Cortical Proximal Tubules Research Articles

Effect of thyroid hormone on gentamicin accumulation in rat proximal tubule lysosomes

T4 decreases gentamicin nephrotoxicity, but the mechanism is unknown. This study investigated whether T4 affects renal processing of gentamicin by examining renal cortical gentamicin accumulation and proximal tubular lysosomal volume after 48 h of gentamicin (30 mg/kg twice daily) in rats (GT4) that had previously received 10 days of T4 (10 micrograms/100 g body wt). The pair-fed control group received only gentamicin. A T4-treated control group and an untreated control group were also pair fed and studied. At the end of the 12-day study the four groups did not differ in food intake, body weight, urine volume, urinary Na+ excretion, or urinary K+ excretion. Plasma creatinine, inulin clearance, and serum clearance of gentamicin were not different among the groups. Gentamicin accumulation was higher in G vs. GT4 (2.1 +/- 0.1 vs. 1.7 +/- 0.1 micrograms/mg protein, P less than 0.01). Blinded, computer-assisted electron microscopic analysis of outer cortical proximal tubules showed that lysosomes occupied a significantly smaller fraction of the cell volume in GT4 (8.1 +/- 0.5 vs. 11.6 +/- 1.1%, P less than 0.02). Thyroxine had no effect on lysosomal volume in non-gentamicin-treated rats. Except for scatter foci of apical vacuolization, proximal tubular cells of both groups were otherwise normal. The results show that chronic T4 administration decreases renal tubular accumulation of gentamicin, and this effect may in part explain its protective effect against gentamicin nephrotoxicity.

Na, K‐ATPase: Comparison of the cellular localization of α‐subunit mRNA and polypeptide in mouse cerebellum, retina, and kidney

A clone encoding mouse brain Na,K-ATPase alpha-subunit was isolated from a mouse brain lambda gt11 cDNA library by using antisera to mouse and bovine brain alpha-subunit. A comparison of the nucleotide sequence of this clone with published sequences of rat brain alpha-subunit isoform clones showed it to be most similar to rat brain alpha 1. An RNA antisense probe prepared from the cDNA insert of the mouse clone detected a single mRNA of approximately 4.5 kb in Northern blots of mouse brain and kidney RNAs. This probe hybridized only to an alpha 1-cDNA insert from rat brain under high stringency conditions on Northern blots. The RNA antisense probe was used for in situ hybridization to sections of mouse kidney, cerebellum, and retina, and the cellular distribution of alpha-subunit mRNA (alpha-mRNA) was compared with that of alpha-subunit polypeptide (alpha-subunit) detected by immunofluorescence in similar sections. In kidney, alpha-mRNA distribution closely paralleled that of the polypeptide with abundant expression in ascending thick limbs and cortical distal tubules and weaker labeling in cortical proximal tubules. The co-distribution of alpha-mRNA and polypeptide in kidney where Na,K-ATPase localization is well established is consistent with the specificity of these probes. In the retina, prominent labeling with both probes was seen in photoreceptor inner segments, inner nuclear layer, and ganglion cell bodies. Plexiform layers and optic fibers expressed abundant alpha-subunit but little mRNA. Light labeling for both was seen in the outer nuclear layer. In cerebellum, alpha-mRNA and alpha-subunit were associated with soma of granule cells, basket cells, and stellate cells. Glomeruli and basket terminals contained abundant alpha-subunit but exhibited little reactivity with the riboprobe. In Purkinje cell bodies, in contrast, the antibody used to identify the cDNA clone did not resolve significant polypeptide in the somal plasmalemma despite abundant somal mRNA expression. Comparison of distribution of the two probes in cerebellum and retina indicates that message accumulation is primarily in cell bodies, while alpha-subunit epitopes may be co-expressed in cell bodies and/or transported to distant sites in cell-specific patterns.

Axial heterogeneity of rabbit proximal tubule luminal H+ and basolateral HCO3- transport

The present in vitro microperfusion study examined whether the rates of the apical membrane Na+-H+ antiporter and basolateral membrane Na(HCO3)3 symporter vary along the length of the proximal tubule. Initial proximal convoluted tubules (PCT obtained within 0.5 mm from the glomerulus), mid-PCT (PCT without glomerular attachments) and cortical proximal straight tubules were examined. The rate of either the apical or basolateral membrane acidification mechanism was measured from the initial rate of change of intracellular pH after a change in either the luminal or bathing solution. Intracellular pH was measured fluorometrically using the pH-sensitive dye (2',7')-bis(carboxyethyl)-(5,6)-carboxyfluorescein. The rate of bicarbonate exit across the basolateral membrane was examined by imposing either a sodium or bicarbonate gradient. There was no difference between initial and mid-PCT, but the rate of change in cell pH was 30% slower in PST in both series. The rate of sodium-dependent apical proton secretion was examined by changing the sodium concentration in the lumen. There was no difference in sodium-dependent apical proton secretion in initial vs. mid-PCT, but the rate fell by 70% in the proximal straight tubule (PST). These differences were not due to a difference in buffer capacity in these segments. These data are consistent with a homogeneous rate of apical Na+-H+ antiporter and basolateral Na(HCO3)3 activity along the rabbit PCT, but a lower rate in the PST.

Effect of cisplatin on proximal straight tubule transport of divalent cations in the rabbit.

Clearance and in vitro microperfusion studies were performed in rabbits to determine the effect of cisplatin on proximal straight tubule transport of calcium and magnesium. Rabbits were injected with cisplatin (2.5 mg/kg i.p. once weekly) for 3 weeks, whereas control rabbits received normal saline solution which served as a diluent for cisplatin. In 5 rabbits, 24-hour clearance studies were performed with the aid of a metabolic cage. Following cisplatin treatment, fractional excretion of magnesium rose significantly (73.3 +/- 11.5 vs. 111.4 +/- 17.5%). Glomerular filtration rate fell with cisplatin treatment (4.05 +/- 0.76 vs. 2.81 +/- 28 ml/min). There was no difference in fractional excretion of calcium (26.3 +/- 9.5 vs. 22.7 +/- 3.2%). The cortical and juxtamedullary proximal straight tubules were perfused in vitro. Net volume absorption was the same in the control and cisplatin-treated rabbits. However, there was a significant reduction in JCa (cortical 0.57 +/- 0.10 vs. -0.10 +/- 0.12 pmol/min/mm; juxtamedullary 0.96 +/- 0.17 vs. 0.31 +/- 0.37 pmol/min/mm) and JMg (cortical 0.43 +/- 0.08 vs. -0.15 +/- 0.07 pmol/min/mm; juxtamedullary 0.40 +/- 0.27 vs. -0.30 +/- 0.28 pmol/min/mm). In contrast to chronic administration, acute addition of cisplatin into the bath had no effect on JCa and JMg in the cortical and juxtamedullary proximal straight tubules. These data indicate that chronic but not acute cisplatin treatment depresses the transport of calcium and magnesium in the cortical and juxtamedullary nephrons of the proximal straight tubule of the rabbit.

Structural and functional development of outer versus inner cortical proximal tubules.

The concept of centrifugal renal development is based on renal embryogenesis. It implies a relationship between nephron age and nephron position along a cortical to medullary axis. In common usage, however, it often also implies a relationship between nephron age or position and nephron maturity. We consider here whether the ideas of centrifugal development and centrifugal maturation should in fact be considered as separate and distinct concepts. That is, we consider the possibility that nephron maturity does not necessarily correlate with nephron age. Unfortunately, pertinent reported data give no clear answer. We conclude only that further study will be required before definitive conclusions about renal developmental stages can be stated with certainty.

Mouse kidney androgen-regulated protein messenger ribonucleic acid is expressed in the proximal convoluted tubules.

Kidney androgen-regulated protein (KAP) mRNA represents the most abundant [approximately 4% of the total poly(A) RNA] mRNA species that is induced by androgens in the mouse kidney. Comparison of the expression of several androgen-regulated mRNAs in this tissue, revealed that the mRNAs were differentially regulated by the hormone. KAP mRNA exhibited unusual sensitivity to low concentrations of the androgen-receptor complex. Because of its unusual characteristics, it was of interest to determine in what cells of the kidney KAP mRNA was being produced. Using the technique of in situ hybridization with single stranded RNA probes, we have identified the epithelial cells of the renal proximal convoluted tubules as the site of synthesis of KAP mRNA. Interestingly, only a subpopulation of these cells, those located in the juxtamedullary region of the renal cortex, contain KAP mRNA in castrated males. After androgen treatment, cortical proximal tubules are also induced to express KAP mRNA. These results suggest that two types of response to androgens occur in these cells. One is the progressive increase of KAP gene expression in the juxtamedullary region while the other involves recruitment of new cells in the cortical proximal tubules to synthesize KAP mRNA.

Effect of Acidosis and Alkalosis on Divalent Ion Transport across the Proximal Straight Tubule of the Rabbit

In vitro microperfusion experiments were performed on the cortical proximal straight tubule of the rabbit to examine the effect of acid base disturbances on calcium and magnesium transport by this segment of the nephron. During acidosis (pH 7.22 +/- 0.01) net calcium reabsorption was reduced, whereas during alkalosis (pH 7.82 +/- 0.02) net calcium reabsorption was increased. Our flux experiments with 45Ca indicate that the efflux of Ca from the lumen to the bath remained the same during acidosis; however, the back-flux (bath to lumen) of calcium increased. On the other hand, during alkalosis the efflux of calcium rose and the influx of calcium remained unchanged. These changes in fluxes during acid-base disturbances can be accounted for mainly by changes in ionized calcium as a result of pH changes. We failed to observe any effect of acid-base changes on net magnesium transport by the superficial proximal straight tubule.

An active pathway for serotonin synthesis by renal proximal tubules

Serotonin (5HT) has significant effects on renal metabolism and glomerular function and is a potent renal vasoconstrictor. In this study we describe and localize a highly active biosynthetic pathway for serotonin in the kidney. Rat kidneys were dissected into cortical and medullary fractions; in some experiments the cortex was also separated into subfractions enriched with glomeruli or proximal tubules. Serotonin and tryptophan hydroxylase (TyOH) were measured by radioenzymatic techniques. (table; see text) Renal denervation did not alter tryptophan hydroxylase activity. In kidneys from human cadaveric donors, cortical tryptophan hydroxylase (4.13 +/- 0.68 nM/30 min/g) exceeded that in the medulla (1.96 +/- 0.86 nM/30 min/g). Aromatic L-amino acid decarboxylase, the remaining enzyme for serotonin synthesis, is present in both rat renal cortex and medulla; however, we found 15-fold greater decarboxylase activity in proximal tubular (2070 nM/30 min/g) as compared to glomerular (131 nM/30 min/g) subfractions. We were able to demonstrate that under physiological conditions, free urine serotonin reflects actual biosynthesis by the kidney. Thus, although serotonin stores retained by the kidney appear small and relatively localized to the medulla, the enzymatic activity for the synthesis of serotonin in the kidney is comparable to that in the brain, with the complete pathway localized to renal cortical proximal tubules. These data suggest that further studies of renal serotonin metabolism may contribute to our understanding of renal function in health and disease.

Comparative toxicity of gentamicin and tobramycin in rats at low multiples of the human therapeutic dose

Previously conducted investigations in rats comparing gentamicin to tobramycin at high multiples of the usual human dose have reported tobramycin to be less nephro- and/or ototoxic than gentamicin. In this study, comparative effects on kidney and hearing/equilibrium between the antibiotics at doses approximating one to three times the daily human therapeutic dose (3 to 5 mg/kg/day) were examined. Gentamicin or tobramycin were given by intramuscular injection to groups of 20 male Sprague-Dawley rats at dose levels of 3, 6, and 9 mg/kg/day for 30 consecutive days. Each rat was tested weekly for changes in auditory function and assessed for effects on vestibular function. Routine clinical laboratory determinations for evaluation of nephrotoxicity were performed throughout the study. Representative portions of the kidney were examined histologically. There were no adverse effects observed on hearing function or equilibrium. Renal responses to the administration of either aminoglycoside included marginal polyuria with a concomitant decrease in urine osmolality at the 6 and 9 mg/kg dose levels. Very mild histomorphological changes of the proximal cortical tubules, typical of an aminoglycoside-induced toxicity, were noted in both drug groups at all dosages. The incidence of these changes was dose-related and was similar between the corresponding aminoglycoside groups. Following single-dose administration, drug serum levels of either antibiotic were similar and compared favorably to those reported in patients receiving gentamicin or tobramycin at usual clinical doses. The results of this study indicate that there were no renal or auditory/vestibular toxicologic differences between gentamicin and tobramycin when given to rats at levels equal to the clinical dose or at low multiples thereof.

Isolated nephron segments in a rabbit model of ischemic acute renal failure.

Renal artery occlusion has been extensively used in animal models to cause acute renal failure. The present isolated tubule microperfusion studies were designed to examine the transport characteristics of multiple nephron segments of the rabbit after 60 min of total renal ischemia. Preliminary studies showed that this maneuver produced significant and persistent elevations of serum creatinine. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate and studied at 37 degrees C. Four nephron segments were examined. Ischemia reduced proximal convoluted tubule fluid reabsorption 77% (0.72 +/- 0.11 vs. 0.14 +/- 0.06 nl . mm-1 . min-1, P less than 0.01) and cortical proximal straight tubule fluid reabsorption 88% (0.54 +/- 0.10 vs. 0.06 +/- 0.03 nl . mm-1 . min-1, P less than 0.005). Ischemia reduced the ability of the thick ascending limb of Henle's loop to lower perfusate chloride ion concentration 60% (-47 +/- 9 vs. -19 +/- 3 meq/liter, P less than 0.02) and its diluting ability 49% (-87 +/- 15 vs. -44 +/- 7 mosmol/kg H2O, P less than 0.01). Ischemia reduced the antidiuretic hormone-dependent osmotic water permeability of the cortical collecting tubule 59% (0.0203 +/- vs. 0.0083 +/- 0.0020 cm/s, P less than 0.01). Morphologic alterations were noted in the proximal segments but not in the distal segments of the nephron. The current studies demonstrate that 60 min of renal ischemia impairs the transport capability of all proximal and distal nephron segments studied.

Scanning and Transmission EM of Rat Kidney Following Low Dose Mercuric Chloride Administration

The development of necrosis and the subsequent regeneration of the proximal tubular epithelium located in the cortex and the outer medulla of the rat kidney following subcutaneous injection of 2 mg of mercuric chloride per kilogram of body weight was observed utilizing light micromicroscopy (LM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The cortical proximal tubules were largely free of injury. In the medullary proximal tubules, the residual, undamaged cells underwent a change in phenotype appearance involving loss of the microvillus border, a decrease in cell height, and an increase in cytoplasmic ribosomes. These cells phagocytosed luminal debris. With time the cell height gradually increased, specialized organelles returned, and the luminal microvilli progressed from short, sparse profiles, to tufts of longer microvilli and finally the brush border was completely reformed. Some kidneys contained calcified tubules which regenerated more slowly than damaged but uncalcified tubules. Similarly, the medullary segments of the proximal tubules regenerated more slowly than the juxtamedullary region. Clusters of epithelial cells were occasionally seen growing into the tubular lumina during regeneration. Marked intersitial leukocyte infiltrations were observed.

General and renal toxicity of phenacetin, paracetamol and some anti-mitotic agents in the rat.

The general and the renal toxicity of large doses of phenacetin, paracetamol, some antimitotic drugs and other constituents of analgesic mixtures was investigated in medium term toxicity tests in a large number of rats. Phenacetin and paracetamol depressed food intake and retarded growth. 800–1200 mg/kg · day paracetamol induced a larger mortality than 1500–3000 mg/kg · day phenacetin. Both analgesics and isophthalanilide, an antimitotic agent, induced hyperchromic anemia. Phenacetin induced methemoglobinemia more readily than paracetamol. Neither the analgesics, nor caffeine, sodium nitrite, isophthalanilide or mercaptopurine depressed GFR, maximal urinary concentration after dehydration plus vasopressin, urinary dilution after hypotonic expansion, or urinary acidification. Phenacetin, paracetamol and isophthalanilide depressed the fractional excretion of urea by the kidney. Very large doses of paracetamol slightly increased the proteinuria and the urinary excretion of tubular cells. Phenacetin and paracetamol induced degenerative histological alterations in cortical proximal and distal tubules, detected and quantitated under blind conditions. There were no inflammatory changes, nor any medullary or papillary lesions. The degenerative lesions could not be explained by the presence of methemoglobinemia or hemolysis. Isophthalanilide actually “improved” the histological appearance of the kidneys. The urinary excretion of tubular cells was not significantly correlated with the severity of the histological changes. It was concluded that neither phenacetin nor paracetamol exert major nephrotoxic effects in rats.