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.     

CO2 along the proximal tubules in the rat kidney.

The proximal intratubular pH of the rat kidney was measured in vivo with an antimony electrode system. PCO2 and bicarbonate concentration of the proximal tubular fluid were determined with an ultramicro equilibration system. The tubular fluid to plasma inulin concentration ratio was evaluated by a microscope fluorometric method. The acid-base parameters and the inulin concentrations were determined under control conditions and during acetazolamide treatment. The intratubular PCO2 was higher than the PCO2 of the systemic arterial blood under control conditions and the difference in PCO2 was increased during acetazolamide treatment. In acetazolamide treated rats the rate of fractional bicarbonate reabsorption was decreased in the early part of the proximal tubule, while it was of about the same in the middle and late parts as compared with control rats. The total bicarbonate reabsorption in the proximal tubule was reduced by 50% due to the carbonic anhydrase inhibition. It seems possible that the bicarbonate is still reabsorbed as CO2 after carbonic anhydrase inhibition, as hydrogen ion secretion is not totally stopped by this treatment. The increase in intratubular PCO2 after acetazolamide treatment is assumed to be due to an inhibition of the carbonic anhydrase facilitating effect on outward diffusion of CO2 from the tubular lumen across the cell wall.     

Histochemical studies on peroxisomes in regenerating proximal tubules of the kidney.

Peroxisomes of the regenerating proximal tubules of the rat kidney were investigated after necrosis induced by mercuric chloride. Slices both for light and electron microscopic examinations were incubated in 3,3''-diaminobenzidine (DAB) medium. Peroxisomes were absent in the necrotic epithelium. They appeared on the fourth day of regeneration and later their number increased reaching the normal distribution in the fourth week. They seem to be needed for the functional differentiation of the proximal tubule cells during regeneration.     

Gentamicin-induced loss of basolateral surface area of rat proximal convoluted tubules

This study was performed to find the quantitative relationship between basolateral surface alterations of proximal convoluted tubules (PCT) and renal failure in 26 Fisher 344 rats receiving 20 mg/kg of gentamicin twice daily up to 28 days. They were sacrificed on day 14 (renal failure, 13 rats) and day 28 (recovering, 13 rats). Control rats were used for morphologic studies (19) and serum creatinine studies (6 of 19). PCTs (14th and 28th day) were graded as to severity with transmission electron microscopy into grades 1 (normal-mild injury), 2 (moderate injury) and 3 (severe injury) using brush border and basolateral surface alterations. Scanning electron microscopy confirmed these grades using kidney slices and isolated tubular cells. With scanning electron microscopy greater than 3400, PCTs were graded and counted from 14th and 28th day kidneys, day 14 (grade 1, 18%, grade 2, 22%, and grade 3, 60%), day 28 (grade 1, 89% grade 2, 4%, and grade 3, 7%). The basolateral surface contour lengths (BSCL) per length of underlying tubular basement membrane were measured using a computer-assisted digitizer on transmission electron microscopy prints. Control PCTs had a mean BSCL of 268 microns, SD +/- 79, SE +/- 19 per 40 microns of tubular basement membrane. Likewise, the mean BSCL of grade 1 tubules measured 249 microns, SD +/- 87, SE +/- 21; the mean BSCL of grade 2 tubules was 125 microns, SD +/- 40, SE +/- 9 and grade 3 tubules BSCL was 90.5 the BSAI values showed that the 14th day rats had 28% of control BSAI and 28th day rats 80%. 2, grade 3, p less than 0.005). Squaring the mean BSCL of each group gave a basolateral surface area index (BSAI). Combining the percentage of the 3 grades of tubules of 14th and 28th day rats with the BSAI values showed that the 14th day rats had 28% of control BSAI and 28th day rats 80%. Mean serum creatinines (milligrams/deciliters) were: 14th day, 0.86; 28th day, 0.54; and control, 0.39. These data indicate loss of function correlates with lower BSAI (decreased basolateral surface area).     

Single channel recordings from basolateral and apical membranes of renal proximal tubules

A new method is described, which enables the recording of single ionic channels from the basolateral as well as the luminal membrane of renal proximal tubules with the patch-clamp technique. Segments of late proximal tubules of rabbit kidney are cannulated and perfused from one end. The other end is open and freely accessible to a patch pipette. The patch electrode can be moved against lateral cell membranes or can be inserted through the open end into the lumen and brought to contact with the brush-border membrane. Both, in the basolateral as well as in the luminal membrane, giga seals can be achieved. In both membranes, K⁺ selective channels could be identified.Part of this work was presented at the 59th Meeting of the Deutsche Physiologische Gesellschaft, March 1984 in Dortmund.     

CO2 along the proximal tubules in the rat kidney

The proximal intratubular pH of the rat kidney was measured in vivo with an antimony electrode system. PCO₂ and bicarbonate concentration of the proximal tubular fluid were determined with an ultramicro equilibration system. The tubular fluid to plasma inulin concentration ratio was evaluated by a microscope fluorometric method. The acid-base parameters and the inulin concentrations were determined under control conditions and during acetazolamide treatment. The intratubular PCO₂., was higher than the PCO₂ of the systemic arterial blood under control conditions and the difference in PCO₂ was increased during acetazolamide treatment. In acetazolamide treated rats the rate of fractional bicarbonate reabsorption was decreased in the early part of the proximal tubule, while it was of about the same in the middle and late parts as compared with control rats. The total bicarbonate reabsorption in the proximal tubule was reduced by 50% due to the carbonic anhydrase inhibition. It seems possible that the bicarbonate is still reabsorbed as CO₂ after carbonic anhydrase inhibition, as hydrogen ion secretion is not totally stopped by this treatment. The increase in intratubular PCO₂ after acetazolamide treatment is assumed to be due to an inhibition of the carbonic anhydrase facilitating effect on outward diffusion of CO₂ from the tubular lumen across the cell wall.     

Ultrastructure of isolated perfused proximal tubules from rabbit kidney. A comparison with proximal tubules fixed by perfusion in vivo

This study was carried out to determine whether the ultrastructure of proximal tubules isolated and perfused in vitro was comparable to the ultrastructure of tubules fixed by perfusion in vivo. The kidneys of female white rabbits were either fixed by perfusion in vivo with glutaraldehyde (controls) or removed for dissection (experimentals) of proximal convoluted tubules and late proximal straight tubules. The isolated tubules were perfused in vitro with Krebs-Ringers bicarbonate solution for 1 hour after which the tubules were fixed with glutaraldehyde. The experimental tubules and the control tubules were processed for electron microscopy, compared qualitatively, and analyzed morphometrically to evaluate the volume densities and surface densities of different cell organelles and the cell membrane. Qualitatively, there were no differences in the appearance of cell organelles in experimental tubules and control tubules, except that cells in some experimental tubules accumulated small lipid droplets which were located close to mitochondria. Only a few quantitative changes were found, the most noteworthy being a 40% decreased surface density of the brush border in experimental proximal convoluted tubules and late proximal straight tubules. Damaged experimental tubules showed an increased number of vacuoles in the inverted microscope. In the electron microscope the vacuoles corresponded to swollen and disintegrated mitochondria and enlarged endocytic vacuoles. However, small degrees of tubular damage were not observable in the inverted microscope, as very small vacuoles could only be seen in the electron microscope. The results show that proximal tubules can be dissected and perfused in vitro for 1 hour without major ultrastructural changes. It should be emphasized that tubules showing an increased vacuolization in the inverted microscope may be damaged and, thus, not function optimally, and even if the tubules appear undamaged in the light microscope they may show signs of cellular degeneration in the electron microscope.     

Acid-induced two-step depolarization in the peritubular membrane of bullfrog kidney proximal tubules.

Using ion-selective microelectrode technique, we investigated acid-induced changes of peritubular membrane potential (EM) and intracellular activity of Na+ and H+ ((Na)i and pHi) in the proximal tubule of perfused bullfrog kidney in vivo. When peritubular pH was reduced at constant PCO2, EM was depolarized in two steps: i.e., an initial sharp fall followed by an additional deeper fall. This was termed as the acid-induced two-step depolarization. During this change, pHi was decreased gradually from 7.4 to 6.9 in response to 1/10 HCO3- reduction (pH from 7.7 to 6.7), whereas (Na)i was increased after a transient decrease. This result supports the peritubular rheogenic HCO3- exit coupled to Na+ movement during the initial depolarization period. Complete removal of peritubular HCO3- at constant pH produced a less marked initial depolarization with a transient rise of pHi, followed by a partial repolarization without appreciable change in (Na)i. Peritubular SITS administration inhibited these depolarization responses to the acid perfusion. The above findings suggest that 1) the initial part of the two-step EM depolarization produced by low HCO3- or HCO(3-)-free perfusion resulted mainly from the peritubular rheogenic exist of HCO3-, 2) the magnitude of initial depolarization would also be affected by pHi, and 3) the subsequent delayed changes of EM were mainly determined by the pHi-sensitive K+ conductance of the peritubular membrane.     

Properties of single K+ channels in the basolateral membrane of rabbit proximal straight tubules

The basolateral membrane of rabbit straight proximal tubules, which were cannulated and perfused on one side, was investigated with the patch clamp technique. Properties of inward and outward directed single K⁺ channel currents were studied in cell-attached and insideout oriented cell-excised membrane patches. In cell-attached patches with NaCl Ringer solution both in pipette and bath, outward K⁺ currents could be detected after depolarization of the membrane patch by about 20–30 mV. The current-voltage (i/V) relationship could be fitted by the Goldman-Hodgkin-Katz (GHK) current equation, with the assumption that these channels were mainly permeable for K⁺ ions. A permeability coefficientP _K of (0.17±0.04) · 10^–12 cm³/s was obtained, the single channel slope conductance at infinite positive potentialg(V ) was 50±12 pS and the single channel conductance at the membrane resting potentialg(V _bl) was 12±3 pS (n=4). In cell-excised patches, with NaCl in the pipette and KCl in the bath, the data could also be fitted to the GHK equation and yieldedP _K = (0.1 ±0.01) ·10^–12 cm³/s,g(V ) = 40 ± 4 pS andg(V _bl) = 7 ± 1 pS (n=8). In cell-attached patches with KCl in the pipette and NaCl in the bath, inward K⁺ channels occurred at clamp potentials 60 mV, whereas outward K⁺ channel current was detected at more positive voltages. The current-voltage curves showed slight inward rectification. The single channel conductance, obtained from the linear part of the i/V curve by linear regression, was 46±3 pS and the reversal potential was 59±6 mV (n=9). In cell-excised patches with KCl in the pipette and NaCl in the bath, inward directed K⁺ channel currents could again be described by the GHK equation. The single channel parameters were similar to those recorded for outward K⁺ currents (see above). In inside-out oriented cell-excised patches with NaCl in the pipette and KCl in the bath, reducing bath (i.e. cytosolic) Ca²⁺ concentration from 10^–6 mol/l to less than 10^–9 mol/l did not affect the open state probability of single channel currents. These results demonstrate that the observed channels are permeable for K⁺ ions in both directions and that these basolateral K⁺ channels in rabbit proximal straight tubule are not directly dependent on Ca²⁺ ions.     

Ouabain decreases apparent potassium-conductance in proximal tubules of the amphibian kidney

According to a previous study from this laboratory, the electrochemical gradient for potassium across the peritubular cell membrane of proximal tubules in the isolated perfused frog kidney increases following the application of ouabain. In order to test, if this phenomenon were due to a decrease of potassium conductance, the effects of ouabain on cell membrane resistances and the sensitivity of the peritubular cell membrane potential difference (PD_pt) to step changes of peritubular potassium and bicarbonate concentration were studied. In the absence of ouabain, PD_pt averaged –60±3 mV (n=25). A step increase of peritubular potassium concentration from 3 to 18 mmol/l (pH 8.07) depolarises PD_pt (PD_k) by +24±2 mV (n=8). An increase of bicarbonate from 20 to 40 mmol/l (pH 8.07) hyperpolarises PD_pt (PD_b) by –2.8±0.4 mV (n=9). The resistance of the luminal and peritubular cell membranes in parallel (R _m) amounts to 45±9 k cm (tubule length) (n=4) and the voltage divider ratio (VDR) to 1.4±0.2 (n=7). The resistance of the cellular cable (cellular core,R _c) approaches 131±37 M/cm (n=4). Peritubular application of 0.1 mmol/l ouabain leads to a gradual decline of PD_pt (t _1/2 approx. 30 min), to an increase ofR _m, a decrease of PD_k and an increase of PD_b. VDR andR _c are not changed significantly. The data point to a functional link between the sodium/potassium ATPase and the potassium conductance of the peritubular cell membrane.     

Transfer of base across the basolateral membrane of cortical tubules of rat kidney

The transfer of HCO ₃ ^– /OH^– across the peritubular membrane of rat cortical tubules was studied by measuring capillary pH during stopped-flow microperfusion of peritubular capillaries with electrolyte solutions containing 3 mM HCO ₃ ^– . The rate of alkalinization of these solutions was significantly delayed when 10^–4 M acetazolamide, 5×10^–4 M SITS or 2 mM Ba²⁺ were added, as well as when chloride was substituted by gluconate. Under these conditions, stationary capillary pH was slightly but significantly increased. In another series, the lumen of proximal tubules was perfused with alkaline solutions while pH was measured in adjacent capillaries. During perfusion, capillary pH rose to a level 0.4 units higher than in free-flow conditions, returning after filling the lumen with oil; the rate of capillary pH return to baseline is a measure of base extrusion from cells, in the absence of influx from the lumen. This rate is also significantly delayed by acetazolamide. The data show that peritubular base extrusion is dependent on carbonic anhydrase, on basolateral membrane voltage, and on interstitial chloride, and delayed by the anion exchange inhibitor SITS; they are compatible with both basolateral HCO ₃ ^– /Cl^– exchange and conductive bicarbonate transfer.Supported by APESP, CNP_q, FINEP and PNUD-UNESCOSupported by CNPq, BrasilSupported by Conicyt, Argentina.     

Electrophysiology of cell volume regulation in proximal tubules of the mouse kidney

The present study has been designed to test for the influence of cell swelling on the potential difference and conductive properties of the basolateral cell membrane in isolated perfused proximal tubules. During control conditions the potential difference across the basolateral cell membrane (PD_bl) is –65±1 mV (n=74). Decrease of peritubular osmolarity by 80 mosmol/l depolarizes the basolateral cell membrane by +7.8±0.5 mV (n=42). An increase of bath potassium concentration from 5 to 20 mmol/l depolarizes the basolateral cell membrane by +25±1 mV (n=11), an increase of bath bicarbonate concentration from 20 to 60 mmol/l hyperpolarizes the basolateral cell membrane by –3.2±0.5 mV (n=13). A decrease of bath chloride concentration from 79.6 to 27 mmol/l hyperpolarizes the basolateral cell membrane by –1.8±0.7 mV (n=6). During reduced bath osmolarity, the influence of altered bath potassium concentration on PD_bl is decreased ( PD_bl=+16±2 mV,n=11), the influence of altered bicarbonate concentration on PDbl is increased ( PD_bl=–6.0±0.8 mV,n=13), and the influence of altered bath chloride concentration on PD_bl is unaffected ( PD_bl=–1.8±0.6 mV,n=6). Barium depolarizes the basolateral cell membrane to –28±2 mV (n=16). In the presence of 1 mmol/l barium, decrease of peritubular osmolarity by 80 mosmol/l leads to a transient hyperpolarization of the basolateral cell membrane by –5.9±0.5 mV (n=16). This transient hyperpolarization is blunted in the absence of extracellular bicarbonate. In conclusion, cell swelling depolarizes straight proximal tubule cells and increases bicarbonate selectivity of the basolateral cell membrane at the expense of potassium selectivity. The data reflect either incrases of bicarbonate conductance or decrease of potassium conductance during exposure of proximal tubule cells to hypotonic media.Parts of this work were presented at the 18th Congress of the Gesellschaft für Nephrologie, Frankfurt/M. 1986 and at the 8th International Symposium on Biochemical Aspects of Kidney Function, Dubrovnik 1986