Quantitative measurement of glucose-6-phosphate dehydrogenase in cortical fractions of the rabbit nephron

Summary A quantitative fluorimetric method is described for estimating the activity of glucose-6-phosphate dehydrogenase in isolated fractions of rabbit nephron from the superficial part of the renal cortex: macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. The mean activity in the macula densa region was 2.5×10^–18 mol/m³/min, which was about twice the mean activity of the proximal and distal tubular cells and four times that of the glomeruli. As glucose-6-phosphate dehydrogenase is located in the cytoplasm, the average cytoplasmic enzyme activity of the different tubular cells was calculated: macula densa activity was 4.0×10^–18 mol/m³/min whilst proximal tubular cells showed about a third, and distal tubular cells about a quarter of this activity.     

Quantitation of glucose-6-phosphate dehydrogenase activity in cortical fractions of the nephron in sodium-depleted and sodium-loaded rabbits

Summary Glucose-6-phosphate dehydrogenase activity was measured quantitatively in isolated cortical fractions of the nephron in sodium-depeleted and sodium-loaded rabbits. The samples consisted of isolated fractions of macula densa, proximal convoluted tubule, distal convoluted tubule and glomerulus. In sodium-depleted rabbits enzyme activity was identical to that of normal rabbits. In sodium-loaded rabbits a significant decrease in enzyme activity was found in the macula densa and proximal convoluted tubule. However, using conventional histochemical incubation methods semiquantitative estimation of glucose-6-phosphate dehydrogenase showed a slight decrease in enzyme activity in the macula densa and distal convoluted tubule, and a slight increase in the proximal convoluted tubule during sodiumdepletion. During sodium-load a pronounced decrease in enzyme activity was seen in the macula densa and distal convoluted tubule. These results show that semiquantitative histochemical evaluation of changes in enzyme activity is less reliable than the more precise quantitative method especially when there are only slight changes in enzyme activity. Only where there were marked changes in histochemical enzyme activity might the results of quantitative and semiquantitative methods be in accord.     

Histochemistry of the juxtaglomerular apparatus in the toad Bufo bufo

Summary An investigation regarding the question of whether there exists a macula densa as part of the juxtaglomerular apparatus in the kidney of amphibians has been carried out. With the aid of a histochemical reaction for glucose-6-phosphate dehydrogenase activity, the presence of a macula densa zone as a specialized part of the distal tubule in the toad Bufo bufo was demonstrated. The functional significance of the high glucose-6-phosphate dehydrogenase activity in the macula densa cells is discussed.     

Acute saline expansion increases nephron filtration and distal flow rate but maintains tubuloglomerular feedback responsiveness: role of adenosine A(1) receptors

Temporal adaptation of tubuloglomerular feedback (TGF) permits readjustment of the relationship of nephron filtration rate [single nephron glomerular filtration rate (SNGFR)] and early distal tubular flow rate (V(ED)) while maintaining TGF responsiveness. We used closed-loop assessment of TGF in hydropenia and after acute saline volume expansion (SE; 10% body wt over 1 h) to determine whether 1) temporal adaptation of TGF occurs, 2) adenosine A(1) receptors (A(1)R) mediate TGF responsiveness, and 3) inhibition of TGF affects SNGFR, V(ED), or urinary excretion under these conditions. SNGFR was evaluated in Fromter-Wistar rats by micropuncture in 1) early distal tubules (ambient flow at macula densa), 2) recollected from early distal tubules while 12 nl/min isotonic fluid was added to late proximal tubule (increased flow to macula densa), and 3) from proximal tubules of same nephrons (zero flow to macula densa). SE increased both ambient SNGFR and V(ED) compared with hydropenia, whereas TGF responsiveness (proximal-distal difference in SNGFR, distal SNGFR response to adding fluid to proximal tubule) was maintained, demonstrating TGF adaptation. A(1)R blockade completely inhibited TGF responsiveness during SE and made V(ED) more susceptible to perturbation in proximal tubular flow, but did not alter ambient SNGFR or V(ED). Greater urinary excretion of fluid and Na(+) with A(1)R blockade may reflect additional effects on the distal nephron in hydropenia and SE. In conclusion, A(1)R-independent mechanisms adjust SNGFR and V(ED) to higher values after SE, which facilitates fluid and Na(+) excretion. Concurrently, TGF adapts and stabilizes early distal delivery at the new setpoint in an A(1)R-dependent mechanism.     

Studies on the histochemistry of phosphatase enzymes in the juxtaglomerular complex

Summary The localisation of alkaline-, adenosine tri-, glucose-6- and acid phosphatase was studied in the juxtaglomerular complexes of rat, mouse and human kidneys. An alkaline-and adenosine triphosphatase active region was observed between the macula densa, Goormaghtigh cell group and in the interstitium of the latter. The adenosine triphosphatase activity extended into the lateral cell membranes of the macular cells and in properly incubated sections it did not appear among other distal tubular cells. The granular juxtaglomerular cells were ATP-ase negative. The cells of the human macula densa and the granular juxtaglomerular cells of the rat and mouse showed acid phosphatase activity. The glucose-6-phosphatase reaction, accomplished at acid and alkaline pH, was negative in the JG complex of all three species. The possible role of these enzymes in the function of the JG complex also has been discussed.     

The transition of the thick ascending limb of Henle's loop into the distal convoluted tubule in the nephron of the rat kidney

Summary Examination of serial semithin sections of rat kidney cortex and a subsequent electron microscopic study of selected areas revealed that the characteristic epithelium of the cortical part of the thick ascending limb of Henle extends for a varying distance beyond the macula densa. The transition from the relatively thin epithelium of the thick ascending limb at this site to the three -or even four-fold thicker epithelium of the convoluted part of the distal tubule is sharply defined and occurs without the interposition of an intermediate cell type.The position of the macula densa at the end but still clearly within the ascending limb of Henle's loop is functionally interpreted to guarantee the separation of the sensor point macula densa from disturbing influences which might arise from the secretory activity of the subsequent tubular portion.Investigations supported by the Deutsche Forschungsgemeinschaft. The skillful technical assistance of Mrs. Saliha Sabanovic is gratefully acknowledged     

Site-specific Expression of IQGAP1, a Key Mediator of Cytoskeleton, in Mouse Renal Tubules

IQGAP1 is a multifunctional junction molecule that is involved in cell migration, proliferation, differentiation, cell polarity, and cell–cell adhesion. It is highly expressed in the kidney and has recently been identified in the glomerular basement membrane as a nephrin-associated protein. However, the distribution of IQGAP1 in renal tubular epithelial cells is unknown. We performed confocal microscopic studies to localize IQGAP1 in each nephron segment using dual immunofluorescence staining with various antibodies against segment-specific markers. We found that IQGAP1 was strongly expressed in the distal convoluted tubule (DCT), collecting duct, and macula densa and moderately in the thick ascending limb and proximal tubule. In the DCT, the IQGAP1–F-actin complex forms a comb-like structure with multiple parallel spikes sitting on the basal membrane. In the macula densa cells, IQGAP1 is strongly expressed in the apical membrane, whereas in type A intercalated cells, IQGAP1 is expressed in the basolateral membrane, where it colocalizes with anion exchanger 1, and in principal cells, it is diffusely expressed. In conclusion, we showed the expression and subcellular localization of IQGAP1 in various nephron segments. The site-specific expression pattern of this potent modulator of multiple biological pathways in the renal tubules suggests that IQGAP1 may have multiple important roles in various renal functions. (J Histochem Cytochem 56:659–666, 2008)     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [35S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Ultrastructural organization of the transition from the distal nephron to the collecting duct in the desert rodent Psammomys obesus

Summary The transition from the nephron to the collecting duct is formed by three tubular segments (convoluted part of the distal tubule, connecting tubule, cortical collecting duct), which in the desert rodent, Psammomys obesus, transform gradually from one segment to the next, due to intermingling of their different cell types.The convoluted part of the distal tubule (DTC) starts abruptly, shortly beyond the macula densa and initially is homogeneously composed of characteristic DTC-cells. Subsequently, the DTC-cells intermingle with intercalated cells. The first appearance of the connecting-tubule cell, which gradually replaces the DTC-cell, is regarded as the beginning of the connecting tubule. The major portion of the connecting tubule is lined by connecting-tubule cells and intercalated cells. The first appearance of the principal cell between them defines the beginning of the cortical collecting duct, which in the medullary ray is lined by principal and intercalated cells only.Each cell type is described in detail and discussed in relation to the assumed function of the tubular segments.Interspecies differences in the cellular composition of the transitional zone from the nephron to the collecting duct are discussed in relation to the different organization of the collecting duct system.     

The normal juxtaglomerular apparatus in the human kidney. A morphological study.

Out of 49 serially studied juxtaglomerular apparatuses, 6 typical variants from two normal human kidneys were reconstructed graphically. The agranular Goormaghtigh cells filled the entire space between the macula densa, the afferent and the efferent arterioles and the glomerular mesangium. The Goormaghtigh cells were always in direct contact with all the other structures. They also invariably continued into the glomerular mesangium. The distal tubule regularly showed widening in the macula densa segment and, at this level, there was considerable variation in the shape of the distal tubule. Direct contact between the macula densa and the hilar arterioles was not always present, the area of contact was usually greater with the afferent than with the efferent arteriole.     

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.     

Preparative free flow electrophoresis for the isolation of two populations of proximal cells from the rabbit kidney

High voltage free flow electrophoresis is a carrier-free method used for analytical and preparative cell separation, based on charge surface properties of cells. Two cell populations from the proximal tubule of the rabbit kidney were isolated by free flow electrophoresis from a suspension of pure proximal cells. This single-cell suspension was obtained through an original method by the combination of a Ca-binder action and gentle mechanical treatment associated with several shifting steps, on a pure suspension of isolated proximal tubules. Before the electrophoretic separation, the proximal cell origin was confirmed by enzymatic marker measurements, and the metabolic capacity was assessed by the cell respiratory activity. The isolated cells were very poor in distal tubule marker enzymes and were enriched in proximal tubule marker enzymes. Respiratory measurement showed a high cell metabolic capacity. After the electrophoretic separation, the origin of the cell populations was assessed by measuring specific marker enzymes. The cells in the slow-moving electrophoresis fractions had a high gamma-glutamyl transpeptidase activity and a low glucose-6-phosphatase activity. The fast moving cells showed a high glucose-6-phosphatase content and a poor gamma-glutamyl transpeptidase activity. Cells isolated by free flow electrophoresis were shown to possess long microvilli. This new methodology, allowed for the first time, the separation of a fast-moving cell population originating from the convoluted portion of the proximal tubule and a slow-moving cell population originating from the straight part of the proximal tubule of the rabbit kidney.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Summary Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [³⁵S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Are there specialized junctions in the pars maculata of the distal tubule?

Summary In the present study the tight junctions at the macula densa were compared to those of the adjacent straight and convoluted segments of the distal tubule using freeze fracturing and thin sectioning techniques. Only insignificant differences were found in the number of strands and the apico-basal depth of the tight junctions in the three distal tubular segments of rat, dog and tree shrew. In experiments with horseradish peroxidase on mice and tree shrews, the tracer did not penetrate the apical junctions in any of the distal tubular segments. Our findings do not support the concept of considerably higher permeability of the tight junctions at the macula densa, as previously reported. Gap junctions were never observed in the distal nephron. The present results suggest that the glomerulo-tubular feedback is more likely to be mediated by transcellular resorption of solutes than by passive diffusion through a leaky paracellular shunt pathway.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Fluid waves in renal tubules.

Autoregulation of renal blood flow is ineffective when arterial pressure perturbations occur at frequencies above 0.05 Hz. To determine whether wave propagation velocity to the macula densa is rate limiting, we estimated compliances of the proximal tubule and the loop of Henle, and used these values in a model of pressure and flow as functions of time and distance in the nephron. Compliances were estimated from measurements of pressures and flows in early proximal, late proximal, and early distal tubules in rats under normal and Ringer-loaded conditions. A model of steady pressure and flow in a compliant, reabsorbing tubule was fitted to these results. The transient model was a set of nonlinear, hyperbolic partial differential equations with split, nonlinear boundary conditions, and was solved with finite difference methods. The loop of Henle compliance was larger than the proximal tubule compliance, and impulses in glomerular filtration rate were attenuated in magnitude and delayed in time in the loop of Henle. Simulated step forcings revealed a similar pattern. Periodic variations of GFR were attenuated at frequencies greater than 0.05 Hz, and there was a delay of 5 s between variations in GFR and macula densa flow rate. The high compliance of the loop slows wave propagation to the macular densa and reduces the amplitude of high frequency waves originating in the glomerulus, but other parts of the signal chain also contribute to the slow response of macula densa feedback.     

Atrial natriuretic peptide elevates cGMP contents in glomeruli and in distal tubules of rat kidney

Effect of synthetic rat atrial natriuretic peptide (1-28) (ANP) on the cGMP content was studied using defined nephron segments of rat kidney. ANP elevates cGMP contents in glomeruli in a concentration and time-dependent manner. The increase of cGMP was observed in glomeruli, distal convoluted tubule (DCT) and cortical collecting tubule (CCT) (delta %; 279 +/- 35, 148 +/- 10 and 152 +/- 18, respectively), and no effect was observed in proximal convoluted (PCT) and straight tubule (PST). These results suggest that ANP may act directly on the tubular cells as well as glomeruli. In glomeruli, effects of ANP and carbamylcholine on cGMP contents were additive suggesting that these two agents may act on different receptors. Angiotensin II and norepinephrine failed to affect the ANP-induced cGMP production in the glomeruli.     

Ultrastructural evaluation of the effect of endosulfan on mice kidney

In this study, the effect of the endosulfan on mice kidney was investigated at ultrastructural level. Moreover, biochemical analyses (G6PD, CAT, SOD, GSH and MDA) were determined in supernatant of kidney tissue. Endosulfan (13mg/kg/day body weight) was administered orally to mices via intragastric-during 10 days. The presence of mitochondrial degeneration in cytoplasm of proximal convoluted tubule cells were a striking feature. Furthermore, there was lipofuscin granules and membranous structures in some of proximal convoluted tubule cells. In some glomeruli, ultrastructural changes such as fusion in pedicels and focal thickening at glomerular basal membrane were seen. There were cytoplasmic bulges in some distal convoluted tubule cells. The biochemical results of the experimental group were significant when compared to the control. The effect of the endosulfan was mainly on the proximal convoluted tubule cells. Morever, the other parts of the nephron were effected. Thus, this degeneration in kidney may be thought that oxidative stress may play a role to the mediator in changing configuration of cell membrane and seem to account for the morphologic alteration of kidney.     

A morphometric study of cellular autophagy including diurnal variations in kidney tubules of normal rats

Cellular autophagy in convoluted tubules of kidney was studied in 24 rats, killed in pairs at constant time intervals during one diurnal cycle, by (a) morphometric evaluation of tubular cells by the point-counting method in randomly sampled micrographs, and (b) selective search for autophagic vacuoles (AV) directly on the electron microscopy screen. The total area of tubular cells recorded in the electron microscopy sections was 93 X 10(-4) mum2. Since the distal convoluted tubules, covering about 12% of the whole tubulocellular area, contained only 3-4% of all AV, they were omitted from the main calculations. The number of AV per area unit and the total amount of segregated material showed a distinct diurnal rhythm, synchronous for the different types of AV which were distinguished from each other according to their contents. The minimum was found during the night, the maximum during the day. This rhythm appears similar to that described elsewhere in liver cells. The mean segregated fractions were calculated from the relation of segregated to nonsegregated material in proximal convoluted tubular cells. The segregated fraction of the mitochondria was 4.4 X 10(-4). This value could account for the degradation of all mitochondria in a cell within 15 days, i.e., the upper limit of the lifetime of mitochondrial DNA in the cortex of the kidney, if one assumes that a mitochondrion is destroyed within 10 min after being segregated. The degregated fraction of microbodies was 11.7 X 10(-4). This suggests a shorter lifetime of these organelles. It is concluded that cellular autophagy plays a significant role in the turnover of cytoplasmic constituents, including the membranes of the endoplasmic reticulum.     

ATP as a mediator of macula densa cell signalling

Within each nephro-vascular unit, the tubule returns to the vicinity of its own glomerulus. At this site, there are specialised tubular cells, the macula densa cells, which sense changes in tubular fluid composition and transmit information to the glomerular arterioles resulting in alterations in glomerular filtration rate and blood flow. Work over the last few years has characterised the mechanisms that lead to the detection of changes in luminal sodium chloride and osmolality by the macula densa cells. These cells are true “sensor cells” since intracellular ion concentrations and membrane potential reflect the level of luminal sodium chloride concentration. An unresolved question has been the nature of the signalling molecule(s) released by the macula densa cells. Currently, there is evidence that macula densa cells produce nitric oxide via neuronal nitric oxide synthase (nNOS) and prostaglandin E₂ (PGE₂) through cyclooxygenase 2 (COX 2)-microsomal prostaglandin E synthase (mPGES). However, both of these signalling molecules play a role in modulating or regulating the macula-tubuloglomerular feedback system. Direct macula densa signalling appears to involve the release of ATP across the basolateral membrane through a maxi-anion channel in response to an increase in luminal sodium chloride concentration. ATP that is released by macula densa cells may directly activate P2 receptors on adjacent mesangial cells and afferent arteriolar smooth muscle cells, or the ATP may be converted to adenosine. However, the critical step in signalling would appear to be the regulated release of ATP across the basolateral membrane of macula densa cells.