Quantitative intravital Ca2+ imaging maps single cell behavior to kidney tubular structure.

Ca2+ is an important second messenger that translates extracellular stimuli into intracellular responses. Although there has been significant progress in understanding Ca2+ dynamics in organs such as the brain, the nature of Ca2+ signals in the kidney is still poorly understood. Here, we show that by using a genetically expressed highly sensitive reporter (GCaMP6s), it is possible to perform imaging of Ca2+ signals at high resolution in the mouse kidney in vivo. Moreover, by applying machine learning-based automated analysis using a Ca2+-independent signal, quantitative data can be extracted in an unbiased manner. By projecting the resulting data onto the structure of the kidney, we show that different tubular segments display highly distinct spatiotemporal patterns of Ca2+ signals. Furthermore, we provide evidence that Ca2+ activity in the proximal tubule decreases with increasing distance from the glomerulus. Finally, we demonstrate that substantial changes in intracellular Ca2+ can be detected in proximal tubules in a cisplatin model of acute kidney injury, which can be linked to alterations in cell structure and transport function. In summary, we describe a powerful new tool to investigate how single cell behavior is integrated with whole organ structure and function and how it is altered in disease states relevant to humans.

Combined Structural and Functional Imaging of the Kidney Reveals Major Axial Differences in Proximal Tubule Endocytosis.

BACKGROUND: The kidney proximal convoluted tubule (PCT) reabsorbs filtered macromolecules via receptor-mediated endocytosis (RME) or nonspecific fluid phase endocytosis (FPE); endocytosis is also an entry route for disease-causing toxins. PCT cells express the protein ligand receptor megalin and have a highly developed endolysosomal system (ELS). Two PCT segments (S1 and S2) display subtle differences in cellular ultrastructure; whether these translate into differences in endocytotic function has been unknown. METHODS: To investigate potential differences in endocytic function in S1 and S2, we quantified ELS protein expression in mouse kidney PCTs using real-time quantitative polymerase chain reaction and immunostaining. We also used multiphoton microscopy to visualize uptake of fluorescently labeled ligands in both living animals and tissue cleared using a modified CLARITY approach. RESULTS: Uptake of proteins by RME occurs almost exclusively in S1. In contrast, dextran uptake by FPE takes place in both S1 and S2, suggesting that RME and FPE are discrete processes. Expression of key ELS proteins, but not megalin, showed a bimodal distribution; levels were far higher in S1, where intracellular distribution was also more polarized. Tissue clearing permitted imaging of ligand uptake at single-organelle resolution in large sections of kidney cortex. Analysis of segmented tubules confirmed that, compared with protein uptake, dextran uptake occurred over a much greater length of the PCT, although individual PCTs show marked heterogeneity in solute uptake length and three-dimensional morphology. CONCLUSIONS: Striking axial differences in ligand uptake and ELS function exist along the PCT, independent of megalin expression. These differences have important implications for understanding topographic patterns of kidney diseases and the origins of proteinuria.

Morphological and histological characterization of sexual segment of the kidney in Notomabuya frenata (Cope, 1862) and Aspronema dorsivittatum (Cope, 1862) (Squamata, Mabuyidae).

The kidneys in two viviparous species of Neotropical lizards, Notomabuya frenata and Aspronema dorsivittatum (Mabuyidae), were investigated by light and scanning electron microscopy in order to determine the presence of the sexual segment of the kidney (SSK) and to study its morphology. The individuals used in this study belong to the Herpetological Collection of the Herpetology Laboratory - Reptiles of the Federal University of Juiz de Fora (CHUFJF-Reptiles) and they were collected between the years 2008 and 2012 from the Cerrado region in the state of Minas Gerais, Brazil. The SSK was present only in sexually mature males (with sperm in the testes / epididymis), whereas it was absent in sexually immature males. The nephron in both species consists of renal corpuscle, proximal convoluted tubule, distal convoluted tubule, collecting duct and sexual segment of the kidney. The SSK of the analyzed species were coated with a simple columnar epithelium, with high cells, basal nucleus and in the apical portion innumerable secretory granules. This study adds to the knowledge on reproductive biology and structures related to reproductive strategies of both lizard species and viviparous Neotropical lizards.

PFE-360-induced LRRK2 inhibition induces reversible, non-adverse renal changes in rats.

Parkinson's disease (PD) is a progressive neurodegenerative disorder for which there is no existing therapeutic approach to delay or stop progression. Genetic, biochemical and pre-clinical studies have provided evidence that leucine-rich-repeat-kinase-2 (LRRK2) kinase is involved in the pathogenesis of PD, and small molecule LRRK2 inhibitors represent a novel potential therapeutic approach. However, potentially adverse target-related effects have been discovered in the lung and kidneys of LRRK2 knock-out (ko) mice and rats. It is unclear if the LRRK2 ko effect in the kidneys and lung is also induced by pharmacological inhibition of the LRRK2 kinase. Here, we show that treatment with the LRRK2 inhibitor PFE-360 in rats induces a morphological kidney phenotype resembling that of the LRRK2 ko rats, whereas no effects were observed in the lung. The PFE-360 treatment induced morphological changes characterised by darkened kidneys and progressive accumulation of hyaline droplets in the renal proximal tubular epithelium. However, no histopathological evidence of renal tubular injury or changes in the blood and urine parameters that would be indicative of kidney toxicity or impaired kidney function were observed after up to 12 weeks of treatment. Morphological changes were detected in the kidney after 2 weeks of treatment and were partially reversible within a 30 day treatment-free period. Our findings suggest that pharmacological LRRK2 inhibition may not have adverse consequences for kidney function.

Expression of bitter taste receptor Tas2r105 in mouse kidney.

The kidney is the most important excretory organ in the body and plays an essential role in maintaining homeostasis in vivo by conserving body fluid and electrolytes and removing metabolic waste. In this study, three types of transgenic system were used to investigate the expression of the bitter taste receptor Tas2r105 in mouse renal tissue (Tas2r105-GFP/Cre, Tas2r105-GFP/Cre-DTA and Tas2r105-GFP/Cre-LacZ). The results suggest that bitter taste receptors Tas2r105 and Tas2r106 are expressed in the renal corpuscle and the renal tubule, including the proximal tubule and distal tubule. Expression of α-gustducin, an important component of taste signal transduction, was also detected in mouse kidney. Meanwhile, conditional diphtheria toxin (DTA) expression in Tas2r105+ cells caused an increase in size of the glomerulus and renal tubule, accompanied by a decrease in cell density in the glomerulus. This indicates that Tas2r105+ cells play an important role in maintaining the structure of the glomerulus and renal tubules. Overall, the current study collectively demonstrates that cells labeled by bitter taste receptor expression may play a critical role in controlling human health, and have properties far beyond the original concept of taste perception.

The small molecule probe PT-Yellow labels the renal proximal tubules in zebrafish.

We report the development of a small fluorescent molecule, BDNCA3-D2, herein referred to as PT-Yellow. Soaking zebrafish embryos in PT-Yellow or intraperitoneal injection into adults results in non-toxic in vivo fluorescent labeling of the renal proximal tubules, the major site of blood filtrate reabsorption and a common target of injury in acute kidney injury. We demonstrate the applicability of this new compound as a rapid and simple readout for zebrafish kidney filtration and proximal tubule reabsorption function.

MicroRNAs are critical regulators of tuberous sclerosis complex and mTORC1 activity in the size control of the Xenopus kidney.

MicroRNAs (miRNAs) are major posttranscriptional regulators of a wide variety of biological processes. However, redundancy among most miRNAs has made it difficult to identify their in vivo functions. We previously demonstrated that global inhibition of miRNA biogenesis in Xenopus resulted in a dramatically smaller pronephric kidney. This suggested that microRNAs play a pivotal role in organ size control. Here we now provide a detailed mechanistic explanation for this phenotype. We identified that the activation of the mechanistic target of rapamycin complex 1 (mTORC1) by Insulin and insulin-like growth factor (Igf) 2 is an important regulator in kidney growth, which in turn is modulated by microRNAs. Molecular analyses demonstrate that microRNAs set a threshold for mTORC1 signaling by down-regulating one of its core negative regulators, tuberous sclerosis 1 (Tsc1). Most importantly, this rheostat can be reprogrammed experimentally. Whereas knockdown of miRNAs causes growth arrest, concomitant knockdown of Tsc1 restores mTORC1 activity and proximal tubular size. Together, these data establish a previously unidentified in vivo paradigm for the importance of posttranscriptional regulation in organ size control.

Immunohistochemical localization of D-aspartate oxidase in porcine peripheral tissues.

D-Aspartate (D-Asp) is an endogenous substance in mammals. Degradation of D-Asp is carried out only by D-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate D-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade D-Asp to prevent an excess of D-Asp from exerting harmful effects on the respective functions of porcine tissues.

The pelvic kidney of male Ambystoma maculatum (Amphibia, urodela, ambystomatidae) with special reference to the sexual collecting ducts.

This study details the gross and microscopic anatomy of the pelvic kidney in male Ambystoma maculatum. The nephron of male Ambystoma maculatum is divided into six distinct regions leading sequentially away from a renal corpuscle: (1) neck segment, which communicates with the coelomic cavity via a ventrally positioned pleuroperitoneal funnel, (2) proximal tubule, (3) intermediate segment, (4) distal tubule, (5) collecting tubule, and (6) collecting duct. The proximal tubule is divided into a vacuolated proximal region and a distal lysosomic region. The basal plasma membrane is modified into intertwining microvillus lamellae. The epithelium of the distal tubule varies little along its length and is demarcated by columns of mitochondria with their long axes oriented perpendicular to the basal lamina. The distal tubule possesses highly interdigitating microvillus lamellae from the lateral membranes and pronounced foot processes of the basal membrane that are not intertwined, but perpendicular to the basal lamina. The collecting tubule is lined by an epithelium with dark and light cells. Light cells are similar to those observed in the distal tuble except with less mitochondria and microvillus lamellae of the lateral and basal plasma membrane. Dark cells possess dark euchromatic nuclei and are filled with numerous small mitochondria. The epithelium of the neck segment, pleuroperitoneal funnel, and intermediate segment is composed entirely of ciliated cells with cilia protruding from only the central portion of the apical plasma membrane. The collecting duct is lined by a highly secretory epithelium that produces numerous membrane bound granules that stain positively for neutral carbohydrates and proteins. Apically positioned ciliated cells are intercalated between secretory cells. The collecting ducts anastomose caudally and unite with the Wolffian duct via a common collecting duct. The Wolffian duct is secretory, but not to the extent of the collecting duct, synthesizes neutral carbohydrates and proteins, and is also lined by apical ciliated cells intercalated between secretory cells. Although functional aspects associated with the morphological variation along the length of the proximal portions of the nephron have been investigated, the role of a highly secretory collecting duct has not. Historical data that implicated secretory activity concordant with mating activity, and similarity of structure and chemistry to sexual segments of the kidneys in other vertebrates, lead us to believe that the collecting duct functions as a secondary sexual organ in Ambystoma maculatum.

Visualization of three-dimensional nephron structure with microcomputed tomography.

The three-dimensional architecture of nephrons in situ and their interrelationship with other nephrons are difficult to visualize by microscopic methods. The present study uses microcomputed X-ray tomography (micro-CT) to visualize intact nephrons in situ. Rat kidneys were perfusion-fixed with buffered formalin and their vasculature was subsequently perfused with radiopaque silicone. Cortical tissue was stained en bloc with osmium tetroxide, embedded in plastic, scanned, and reconstructed at voxel resolutions of 6, 2, and 1 microm. At 6 microm resolution, large blood vessels and glomeruli could be visualized but nephrons and their lumens were small and difficult to visualize. Optimal images were obtained using a synchrotron radiation source at 2 microm resolution where nephron components could be identified, correlated with histological sections, and traced. Proximal tubules had large diameters and opaque walls, whereas distal tubules, connecting tubules, and collecting ducts had smaller diameters and less opaque walls. Blood vessels could be distinguished from nephrons by the luminal presence of radiopaque silicone. Proximal tubules were three times longer than distal tubules. Proximal and distal tubules were tightly coiled in the outer cortex but were loosely coiled in the middle and inner cortex. The connecting tubules had the narrowest diameters of the tubules and converged to form arcades that paralleled the radial vessels as they extended to the outer cortex. These results illustrate a potential use of micro-CT to obtain three-dimensional information about nephron architecture and nephron interrelationships, which could be useful in evaluating experimental tubular hypertrophy, atrophy, and necrosis.

Fractal Dimension of Dog Kidney Proximal Convoluted Tubuli Sections by Mean Box-Counting Algorithm / Dimensión Fractal de Secciones de los Túbulos Contorneados Proximales del Riñón Mediante un Algoritmo de Recuento de Cajas

The possibility of characterizing the proximal convoluted tubuli of the dog kidney by means of a unique and objective value, is an attractive idea in order to automate its recognition in anatomy and patology. For this, we obtained a fractal dimension of the proximal convoluted tubuli in the dog kidney by mean box-counting method. The fractal value we obtained is a flat dimension, and it is different than the line fractal dimension of the dog kidney arterial pattern, which was previously calculated by us (Gil et al., 2006). Thus, from optical microscopy images, we are able to obtain a single quantitative measure to discriminate these dog kidney components. This measures can use to automate its recognition. Fractal geometry provides many advantages when examining the complex microscopical images of natural objects.

La posibilidad de caracterizar los túbulos contorneados proximales del riñón de perro, mediante un valor único y objetivo, es una idea interesante para automatizar su reconocimiento en anatomía y en patología. Para ello, hemos obtenido la dimensión fractal de los túbulos contorneados proximales del riñón de perro, mediante la técnica de recuento de cajas. El valor fractal obtenido es una dimensión de superficie, diferente de la dimensión fractal lineal que calculamos previamente (Gil et al., 2006). De esta forma, desde imágenes microscópicas, nos es posible obtener una medida simple y cuantitativa para diferenciar estos compontes del riñón de perro. Esta medida puede usarse para automatizar su reconocimiento.

Localization of tubular uptake segment of filtered Cystatin C and Aprotinin in the rat kidney.

AIM: The renal tubular uptake of 125I-Aprotinin (*Ap) is on average located more superficially than its filtration site, causing transfer of some of *Ap filtered in deep to more superficial cortical zones. 125I-Cystatin C (*Cy) showed less uptake in deep cortical zones than Ap, suggesting a longer and/or a more superficial tubular uptake site. To test that hypothesis and to quantify the outward transfer of the filtered polypeptides, we estimated the tubular uptake pattern of the tracers in perfusion fixed rat kidneys after intravenous injection of *Cy and *Ap. METHODS: Autoradiographs were made from 10 mum thick slices of Microfil nephron casts from outer (OC), middle (MC) and inner (IC) cortical zones to quantify cortical border-crossing *Ap transfer. Single nephron glomerular filtration rate (snGFR) was estimated as the zonal uptake of *Ap corrected for *Ap transfer, divided by its time-integrated plasma concentration and the zonal number of glomeruli. RESULTS: *Ap and *Cy uptake fell exponentially along the proximal convoluted tubule (PCT), indicating an uptake proportional to luminal concentration. Uptake in IC exceeded that in MC and OC nephrons. The per cent PCT length with *Cy uptake (67.2 +/- 1.6) exceeded that of *Ap (54.6 +/- 1.8). The zonal border-crossing PCT length (29-34% of total PCT) from deep to more superficial cortical zones transferred 4-6% more *Cy than *Ap. CONCLUSION: Greater tubular uptake length of *Cy than of *Ap causes more cortical border-crossing of *Cy. The zonal snGFR estimated from Aprotinin uptake corrected for border-crossing agreed well with that obtained with the Hanssen ferrocyanide technique.

Expression of angiotensinogen in proximal tubule as a function of glomerular filtration rate.

BACKGROUND: Proximal tubule (PT) angiotensinogen (AGT) is part of a tubular renin-angiotensin system (RAS) that participates in the regulation of sodium reabsorption along the entire nephron. Physiologic maneuvers affecting AGT expression in PT also affect systemic RAS. Here, we tested the hypothesis that PT AGT is regulated by increased glomerular filtration rate (GFR). METHODS: Complete unilateral nephrectomy (UNX) in mice was used to induce a sustained increase in GFR in the remaining kidney. AGT expression was monitored by quantitative reverse transcription-polymerase chain reaction (RT-PCR). AGT protein in PT was investigated by semiquantitative histology. We also measured AGT concentration in plasma and in 24-hour urine by a specific enzyme-linked immunosorbent assay (ELISA). RESULTS: Seven weeks after nephrectomy, UNX animals exhibited a 2-fold increase in tubular AGT mRNA (P <.001) compared with sham-operated control animals. The proportion of PT sections exhibiting AGT immunostaining was significantly increased at day 3 (P <.05), and remained elevated at seven weeks (UNX = 0.63 +/- 0.09, sham = 0.38 +/- 0.02, P <.01), revealing recruitment of AGT-producing cells along the PT. AGT excretion in final urine corrected for creatinine and kidney weight was also elevated by UNX at seven weeks (UNX = 209 +/- 42 pmol/mg/g, sham = 147 +/- 29 pmol/mg/g, P <.05), with no difference in plasma AGT between UNX and control animals. CONCLUSION: These observations suggest that AGT expression in PT adapts in the long-term to changes in GFR. In the UNX model, urinary AGT excretion is also elevated as a consequence of increase in net tubular flow.

Giant lysosomes in the renal proximal tubules--a morphological characteristic of DBA/2 and DBA/1 mouse kidneys.

The DBA/2Cr mouse is characterized by the presence of giant lysosomes located in the proximal convoluted tubules of males and proximal straight tubules of females. However, it remains unclear whether these giant lysosomes in the proximal tubules are characteristic of DBA/2Cr specifically, or are common to other DBA/2 substrains and DBA/1. The present study investigated the morphology of kidneys from DBA/2CrSlc, DBA/2JJcl, DBA/2NCrj and DBA/1JNCrj mice of both sexes. Giant lysosomes in the renal proximal tubules were found to represent common morphological characteristic of both DBA/2 and DBA/1JN.

[Functional and structural characteristics of the nephron segments]. / Funktsional'no-strukturna kharakterystyka sehmentiv nefronu.

The review presents a modern view on the functional--morphological peculiarities of 12 segments of the nephron canaliculi possessing some functional, biochemical and histologic distinctions that should be taken into account to study physiology, biochemistry, morphology and pathophysiology of the kidney.

Sex- and strain-dependent histological features of the proximal convoluted tubular epithelium of mouse kidney: association with lysosomes containing apolipoprotein B.

In the present study, we performed comparative histological observations of ICR, BALB/c, C57BL/6, C3H/HeN and DBA/2 mice kidneys. Sex and strain differences were observed in the appearance of vacuolar structures of the proximal convoluted tubules (toluidine blue-positive granules in osmium-postfixed epoxy-resin sections). These features were especially remarkable in male DBA/2 mice. The vacuolar structures in male DBA/2 mice showed heterogeneous staining with Sudan B in frozen sections and appeared under an electron microscope as multilammelar giant dense bodies. In addition, these dense bodies showed heterogeneous acid phosphatase reactions. Immunohistochemical analyses of these structures for apolipoprotein B showed strong positive reactions. These results suggested that vacuolar structures in the proximal convoluted tubules, which were remarkable in male DBA/2 mice, were giant lysosomes containing apolipoprotein B.

Regulation of proximal tubule sodium/hydrogen antiporter with chronic volume contraction.

We developed a model of volume contraction in rabbits by using a furosemide/low-salt diet to follow changes, if any, in proximal tubule Na+/H+ exchanger 3 (NHE3) mRNA and brush-border protein. The rabbits' plasma renin, aldosterone, and urine sodium content confirmed the volume-contracted state. RNase protection assays demonstrated increases in treated-animal NHE3 mRNA as a percentage of control with 172 +/- 23, 154 +/- 15, 153 +/- 14, and 141 +/- 7 (SE) % (P < 0.05) at 1, 5, 10, and 31 days, respectively. Western analysis of brush-border membrane with NHE3 antibody revealed increased immunoreactivity in treated animals as a percentage of control with 120 +/- 30, 190 +/- 59, 307 +/- 72, and 427 +/- 41% (P < 0.05) at 1, 5, 10, and 31 days, respectively. There was no significant difference in serum potassium, bicarbonate, and cortisol in control vs. experimental animals. These data suggest that there is chronic upregulation of NHE3 in the volume-contracted state.

Effects of season on kidney morphology in house sparrows.

Seasonal variability in kidney morphology of the house sparrow Passer domesticus was examined using light microscopy. Sparrows were captured from the wild in winter, spring, summer and autumn. The kidneys were perfused with half-strength Karnovsky's fixative and processed for light microscopy by embedding in either paraffin wax or JB4 acrylic resin. Absolute volumes of the kidneys, their components (cortex, medulla and blood vessels), components of the nephron (renal corpuscles, proximal tubules, loops of Henle, distal tubules and collecting ducts) and the capillaries surrounding the nephron were quantified using stereology. Tissue processed in paraffin wax had a mean shrinkage of 17.7 % compared with 10.1 % in JB4 resin. Absolute volumes of the kidneys and nephrons were compared statistically between tissue processing methods and among seasons. Absolute volumes of the structures within the kidneys were not significantly different between treatments or among seasons. Within the nephron, the only measured variables to show significant differences were the absolute volumes of the distal tubules and cortical collecting ducts between tissue processing treatments. Thus, kidney morphology was relatively unaffected by changes in season. In addition, the results show that embedding tissue using acrylic resin causes less shrinkage, and it should therefore be the preferred embedding medium for quantitative morphologists.

Functional morphology of the avian medullary cone.

The organization of the renal medulla of the Gambel's quail, Callipepla gambelii, kidney was examined to determine the number of loops of Henle and collecting ducts and the surface area occupied by the different nephron segments as a function of distance down the medullary cones. Eleven medullary cones were dissected from the kidneys of four birds, and the tissue was processed and sectioned for light microscopy. In addition, individual nephrons were isolated on which total loop thin descending segment and thick prebend segment lengths were measured. The results show no correlation between the absolute number of loops of Henle and the length of the medullary cones. The number of thick and thin limbs of Henle and collecting ducts decrease exponentially with distance toward the apex of the cones and the rate of decrease is similar for cones of different lengths. Initially there is a rapid decrease in the number of thin limbs of Henle, indicating that most nephrons do not penetrate the cones a great distance. Thick descending limbs of Henle (prebend segment) ranged in length from 50 to 770 microm, and there was little correlation with the total length of the loop of Henle. However, the length of the thin limb of Henle correlated well with total loop length. The cell surface areas of the limbs of the loop of Henle and the collecting ducts decreased toward the apex of the cones.

Glutamine synthesis is heterogeneous and differentially regulated along the rabbit renal proximal tubule.

Glutamine synthesis, a major process for ammonia detoxification and the control of acid-base balance, occurs from various precursors in suspensions of rabbit proximal tubules. However, no data are currently available on the distribution of glutamine synthesis along the rabbit proximal tubule, and its modulation by changes of substrate concentration. Therefore we have microdissected and incubated the three parts (S1, S2 and S3) of rabbit proximal tubules and measured glutamine synthesis from alanine and aspartate. With a physiological concentration of alanine (0.25 mM) or aspartate (0.05 mM), glutamine synthesis in the S1 segment was about half of that in the S2 and S3 segments, and was greater from alanine than from aspartate along the entire proximal tubule. Elevation of alanine and aspartate concentrations to 5 mM increased glutamine synthesis in both a substrate- and segment-dependent manner. It is concluded that glutamine synthesis occurs from alanine and aspartate along the entire rabbit proximal tubule; however, contrary to what might have been expected on the basis of measurement of glutamine synthetase activity, the basal rate of glutamine synthesis and its adaptation to increased substrate availability are heterogeneous along this nephron segment.