Jung Julie Kang

Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney

Diabetes mellitus is the most common and rapidly growing cause of end-stage renal disease in developed countries. A classic hallmark of early diabetes mellitus includes activation of the renin-angiotensin system (RAS), which may lead to hypertension and renal tissue injury, but the mechanism of RAS activation is elusive. Here we identified a paracrine signaling pathway in the kidney in which high levels of glucose directly triggered the release of the prohypertensive hormone renin. The signaling cascade involved the local accumulation of succinate and activation of the kidney-specific G protein-coupled metabolic receptor, GPR91, in the glomerular endothelium as observed in rat, mouse, and rabbit kidney sections. Elements of signal transduction included endothelial Ca2+, the production of NO and prostaglandin (PGE2), and their paracrine actions on adjacent renin-producing cells. This GPR91 signaling cascade may serve to modulate kidney function and help remove metabolic waste products through renal hyperfiltration, and it could also link metabolic diseases, such as diabetes, or metabolic syndrome with RAS overactivation, systemic hypertension, and organ injury.

The Collecting Duct Is the Major Source of Prorenin in Diabetes

In addition to the juxtaglomerular apparatus, renin is also synthesized in renal tubular epithelium, including the collecting duct (CD). Angiotensin (Ang) II differentially regulates the synthesis of juxtaglomerular (inhibition) and CD (stimulation) renin. Because diabetes mellitus, a disease with high intrarenal renin-Ang system and Ang II activity, is characterized by high prorenin levels, we hypothesized that the CD is the major source of prorenin in diabetes. Renin granular content was visualized using in vivo multiphoton microscopy of the kidney in diabetic Munich-Wistar rats. Diabetes caused a 3.5-fold increase in CD renin, in contrast to less pronounced juxtaglomerular changes. Ang II type 1 receptor blockade with Olmesartan reduced CD renin to control levels but significantly increased juxtaglomerular renin. Using a fluorogenic renin assay, the prorenin component of CD renin content was measured by assessing the difference in enzymatic activity of medullary homogenates before and after trypsin activation of prorenin. Trypsinization caused no change in control renin activity but a 5-fold increase in diabetes. Studies on a CD cell line (M1) showed a 22-fold increase in renin activity after trypsinization and a further 35-fold increase with Ang II treatment. Therefore, prorenin significantly contributes to baseline CD renin. Diabetes, possibly via Ang II, greatly stimulates CD prorenin and causes hyperplasia of renin-producing connecting segments. These novel findings suggest that, in a rat model of diabetes, prorenin content and release from the CD may be more important than the juxtaglomerular apparatus in contrast to the existing paradigm.

Activation of the Succinate Receptor GPR91 in Macula Densa Cells Causes Renin Release

Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) are salt sensors and generate paracrine signals that control renal blood flow, glomerular filtration, and release of the prohypertensive hormone renin. We hypothesized that the recently identified succinate receptor GPR91 is present in MD cells and regulates renin release. Using immunohistochemistry, we identified GPR91 in the apical plasma membrane of MD cells. Treatment of MD cells with succinate activated mitogen-activated protein kinases (MAPKs; p38 and extracellular signal-regulated kinases 1/2) and cyclooxygenase 2 (COX-2) and induced the synthesis and release of prostaglandin E(2), a potent vasodilator and classic paracrine mediator of renin release. Using microperfused JGA and real-time confocal fluorescence imaging of quinacrine-labeled renin granules, we detected significant renin release in response to tubular succinate (EC(50) 350 microM). Genetic deletion of GPR91 (GPR91(-/-) mice) or pharmacologic inhibition of MAPK or COX-2 blocked succinate-induced renin release. Streptozotocin-induced diabetes caused GPR91-dependent upregulation of renal cortical phospho-p38, extracellular signal-regulated kinases 1/2, COX-2, and renin content. Salt depletion for 1 wk increased plasma renin activity seven-fold in wild-type mice but only 3.4-fold in GPR91(-/-) mice. In summary, MD cells can sense alterations in local tissue metabolism via accumulation of tubular succinate and GPR91 signaling, which involves the activation of MAPKs, COX-2, and the release of prostaglandin E(2). This mechanism may be integral in the regulation of renin release and activation of the renin-angiotensin system in health and disease.

Connexin 40 and ATP-dependent intercellular calcium wave in renal glomerular endothelial cells

Endothelial intracellular calcium ([Ca(2+)](i)) plays an important role in the function of the juxtaglomerular vasculature. The present studies aimed to identify the existence and molecular elements of an endothelial calcium wave in cultured glomerular endothelial cells (GENC). GENCs on glass coverslips were loaded with Fluo-4/Fura red, and ratiometric [Ca(2+)](i) imaging was performed using fluorescence confocal microscopy. Mechanical stimulation of a single GENC caused a nine-fold increase in [Ca(2+)](i), which propagated from cell to cell throughout the monolayer (7.9 +/- 0.3 microm/s) in a regenerative manner (without decrement of amplitude, kinetics, and speed) over distances >400 microm. Inhibition of voltage-dependent calcium channels with nifedipine had no effect on the above parameters, but the removal of extracellular calcium reduced Delta[Ca(2+)](i) by 50%. Importantly, the gap junction uncoupler alpha-glycyrrhetinic acid or knockdown of connexin 40 (Cx40) by transfecting GENCs with Cx40 short interfering RNA (siRNA) almost completely eliminated Delta[Ca(2+)](i) and the calcium wave. Breakdown of extracellular ATP using a scavenger cocktail (apyrase and hexokinase) or nonselective inhibition of purinergic P2 receptors with suramin, had similar blocking effects. Scraping cells off along a line eliminated physical contact between cells but did not effect calcium wave propagation. Using an ATP biosensor technique, we detected a significant elevation in extracellular ATP (Delta = 76 +/- 2 microM) during calcium wave propagation, which was abolished by Cx40 siRNA treatment (Delta = 6 +/- 1 microM). These studies suggest that connexin 40 hemichannels and extracellular ATP are key molecular elements of the glomerular endothelial calcium wave, which may serve important juxtaglomerular functions.

Activation of the renal renin–angiotensin system in diabetes—new concepts

The incidence of diabetes mellitus, obesity and the metabolic syndrome is rapidly rising to epidemic levels worldwide. Hyperglycemia, the metabolic hallmark of the pathology, is a significant causative factor for the complications of diabetes mellitus which result in significant morbidity and mortality for millions. Hyperglycemia is clearly associated with microvascular complications in many organs including the kidney, and diabetic nephropathy is the leading cause of end-stage renal disease (ESRD) in developed countries [1,2]. In addition, diabetes may lead to other vascular complications, including systemic hypertension [1,2]. Recent advances on the cellular and kidney-specific effects of hyperglycemia place activation of the local, intrarenal renin–angiotensin system (RAS) as a strong candidate for the core abnormality that leads to renal tissue injury [1–3]. The nature of RAS activation in diabetes is, however, controversial [3]. It has been difficult to isolate the acute and direct actions of hyperglycemia per se from the many other systemic factors and intra-renal, macula densa-mediated feedback mechanisms that can indirectly activate the intrarenal RAS. Therefore, the primary cause and exact mechanism of RAS activation in early diabetes have been unknown. The prevailing paradigm, the ‘tubular hypothesis of glomerular filtration’ [4], argues that the two hallmarks of early changes, glomerular hyperfiltration and renin activation, originate from the primary effects of glucose on proximal tubule salt reabsorption that secondarily activate macula densa-mediated feedback mechanisms. However, the development of diabetes-induced glomerular hyperfiltration was intact, or even augmented, in mice that lacked tubuloglomerular feedback (TGF), demonstrating that the TGF mechanism could not be the major cause of the development of hyperfiltration [5,6].

Imaging Renin Content and Release in the Living Kidney

Renin release is the first, and at least initially, the rate-limiting step in the activation of the renin-angiotensin system, which helps to maintain body salt and water balance. Recent advances in our understanding of pathophysiology have generated a renewed interest in the multiple roles of renin and prorenin as a hormone, enzyme, and signaling molecule. The assays available to measure renin content, release and tissue activity are complex, indirect and work with significant internal errors. We developed an imaging approach to directly visualize renin content and study the dynamics of both the release and tissue activity of renin. Our experimental model uses multiphoton fluorescence microscopy, which is ideal for deep optical sectioning of the living renal tissue. Here we review the application of this renin imaging approach to the dissected, in vitro microperfused glomerulus as well as in the intact kidney in vivo.

From In Vitro to In Vivo: Imaging from the Single Cell to the Whole Organism

This unit addresses the applications of fluorescence microscopy and quantitative imaging to study multiple physiological variables of living tissue. Protocols are presented for fluorescence‐based investigations ranging from in vitro cell and tissue approaches to in vivo imaging of intact organs. These include the measurement of cytosolic parameters both in vitro and in vivo (such as calcium, pH, and nitric oxide), dynamic cellular processes (renin granule exocytosis), FRET‐based real‐time assays of enzymatic activity (renin), physiological processes (vascular contraction, membrane depolarization), and whole organ functional parameters (blood flow, glomerular filtration). Multi‐photon microscopy is ideal for minimally invasive and undisruptive deep optical sectioning of the living tissue, which translates into ultra‐sensitive real‐time measurement of these parameters with high spatial and temporal resolution. With the combination of cell and tissue cultures, microperfusion techniques, and whole organ or animal models, fluorescence imaging provides unmatched versatility for biological and medical studies of the living organism. Curr. Protocol. Cytom. 44:12.12.1‐12.12.26.