Localization of immunoreactive vitamin D-dependent calcium binding protein in chick nephron.

Immunoreactive vitamin D-dependent calcium binding protein (iCaBP) has been localized in the chicken nephron, using microdissection and subsequent radioimmunoassay (RIA). Six to ten weeks old vitamin D-replete chicks were sacrificed and the kidneys removed. The various segments of the nephron, i.e. the proximal convoluted tubule (PCT), the thick part of the hairpin loops (thick loops), the thin part of the hairpin loops (thin loops), the distal convoluted tubules (DCT) and the cortical collecting tubules (CCT) were microdissected and grouped according to their morphological characteristics. Because the transition between DCT and CCT was not evident, these two segments were gathered together. The samples were assayed for iCaBP by a microradioimmunoassay, using antisera against the vitamin D-dependent chicken intestinal CaBP. iCaBP was localized in the thin loops and in the DCT-CCT segments (24.7 +/- 5 and 20.8 +/- 3 ng/mg protein, respectively). No iCaBP was detected either in the PCT using up to 32 micrograms protein, or in fragments of medullary papilla, assaying up to 4 micrograms protein.

Immunocytochemical localization of vitamin D-dependent calcium binding protein in mammalian nephron.

Immunoreactive calcium binding protein (iCaBP) has been localized in the rat nephron using the unlabeled antibody peroxidase-antiperoxidase immunocytochemical technique. Kidneys from normal young adult, vitamin D-deficient, and 12 day old rats were prepared by either freeze-substitution or 1% glutaraldehyde-Bouin's fixation. CaBP was localized with rabbit antiserum to chicken vitamin D-dependent intestinal CaBP. iCaBP was found specifically in the distal convoluted tubules (DCT); however, not all cells of the DCT were positive. In adult nephrons, a few scattered cells apparently belonging to the collecting tubules were positive. In the neonatal nephrons, there was also localization of iCaBP to the thick limb of Henle, suggesting a difference in the regulation of intracellular calcium during maturation. Proximal tubules, renal corpuscles, macula densa, and thin limbs of Henle gave no specific localization of iCaBP. These results present for the first time the localization of iCaBP in the mammalian nephron both in the neonate and in the young adult.

Anatomical relationship between kallikrein-containing tubules and the juxtaglomerular apparatus in the human kidney.

Current evidence suggests a functional and biochemical link between the renin and the kallikrein systems. The purpose of this work was to study the localization of kallikrein along the human nephron to elucidate whether there exists an anatomical base for such interrelation. Serial sections of human kidney tissue were stained by immunocytochemical methods with antisera against kallikrein. Kallikrein immunostaining was observed exclusively in segments of the distal nephron lying in the cortical labyrinths and forming arcades in its distal portion. Consistently the tubules containing kallikrein established a close anatomical relationship with the afferent arteriole of the juxtaglomerular apparatus providing an anatomical base for an interaction between the renin and kallikrein systems in the human kidney.

IgA nephropathy in Pakistan.

A light, electron and immunofluorescence microscopy study was performed on 102 consecutive patients on whom suitable percutaneous renal biopsies were obtained. In this selected group of patients primary IgA glomerulonephritis was diagnosed in 6 (5.9%) cases. On light microscopy the glomerular lesions were predominantly focal (WHO class III) and diffuse mesangial proliferative glomerulonephritis (Class IV). The mesangial deposits showed high association with IgM deposits and presence of early complement components (C1q, C4) indicative of both classical and alternative pathways of C3 activation in our patients. The high incidence of nephrotic syndrome with microhaematuria (5 cases) is due to patient selection when compared to other studies. This study shows the existence of IgA nephropathy in Pakistan and larger number of cases need to be investigated to determine the true prevalence of this disease and its clinical manifestations and importance in Pakistan.

Electrolyte transport in a central core model of the renal medulla.

Transport of Na+, K+, Cl-, urea, and water is described in a central core model of the renal medulla. Equations for mass balance, Poiseuille flow, and the Nernst-Planck equation describe the continuous behavior of the system along the medullary axis and along the distal nephron; the Kedem and Katchalsky phenomenology describes passive transmural transport; active transmural transport obeys Michaelis-Menten kinetics. Numerical solution of the differential equations shows that to a close approximation any combination of active Na+ and active Cl- transport can generate the same concentration profiles but will generate very different potential profiles, and consequently, very different K+ absorption in thick ascending limb of Henle's loop. If a net transport stoichiometry of 2 Cl- ions to 1 Na+ ion is selected for the pumps, an active Cl- transport rate of approximately 10,000 peq.s-1.cm-2 gives K+ and Na+ concentrations in early distal nephron and a medullary osmolality profile in reasonable agreement with experimental data.

Aldosterone binding sites along nephron of Xenopus and rabbit.

Distal segment of several amphibians exhibits aldosterone-modulated ion transport properties. On the other hand, A6 cells, derived from Xenopus laevis (XL) kidney, are aldosterone sensitive. We examined the distribution of aldosterone binding sites in isolated tubules of XL compared with rabbit. After incubation with 2 nM [3H]aldosterone, microdissected tubular segments from proximal (PT), distal straight segment (DST), and flask cell collecting (CT) tubules from XL and from rabbit cortical thick ascending limb (CTAL), connecting (CNT), and collecting (CCD) tubules were processed for dry film autoradiography. In XL, specific nuclear labeling of type I (mineralocorticoid) sites was restricted to DST. Labeling of type II (glucocorticoid) sites was present all along the tubule. No specific cytoplasmic labeling was observed, except for type II sites in PT. In the rabbit, aldosterone binds to both type I and type II sites in the three tubular segments studied. In these segments, the binding was about fourfold higher than in DST of XL. These results bring direct evidence in designating the distal tubule of amphibians as a target epithelium for aldosterone. In addition, they suggest that A6 cell line may derive from DST of the Xenopus nephron.

B-cell markers in the human kidney.

This report describes the reactivity of a panel of 89 monoclonal antibodies with the normal human kidney. The antibodies were all submitted to the B-cell panel of the Third Leukocyte Differentiation Workshop. Several different patterns of staining of renal structures were identified. Most antibodies clustered in defined CD groups showed the same pattern of reactivity in the kidney, but some, notably CD19 antibodies, showed heterogeneity of binding to the kidney. These findings are important for the use of monoclonal antibodies to define phenotype in cell culture and immunohistology experiments.

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron.

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.

Localization of Tamm-Horsfall glycoprotein in the human kidney using immuno-fluorescence and immuno-electron microscopical techniques.

Small pieces of tissue obtained from apparently normal areas of four surgically removed adult human kidneys were used in the present study. The results obtained by immuno-fluorescence and immuno-electron microscopical techniques show that Tamm-Horsfall glycoprotein (THP) is present in the thick ascending limbs of the loops of Henle and the distal convoluted tubules. Within the cells concerned, the protein is associated with the luminal, lateral as well as basal, plasma membranes and their infoldings. The cells of the macula densa are completely negative as are those of proximal convoluted tubules, glomeruli and collecting ducts. The possible significance of these findings in relation to the process of urine dilution in the nephron is discussed.

Changes in single nephron renin release are mediated by tubular fluid flow rate.

In vivo renin release from single nephrons microperfused with artificial tubular fluid was studied in recollection experiments. Renin concentration was measured in systemic arterial plasma (A-PRC) and in either early proximal tubular fluid (TFR), or in plasma from the welling point of the efferent arteriole (SV-PRC) belonging to the microperfused nephron. Micropuncture collections were controlled to maintain the proximal intratubular pressure equal to the control free-flow pressure. Increasing the Henle loop flow rate from 12 to 18, or to 34 nl/min, was followed by a significant decrease in TFR, while reducing the flow rate from 12 to 6 nl/min caused a significant increase in TFR. Similarly, increasing the Henle loop free-flow rate by 6 to 8 nl/min depressed SV-PRC, while reducing the flow rate by fluid aspiration at 8 to 10 nl/min caused a significant increase in SV-PRC. These data indicate: that renin release, to a significant part at least, occurs into the vascular lumen of the afferent arteriole: that modest changes in early distal flow rate may control renin release from the JG-cells; and that increasing the flow rate depresses renin release, and vice versa. It is suggested that the renin system is directly involved in an additional TGF mechanism controlling postglomerular vascular resistances.

Nephron functional heterogeneity in the postobstructive kidney.

The purpose of this study was to determine the distribution of nephron filtration rates between superficial and juxtamedullary nephrons (S/J ratio) in kidneys studied immediately after relief of 24 hr total obstruction (acute) and after relief of prolonged partial obstruction (chronic). Injection of 14C-ferrocyanide and microdissection (modified Hanssen's technique) was used to provide an index of superficial and deep nephron glomerular filtration rate (GFR), and standard clearance determinations were done. In normal kidneys S/J ratio of 14C content (nephron GFR) was 0.73 plus or minus 0.03, a value similar to those obtained by other workers. After relief of acute obstruction, nephron GFR was too low for accurate measurement in 22% of superficial and 13% of deep nephrons. The mean S/J ratio of 14C content was similar to control, being 0.67 plus or minus 0.07, with only three of seven kidney showing loss of the normal S/J ratio. Since redistribution of nephron GFR was an inconsistent finding, while marked diuresis and natriuresis occurred in all rats, it appears that redistribution of nephron GFR is not an important factor in the phenomenon of postobstructive diuresis. After relief of chronic obstruction, diuresis and natriuresis were less marked but the mean S/J ratio of 14C content was 0.95 plus or minus 0.11, and in the majority of kidneys (six of eight), there was loss of distinction between superficial and deep nephron GFR. This redistribution of nephron function after relief of chronic, rather than acute, obstruction may be due to the more severe structural damage to the renal medulla of such kidneys and, although not responsible for postobstructive diuresis, it may be important in the diminished capacity of the chronically hydronephrotic kidney to conserve salt and water.

Localization of collagen types IV and V, laminin, and heparan sulfate proteoglycan to the basal lamina of kidney epithelial cells in transfilter metanephric culture.

Mouse metanephric mesenchyme and embryonic spinal cord were cultured on opposite sides of a filter membrane. This resulted in formation of prenephronic vesicles after 36 hours in culture, S-shaped bodies after 2-3 days, and glomeruli and tubules after 4-7 days. The glomeruli consisted of an arborizing tuft of podocytes lying on a basement membrane without vascularization or a mesangial ingrowth. We have used antibodies to study the molecular composition of the nephron basement membrane at each stage of development. By immunofluorescence light microscopy, collagen Types IV and V, laminin, and heparin sulfate proteoglycan were expressed within the pericellular/intercellular matrix at the onset of morphologic differentiation. The molecules were organized into a linear basement membrane associated with epithelial cells during the prenephronic vesicle, S-shaped body, and glomerulus and tubule stages of development.

Avian and mammalian vitamin D-dependent calcium binding protein in reptilian nephron.

A calcium binding protein (CaBP) with an apparent relative molecular weight of 28,000 was localized in the kidney of Anolis carolinensis with antisera directed against vitamin D-dependent CaBP from either rat kidney (RRCaBP) or chick intestine (CICaBP). When extracts of female saurian kidneys were fractionated by gel filtration on Sephadex G-100, both calcium binding activity, as measured by the chelex resin assay, and CaBP immunoreactivity, as measured by radioimmunoassay for RRCaBP, were observed near the 28,000-Da region similar to RRCaBP and CICaBP. Utilization of the immunoblot technique following SDS-polyacrylamide slab gel electrophoresis resulted in cross-reactivity for CaBP in the Mr 28,000 region for both rat and anolian kidneys. Immunoreactive CaBP was localized in the nephron using the unlabeled antibody peroxidase-antiperoxidase technique. Distal tubules (DT) gave a strong, specific reaction with either antiserum, but not all cells of the DT reacted with equal intensity. Cells in the transition zone between the DT and the terminal tubules, and cells in the collecting tubules, were occasionally positive. Renal corpuscles, proximal tubules, and thin segments gave no specific localization of CaBP. The sexual segment of male kidneys was also negative. The results indicate that a calcium binding protein with an apparent molecular weight of 28K is highly conserved during vertebrate evolution, and thus may have widespread but specific functional significance. The localization of CaBP to the DT suggests that this protein may be involved in the selective reabsorption and/or excretion of calcium.

Detection of specific mRNAs in single nephron segments by use of the polymerase chain reaction.

We have developed a procedure to detect specific mRNAs in single renal nephron segments. This approach combines microdissection, reverse transcription (RT) of the target mRNA, and amplification of the resulting cDNA using the polymerase chain reaction (PCR). After microdissection, the sample is placed in a tube where it is permeabilized and where all reactions are performed directly without the need for isolation of the RNA. Our model target was the mRNA for aldose reductase. This enzyme catalyzes the conversion of glucose to sorbitol. Its expression is modulated by changes in extracellular osmolality in the renal medulla. RT-PCR of inner medullary collecting duct (1 mm) and glomeruli (6-10) yielded a product of the predicted length (670 base pairs) defined by the PCR primers. Its identity was confirmed by a specific oligonucleotide probe that differed from the primers. RT-PCR of proximal tubules (1 mm) resulted in no aldose reductase-specific amplification product. RT-PCR is generally applicable for measuring specific gene expression in single nephron segments or small numbers of cultured cells. Utility, limitations, and refinements of this approach are discussed.

Evolution of experimental mild and severe chronic renal failure in the rat. Mathematical models.

Two degrees of renal insufficiency were obtained in female Wistar rats, one mild (M) and the other severe (S) by surgical resection of 2/3 or 3/4 of one kidney, followed by contralateral nephrectomy. The serum creatinine concentration (Cr) s was evaluated as an index of renal function and followed in the two mentioned groups under a normal protein diet and in "S" also under a low protein diet. The evolution was assessed by two functions of time: 1/(Cr) s and log e (Cr) s. Several differences between M and S were detected. In M a smaller initial and maximal increase in (Cr) s than in S followed by a plateau was observed; in S a decrease in (Cr) s posterior to the initial increase was detected attributed to hyperfiltration followed by a sustained increment. The lowering of protein content in the diet caused a decrement in (Cr) s. In both groups a better adjustment with the logarithmic than with the inverse function was obtained, although poorer in S rats owing to a larger variability. In order to look for a mathematical link between inulin clearance (Cl In) and (Cr) s, the relationship between Cl In vs. 1/(Cr) s, and log e Cl In vs. log e (Cr) s was studied in normal and in rats in renal insufficiency. By regression analysis we found a good adjustment with both functions from 0.58 to 1.60 mg % (Cr) s.

Measurement of Na-K-ATPase-mediated rubidium influx in single segments of rat nephron.

To determine the functioning rate of Na-K-ATPase in the rat nephron, a micromethod was developed to measure the rate of rubidium uptake in single nephron segments microdissected from collagenase-treated kidneys. Because the hydrolytic activity of Na-K-ATPase displayed the same apparent affinity for K and Rb ions, whereas the Vmax elicited by K was higher than that in the presence of Rb, experiments were performed in the presence of cold Rb plus 86Rb. Before the assay, tubules were preincubated for 10 min at 37 degrees C to restore the normal transmembrane cation gradients. 86Rb uptake was measured after washing out extracellular cations by rinsing the tubules in ice-cold choline chloride solution containing Ba2+. Rb uptake increased quasi-linearly as a function of incubation time up to 30 s in the thick ascending limb, 1 min in the proximal convoluted tubule, and 5 min in the collecting tubule, and reached an equilibrium after 5-30 min. The initial rates of Rb uptake increased in a saturable fashion as Rb concentration in the medium rose from 0.25 to 5 mM. In medullary thick ascending limb, the initial rate of Rb uptake was inhibited by greater than 90% by 2.5 mM ouabain and by 10(-5) M of the metabolic inhibitor carbonyl cyanide trifluoromethoxyphenylhydrazone. Correlation of Na-K-ATPase hydrolytic activity at Vmax and initial rates of ouabain-sensitive Rb uptake in the successive segments of nephron indicates that in intact cells the pump works at approximately 20-30% of its Vmax. Increasing intracellular Na concentration by tubule preincubation in a Rb- and K-free medium increased the initial rates of Rb intake up to the Vmax of the hydrolytic activity of the pump.

Alpha2-adrenoceptors and cellular cAMP levels in single nephron segments from the rat.

A functional role for the numerically predominant renal alpha2-adrenoceptors, which in other tissues inhibit adenylate cyclase, remains undefined. We therefore examined the effect of alpha2-adrenoceptor stimulation with (-)-epinephrine (E) on cell cAMP content in the isolated proximal convoluted tubule (PCT), medullary and cortical thick ascending limb of Henle, and collecting tubule (MTAL, CTAL, MCT, and CCT, respectively). Parathyroid hormone (1-34 PTH), in PCT or CTAL, or arginine vasopressin (AVP), in MTAL, CTAL, MCT, or CCT, was used to activate adenylate cyclase in intact cells from these microdissected nephron segments in the presence of 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor) and propranolol. Alpha2-Adrenoceptors were activated using varying concentrations of E (37 degrees C, 2 min). Alpha2-Adrenoceptor activation with E (5 X 10(-7) to 5 X 10(-6) M) suppressed cellular cAMP stimulation by PTH by 35% in PCT and stimulation by AVP in CCT by 50%. This suppression by E in PCT and CCT was inhibited by 5 X 10(-6) M yohimbine or 5 X 10(-7) M phentolamine but not by 5 X 10(-6) M prazosin. E also suppressed cAMP stimulated by AVP in MCT, but it did not suppress the PTH-or AVP-stimulated increase in cellular cAMP in CTAL and MTAL. These studies show that there are alpha2-adrenoceptors in the rat nephron. Activation of these alpha 2-adrenoceptors can inhibit cAMP formation stimulated by PTH in PCT and by AVP in the CCT and MCT but not in the CTAL and MTAL. A pathophysiological role of altered regulation of these receptors is yet to be described.

Lectin- and immunohistochemical studies on acquired cystic kidney and associated renal cell carcinoma.

Nine specimens of acquired cystic kidney were studied by means of lectin- and immunohistochemistry. The cysts appeared to originate from any part of the nephron, but mostly from the proximal tubule. The cause of multicyst formation is still unclear, but the results of this study suggested that obstruction of the lower nephron might play an important role. Many microscopic carcinomas or dysplastic epithelial cells were observed in the cyst wall. All these carcinomas originated from the proximal tubule, except for one which was of Bellini duct origin and showed papillary growth. The reasons why almost all of the carcinomas originated from the proximal tubule were: 1) the majority of the cysts originated from the proximal tubule, and 2) dysplastic epithelial cells in the cysts originating from the proximal tubules were more frequent in number than those in cysts originating from other parts of the nephron. Histological observation showed that stimulation due to oxalate crystals in the proximal cysts was one of the causes of dysplastic epithelial cell hyperplasia.