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.

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.

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 ultrastructure of peroxisomes in the kidney of the camel (Camelus dromedarius).

Dromedary camels can survive and reproduce in desert areas. The unique anatomical structure of the kidney enables the camel to prevent water loss. The present study aimed to investigate the ultrastructure of the peroxisomes in the normal kidney of the adult dromedary camel. Tissue samples were taken from the cortex and outer medulla of the kidney of eight camels. The samples were then processed for histological and ultrastructural investigations. The epithelial cells of the proximal tubules displayed peroxisomes with varying sizes and shapes. The peroxisomes were observed in either dispersed or clustered arrangement. Each peroxisome exhibited a homogenous matrix enveloped by a single membrane. Several peroxisomes exhibited one or more dark marginal plates that were always strongly associated with the smooth endoplasmic reticulum. The intensity of the peroxisomal matrix differed significantly, either within the same cell or across different cells. The intensity was light or dark, with a few peroxisomes presenting a similar intensity to that of the mitochondria. Some peroxisomes contained nucleoids within their matrix. The peroxisomes in the first and second sections of proximal convoluted tubules were scattered and primarily located in the region between the microvilli and the underlying mitochondria. The peroxisomes in the third region were abundant and frequently aggregated in clusters throughout the cytoplasm. In the fourth region, the number of peroxisomes was low. The proximal straight tubule had a limited quantity of peroxisomes. In conclusion, peroxisomes in the proximal tubule in kidney of normal dromedary camel were similar in shape and size to other mammals; however, heterogeneity exists as a result of differences in species-specific peroxisomal proteins. Peroxisomes are suggested to be a major source of metabolic energy and act as hydrogen peroxide (H2O2) scavengers, resulting in the release of water and oxygen.

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.

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.

A definition of proximal and distal tubular compliance. Practical and theoretical implications.

Micropuncture studies were carried out in the rat to evaluate the in situ distensibility characteristics of the proximal and distal tubules under a variety of experimental conditions. In the first phase, we determined the response of tubular diameter (D) to changes in tubular pressure (P) induced by partially obstructing single tubules. The response observed under these conditions (i.e., when interstitial pressure is presumed to be constant) has been defined as the compliance of the tubule. Over the range of tubular pressures studied (10-35 mm Hg for the proximal tubule, 5-25 mm Hg for the distal tubule) the compliance characteristics of the proximal and distal tubule were found to be markedly different; the proximal tubular pressure-diameter relationship was linear, DeltaD/DeltaP = 0.45 mum/mm Hg, whereas the distal pressure-diameter relationship was curvilinear, DeltaD/DeltaP = c(-0.1xP+2.2). In the second phase we used the compliance data to construct a series of theoretical pressure-diameter curves that define the response of the tubule to increments in interstitial as well as intratubular pressure. These curves indicate that changes in distal diameter should provide a sensitive index of a rise in interstitial pressure under conditions in which the transtubular pressure gradient is increased by a small amount, but that proximal diameter should provide a more sensitive index of changes in interstitial pressure when the transtubular pressure gradient is increased by a large amount. In subsequent experiments in which furosemide was administered, we observed that the pressure-diameter relationships for both the proximal and distal tubule were indistinguishable from the compliance curves, a finding consistent with the interpretation that interstitial pressure was not appreciably changed from control. By contrast, when mannitol was administered, both proximal and distal tubular pressure-diameter relationships were significantly altered in a fashion consistent with a large increase in interstitial pressure. Neither with furosemide nor mannitol administration did it appear likely that significant changes in tubular compliance could account for the observed behavior of the tubule.Finally, we propose that a knowledge of tubular compliance will be useful in exploring the interrelationships between tubular and peritubular pressures, tubular anatomy, and transtubular ionic permeability. Recent studies linking changes in the geometry of lateral intercellular spaces of the tubule to changes in passive ion movement suggest that an investigation of such anatomical-functional correlates should be productive.

Tracer microinjection study of renal tubular phosphate reabsorption in the rat.

To determine the sites of tubular phosphate reabsorption in the nephron, microinjection studies were undertaken, utilizing isotonic electrolyte solutions, containing either 1.4 or 8.0 mM phosphate and radioactive PO(4)-(33)P and inulin-(3)H, in rats made mildly diuretic by infusion of mannitol. The injected sites were localized by the technique of latex dissection. The relation between proximal tubular length and per cent (33)P recovery for injections of 1.4 mM phosphate (physiological amounts) suggest that relatively little reabsorption of phosphate occurs in the distal 30% of the proximal tubule compared with the proximal portion of the tubule. The corresponding recoveries for proximal tubular microinjections of 8.0 mM phosphate fall along a smooth curve tending to plateau with essentially complete (33)P recovery (> 95%) beyond 50% of the tubule. Absolute reabsorption of injected phosphate for both concentrations (i.e., absolute efflux per unit tubular length in the proximal tubule) was independent of phosphate delivery, since the relationship between reabsorption and site of injection was no different for the two concentrations. Distal convoluted tubular microinjections for both phosphate concentrations showed complete recovery of (33)P from all injection sites. THE DATA INDICATE THAT: (a) no phosphate reabsorption occurs in the distal convoluted tubule or in the collecting duct, (b) phosphate efflux per unit tubular length is greater in the first one-third of the proximal tubule than in the remaining two-thirds, and (c) in the last two-thirds of the proximal tubule, absolute phosphate reabsorption is relatively small and might be limited by factors other than the amount or concentration of injected phosphate.

Characteristics of volume reabsorption in rabbit superficial and juxtamedullary proximal convoluted tubules.

Segments of superficial and juxtamedullary proximal convoluted tubules of the rabbit were perfused in vitro to examine the mechanisms responsible for net volume reabsorption. The very early postglomerular segments were not studied. Fluid reabsorptive rates and transepithelial potential differences were compared under various conditions: (a) with perfusate that simulated glomerular filtrate; (b) with perfusate that lacked glucose, amino acids, and acetate and that had HCO(3) and Cl concentrations of 5 and 140 mM, respectively; (c) with perfusate that lacked glucose, amino acids, and acetate but with 20 meq of NaHCO(3) replaced with 20 meq of Na cyclamate; (d) with the same perfusate as in b but in the presence of ouabain in the bath; (e) with ultrafiltrate of rabbit serum titrated with HCl to final HCO(3) and Cl concentrations of 2 and 134 mM, respectively. Tubules were perfused with this titrated ultrafiltrate at 37 degrees C, 21 degrees C, and in the presence of 0.1 mM ouabain in the bath. Bath fluid in all experiments was regular rabbit serum. Under conditions a and b superficial proximal convoluted tubule (SFPCT) and juxtamedullary proximal convoluted tubule (JMPCT) behaved similarly with the exception that SFPCT exhibited a lumen-positive and JMPCT a lumen-negative electrical potential under condition b. However, under condition c SFPCT failed to exhibit net volume reabsorption, whereas reabsorption in JMPCT continued unchanged. Ouabain did not affect volume reabsorption in SFPCT under condition d, whereas neither ouabain nor hypothermia affected SFPCT under condition e. In contrast, ouabain and hypothermia totally inhibited volume reabsorption in JMPCT under conditions d and e. These studies document heterogeneous mechanisms responsible for volume reabsorption in the major portions of SFPCT and JMPCT with passive forces predominating in SFPCT and active forces in JMPCT.

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.

Segmental distribution of the endocytosis receptor gp330 in renal proximal tubules.

The subcellular distribution and segmental variations in location of gp330, a scavenger receptor for filtered proteins in renal proximal tubules, was analyzed. Kidney tissue from rats (4 different strains), rabbits and humans were analyzed by light- and electron microscope immunocytochemistry, using cryosections or Lowicryl sections from cryosubstituted tissue. Gp330 was located mainly in apical coated pits, small and large endocytic vacuoles and in dense apical tubules in the proximal tubule cells. The labeling density was markedly higher in segments 1 and 2 as compared to segment 3 of the proximal tubule. In addition to the location in the early part of the endocytic pathway, gp330 was also present in lysosomes, especially in segments 1 and 2. The lysosomal labeling was not restricted to the membrane, but was also seen in the matrix. Localization of gp330 in lysosomes was confirmed on sections from purified lysosomal fractions from rat renal cortex. The brush border localization of gp330 in proximal tubules exhibited a characteristic segmental variation. In the initial part of segment 1, there was virtually no brush border labeling. In the remaining part of segment 1 and in segment 2, there was a distinct but sometimes patchy labeling of the brush border. In segment 3, groups of microvilli of approximately 10 as seen in sections were intensively labeled from bottom to tip and there were often more than one of these groups on a single cell, the remaining microvilli were unlabeled. No differences in the cellular and subcellular localization of gp330 were observed between species or rat strains. In conclusion, the present study demonstrates that in addition to its location in the early endocytic and recycling pathway, gp330 is also present in microvilli and the protein and degradation products thereof is present in lysosomes, consistent with its role as a protein scavenger receptor.

The effect of Collins' solution on the function and structure of isolated proximal convoluted tubules from rabbit kidneys.

Proximal convoluted tubules (PCTs) from rabbit cortical slices were perfused after preservation in Collins' solution with the in vitro microperfusion technique. Under these conditions, after maneuvers of in vitro preservation, the following findings were observed: Tubules were best preserved, functionally and morphologically, when bathed with Collins' solution peritubularly. Tubular preservation was inadequate when the Collins' solution contacted only the luminal or luminal and peritubular sides. These observations indicate a difference in the reactions of various cellular sides to the preservation process, suggesting that during preservation of the whole kidney, there are differences in the preservation of the various tissues of the organ.

Transepithelial endoplasmic reticulum in rat proximal convoluted tubule.

Inosine 5'-diphosphatase (IDPase) activity was demonstrated cytochemically in the endoplasmic reticulum of rat kidney proximal tubule cells in tissue fixed by perfusion with glutaraldehyde--formaldehyde. Incubation for IDPase activity at pH 7.2 was performed with and without 0.5 mM levamisole, a potent inhibitor of alkaline phosphatase (AlkPase) (M Borgers, J Histochem Cytochem 21:812, 1973). Levamisole treatment of sections eliminated all reaction product in the brush border, but did not affect the IDPase activity the endoplasmic reticulum (ER). The ER appears as a basilar-luminal-oriented transcellular structure, suggesting a possible cellular transport route. This study supports and extends earlier observations made by others that suggest a transport role for the ER in these cells. It also emphasizes the value of thick section cytochemistry.

Progressive enzyme changes within anatomically defined segments of rabbit nephron: demonstration with a new technique.

The kidney is an extremely heterogeneous organ, with morphological, physiological, and metabolic changes occurring from segment to segment along each nephron. To determine the heterogeneity that might exist within discrete anatomical segments of rabbit nephron, we developed a technique for making quantitative enzyme assays in serial samples, about 100 micron long, along identified segments of the nephron. Results for three enzymes in proximal convoluted and straight tubules show that adenylate kinase, an enzyme of high-energy phosphate metabolism, gradually decreases along the S1 and S2 segments of the proximal tubule, with no abrupt changes. Fructose bisphosphatase, a gluconeogenic enzyme, is high along the major portion of the proximal tubule but plummets along the final millimeter of S3. Conversely, phosphofructokinase, a glycolytic enzyme, is very low along the proximal tubule but increases sharply within the final millimeter. These data underscore the biochemical heterogeneity of the nephron, illustrating the enzyme levels may change markedly even within anatomically defined regions. They also suggest the importance of further studies of this type and demonstrate a practical means for such studies.

Diabetic nephropathy: the modulating influence of glucose on transforming factor beta production.

Diabetes in now the commonest cause of renal failure in the western world. Furthermore the survival of diabetic patients requiring dialysis treatment for renal failure is far less than patients with renal failure secondary to all other diseases. It is therefore important to identify the factors that control the development of progressive renal disease to allow targeted therapeutic interventions which would have major implications both to patient well-being and also to the provision of health care world wide. In this review we discuss possible metabolic consequences of hyperglycemia and their role in the pathogenesis of diabetic nephropathy. We also focus on the involvement of the pro-fibrotic cytokine Transforming Growth Factor beta, and contrast its role in the pathogenesis of glomerular and tubulo-interstitial changes seen in diabetic nephropathy.

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.

Increased alpha 1B-adrenoreceptor mRNA levels in the rat kidney after thyroidectomy.

Oligonucleotide probes were designed to sequences of the rat alpha 1B- and alpha 2B-adrenergic receptor mRNA and used for in situ hybridization histochemistry on tissue sections of kidneys from control and thyroidectomized rats. Both alpha 1B- and alpha 2B-receptor mRNA labelling was demonstrated in proximal tubule cells in the outer stripe of the outer medulla, with tubular rays radiating into the cortex. Thyroidectomy induced a more than 4-fold increase in mRNA for the alpha 1B-receptor in the kidney, whereas no change in alpha 2B-receptor mRNA levels could be demonstrated in thyroidectomized rats as compared to control animals. The results suggest that thyroid hormone plays an important role in regulating expression of alpha 1B-receptors in renal tubule cells.

Preparation of precision-cut renal slices and renal proximal tubular fragments for evaluating segment-specific nephrotoxicity.

Previous research in animals and humans has demonstrated that many nephrotoxic chemicals induce selective injury within the kidney affecting either renal proximal straight (PST) or proximal convoluted (PCT) tubules. Selective injury has also been observed following in vitro nephrotoxicant exposure to precision-cut renal slices and isolated PCT and PST segments. These in vitro models provide a means of comparing and contrasting basic mechanistic differences which render these segments innately susceptible to nephrotoxicant injury. In this article, methods for preparing precision-cut slices and isolating PST and PCT segments will be reviewed.