of glomerular blood flow and filtration, developed previously by us. We parameterized the renal autoregulation model based on data from previous literature, and we found evidence for an increased myogenic mechanism sensitivity when TGF is operant, as has been reported previously. We examined the mechanical effects of each autoregulatory mechanism (the myogenic, TGF and modified myogenic) by simulating blood flow through the glomerular capillary network with and without each mechanism operant. Results: Our model results indicate that the myogenic mechanism plays a central role in maintaining glomerular mechanical homeostasis, by providing the most protection to the glomerular capillaries. However, at higher perfusion pressures, the modulation of the myogenic mechanism sensitivity by TGF is crucial for the maintenance of glomerular mechanical homeostasis. Overall, a loss of renal autoregulation increases mechanical strain by up to twofold in the capillaries branching off the afferent arteriole. This further corroborates our previous simulation studies, that have identified glomerular capillaries nearest to the afferent arteriole as the most prone to mechanical injury in cases of disturbed glomerular hemodynamics. Discussion: Renal autoregulation is a complex process by which multiple feedback mechanisms interact to control blood flow and filtration in the glomerulus. Importantly, our study indicates that another function of renal autoregulation is control of the mechanical stresses on the glomerular cells, which indicates that loss or inhibition of renal autoregulation may have a mechanical effect that may contribute to glomerular injury in diseases such as hypertension or diabetes. This study highlights the utility of mathematical models in integrating data from previous experimental studies, estimating variables that are difficult to measure experimentally (i.e. mechanical stresses in microvascular networks) and testing hypotheses that are historically difficult or impossible to measure.

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes.

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Using 2-Photon Microscopy to Quantify the Effects of Chronic Unilateral Ureteral Obstruction on Glomerular Processes

Applying novel microscopy methods to suitable animal disease models to explore the dynamic physiology of the kidney remains a challenge. Rats with surface glomeruli provide a unique opportunity to investigate physiological and pathophysiological processes using intravital 2-photon microscopy. Quantification of glomerular capillary blood flow and vasoconstriction and dilatation in response to drugs, permeability, and inflammation are just some of the processes that can be studied. In addition, transgenic rats, i.e., podocytes labeled with fluorescent dyes and other molecular biomarker approaches, provide increased resolution to directly monitor and quantify protein-protein interactions and the effects of specific molecular alterations. In mice, which lack surface glomeruli after four weeks of age, unilateral ureteral obstruction (UUO) for several weeks has been used to induce surface glomeruli. As this induction model does not allow for baseline studies, we quantified the effects of UUO on glomerular processes in the UUO model in Munich Wistar Frömter (MWF) rats, which have surface glomeruli under physiologic conditions. The UUO model for five weeks or more induced significant alterations to gross renal morphology, the peritubular and glomerular microvasculature, as well as the structure and function of tubular epithelia. Glomerular and peritubular red blood cell (RBC) flow decreased significantly (p < 0.01), probably due to the significant increase in the adherence of white blood cells (WBCs) within glomerular and peritubular capillaries. The glomerular sieving coefficient of albumin increased from 0.015 ± 0.002 in untreated MWFs to 0.045 ± 0.05 in 5-week-old UUO MWF rats. Twelve weeks of UUO resulted in further increases in surface glomerular density and glomerular sieving coefficient (GSC) for albumin. Fluorescent albumin filtered across the glomeruli was not reabsorbed by the proximal tubules. These data suggest that using UUO to induce surface glomeruli limits the ability to study and interpret normal glomerular processes and disease alterations.

Renal microvascular endothelial cell responses in sepsis-induced acute kidney injury.

Microvascular endothelial cells in the kidney have been a neglected cell type in sepsis-induced acute kidney injury (sepsis-AKI) research; yet, they offer tremendous potential as pharmacological targets. As endothelial cells in distinct cortical microvascular segments are highly heterogeneous, this Review focuses on endothelial cells in their anatomical niche. In animal models of sepsis-AKI, reduced glomerular blood flow has been attributed to inhibition of endothelial nitric oxide synthase activation in arterioles and glomeruli, whereas decreased cortex peritubular capillary perfusion is associated with epithelial redox stress. Elevated systemic levels of vascular endothelial growth factor, reduced levels of circulating sphingosine 1-phosphate and loss of components of the glycocalyx from glomerular endothelial cells lead to increased microvascular permeability. Although coagulation disbalance occurs in all microvascular segments, the molecules involved differ between segments. Induction of the expression of adhesion molecules and leukocyte recruitment also occurs in a heterogeneous manner. Evidence of similar endothelial cell responses has been found in kidney and blood samples from patients with sepsis. Comprehensive studies are needed to investigate the relationships between segment-specific changes in the microvasculature and kidney function loss in sepsis-AKI. The application of omics technologies to kidney tissues from animals and patients will be key in identifying these relationships and in developing novel therapeutics for sepsis.

Laminar flow substantially affects the morphology and functional phenotype of glomerular endothelial cells.

Shear stress induced by laminar blood flow has a profound effect on the morphology and functional phenotype of macrovascular endothelial cells. The influence of laminar flow on the glomerular microvascular endothelium, however, remains largely elusive. The glomerular endothelium, including its glycocalyx, is a crucial part of the glomerular filtration barrier, which is involved in blood filtration. We therefore investigated the influence of laminar flow-induced shear stress on the glomerular endothelium. Conditionally immortalized mouse glomerular endothelial cells were cultured for 7 days under a laminar flow of 5 dyn/cm2 to mimic the glomerular blood flow. The cells were subsequently analysed for changes in morphology, expression of shear stress-responsive genes, nitric oxide production, glycocalyx composition, expression of anti-oxidant genes and the inflammatory response. Culture under laminar flow resulted in cytoskeletal rearrangement and cell alignment compared to static conditions. Moreover, production of nitric oxide was increased and the expression of the main functional component of the glycocalyx, Heparan Sulfate, was enhanced in response to shear stress. Furthermore, glomerular endothelial cells demonstrated a quiescent phenotype under flow, characterized by a decreased expression of the pro-inflammatory gene ICAM-1 and increased expression of the anti-oxidant enzymes HO-1 and NQO1. Upon exposure to the inflammatory stimulus TNFα, however, glomerular endothelial cells cultured under laminar flow showed an enhanced inflammatory response. In conclusion, laminar flow extensively affects the morphology and functional phenotype of glomerular endothelial cells in culture. Furthermore, glomerular endothelial cells respond differently to shear stress compared to macrovascular endothelium. To improve the translation of future in vitro studies with glomerular endothelial cells to the in vivo situation, it appears therefore crucial to culture glomerular endothelial cells under physiological flow conditions.

Abstract 12882: Renal Hemodynamics in Chronic Heart Failure With Preserved and Reduced Ejection Fraction

Introduction: Chronic heart failure (CHF) and impaired renal function are two co-existing medical conditions and known to be associated with adverse outcome. The cardiorenal interaction has not yet been analyzed thoroughly. The aim of this study was to assess renal and intraglomerular hemodynamics by constant infusion input clearance technique in subjects with CHF compared to healthy controls. Methods: This was a cross-sectional observational study including 85 subjects. The group of subjects with CHF consisted of 27 individuals with HFpEF and 27 individuals with HFrEF, who were compared to 31 controls. All subjects underwent renal clearance examination to determine measured -not estimated- glomerular filtration rate (GFR), renal blood and plasma flow (RBF, RPF) and to calculate renal hemodynamic parameters such as filtration fraction (FF), renal vascular resistance (RVR), intraglomerular pressure (P glom ) and resistances of the afferent (R A ) and efferent arterioles (R E ). Results: GFR was lower in subjects with CHF (88.6±13.1ml/min/1.73m 2 ) compared to controls (108.6±17. ml/min/1.73m 2 ) after adjustment for age and BP (p adj =0.037). There were no significant differences regarding RPF, RBF, FF, RVR, P glom , R A as well as R E after adjustment for age and BP. Similarly, there were no significant differences regarding renal hemodynamic parameters between HFpEF and HFrEF subjects. Bivariate correlation analysis in the group of subjects with CHF revealed an inverse association between NT-proBNP and RPF (R=-0.421, p=0.002), RBF (R=-0.414, p=0.002) and a positive association with FF (R=0.324, p=0.019), RVR (R=0.346, p=0.012) and R E (R=0.318, p=0.022). Conclusions: The findings of this study indicate that in CHF renal function is slightly reduced even though renal perfusion is preserved. With progressive severity of CHF as indicated by increasing NT-proBNP, renal vascular resistance in particular at the postglomerular side increases. Our data are in accordance with neuroendocrine activation in CHF since vasoconstriction at the postglomerular site points towards angiotensin II as mediator. The association between NT-proBNP and renal hemodynamics documents a close cardiorenal interaction in CHF.

Purinoceptors, renal microvascular function and hypertension.

Proper renal blood flow (RBF) and glomerular filtration rate (GFR) are critical for maintaining normal blood pressure, kidney function and water and electrolyte homeostasis. The renal microvasculature expresses a multitude of receptors mediating vasodilation and vasoconstriction, which can influence glomerular blood flow and capillary pressure. Despite this, RBF and GFR remain quite stable when arterial pressure fluctuates because of the autoregulatory mechanism. ATP and adenosine participate in autoregulatory control of RBF and GFR via activation of two different purinoceptor families (P1 and P2). Purinoceptors are widely expressed in renal microvasculature and tubules. Emerging data show altered purinoceptor signaling in hypertension-associated kidney injury, diabetic nephropathy, sepsis, ischemia-reperfusion induced acute kidney injury and polycystic kidney disease. In this brief review, we highlight recent studies and new insights on purinoceptors regulating renal microvascular function and renal hemodynamics. We also address the mechanisms underlying renal microvascular injury and impaired renal autoregulation, focusing on purinoceptor signaling and hypertension-induced renal microvascular dysfunction. Interested readers are directed to several excellent and comprehensive reviews that recently covered the topics of renal autoregulation, and nucleotides in kidney function under physiological and pathophysiological conditions (Inscho 2009, Navar et al. 2008, Carlstrom et al. 2015, Vallon et al. 2020).

P0146MESENCHYMAL STEM CELLS AND THEIR EXTRACELLULAR VESICLES PROGENY DECREASE RENAL INJURY IN EXPERIMENTAL SWINE RENAL ARTERY STENOSIS THROUGH DIFFERENT MECHANISMS

Abstract Background and Aims Novel therapies are needed to address the increasing prevalence of chronic kidney disease. Mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles (EVs) augment tissue repair. EVs have practical benefits, but their efficacy relative to MSCs is incompletely understood. We tested the hypothesis that EVs are as effective as MSCs in protecting the stenotic kidney but target different injury pathways. Method Pigs were studied after 16 weeks of renal injury achieved by diet-induced metabolic syndrome (Mets) and renal artery stenosis (RAS). Pigs were untreated or treated four weeks earlier with a single intrarenal delivery of autologous adipose tissue-derived MSCs (1 × 107) or EVs (1 × 1010). Lean pigs and sham RAS served as controls (n=6 each). Stenotic kidney function was studied in-vivo using CT. Histopathology and expression of necroptosis markers (RIPK-1 and RIPK-3), inflammatory and growth factors (angiopoeitin-1 and VEGF) was studied ex-vivo. Results Stenotic-kidney glomerular filtration rate and blood flow in Mets+RAS were both lower than Mets, increased in Mets+RAS+MSCs, and further improved in Mets+RAS+EV. Both MSCs and EVs improved renal function, hypoxia, and decreased renal fibrosis and apoptosis. MSC were slightly more effective in preserving microvascular density (0.02-0.2mm in diameter), and prominently attenuated renal inflammation. However, EVs more significantly upregulated growth-factor expression and decreased necropotosis. Conclusion: Adipose tissue-derived MSCs and their EVs both improve stenotic kidney function and decrease tissue injury in Mets+RAS. MSCs more effectively preserve the microcirculation, while EVs bestow better preservation of renal cellular integrity. These findings encourage further exploration of this novel approach to attenuate renal injury.

Quantifying Mechanical Stress in the Remaining Glomeruli in 5/6‐Nephrectomy

The purpose of the present study was to estimate the increases in mechanical stress in remnant rat glomeruli after 5/6‐nephrectomy. In cases of reduction of renal mass there is increased glomerular blood flow and pressure in the remnant glomeruli. These altered hemodynamic conditions are believed to result in increased shear stress on and strain of the glomerular capillary walls, however the actual magnitudes of these stresses have not been estimated. These magnitudes are of importance given the inflammatory reaction of endothelial cells and podocytes to excess shear stress and strain, respectively. In response to increased shear stress, vascular endothelial cells increase production of TGF‐β1, a pro‐fibrotic factor implicated in numerous kidney diseases. Sustained increased fluid flow shear stress also raises endothelial cell production of ICAM‐1 which contributes to the initiation of the inflammatory process. TGF‐β and cyclic stretch cause podocyte detachment from the glomerular capillary and hypertrophy of the remaining podocytes.We developed an anatomically accurate mathematical model of the rat glomerulus which calculates blood flow and filtration on each segment of a rat glomerular capillary network. The model takes into account conservation of plasma protein mass and hematocrit distribution, allowing for simulation of the non‐Newtonian aspects of blood flow in calculating shear stresses on the vessel walls. Hemodynamic parameters were taken from glomerular micropuncture studies of 5/6‐nephrectomized animals to simulate these disease states (Kasiske, Bertram L., et al. Circulation Research 62.2 (1988): 367–374). Our 5/6‐nephrectomy results were validated by comparison to a study which utilized intravital imaging to estimate glomerular capillary wall shear stress in vivo (Ferrell, Nicholas, et al. Amer. J. of Physiology‐Renal 308.6 (2015): F588–F593).The model demonstrates that most of the filtration occurs near the afferent arteriole. Hoop stress, an index of strain, is highest in the large diameter vessels branching off the afferent arteriole, whereas shear stress is highest in the vessels closer to the efferent arteriole, primarily due to a concentration of hematocrit in those vessels from fluid volume loss via filtration. Despite an increase in capillary diameter via a hypertrophic response in the 5/6‐nephrectomy model, the shear stress was significantly elevated. Hoop stress was elevated due to the increased pressure in 5/6‐nephrectomy.Through the of use of an anatomically‐accurate mathematical model of the rat glomerulus we calculated significant increases of hoop and shear stresses in 5/6‐nephrectomy. These results indicate that increased mechanical stress may play a significant role in the cause and/or progression of remnant glomerular injury in cases of reduced renal mass.Support or Funding InformationThis work was supported by fellowship grant NIH F31 DK121445 and the NIGMS IDeA Program (CoBRE P30GM103337). Owen Richfield is a graduate student in the Tulane University Bioinnovation PhD program supported by grants NSF IGERT DGE‐1144646 and NIH T32 EB027632.

Injured Podocytes Are Sensitized to Angiotensin II-Induced Calcium Signaling.

Inhibition of angiotensin II (AngII) signaling, a therapeutic mainstay of glomerular kidney diseases, is thought to act primarily through regulating glomerular blood flow and reducing filtration pressure. Although extravascular actions of AngII have been suggested, a direct effect of AngII on podocytes has not been demonstrated in vivo. To study the effects of AngII on podocyte calcium levels in vivo, we used intravital microscopy of the kidney in mice expressing the calcium indicator protein GCaMP3. In healthy animals, podocytes displayed limited responsiveness to AngII stimulation. In contrast, in animals subjected to either adriamycin-induced acute chemical injury or genetic deletion of the podocin-encoding gene Nphs2, the consequent podocyte damage and proteinuria rendered the cells responsive to AngII and resulted in AngII-induced calcium transients in significantly more podocytes. The angiotensin type 1 receptor blocker losartan could fully inhibit this response. Also, responsiveness to AngII was at least partly mediated through the transient receptor potential channel 6, which has been implicated in podocyte calcium handling. Interestingly, loss of a single Nphs2 allele also increased podocytes' responsiveness to AngII signaling. This direct effect of AngII on injured podocytes results in increased calcium transients, which can further aggravate the underlying kidney disease. Our discovery that podocytes become sensitized to AngII-induced calcium signaling upon injury might explain results from large, randomized, controlled trials in which improved renal outcomes occur only in the subgroup of patients with proteinuria, indicating podocyte damage. Our findings also emphasize the need to treat every patient with a glomerular disease with either an angiotensin-converting enzyme inhibitor or an angiotensin type 1 receptor blocker.

Integration of purinergic and angiotensin II receptor function in renal vascular responses and renal injury in angiotensin II-dependent hypertension.

Glomerular arteriolar vasoconstriction and tubulointerstitial injury are observed before glomerular damage occurs in models of hypertension. High interstitial ATP concentrations, caused by the increase in arterial pressure, alter renal mechanisms involved in the long-term control of blood pressure, autoregulation of glomerular filtration rate and blood flow, tubuloglomerular feedback (TGF) responses, and sodium excretion. Elevated ATP concentrations and augmented expression of P2X receptors have been demonstrated under a genetic background or induction of hypertension with vasoconstrictor peptides. In addition to the alterations of the microcirculation in the hypertensive kidney, the vascular actions of elevated intrarenal angiotensin II levels may be mitigated by the administration of broad purinergic P2 antagonists or specific P2Y12, P2X1, and P2X7 receptor antagonists. Furthermore, the prevention of tubulointerstitial infiltration with immunosuppressor compounds reduces the development of salt-sensitive hypertension, indicating that tubulointerstitial inflammation is essential for the development and maintenance of hypertension. Inflammatory cells also express abundant purinergic receptors, and their activation by ATP induces cytokine and growth factor release that in turn contributes to augment tubulointerstitial inflammation. Collectively, the evidence suggests a pathophysiological activation of purinergic P2 receptors in angiotensin-dependent hypertension. Coexistent increases in intrarenal angiotensin II and activates Ang II AT1 receptors, which interacts with over-activated purinergic receptors in a complex manner, suggesting convergence of their post-receptor signaling processes.

Glomerular Hypertrophy and Alterations in Renin-Angiotensin System Activation Are Associated with Diminished Systemic Blood Pressure in Aging Mice with Sickle Cell Anemia

Sickle cell nephropathy is a major and severe clinical complication of sickle cell anemia (SCA). The hypoxic microenvironment of the renal medulla leads to hyperosmolarity and acidosis, which favors deoxygenated hemoglobin S (HbS) polymerization and subsequent sickling of erythrocytes, promoting vaso-occlusive processes in the organ. Renal ischemia and glomerular blood flow can modulate angiotensin II (AngII) production, a vasoconstrictor hormone derivative of the Renin-Angiotensin System (RAS), which regulates blood pressure (BP), among other functions. Considering the elevated blood volume and cardiac output in individuals with SCA, an increase in BP might be expected; however SCA patients often present lower PB (Pikilidou et al., J Clin Hypertens., 2015), which modifies with aging. Reduced plasma AngII in male mice with SCA has been reported previously (Dos Santos et al., Life Sci., 2014), thus we aimed to investigate changes in PB and RAS, in association with aging and renal alterations in an animal model of SCA. Male chimeric mice without SCA (QCON) and male chimeric mice with SCA (QSCA) were generated by the irradiation of 8-week old C57BL6 mice followed by bone marrow cell transplantation from C57BL6 or transgenic Berkeley SCA mice, respectively. As circulating and tissue RAS are markedly affected by estrogens, we used only male mice for all protocols. Animals were subdivided into young adults (5 months of age) and adults (8 - 9 months of age). After confirming the phenotype of the transplanted SCA mice, BP was measured by a noninvasive method (NBP - AdInstruments) and animals were sacrificed at the age of 5/9 months for biological material collection. Plasma AngII and Angiotensin Convertor Enzyme (ACE) were quantified by ELISA. For histological analysis of the kidneys, organ specimens were fixed and embedded in historesin, sectioned and stained with hematoxylin and eosin. Renal AngI/II was quantitated by Western Blot from snap-frozen kidneys. The mean BPs of QSCA at 5 and 8 months old were significantly lower (87.9 ± 1.9 mmHg, n=12; and 94.31 ± 4.4 mmHg, n=2; respectively) than those of QCON of the same age (108.9 ± 5 mmHg, n=10; and 110.6 ± 2.2 mmHg, n=9; respectively), indicating that this model mimics the pathological changes in the blood flow that occur in SCA patients. Given the close relationship between AngII and PB regulation, we investigated whether the RAS is altered in QSCA mice at these ages. Both young adult and adult male QSCA mice presented lower plasma AngII concentrations (young adult, 24.18 ± 3 pg/ml, n= 11; adult, 16.44 ± 2 pg/ml, n=4), compared to QCON animals (young adult, 38.77 ±9, n= 10; adult, 35.52 ± 5.3 pg/ml, n=10, P<0.05, compared to QSCA); however no significant correlation between reduced BP and reduced plasma AngII was observed. AngII is produced by the cleavage of AngI by ACE; plasma ACE concentrations were also reduced in young adult QSCA mice (12.5 ± 1.8 pg/ml, n=11), compared to young adult QCON mice (21.9 ± 4.9 pg/ml, n=9). Moreover, reduced ACE concentrations in young adult/adult QSCA correlated with plasma levels of AngII (rs=0.785, P<0.001, n=15). Findings imply that AngII generation could be compromised in the organs of SCA mice; indeed, intra-renal AngI/II protein expression analysis showed that adult QSCA mice presented a significantly reduced expression of renal AngI/II (0.71 ± 0.2 AU, normalized to β-actin) compared to adult QCON mice (1.6 ± 0.2 AU), suggesting a low production of this hormone at advanced age in SCA. Glomerular hypertrophy increases in cardiovascular and renal disease models, including in hypertension and SCA. Quantitative morphometric analyses showed expansion of the glomerular-capillary tuff area of QSCA mice, which increased with age (young-adult: 372.2 ± 12.8 µm2; adult: 412.9 ±12.1 µm2, n=3), compared to young-adult and adult QCON mice (324.1 ± 10.8 µm2; 332.8 ± 12.4 µm2, n=3, p<0.001). As such, glomerular hypertrophy may reflect hyperfiltration and renal damage in the SCA mice, which apparently augments with age. Subsequent alterations in glomerular pressure may incur adaptive responses in the RAS, in turn diminishing ACE and AngII production, in association with alterations in systemic blood pressure. An understanding of the RAS in SCA may improve our understanding of renal damage and its systemic consequences in SCA and will be important for determining how ACE inhibitor and AngII receptor blockers drugs may be best utilized in these patients. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of the Effect of Aminophylline in Reducing the Incidence of Acute Kidney Injury After Cardiac Surgery.

ObjectivesAcute renal failure is a common complication of major cardiovascular surgeries (One-third of patients). Adenosine release as a vascular vasodilator increases after cardiac surgery, which reduces renal and glomerular blood flow and subsequently causes kidney ischemic damage. The present study aimed at evaluating the impact of aminophylline as an adenosine receptor antagonist on renal function after cardiac surgery hoping to find an appropriate method to reduce acute kidney injury.MethodsThe patients in the intervention group received 5 mg/kg aminophylline bolus after induction of anesthesia; then, 0.25 mg/kg/hr of the drug was administered intraoperatively and up to 48 hours after surgery in the ICU cardiac surgery. Similar volume of normal saline was injected to the patients of the second group. Serum BUN, Cr, and GFR were measured pre- and postoperatively and 3 days postsurgery. Patients’ 24- hour urine output and RIFLE were also calculated.ResultsThose patients who received medication were extubated earlier (P = 0.018) and received lower amount of inotropic drugs (P < 0001). According to the RIFLE criteria, most of the patients experienced no change or even improved GFR and Cr amounts compared to the control group (p < 0.05). GFR and Cr value of all the patients with Cleveland score, less and more than 6, showed a significant difference between the 2 groups (P = 0.001 and P = 0.01, respectively). According to the RIFLE criteria, most of the patients experienced no change or even improved GFR.ConclusionsAminophylline in cardiac surgery can reduce the frequency of acute kidney injury according to RIFLE criteria and could be used in the prevention of AKI as a safe and efficient modality in high-risk patients. Also, the use of this drug may reduce the need for inotropic medication at the time of surgery, intensive care unit stay length, and extubation time.

Abstract P090: Agonistic Autoantibodies to the Angiotensin II Type 1 Receptor Enhances ANG II Induced Renal Vascular Sensitivity and Reduces Renal Function During Pregnancy

Preeclamptic women produce agonistic autoantibodies to the Angiotensin II type 1 receptor (AT1-AA) and exhibit increased blood pressure (BP) and vascular sensitivity to angiotensin II (ANG II). Although, together AT1-AAs and ANGII increase the BP, renal artery resistant index, and vasoconstriction of renal afferent arterioles in pregnant rats; the renal hemodynamics in the presence of the AT1-AAs during pregnancy has not been examined. Thus the objective of this study was to examine the changes in the glomerular filtration rate (GFR) and renal blood flow (RBF) during pregnancy in the presence of AT1-AAs and/or ANGII. Methods: Pregnant Sprague Dawley rats were divided into 4 groups: Normal Pregnant (NP, n=6), Pregnant + ANG II (Preg + ANG II, n=6), Pregnant + AT1-AA (Preg + AT1-AA, n=8), and Pregnant + ANG II + AT1-AA (Preg + ANGII + AT1-AA, n= 6). On day 13 of pregnancy, rats were implanted with mini-pumps infusing ANG II (50 ng/kg/min) and/or AT1-AA (1:40 dilution). On day 19 of pregnancy, rats were subjected to terminal renal function surgeries using FITC labeled Inulin. During the surgery, the BP was recorded and a transonic flowmeter probe was placed on the left renal artery to measure RBF. Results: BP was elevated in all pregnant rats administered ANG II and/or the AT1-AA. Although GFR was reduced, it was not significant between Preg + ANG II and Preg + AT1-AA vs. NP rats (1.5 ±0.24, 1.60 ±0.17 vs. 1.90 ±0.16 ml/min). However, the GFR was further decreased in Preg + ANGII + AT1-AA rats (1.20 ±0.08). No difference was observed with the RBF between Preg + ANG II and Preg + AT1-AA vs NP rats (14.4 ±2.96, 14.4 ±1.48 vs. 15.4 ±1.75 ml/min). RBF was decreased in Preg + ANGII + AT1-AA vs NP rats (7.4 ±1.09 vs. 15.4 ±1.75 ml/min). No change in RVR between Preg + ANG II and Preg + AT1-AA vs. NP rats (9.7 ±2.69, 8.3 ±0.58 vs. 6.4 ±0.77). However, the RVR was drastically increased between Preg + ANGII + AT1-AA vs NP rats (18.4 ±2.91 vs. 6.4 ±0.77). Conclusion: Together ANG II and AT1-AA drastically decreases renal function by 37%, RBF by 50%, and caused a 3 fold increase in RVR vs NP rats. These data indicate the importance of AT1-AAs to drastically enhance ANG II induced renal vascular sensitivity and reduce renal function during preeclampsia. Research Supported by T32HL105324 and RO1HD067541

Shear stress is normalized in glomerular capillaries following ⅚ nephrectomy.

Loss of significant functional renal mass results in compensatory structural and hemodynamic adaptations in the nephron. While these changes have been characterized in several injury models, how they affect hemodynamic forces at the glomerular capillary wall has not been adequately characterized, despite their potential physiological significance. Therefore, we used intravital multiphoton microscopy to measure the velocity of red blood cells in individual glomerular capillaries of normal rats and rats subjected to ⅚ nephrectomy. Glomerular capillary blood flow rate and wall shear stress were then estimated using previously established experimental and mathematical models to account for changes in hematocrit and blood rheology in small vessels. We found little change in the hemodynamic parameters in glomerular capillaries immediately following injury. At 2 wk postnephrectomy, significant changes in individual capillary blood flow velocity and volume flow rate were present. Despite these changes, estimated capillary wall shear stress was unchanged. This was a result of an increase in capillary diameter and changes in capillary blood rheology in nephrectomized rats.

Functional assessment of transplanted kidneys with magnetic resonance imaging.

Kidney transplantation has emerged as the treatment of choice for many patients with end-stage renal disease, which is a significant cause of morbidity and mortality. Given the shortage of clinically available donor kidneys and the significant incidence of allograft dysfunction, a noninvasive and accurate assessment of the allograft renal function is critical for postoperative management. Prompt diagnosis of graft dysfunction facilitates clinical intervention of kidneys with salvageable function. New advances in magnetic resonance imaging (MRI) technology have enabled the calculation of various renal parameters that were previously not feasible to measure noninvasively. Diffusion-weighted imaging provides information on renal diffusion and perfusion simultaneously, with quantification by the apparent diffusion coefficient, the decrease of which reflects renal function impairment. Diffusion-tensor imaging accounts for the directionality of molecular motion and measures fractional anisotropy of the kidneys. Blood oxygen level-dependent MR evaluates intrarenal oxygen bioavailability, generating the parameter of R2* (reflecting the concentration of deoxyhemoglobin). A decrease in R2* could happen during acute rejection. MR nephro-urography/renography demonstrates structural data depicting urinary tract obstructions and functional data regarding the glomerular filtration and blood flow. MR angiography details the transplant vasculature and is particularly suitable for detecting vascular complications, with good correlation with digital subtraction angiography. Other functional MRI technologies, such as arterial spin labeling and MR spectroscopy, are showing additional promise. This review highlights MRI as a comprehensive modality to diagnose a variety of etiologies of graft dysfunction, including prerenal (e.g., renal vasculature), renal (intrinsic causes) and postrenal (e.g., obstruction of the collecting system) etiologies.

L-Arginine Supplementation in Type II Diabetic Rats Preserves Renal Function and Improves Insulin Sensitivity by Altering the Nitric Oxide Pathway

Rat studies demonstrated that type II diabetes mellitus (T2DM) decreases both the production and bioavailability of nitric oxide (NO). L-arginine (LA) provides the precursor for the production of NO. We hypothesized that LA dietary supplementation will preserve NO production via endothelial nitric oxide synthase (eNOS) causing renal microvascular vasodilation and increased glomerular blood flow and thus increasing glomerular filtration rate (GFR). This would impede the formation of reactive oxygen species which contributes to cell damage and death. LA supplementation preserved GFR in the treated diabetic rats compared to untreated diabetic rats. We provide evidence that this effect may be due to increased levels of eNOS and urinary cyclic guanosine monophosphate, which leads to renal microvascular vasodilation. Plasma nitrotyrosine was decreased in the LA treated rats; however, plasma nitrite levels remained unaffected as expected. Marked improvements in glucose tolerance were also observed in the LA treated diabetic rats. These results demonstrate that LA supplementation preserves NO activity and may delay the onset of insulin resistance and renal dysfunction during hyperglycemic stress. These results suggest the importance of the NO pathway in consequent renal dysfunction and in the development of insulin resistance in diabetic rats.

Abstract 473: Compound-21 Acts at Renal Proximal Tubule AT2 Receptors to Inhibit Na+ Reabsorption via Bradykinin, Nitric Oxide, and Cyclic GMP

Angiotensin (Ang) AT2 receptors (AT2Rs) are expressed in the kidney at vascular and tubular sites. Previous studies from our lab have shown that renal AT2R activation induces natriuresis and that Ang III is the endogenous agonist for this response. Compound 21 (C21) is a highly selective non-peptide AT2R agonist with approx. 25,000-fold selectivity for AT2Rs over AT1 receptors (AT1Rs). However, C21, particularly at high concentrations, has recently been shown to have AT2R-independent effects in the vasculature. We hypothesized that C21 induces natriuresis via AT2R translocation to the apical membrane and by activating a bradykinin (BK)-nitric oxide (NO)-cyclic GMP (cGMP) signaling pathway in the renal proximal tubule (RPT). In anesthetized, uninephrectomized, volume-expanded rats, intravenous (iv) C21 infusion (100, 200 and 300 ng/kg/min; each dose for 30 min) increased urinary Na+ excretion (UNaV) from 0.16±0.03 to 1.12±0.19, 1.51±0.08 and 2.03±0.21 μmol/min, respectively; all P &lt;0.001). This natriuretic response was not altered by 24 h subcutaneous infusion of AT1R antagonist candesartan (CAND; 0.01 mg/kg/min), but was abolished by renal interstitial (RI) infusion of specific AT2R antagonist PD-123319 (PD). Blood pressure was unaltered by C21 or PD, but was lower in CAND-infused rats (P &lt;0.0001). C21 infusion increased fractional excretion of Na+ (FENa) from 0.36±0.07 to 0.90±0.18% (P&lt;0.05) and lithium (FELi) from 30.2±3.3 to 57.2±5.2% (P &lt;0.01) without altering glomerular filtration rate or renal blood flow. Compared to control, C21 infusion increased RPT cell apical membrane AT2R protein (0.11±0.006 vs. 0.08±0.004 DU respectively) without changing total cell AT2R. C21-induced natriuresis was also accompanied by an increase in RI cGMP from 3.78±0.82 to 17.1±3.39 pmol/ml (P&lt;0.01). The C21-induced increases in both UNaV and RI cGMP were abolished by RI infusion of NO synthase inhibitor L-NAME or BK B2 receptor antagonist icatibant. The results demonstrate that C21 initiates AT2R translocation to the RPT cell apical membrane inducing natriuresis in a BK-NO-cGMP-dependent manner in the absence of concurrent AT1R blockade. C21 holds promise as a natriuretic/diuretic agent for the treatment of fluid retaining states and hypertension.