Renoprotective effects of GLP-1 receptor agonists and SGLT-2 inhibitors-is hemodynamics the key point?

Two novel treatments for diabetic kidney disease have emerged after decades with little progression. Both agents were developed for improved glycemic control in patients with type-2 diabetes. However, large clinical trials showed renoprotective effects beyond their ability to lower plasma glucose levels, body weight, and blood pressure. How this renal protection occurs is unknown. We will discuss their physiological effects, with special focus on the renal effects. We discuss how these drugs affect the function of the diabetic and nondiabetic kidneys to elucidate mechanisms by which the renoprotection could arise. Diabetic kidney disease affects the glomerular capillaries, which are usually protected by the renal autoregulatory mechanisms, the myogenic response, and the tubuloglomerular feedback mechanism. Animal models with reduced renal autoregulatory capacity develop chronic kidney disease. Despite different cellular targets, both drugs are suspected to affect renal hemodynamics through changes in the renal autoregulatory mechanisms. The glucagon-like peptide-1 receptor agonists (GLP-1RAs) exert a direct vasodilatory effect on the afferent arteriole (AA) positioned just before the glomerulus. Paradoxically, this effect is expected to increase glomerular capillary pressure, causing glomerular injury. In contrast, the sodium-glucose transporter-2 inhibitors (SGLT2i) are believed to activate the tubuloglomerular feedback mechanism to elicit vasoconstriction of the afferent arteriole. Because of their opposing effects on the renal afferent arterioles, it appears unlikely that their renoprotective effects can be explained by common effects of renal hemodynamics, but both drugs appear to add protection to the kidney beyond what can be obtained with classical treatment targeted at lowering blood glucose levels and blood pressure.

The nephron-arterial network and its interactions.

Tubuloglomerular feedback and the myogenic mechanism form an ensemble in renal afferent arterioles that regulate single-nephron blood flow and glomerular filtration. Each mechanism generates a self-sustained oscillation, the mechanisms interact, and the oscillations synchronize. The synchronization generates a bimodal electrical signal in the arteriolar wall that propagates retrograde to a vascular node, where it meets similar electrical signals from other nephrons. Each signal carries information about the time-dependent behavior of the regulatory ensemble. The converging signals support synchronization of the nephrons participating in the information exchange, and the synchronization can lead to formation of nephron clusters. We review the experimental evidence and the theoretical implications of these interactions and consider additional interactions that can limit the size of nephron clusters. The architecture of the arterial tree figures prominently in these interactions.

A simple method to ensure homogeneous drug distribution during intrarenal infusion.

Intrarenal drug infusion plays an important role in renal experimental research. Laminar flow of the blood can cause streaming and inhomogeneous intrarenal distribution of infused drugs. We suggest a simple method to achieve a homogeneous intravascular distribution of drugs infused into the renal artery of anesthetized rats. The method employs a multiple sidehole catheter inserted into the renal artery, which enables an efficient drug mixing with the arterial blood. To verify the efficiency of this method, we use laser speckle imaging and renal artery flowmetry. The results show that, compared with the conventional single-hole catheter, the multiple sidehole catheter provides a more uniform drug distribution and a homogenous vascular response on the surface of the kidney.

Architecture of the rat nephron-arterial network: analysis with micro-computed tomography.

Among solid organs, the kidney's vascular network stands out, because each nephron has two distinct capillary structures in series and because tubuloglomerular feedback, one of the mechanisms responsible for blood flow autoregulation, is specific to renal tubules. Tubuloglomerular feedback and the myogenic mechanism, acting jointly, autoregulate single-nephron blood flow. Each generates a self-sustained periodic oscillation and an oscillating electrical signal that propagates upstream along arterioles. Similar electrical signals from other nephrons interact, allowing nephron synchronization. Experimental measurements show synchronization over fields of a few nephrons; simulations based on a simplified network structure that could obscure complex interactions predict more widespread synchronization. To permit more realistic simulations, we made a cast of blood vessels in a rat kidney, performed micro-computed tomography at 2.5-µm resolution, and recorded three-dimensional coordinates of arteries, afferent arterioles, and glomeruli. Nonterminal branches of arcuate arteries form treelike structures requiring two to six bifurcations to reach terminal branches at the tree tops. Terminal arterial structures were either paired branches at the tops of the arterial trees, from which 52.6% of all afferent arterioles originated, or unpaired arteries not at the tree tops, yielding the other 22.9%; the other 24.5% originated directly from nonterminal arteries. Afferent arterioles near the corticomedullary boundary were longer than those farther away, suggesting that juxtamedullary nephrons have longer afferent arterioles. The distance separating origins of pairs of afferent arterioles varied randomly. The results suggest an irregular-network tree structure with vascular nodes, where arteriolar activity and local blood pressure interact.

Modeling of Kidney Hemodynamics: Probability-Based Topology of an Arterial Network.

Through regulation of the extracellular fluid volume, the kidneys provide important long-term regulation of blood pressure. At the level of the individual functional unit (the nephron), pressure and flow control involves two different mechanisms that both produce oscillations. The nephrons are arranged in a complex branching structure that delivers blood to each nephron and, at the same time, provides a basis for an interaction between adjacent nephrons. The functional consequences of this interaction are not understood, and at present it is not possible to address this question experimentally. We provide experimental data and a new modeling approach to clarify this problem. To resolve details of microvascular structure, we collected 3D data from more than 150 afferent arterioles in an optically cleared rat kidney. Using these results together with published micro-computed tomography (µCT) data we develop an algorithm for generating the renal arterial network. We then introduce a mathematical model describing blood flow dynamics and nephron to nephron interaction in the network. The model includes an implementation of electrical signal propagation along a vascular wall. Simulation results show that the renal arterial architecture plays an important role in maintaining adequate pressure levels and the self-sustained dynamics of nephrons.

Multinephron dynamics on the renal vascular network.

Tubuloglomerular feedback (TGF) and the myogenic mechanism combine in each nephron to regulate blood flow and glomerular filtration rate. Both mechanisms are nonlinear, generate self-sustained oscillations, and interact as their signals converge on arteriolar smooth muscle, forming a regulatory ensemble. Ensembles may synchronize. Smooth muscle cells in the ensemble depolarize periodically, generating electrical signals that propagate along the vascular network. We developed a mathematical model of a nephron-vascular network, with 16 versions of a single nephron model containing representations of both mechanisms in the regulatory ensemble, to examine the effects of network structure on nephron synchronization. Symmetry, as a property of a network, facilitates synchronization. Nephrons received blood from a symmetric electrically conductive vascular tree. Symmetry was created by using identical nephron models at each of the 16 sites and symmetry breaking by varying nephron length. The symmetric model achieved synchronization of all elements in the network. As little as 1% variation in nephron length caused extensive desynchronization, although synchronization was maintained in small nephron clusters. In-phase synchronization predominated among nephrons separated by one or three vascular nodes and antiphase synchronization for five or seven nodes of separation. Nephron dynamics were irregular and contained low-frequency fluctuations. Results are consistent with simultaneous blood flow measurements in multiple nephrons. An interaction between electrical signals propagated through the network to cause synchronization; variation in vascular pressure at vessel bifurcations was a principal cause of desynchronization. The results suggest that the vasculature supplies blood to nephrons but also engages in robust information transfer.

The assessment of cortical hemodynamic responses induced by tubuloglomerular feedback using in vivo imaging.

The tubuloglomerular feedback (TGF) mechanism modulates renal hemodynamics and glomerular filtration rate in individual nephrons. Our study aimed to evaluate the TGF-induced vascular responses by inhibiting Na-K-2Cl co-transporters and sodium-glucose co-transporters in rats. We assessed cortical hemodynamics with high-resolution laser speckle contrast imaging, which enabled the evaluation of blood flow in individual microvessels and analysis of their dynamical patterns in the time-frequency domain. We demonstrated that a systemic administration of furosemide abolishes TGF-mediated hemodynamic responses. Furthermore, we showed that the local microcirculatory blood flow decreased, and the TGF-induced hemodynamic oscillations were sustained but weakened after inhibiting sodium-glucose co-transporters in Sprague-Dawley rats.

In vivo mapping of hemodynamic responses mediated by tubuloglomerular feedback in hypertensive kidneys.

The kidney has a sophisticated vascular structure that performs the unique function of filtering blood and managing blood pressure. Tubuloglomerular feedback is an intra-nephron negative feedback mechanism stabilizing single-nephron blood flow, glomerular filtration rate, and tubular flow rate, which is exhibited as self-sustained oscillations in single-nephron blood flow. We report the application of multi-scale laser speckle imaging to monitor global blood flow changes across the kidney surface (low zoom) and local changes in individual microvessels (high zoom) in normotensive and spontaneously hypertensive rats in vivo. We reveal significant differences in the parameters of TGF-mediated hemodynamics and patterns of synchronization. Furthermore, systemic infusion of a glucagon-like-peptide-1 receptor agonist, a potential renoprotective agent, induces vasodilation in both groups but only alters the magnitude of the TGF in Sprague Dawleys, although the underlying mechanisms remain unclear.

A hybrid approach to full-scale reconstruction of renal arterial network.

The renal vasculature, acting as a resource distribution network, plays an important role in both the physiology and pathophysiology of the kidney. However, no imaging techniques allow an assessment of the structure and function of the renal vasculature due to limited spatial and temporal resolution. To develop realistic computer simulations of renal function, and to develop new image-based diagnostic methods based on artificial intelligence, it is necessary to have a realistic full-scale model of the renal vasculature. We propose a hybrid framework to build subject-specific models of the renal vascular network by using semi-automated segmentation of large arteries and estimation of cortex area from a micro-CT scan as a starting point, and by adopting the Global Constructive Optimization algorithm for generating smaller vessels. Our results show a close agreement between the reconstructed vasculature and existing anatomical data obtained from a rat kidney with respect to morphometric and hemodynamic parameters.

SERS uncovers the link between conformation of cytochrome c heme and mitochondrial membrane potential.

The balance between the mitochondrial respiratory chain activity and the cell's needs in ATP ensures optimal cellular function. Cytochrome c is an essential component of the electron transport chain (ETC), which regulates ETC activity, oxygen consumption, ATP synthesis and can initiate apoptosis. The impact of conformational changes in cytochrome c on its function is not understood for the lack of access to these changes in intact mitochondria. We have developed a novel sensor that uses unique properties of label-free surface-enhanced Raman spectroscopy (SERS) to identify conformational changes in heme of cytochrome c and to elucidate their role in functioning mitochondria. We have verified that molecule bond vibrations assessed by SERS are a reliable indicator of the heme conformation during changes in the inner mitochondrial membrane potential and ETC activity. We have demonstrated that cytochrome c heme reversibly switches between planar and ruffled conformations in response to the inner mitochondrial membrane potential (ΔΨ) and H+ concentration in the intermembrane space. This regulates the efficiency of the mitochondrial respiratory chain, thus, adjusting the mitochondrial respiration to the cell's consumption of ATP and the overall activity. We have found that under hypertensive conditions cytochrome c heme loses its sensitivity to ΔΨ that can affect the regulation of ETC activity. The ability of the proposed SERS-based sensor to track mitochondrial function opens broad perspectives in cell bioenergetics.

Multi-scale laser speckle contrast imaging of microcirculatory vasoreactivity.

Laser speckle contrast imaging is a robust and versatile blood flow imaging tool in basic and clinical research for its relatively simple construction and ease of customization. One of its key features is the scalability of the imaged field of view. With minimal changes to the system or analysis, laser speckle contrast imaging allows for high-resolution blood flow imaging through cranial windows or low-resolution perfusion visualization of perfusion over large areas, e.g. in human skin. We further utilize this feature and introduce a multi-scale laser speckle contrast imaging system, which we apply to study vasoreactivity in renal microcirculation. We combine high resolution (small field of view) to segment blood flow in individual vessels with low resolution (large field of view) to monitor global blood flow changes across the renal surface. Furthermore, we compare their performance when analyzing blood flow dynamics potentially associated with a single nephron and show that the previously published approaches, based on low-zoom imaging alone, provide inaccurate results in such applications.

Synchronization in renal microcirculation unveiled with high-resolution blood flow imaging.

Internephron interaction is fundamental for kidney function. Earlier studies have shown that nephrons signal to each other, synchronize over short distances, and potentially form large synchronized clusters. Such clusters would play an important role in renal autoregulation, but due to the technological limitations, their presence is yet to be confirmed. In the present study, we introduce an approach for high-resolution laser speckle imaging of renal blood flow and apply it to estimate the frequency and phase differences in rat kidney microcirculation under different conditions. The analysis unveiled the spatial and temporal evolution of synchronized blood flow clusters of various sizes, including the formation of large (>90 vessels) and long-lived clusters (>10 periods) locked at the frequency of the tubular glomerular feedback mechanism. Administration of vasoactive agents caused significant changes in the synchronization patterns and, thus, in nephrons' co-operative dynamics. Specifically, infusion of vasoconstrictor angiotensin II promoted stronger synchronization, while acetylcholine caused complete desynchronization. The results confirm the presence of the local synchronization in the renal microcirculatory blood flow and that it changes depending on the condition of the vascular network and the blood pressure, which will have further implications for the role of such synchronization in pathologies development.

Detection of Hypertension-Induced Changes in Erythrocytes by SERS Nanosensors.

Surface-enhanced Raman spectroscopy (SERS) is a promising tool that can be used in the detection of molecular changes triggered by disease development. Cardiovascular diseases (CVDs) are caused by multiple pathologies originating at the cellular level. The identification of these deteriorations can provide a better understanding of CVD mechanisms, and the monitoring of the identified molecular changes can be employed in the development of novel biosensor tools for early diagnostics. We applied plasmonic SERS nanosensors to assess changes in the properties of erythrocytes under normotensive and hypertensive conditions in the animal model. We found that spontaneous hypertension in rats leads (i) to a decrease in the erythrocyte plasma membrane fluidity and (ii) to a decrease in the mobility of the heme of the membrane-bound hemoglobin. We identified SERS parameters that can be used to detect pathological changes in the plasma membrane and submembrane region of erythrocytes.

Nephron blood flow dynamics measured by laser speckle contrast imaging.

Tubuloglomerular feedback (TGF) has an important role in autoregulation of renal blood flow and glomerular filtration rate (GFR). Because of the characteristics of signal transmission in the feedback loop, the TGF undergoes self-sustained oscillations in single-nephron blood flow, GFR, and tubular pressure and flow. Nephrons interact by exchanging electrical signals conducted electrotonically through cells of the vascular wall, leading to synchronization of the TGF-mediated oscillations. Experimental studies of these interactions have been limited to observations on two or at most three nephrons simultaneously. The interacting nephron fields are likely to be more extensive. We have turned to laser speckle contrast imaging to measure the blood flow dynamics of 50-100 nephrons simultaneously on the renal surface of anesthetized rats. We report the application of this method and describe analytic techniques for extracting the desired data and for examining them for evidence of nephron synchronization. Synchronized TGF oscillations were detected in pairs or triplets of nephrons. The amplitude and the frequency of the oscillations changed with time, as did the patterns of synchronization. Synchronization may take place among nephrons not immediately adjacent on the surface of the kidney.

Coupling-induced complexity in nephron models of renal blood flow regulation.

Tubular pressure and nephron blood flow time series display two interacting oscillations in rats with normal blood pressure. Tubuloglomerular feedback (TGF) senses NaCl concentration in tubular fluid at the macula densa, adjusts vascular resistance of the nephron's afferent arteriole, and generates the slower, larger-amplitude oscillations (0.02-0.04 Hz). The faster smaller oscillations (0.1-0.2 Hz) result from spontaneous contractions of vascular smooth muscle triggered by cyclic variations in membrane electrical potential. The two mechanisms interact in each nephron and combine to act as a high-pass filter, adjusting diameter of the afferent arteriole to limit changes of glomerular pressure caused by fluctuations of blood pressure. The oscillations become irregular in animals with chronic high blood pressure. TGF feedback gain is increased in hypertensive rats, leading to a stronger interaction between the two mechanisms. With a mathematical model that simulates tubular and arteriolar dynamics, we tested whether an increase in the interaction between TGF and the myogenic mechanism can cause the transition from periodic to irregular dynamics. A one-dimensional bifurcation analysis, using the coefficient that couples TGF and the myogenic mechanism as a bifurcation parameter, shows some regions with chaotic dynamics. With two nephrons coupled electrotonically, the chaotic regions become larger. The results support the hypothesis that increased oscillator interactions contribute to the transition to irregular fluctuations, especially when neighboring nephrons are coupled, which is the case in vivo.

The role of the myeloperoxidase-derived oxidant hypothiocyanous acid (HOSCN) in the induction of mitochondrial dysfunction in macrophages.

A host of chronic inflammatory diseases are accelerated by the formation of the powerful oxidant hypochlorous acid (HOCl) by myeloperoxidase (MPO). In the presence of thiocyanate (SCN-), the production of HOCl by MPO is decreased in favour of the formation of a milder oxidant, hypothiocyanous acid (HOSCN). The role of HOSCN in disease has not been fully elucidated, though there is increasing interest in using SCN- therapeutically in different disease settings. Unlike HOCl, HOSCN can be detoxified by thioredoxin reductase, and reacts selectively with thiols to result in reversible modifications, which could potentially reduce the extent of MPO-induced damage during chronic inflammation. In this study, we show that exposure of macrophages, a key inflammatory cell type, to HOSCN results in the reversible modification of multiple mitochondrial proteins, leading to increased mitochondrial membrane permeability, decreased oxidative phosphorylation and reduced formation of ATP. The increased permeability and reduction in ATP could be reversed by pre-treatment of the macrophages with cyclosporine A, implicating a role for the mitochondrial permeability transition pore. HOSCN also drives cells to utilise fatty acids as an energetic substrate after the inhibition of oxidative phosphorylation. Raman imaging studies highlighted the ability of HOSCN to perturb the electron transport chain of mitochondria and redistribute these organelles within the cell. Taken together, these data provide new insight into the pathways by which HOSCN can induce cytotoxicity and cellular damage, which may have relevance for the development of inflammatory disease, and therapeutic strategies to reduce HOCl-induced damage by supplementation with SCN-.

New insight into erythrocyte through in vivo surface-enhanced Raman spectroscopy.

The article presents a noninvasive approach to the study of erythrocyte properties by means of a comparative analysis of signals obtained by surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RS). We report step-by-step the procedure for preparing experimental samples containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living erythrocyte: submembrane and cytosolic hemoglobin (Hb(sm) and Hb(c)). We show that the conformation of Hb(sm) differs from the conformation of Hb(c). This finding has an important application, as the comparative study of Hb(sm) and Hb(c) could be successfully used in biomedical research and diagnostic tests.

Novel chiroptical analysis of hemoglobin by surface enhanced resonance Raman optical activity spectroscopy.

The metalloprotein hemoglobin (Hb) was studied using surface enhanced resonance Raman spectroscopy (SERRS) and surface enhanced resonance Raman optical activity (SERROA). The SERROA results are analyzed and compared with the SERRS, and the later to the resonance Raman (RRS) performed on Hb. The SERRS measurements careful optimization, with respect to the concentration and volume ratio of the analyte to colloids, enables for the first time SERROA of this molecule. We observed that the most intense SERROA signals were attributed the nu(4), nu(20), and nu(21) vibrations, which are sensitive to the redox state of the heme's iron ion, and to the presence of its sixth site, bound to exogenous ligand; O(2), NO or CO. However, in this study, the SERROA signals corresponding to these vibrations appear more sensitive to the Hb oxygen-binding properties than they appear in the SERRS or RRS. Moreover, the SERROA signal of Hb has successfully been monitored as a function of time, and was observed to be stable for 4-5 min. To our knowledge, the SERROA results of Hb, and its comparison to SERRS and RRS, are here reported for the first time.

Phase-modulation laser interference microscopy: an advance in cell imaging and dynamics study.

We describe how phase-modulation laser interference microscopy and wavelet analysis can be applied to noninvasive nonstained visualization and study of the structural and dynamical properties of living cells. We show how phase images of erythrocytes can reveal the difference between various erythrocyte forms and stages of hemolysis and how phase images of neurons reveal their complex intracellular structure. Temporal variations of the refractive index are analyzed to detect cellular rhythmic activity on different time scales as well as to uncover interactions between the cellular processes.

Dairy products viscosity estimated by laser speckle correlation.

Dairy products exhibit several physical properties that are crucial to define whether we like the food or not: firmness, creaminess, thickness, or lightness. Viscosity changes the flow properties of food and influences the appearance and the consistency of a product; this control variable is important in most production stages-manufacture, processing, and storage. Viscosity of heterogeneous products at a given temperature depends on its composition and physical state of its substances. Although rheology provides a method to access the product viscosity, it lacks non-contact full-field monitoring. We apply a simple correlation analysis of laser speckle images to evaluate viscosity properties of dairy products. Our approach ensures robust measurements with high degree of detectability.