Nonfiltering Kidney Research Articles

Transcriptional response in the unaffected kidney after contralateral hydronephrosis or nephrectomy

Unilateral loss of kidney function is followed by compensatory contralateral growth. The early, genome-wide transcriptional response of the untouched kidney to unilateral ureteral obstruction (UUO) or unilateral nephrectomy is unknown. Twelve adult male Sprague-Dawley rats were subjected to UUO and twelve rats to unilateral nephrectomy. At time points 12, 24, and 72 hours after insult four rats each were sacrificed and the contralateral kidney harvested for genome-wide gene expression analysis, transcription factor analysis, and histomorphology. Microarray studies revealed that the majority of differentially expressed transcripts were suppressed in UUO and unilateral nephrectomy compared to control kidneys. The function of these suppressed genes is predominantly growth inhibition and apoptosis suggesting a net pro-hypertrophic response. Insulin-like growth factor-2 (IGF-2)-binding protein was one of the few activated genes. We observed a distinctly different molecular signature between UUO and unilateral nephrectomy at the three time points investigated. The early response in UUO rats suggests a counterbalance to the nonfiltering kidney by activation of transport pathways such as the aquaporins. Unilateral nephrectomy kidneys, on the other hand, respond immediately to contralateral nephrectomy by activation of cell cycle regulators such as the cyclin family. Several genes with weakly defined function were found to be associated with either UUO or unilateral nephrectomy. Transcription factor analysis of the identified transcripts suggests common regulation at least of some of these genes. All kidneys showed normal histology. Release of growth inhibition by nephrectomy leads to immediate cell cycle activation after unilateral nephrectomy, whereas UUO kidneys counterbalance filtration failure by activation of several transporters.

Exogenous Albumin Peptides Influence the Processing of Albumin during Renal Passage

Exogenous albumin peptides influence the processing of albumin during renal passage. Background/Aims: This study investigates the hypothesis that there will be peptide regions in albumin that will effectively compete for the receptors associated with the renal processing of albumin. Methods: We employ albumin peptides prepared from albumin by trypsin digestion. The presentation of the exogenous peptides along with intact [³H]albumin to the kidney was made by intravenous injection into rats. The excretion rate and integrity of urinary [³H]albumin was measured. Similar experiments were performed with the use of gelatin peptides produced by trypsin digestion as controls. The formation of [³H]albumin derived fragments by extrarenal sources was also examined in rats with nonfiltering kidneys. Results: In the presence of exogenous albumin-derived peptides there was a significant increase in the proportion of larger [³H]albumin fragments in the urine. This is a reversible effect. There was no significant change when gelatin peptides were used. The albumin peptides also increase the fractional clearance of [³H]albumin. There were no [³H]albumin-derived fragments produced in plasma over a 4-hour circulation period in rats with nonfiltering kidneys. Conclusions: This study demonstrates that albumin fragments, which are produced by the kidney and not by extrarenal sources, are exclusively excreted in the urine. Exogenous albumin peptides were able to specifically exert a competitive effect on the renal enzyme cleavage of intact albumin.

THE RETURN OF GLOMERULAR FILTERED ALBUMIN TO THE RAT RENAL VEIN – THE ALBUMIN RETRIEVAL PATHWAY

Background. Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high. This has been confirmed in studies where albumin uptake by the tubules has been inhibited. Therefore, there must be a high capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. Methods. This study identifies the presence of glomerular filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium labeled albumin into the renal artery in vivo and in the isolated perfused kidney (IPK). Results. The glomerular filtered albumin is returned to the blood supply by a high capacity pathway that transports this albumin at a rate of 1830 ± 292 μg/min rat kidney (n = 14) (mean ± SEM). This pathway has been identified under physiological conditions in vivo and in the IPK. The pathway is specific for albumin as it does not occur for horseradish peroxidase (HRP). The pathway is inhibited in a non-filtering kidney. The pathway is also inhibited by NH4Cl, an inhibitor of protein uptake. Conclusions. The high capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

The return of glomerular-filtered albumin to the rat renal vein

Recent studies have demonstrated that the normal glomerular capillary wall (GCW) is not charge selective to albumin. This means that albumin flux across the GCW is high, and this has been confirmed in studies in which albumin uptake by the tubules has been inhibited. Therefore, there must be a high-capacity postglomerular retrieval pathway in normal kidneys that returns filtered albumin back to the blood supply. This study identifies the presence of glomerular-filtered albumin in the renal vein from the analysis of the decrease of radioactivity in the venous effluent after the injection of a pulse of tritium-labeled albumin into the renal artery in vivo and in the isolated perfused kidney. The postglomerular filtered albumin is returned to the blood supply by a high-capacity pathway that transports this albumin at a rate of 1830 +/- 292 micrograms/min.rat kidney (N = 14, mean +/- SEM). This pathway has been identified under physiological conditions in vivo and in the isolated perfused kidney. The pathway is specific for albumin, as it does not occur for horseradish peroxidase. The pathway is inhibited in a nonfiltering kidney. The pathway is also inhibited by ammonium chloride (an agent that inhibits tubular protein uptake but does not alter glomerular size selectivity) and by albumin peptides (which compete for the tubular albumin receptor). The high-capacity retrieval pathway for albumin is most likely associated with transtubular cell transport. It is also apparent that most albuminuric states could be accounted for by the malfunctioning of this pathway without resorting to any change in glomerular permselectivity.

Inhibition of intrarenal NO stimulates renin secretion through a macula densa-mediated mechanism.

Because endothelium-derived factors are known to have multiple actions throughout the body, the role of nitric oxide (NO) produced within the kidney in the regulation of renin release is still unclear. Therefore, the objectives of this study were to determine the effect of local NO synthesis inhibition within the kidney on renin secretion rate (RSR) and to determine whether the macula densa mechanism mediates the effect of NO on renin secretion rate in dogs. The NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) was administered via the renal artery at 5 microg x kg(-1) x min(-1) to dogs with normal kidney function and to dogs with the macula densa mechanism blocked, established by using the nonfiltering kidney model. In dogs with normal kidney function, renal arterial pressure (RAP) and glomerular filtration rate (GFR) remained constant throughout the experiment (131 +/- 5 mmHg and 22.6 +/- 3.0 ml/min, respectively). However, intrarenal NO synthesis inhibition decreased renal blood flow (RBF) by 16% (240 +/- 22 to 201 +/- 23 ml/min) and increased renal vascular resistance (RVR) by 24% (0.59 +/- 0.08 to 0.73 +/- 0.09 mmHg x ml(-1) x min). In addition, L-NAME decreased the fractional excretion of lithium by 27% (30.0 +/- 3.7 to 21.6 +/- 4.3%) and decreased the fractional excretion of sodium by 35% (0.86 +/- 0.29 to 0.56 +/- 0.21%). Associated with these changes in renal function, renin secretion rate increased by 194 and 235%. In marked contrast, renin secretion rate remained constant in dogs with the macula densa mechanism blocked. Intrarenal NO synthase blockade decreased RSR by 4 and 10% in dogs with the macula densa mechanism blocked. The RAP, RBF, and RVR responses to intrarenal NO synthesis inhibition in dogs with the macula densa mechanism blocked were similar to the renal hemodynamic response in dogs with normal kidney function. In summary, we have demonstrated that intrarenal NO synthesis blockade enhances renin secretion in dogs. The macula densa mechanism appears to play an important role in mediating the effect of intrarenal NO synthesis inhibition on renin release.

Metabolism of diglycine and triglycine by non-filtering kidneys.

We have studied the metabolism of diglycine and triglycine in the isolated non-filtering rat kidney. Kidneys from adult male Wistar Kyoto rats weighing 250-350 g were perfused with Krebs-Henseleit solution containing either 1 mM diglycine or triglycine. The analysis of the peptide residues and their components was performed using an amino acid microanalyzer utilizing ion exchange chromatography. Diglycine was degraded to a final concentration of 0.09 mM after 120 min (91%); this degradation occurred predominantly during the first hour, with a 56% reduction of the initial concentration. The metabolism of triglycine occurred similarly, with a final concentration of 0.18 mM (82%); during the first hour there was a 67% reduction of the initial concentration of the tripeptide. Both peptides produced glycine in increasing concentrations, but there was a slightly lower recovery of glycine, suggesting its utilization by the kidney as fuel. The hydrolysis of triglycine also produced diglycine, which was also hydrolyzed to glycine. The results of the present study show the existence of functional endothelial or contraluminal membrane peptidases which may be important during parenteral nutrition.

Renal disposition of recombinant human interleukin-11 in the isolated perfused rat kidney.

To clarify the mechanism of the renal clearance of recombinant human interleukin- 11 (rhIL- 1), we investigated the renal disposition characteristics of rhIL-11 in the perfused rat kidney. The disposition characteristics of (111)In-labeled rhIL-11 were analyzed using a single-pass indicator dilution technique and statistical moment analysis in the perfused rat kidney under filtering and nonfiltering conditions. Steady-state distribution volume (Vd) calculated from the venous outflow patterns of rhIL-11 at the doses of 0.3 to 10 microg/kidney was between 0.35 and 0.40 ml/g kidney. However, Vd at the highest dose decreased to a value almost identical to that of bovine serum albumin, suggesting that there is a reversible and saturable interaction between the capillary wall and rhIL-11 molecule. In filtering kidney, a remarkable accumulation of rhIL-11 was observed while its urinary excretion was highly restricted at all doses. In nonfiltering kidney, rhIL-11 showed a decreased but still significant renal uptake. Taken together, the marked renal uptake of rhIL-11 may be explained by both efficient tubular reabsorption and significant uptake from the capillary side. These processes were not saturable within the tested dose range. These characteristics of rhIL-11 are likely based on non-specific electrostatic interaction with the tissues due to its cationic charge in the cytokine. The renal disposition processes of rhIL-11 were clarified at organ level in a quantitative manner. These findings agree well with previous observations in an in vivo disposition study in mice.

Roles of prostaglandins and nitric oxide in the effect of endothelin-1 on renal hemodynamics.

It is known that endothelin-1 stimulates the release of nitric oxide and prostaglandins in various vascular beds. We designed the present study to analyze the roles of prostaglandins and nitric oxide in the effect of endothelin-1 on the regulation of renal hemodynamics and renin release. We used N omega-nitro-L-arginine methyl ester (L-NAME) and meclofenamic acid to inhibit the production of nitric oxide and prostaglandins, respectively. With a nonfiltering kidney model, renal blood flow was reduced 21% in dogs treated with L-NAME and 18% in dogs treated with meclofenamic acid. Inhibition of nitric oxide and prostaglandins, however, produced opposite effects on estimated glomerular hydraulic pressure: L-NAME increased glomerular hydraulic pressure from 63.1 +/- 0.9 to 64.6 +/- 1.3 mm Hg (P < .01), and meclofenamic acid reduced glomerular hydraulic pressure from 63.3 +/- 1.4 to 59.8 +/- 1.6 mm Hg (P < .01). Endothelin-1 infusion produced a dose-dependent reduction in renal blood flow after blockade of nitric oxide and prostaglandins. The responses of glomerular hydraulic pressure were different in the two groups during endothelin-1 infusion. Endothelin-1 progressively reduced glomerular hydraulic pressure in a dose-dependent fashion in the meclofenamic acid group. However, endothelin-1 slightly increased glomerular hydraulic pressure until the infusion rate reached 5.0 ng/kg per minute. At that rate, endothelin-1 reduced glomerular hydraulic pressure from 63.3 +/- 1.4 to 47.0 +/- 1.4 mm Hg in the meclofenamic acid group (P < .01), a more than 25% reduction, whereas at the same dose, endothelin-1 reduced glomerular hydraulic pressure only less than 2% in the L-NAME group. In addition, blockade of nitric oxide and prostaglandins did not alter the inhibitory effect of endothelin-1 on renin release in the non-filtering kidney. Therefore, the present study demonstrates that the release of nitric oxide and prostaglandins might modulate the effects of endothelin-1 on the renal circulation. The present findings suggest that the differential vasoconstrictive effects of endothelin-1 on preglomerular and postglomerular vessels are associated with its stimulation of nitric oxide and prostaglandin production.

Influence of trifluoperazine and verapamil on the isolated perfused rat kidney

1. 1. Isolated rat filtering kidneys were perfused with Ringer bicarbonate solutions containing either trifluoperazine (TFP, 50 μM) or verapamil (VER, 100 μM) to prevent tissue dysfunctions observed during perfusion. 2. 2. Water, sodium and chloride kidney contents diminished under both treatments as compared with control preparations, and potassium content increased. 3. 3. When albumin concentration was increased (10 g %) in the perfusion medium (nonfiltering kidney preparation) these effects of TFP or VER were not observed. 4. 4. Lipid peroxidation and LDH release diminished significantly under 50 μM TFP but only slightly under 100 μM VER.

Relationship between the diuretic effect of radiocontrast media and their ability to increase renal vascular resistance

The relationship between diuresis and natriuresis induced by radiocontrast media (RCM) and their renal haemodynamic effects were investigated. The effects of the iso-osmolar iotrolan and the hyperosmolar diatrizoate on the renal vascular resistance (RVR) were studied in the filtering and non-filtering variants of the isolated perfused rat kidney (IPRK) preparation. In the non-filtering model, no tubular regulatory process can be activated. The effect of diatrizoate on the RVR of the filtering IPRK in the presence of fursemide (0.3 mmol l-1) an inhibitor of the tubuloglomerular feedback (TGF) was also investigated. There was no significant difference (p > 0.05) in the response of the filtering (n = 6) and non-filtering (n = 6) IPRK to iotrolan. The induced reduction in the renal perfusate flow (RPF) by iotrolan was 20.5 +/- 3.05% and 22.9 +/- 3.03%, respectively. The reduction in the RPF which was observed with diatrizoate in the non-filtering IPRK (n = 5, 17.5 +/- 3.04%) was significantly less (p < 0.05) in comparison to that of the filtering IPRK (n = 6, 26.9 +/- 4.28%). In the frusemide experiments, a reduction in the RPF comparable to that of the non-filtering kidney was observed (n = 5, 13.7 +/- 4.34%). This study demonstrates that the renal vascular effect of diatrizoate is partially dependent on the TGF response. No tubular regulatory mechanism was accountable for the haemodynamic effect of iotrolan. The activation of the tubular response is osmolarity dependent.

Metabolism of N-terminal and C-terminal parathyroid hormone fragments by isolated perfused rat kidney and liver.

The metabolism of added N-terminal and C-terminal synthetic human PTH (synhPTH) fragments was studied in isolated perfused rat livers and in filtering and nonfiltering kidneys. Initial concentrations: synhPTH-(1-34), 14 pmol/liter, synhPTH-(39-84) 85 pmol/liter, synhPTH-(1-84) 3 pmol/liter, or 1000 pmol/liter of each fragment and intact PTH. Clearances were measured by assays specific for intact PTH, N-terminal, midmolecule, and C-terminal immunoreactive PTH. Metabolism of the added PTH into smaller circulating fragments was analyzed by HPLC. The clearance of synhPTH-(1-34) in the filtering kidneys was not significantly different from the clearance of synhPTH-(39-84) and inulin, and HPLC demonstrated no metabolism of synhPTH-(1-34) or synhPTH-(39-84) in the kidneys. The livers did not clear synhPTH-(39-84), while the clearance of synhPTH-(1-34) was significant (P < 0.005). HPLC demonstrated no metabolism of synhPTH-(39-84) by the livers, but extensive metabolism of synhPTH-(1-34). The livers cleared synhPTH-(1-34) significantly (P < 0.05) faster than synhPTH-(1-84). In conclusion, the kidneys cleared N-terminal PTH fragments at the same rate as C-terminal fragments mainly by filtration. The livers cleared N-terminal fragments faster than intact PTH, but did not clear C-terminal fragments. This differential hepatic clearance may play a major role in maintaining the differences between the circulating levels of N-terminal and C-terminal immunoreactive PTH.

Manipulation of renal disposition of human recombinant superoxide dismutase by chemical modification.

The renal disposition characteristics of superoxide dismutase (SOD) and its derivatives, including macromolecular conjugates with polyethylene glycol and carboxymethyl-dextran, a cationized derivative, and glycosylated derivatives with galactose and mannose, were studied in the isolated perfused rat kidney. Renal disposition processes, such as glomerular filtration, tubular reabsorption, and uptake from the capillary side, were quantitatively determined by single-pass indicator dilution experiments under filtering and nonfiltering kidney conditions. Native SOD had a high glomerular filtration rate (40% of that of inulin) and was effectively reabsorbed in the tubules, while no significant uptake was observed from capillary side. Macromolecular conjugates showed restricted glomerular filtration due to an increase in molecular size. Cationization of SOD greatly enhanced its association with the tissue, not only from the luminal side but also from the capillary side, based upon electrostatic interaction. Galactosylated and mannosylated SOD showed reduced tubular reabsorption and increased exposure of the luminal surface to the enzyme. In addition, a small but significant uptake of mannosylated SOD from the capillary side was observed. This uptake was dose-dependent and completely inhibited by mannan, suggesting that mannose receptor-mediated endocytosis existed in the capillary side of the kidney. Thus, we can manipulate the renal disposition profiles of SOD by changing its physicochemical or biological properties through chemical modification.

Renin stimulating properties of parathyroid hormone-related peptide in the isolated perfused rat kidney

Previous studies showed that PTHrP exhibits renal vasodilating, arteriolar cAMP stimulating and receptor binding properties. The present experiments were designed to study whether PTHrP may influence renin secretion. Rat kidneys were isolated and single-pass perfused at constant flow and stabilized pressure. Exposures to PTHrP or PTH stimulated a dose-dependent renin release reaching similar Vmax. The affinity (0.1 nM) and threshold concentration (0.01 nM) for PTHrP were about 10 times lower than for PTH. Compared to 10 microM isoproterenol, the maximum renin responses to PTHrP were similar but of shorter duration. The PTHrP dose-response curve was not affected by 10 microM indomethacin. Administered simultaneously, PTHrP and PTH displayed no additive effects. PTHrP-induced renin release as well as the role of extracellular calcium were further studied in nonfiltering kidneys, which were perfused at a constant flow and stable pressure in a closed circuit. Basal renin release was inversely related with perfusate calcium and was depressed by the calcium ionophore BAY-K8644. PTHrP (100 nM) induced a 1.6-fold increase of basal renin release in normocalcic perfusate. Removing calcium abolished renin responses. PTHrP reversed the inhibiting effects of hypercalcic media or BAY-K8644 on basal renin release. The results support calcium-mediated renin stimulating properties for PTHrP, via PTH receptors, independently from baroreceptors, macula densa and prostaglandins.

Effect of endothelin-1 on glomerular hydraulic pressure and renin release in dogs.

The present study was designed to analyze quantitatively the effects of a wide range of endothelin-1 levels on renal hemodynamics and renin release in the canine nonfiltering kidney, including their effects on glomerular hydraulic pressure. Intrarenal infusion of endothelin-1 produced dose-dependent reductions in renal blood flow, but it did not affect glomerular hydraulic pressure until the infused dose reached high rates. At the rate of 1.0 ng/kg per minute, endothelin-1 reduced renal blood flow by 23% (p < 0.01), whereas glomerular hydraulic pressure was not significantly changed from 68.1 +/- 1.3 to 67.4 +/- 1.2 mm Hg. However, with a higher rate of endothelin-1 infusion (5.0 and 10.0 ng/kg per minute), glomerular hydraulic pressure fell to 59.5 +/- 1.3 and 51.5 +/- 1.8 mm Hg (p < 0.01), whereas renal blood flow was reduced from 154.5 +/- 15 to 83.0 +/- 9.5 and 53.5 +/- 9.9 mL/min, respectively. Endothelin-1 infusion also produced an inhibitory effect on renin release. With infusion at 1.0 ng/kg per minute, renin release fell from the control level of 47.9 +/- 5.6 to 26.6 +/- 4.9 units/min per gram kidney weight (p < 0.01), and it fell further to 16.1 +/- 4.3 units/min per gram kidney weight with infusion at 10.0 ng/kg per minute. In summary, endothelin-1 infusion did not affect glomerular hydraulic pressure despite a fall in renal blood flow at low doses, but at high doses it reduced both, suggesting that endothelin-1 exerts separate, dose-dependent effects on preglomerular and postglomerular resistances.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoregulation of Renal Blood Flow During Ovine Pregnancy

Since the renal vasculature is vasodilated and the renal perfusion pressure (RPP) is reduced during pregnancy, the capacity to autoregulate renal blood flow (RBF) during pregnancy is unclear. Therefore, we assessed RBF auto-regulation in nonpregnant and pregnant sheep at RPP of 80, 75, 70, and 60 mmHg under control conditions, during amino acid infusion (SAP), and in sheep with nonfiltering kidneys in order to stress the normal feedback regulations of RBF. In nonpregnant animals, RBF was unchanged until RPP was reduced below 70 mmHg, but in pregnant animals the breakpoint occurred at 75-80 mmHg. The autoregulatory index (AI) was significantly higher in pregnant than in nonpregnant animals at all levels of RPP below 80 mmHg, indicating a greater impairment of RBF autoregulation during pregnancy. Amino acid infusion (SAP) elevated basal RBF similarly in all sheep and suppressed the autoregulatory curves of both groups, although to a greater extent in the pregnant sheep. Basal RBF was increased with nonfilte...

Disposition characteristics of model macromolecules in the perfused rat kidney.

The disposition characteristics of model macromolecules such as dextran (70 kDa), bovine serum albumin (BSA), and their charged derivatives were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery patterns and tissue accumulation of radiolabeled compounds were evaluated under filtering or nonfiltering conditions. In the filtering kidney, cationic macromolecules such as diethylaminoethyl-dextran (DEAE-dex) and cationized BSA (cBSA) accumulated in the kidney to a great extent whereas anionic and neutral macromolecules such as BSA, carboxymethyl-dextran (CM-dex), and dextran showed only small uptake. DEAE-dex and cBSA were distributed to both the medulla and cortex regions of the kidney and their recoveries in the kidney decreased as the injected dose increased. Similar tissue uptake was observed in the nonfiltering kidney perfusion system suggesting that they were mainly taken up by the kidney from the renal capillary side based on electrostatic interaction. In addition, the steady-state distribution volumes of cationic macromolecules calculated from venous outflow patterns were larger than those of the intravascular volume estimated from the distribution volumes of neutral and anionic macromolecules, suggesting their reversible interaction with the vascular wall. On the other hand, dextran derivatives with molecular weight distribution were excreted into urine based on glomerular permselectivity; i.e., cationic DEAE-dex and anionic CM-dex showed enhanced and restricted urinary excretion, respectively, compared with neutral dextran. In contrast, no significant excretion was observed for BSA and cBSA. The utility of the isolated rat kidney perfusion experiment for studying the renal disposition of macromolecular drugs was thus demonstrated.

Disposition characteristics of protein drugs in the perfused rat kidney.

The renal disposition characteristics of 111In-labeled neocarzinostatin (NCS), soybean trypsin inhibitor (STI), and superoxide dismutase (SOD) were studied in the perfused rat kidney. In a single-pass indicator dilution experiment, venous and urinary recovery profiles and tissue accumulation of proteins were determined under filtering or nonfiltering conditions. In the nonfiltering kidney perfusion experiment, no significant tissue accumulation was observed, suggesting minimal uptake from the glomerular and peritubular capillary sides. Therefore, tissue recovery corresponded to that with tubular reabsorption after glomerular filtration. The total amount of NCS or STI being filtrated through glomeruli, the sum of tissue and urinary recoveries, was similar to that of inulin, but that of SOD was about half. Similarly, the steady-state distribution volumes (Vd) of NCS and STI obtained by moment analysis of their venous outflow curves were similar to that of inulin, while the Vd value of SOD was significantly lower. These results suggest the restricted passage of SOD through the glomerular and postglomerular capillary wall. The tubular reabsorption ratio of proteins against the total filtrated amount decreased with an increase in the administered dose, suggesting nonlinearity of reabsorption. SOD had the largest reabsorption ratio. Thus, this experimental system is useful for quantitative analysis of renal disposition of proteins.

Luminal and basolateral uptake and degradation of insulin in the proximal tubules of the dog kidney.

In order to determine the major routes of insulin degradation in the body, insulin was labelled with a 'trapped' or 'residualizing' label: [125I]tyramine-cellobiose ([125I]TC) and injected intravenously in dogs. In contrast to conventional iodine-labelled insulin (131I-insulin), the [125I]TC-insulin allows measurements of total uptake in specific organs in vivo because the radioactive degradation products do not leave the cells. One h after the injection of trace doses, the amount of radioactivity recovered in the kidney from [125I]TC-insulin was nine times higher than when conventional [131I]insulin was used. In the blood, the amount of acid-precipitable radioactivity was the same for both labelled preparations, indicating similar clearance rates. A comparison of the uptake of insulin in filtering vs. non-filtering (ureter-occluded) kidneys indicated that the uptake of insulin is twice as high through the luminal than through the basolateral cell membrane; after 60 min, 8.9 +/- 0.8% of the injected [125I]TC-insulin dose remained in the filtering kidney and 3.2 +/- 0.2% of the dose was accumulated in the contralateral kidney, with occluded ureter but normal blood perfusion. In both filtering and non-filtering (ureter-occluded) kidneys, the subcellular distributions of [125I]TC-insulin were studied after various times by isopycnic sedimentation in sucrose gradients. No difference between peritubular and tubular uptake was discernible. The intracellular transport was rapid, leading to accumulation of radioactive label in dense lysosomes within 10 min.

Renal tubular handling of p-aminohippurate and epidermal growth factor (EGF) in filtering and nonfiltering perfused rat kidneys.

We examined the integrity of renal tubular function in filtering and nonfiltering isolated perfused rat kidneys by using p-amino-3H-hippurate (3H-PAH) and the multiple indicator dilution method with 14C-creatinine as a reference. The influx clearance (PSu,1) of unbound 3H-PAH was 0.37 and 0.38 ml/sec in the filtering and nonfiltering kidneys, respectively. The efflux rate constants were comparable between filtering and nonfiltering kidneys, while the sequestration rate constant in the filtering kidney was approximately three times larger than that in the nonfiltering kidney. These data suggest that the nonfiltering kidney maintains 3H-PAH transporting ability through the antiluminal plasma membrane. The renal handling of epidermal growth factor (EGF) by filtering and nonfiltering kidneys was compared. The ratio of the total uptake of tracer 125I-EGF over 20 min in the nonfiltering kidney to that in the filtering kidney was 0.8. This ratio was reduced to 0.2 when the kidneys were perfused with tracer 125I-EGF plus 20 nM EGF. Furthermore, the total uptake of tracer 125I-EGF in the nonfiltering kidney was reduced 20-fold in the presence of 20 nM unlabeled EGF. These findings suggest that the tubular uptake of tracer 125I-EGF by filtering kidney takes place mainly via the antiluminal plasma membrane and that this uptake is a saturable process.

Localization of epidermal growth factor (EGF) binding sites on antiluminal plasma membrane of rat kidney: autoradiographic study using nonfiltering perfused rat kidney.

We previously demonstrated that the specific binding of EGF to the antiluminal plasma membrane was a prerequisite step for the renal uptake of EGF. In the present study, the localization of 125I-EGF binding sites on the antiluminal plasma membrane was investigated by tissue sampling and X-ray autoradiography in the nonfiltering kidney. The binding of 125I-EGF was recognized over the whole kidney and was highest in the inner medulla followed by the cortex and outer medulla. The binding of 125I-EGF in the nonfiltering kidney was completely inhibited in the presence of 20 nM unlabeled EGF, suggesting specific binding of 125I-EGF to its receptor. Further, we used a histologic tissue staining method to confirm the location of the 125I-EGF binding sites. Binding of 125I-EGF was demonstrated on the proximal straight tubules (PST), cortical collecting ducts (CCD), inner medullary collecting ducts (IMCD), and thin limb of Henle in the inner medulla (IMTLH). We found that the binding of 125I-EGF was high in the IMTLH. In addition, we determined the grain density both on the cell surface membrane and in the intracellular space of the proximal straight tubules, where the grain density on the antiluminal plasma membrane was approximately 50% that in the intracellular space at 20 min after the start of 125I-EGF perfusion, suggesting the internalization of 125I-EGF from the antiluminal plasma membrane to the intracellular compartment. In conclusion, the binding sites of 125I-EGF, which were accessible from the antiluminal side, were broadly distributed over the whole kidney and were most dense around the IMTLH.