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Functional Anatomy of the Kidney
Abstract
The kidney is one of the two major excretory organs in the body. It is involved in numerous vital bodily functions, including regulation of acid-base balance; maintenance of blood pressure via regulation of sodium excretion; maintenance of plasma osmolality via regulation of water excretion; regulation of plasma potassium, calcium, and phosphorus; elaboration of erythropoietin to maintain hematocrit; and hydroxylation of vitamin D. This chapter provides an overview of the functional anatomy of the kidney, with an emphasis upon the major ultrastructural and functional features of each nephron segment in order to provide an anatomic and functional framework for understanding renal function and toxicology. The kidney is composed of a variety of highly specialized nephron segments that are composed of distinct epithelial cell types, which engender specific transport properties to nephron segments where they reside. The kidney is uniquely sensitive to toxins and toxicants because they receive and filter an enormous quantity of blood.
... But, the efficiency of the kidney is a decline when there is a loss of nephron function. (2) A chronic renal failure which is also known uremia is a drastically high level of urea in the blood which may be the end result of acute glomerulonephritis and nephrotic syndrome. (3 ,4) CRF is a slowly progressive loss of renal function over a period of month or year resulting in abnormally low glomerular filtration rate which is usually determined indirectly by the creatinine level in the blood serum. ...
ABSTRACT
End-stage renal disease (ESRD) can result from a wide variety of different kidney diseases. Currently, 90% of
patients reaching CKD have chronic diabetes mellitus, glomerulonephritis or hypertension. With CKD comes
a myriad of problems related to the kidney's inability to excrete waste products leads to symptoms of uraemia.
The treatments of CKD require dialysis or kidney transplantation. In this review, an attempt has been made to
explain the nutritional management of CKD along with various dialysis treatment and the complications
related to the dialysis method. It is important to maintain optimal nutritional status so that the patient will be
a good candidate to respond to the treatment effectively.Kidney Patients necessitate following a blanced diet
plan to retain normal protein stores and to avoid metabolic complications. This article deals with the
therapeutic aspects of nutrition in CKD patients and will improve the quality of life
Keywords: ESRD, CKD, Dialysis, Nutritional management.
... Thus our findings on nNOS and eNOS restoration after Sch B pre-cotreatment plus mercury, might indicate a vasoregulatory effect of Sch B and control on vascular perfusion according to Park et al. (2009). From an anatomic point of view, each nephron is a nephro-vascular unit combined with its glomerulus and regulated by juxtaglomerular apparatus (Lash, 2010;Sands and Verlander, 2010). The apparatus consisted of three cooperating sites: juxtaglomerular cells, with a specific endocrine role, the mesangium, and the macula densa. ...
Mercuric chloride (HgCl₂) causes acute oxidant renal failure that affects mainly proximal tubules. Schisandrin B (Sch B), an active lignan from the fruit of Schisandra chinensis, has been successfully used to treat gentamicin nephrotoxicity, but its role against mercury damage is still largely unknown. Here we analysed in vivo and in vitro the efficacy of Sch B supplementation against HgCl₂ nephrotoxicity, focusing on histopathology, stress proteins, oxidative (cytochrome c oxidase) and nitrosactive markers (eNOS, nNOS). Wistar rats were treated with Sch B (10 mg/kg/day p.o.) or vehicle (olive oil) for 9 days, then coadministered with a single HgCl₂ nephrotoxic dose (3.5 mg/kg i.p.) and killed after 24 h. The tubular and mitochondrial damage induced by mercury was limited by Sch B coadministration in vivo. Remarkably, after Sch B and mercury challenge, HSP25, HSP72, GRP75 were reduced in the renal cortex, cytochrome c oxidase increased and eNOS and nNOS were restored in glomeruli. In contrast, NRK-52E proximal tubular cells treated with Sch B 6.25 μM plus HgCl₂ 20 μM did not show any amelioration on viability and oxidative stress in respect to HgCl₂ 20 μM alone. Moreover, after Sch B plus mercury in vitro treatment, HSP72 staining persisted while HSP25 further increased. Thus, in our experimental conditions, Sch B cotreatment afforded better protection against mercury poisoning in vivo than in vitro. This discrepancy might be partly attributable to Sch B influence on glomerular perfusion as corroborated by the recovery of vasoactive markers like macular and endothelial nitric oxide isoforms.
Three-dimensional (3D) histopathology involves the microscopic examination of a specimen, which plays a vital role in studying tissue's 3D structures and the signs of diseases. However, acquiring high-quality histological images of a whole organ is extremely time-consuming (e.g., several weeks) and laborious, as the organ has to be sectioned into hundreds or thousands of slices for imaging. Besides, the acquired images are required to undergo a complicated image registration process for 3D reconstruction. Here, by incorporating a recently developed vibratome-assisted block-face imaging technique with deep learning, we developed a pipeline termed HistoTRUST that can rapidly and automatically generate subcellular whole organ's virtual hematoxylin and eosin (H&E) stained histological images which can be reconstructed into 3D by simple image stacking (i.e., without registration). The performance and robustness of HistoTRUST have been successfully validated by imaging all vital mouse organs (brain, liver, kidney, heart, lung, and spleen) within 1-3 days depending on the size. The generated 3D dataset has the same color tune as the traditional H&E stained histological images. Therefore, the virtual H&E stained images can be directly analyzed by pathologists. HistoTRUST has a high potential to serve as a new standard in providing 3D histology for research or clinical applications.
Anatomical texts describe the neck musculature without measurements of muscle locations or quantitative estimates of pulling actions (torques). This study is based on measurements in stereotaxic coordinates of cat neck muscle origins and insertions, and neck intervertebral rotation axes. Torque vectors in three dimensions were calculated for 14 pairs of dorsal and ventral muscles that insert on the skull or first cervical vertebra. Predicted torque vectors were in general agreement with qualitative statements in the literature. Biventer cervicis and the rectus capitis major, medius, and minor muscles act mainly to raise the head, and longus capitis acts almost exclusively to lower the head. Longissimus capitis, sternomastoid, and cleidomastoid act mainly to roll the head. Complexus acts about equally to raise the head and rool it. Splenius and occipitoscapularis have torque in all three coordinate directions. Torques were altered by changing the pitch of the head with respect to the neck. The calculated neck muscle torques did not correspond to previously reported directions of neck muscle excitation during the vestibulocollic reflex. The neck musculature appears to be a complex, multidimensional system that presents interesting problems in motor control.
A formula has been developed to predict creatinine clearance (Ccr) from serum creatinine (Scr) in adult males: (see article)(15% less in females). Derivation included the relationship found between age and 24-hour creatinine excretion/kg in 249 patients aged 18-92. Values for Ccr were predicted by this formula and four other methods and the results compared with the means of two 24-hour Ccr's measured in 236 patients. The above formula gave a correlation coefficient between predicted and mean measured Ccr's of 0.83; on average, the difference predicted and mean measured values was no greater than that between paired clearances. Factors for age and body weight must be included for reasonable prediction.
Proton secretion in the renal medullary collecting duct is thought to occur via a luminal proton-ATPase. In order to determine what mechanism(s) participate in proton transport across medullary collecting duct (MCD) cells membranes, intracellular pH (pHi) regulation and proton extrusion rates were measured in freshly prepared suspensions of rabbit outer MCD cells. Cells were separated by protease digestion and purified by Ficoll gradient centrifugation. pHi was estimated fluorometrically using the entrapped intracytoplasmic pH indicator, 6-carboxyfluorescein. Proton extrusion rates were measured using a pH stat. The resting pHi of MCD cells was 7.19 +/- 0.05 (SE) in a nonbicarbonate medium of pH 7.30. When cells were acidified by exposure to acetate salts or by abrupt withdrawal of ammonium chloride, they exhibited pHi recovery to the resting pHi over a 5-min time-course. Depletion of greater than 95% of cellular ATP content by poisoning with KCN in the absence of glucose inhibited pHi recovery. ATP depletion inhibited proton extrusion from MCD cells. Treatment with N-ethylmaleimide also inhibited pHi recovery. In addition, cellular ATP content was dependent on transmembrane pH gradients, suggesting that proton extrusion stimulated ATP hydrolysis. Neither removal of extracellular sodium nor addition of amiloride inhibited pHi recovery. These results provide direct evidence that a plasma membrane proton-ATPase, but not a Na+/H+ exchanger, plays a role in proton transport and pHi regulation in rabbit MCD.
The cellular distributions of the kidney form of the erythrocyte band 3 chloride/bicarbonate exchanger and the kidney vacuolar H+-transporting ATPase were examined in rat kidney collecting duct by immunocytochemical staining of adjacent semithin sections. Polyclonal anti-peptide antibodies directed against two regions of murine erythroid band 3 gave a pattern of basolateral labeling similar to that seen with antibodies directed against the entire protein. In the medullary collecting duct almost all intercalated cells expressed basolateral membrane band 3 and displayed apical membrane H+-ATPase. In the cortical collecting duct and the connecting segment, band 3 labeling was restricted to a subpopulation of intercalcated cells. In the cortical collecting duct 46% of intercalated cells had apical H+-ATPase and basolateral band 3. Cells that had either basolateral or diffuse cytoplasmic staining for H+-ATPase were all band 3-negative and accounted for 53% of the intercalated cells. In addition, occasional intercalated cells with apical H+-ATPase appeared to lack basolateral band 3. These results demonstrate the coexpression of H+-ATPase and band 3 in opposite plasma membrane domains of a subpopulation of intercalated cells that are probably the acid-excreting (type A) cells. All other intercalated cells lacked immunoreactive band 3 and probably include the bicarbonate-excreting (type B) cells.
The collecting duct of the inner stripe outer medulla (OMCDi) is a major site of distal nephron acidification. Using the pH sensitive fluorescent dye 2'-7'-bis(carboxyethyl)-5,6,-carboxyfluorescein (BCECF) and quantitative spectrofluorometry to measure intracellular pH in isolated perfused OMCDi, we have characterized basolateral transport processes responsible for regulation of intracellular pH. Experiments suggesting the existence of basolateral Cl-/base exchange were performed. In HCO3- containing buffers, bath Cl- replacement resulted in reversible alkalinization of the OMCDi from 7.22 +/- 0.05 to 7.57 +/- 0.12. Similarly 0.1 mM bath 4',4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) alkalinized the OMCDi from 7.14 +/- 0.09 to 7.34 +/- 0.09 and blocked further alkalinization by bath Cl- removal (delta = + 0.02 pH units). The concentration dependence kinetics of Cl-/base exchange revealed a K1/2 of 10 mM for external Cl- with a Vmax of 0.50 pH U/min. Experiments suggesting the existence of basolateral Na+/H+ exchange were also performed. Replacement of bath Na+ by tetramethylammonium resulted in reversible cell acidification (7.14 +/- 0.09 to 6.85 +/- 0.1). Tubules that were acidified by a brief exposure to NH4Cl displayed recovery of cell pH back to baseline at a rate that was highly dependent on bath Na+ concentration. Half maximal recovery rate was achieved at 7 mM bath Na+ and Vmax was 0.605 pH U/min. The Na+-dependent rate of cell pH recovery after acidification was blocked by 0.2 mM bath amiloride. These results suggest that intracellular pH in the OMCDi is regulated by parallel basolateral Na+/H+ exchange and Cl-/base exchange.
Chemotherapy often fails because a tumor develops resistance to an array of different drugs. A single glycoprotein turns out to be responsible: it proliferates in some cells and pumps out the drugs. Now that the protein pump has been identified it may be possible to interfere with its action or to make it the target for drugs that destroy the cancer cell.
We determined whether a spontaneous luminal disequilibrium pH, pHdq (pH measured - pH equilibrium), was present in isolated perfused rabbit S2 and S3 proximal tubules. Luminal pH was measured by perfusing with the fluorescent pH probe 1,4-DHPN, and the equilibrium pH was calculated from the measured collected total CO2 and dissolved CO2 concentrations. S2 tubules failed to generate a spontaneous pHdq. S3 tubules generated a spontaneous acidic pHdq of -0.46 +/- 0.15 (P less than 0.05), which was obliterated following the addition of carbonic anhydrase (0.1 mg/ml) to the perfusate. In S3 tubules perfused and bathed in 4 mM total ammonia, luminal total ammonia rose from 4.08 +/- 0.05 mM (perfusate) to 4.95 +/- 0.20 mM (collected fluid) (P less than 0.02). Carbonic anhydrase added to the perfusate prevented the rise in the collected total ammonia concentration. We conclude that the rabbit S3 proximal tubule lacks functional luminal carbonic anhydrase. The acidic pHdq in the S3 segment enhances the diffusion of NH3 into the lumen. In contrast, the S2 segment has functional luminal carbonic anhydrase.
The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.
Background: Serum creatinine concentration is widely used as an index of renal function, but this concentration is affected by factors other than glomerular filtration rate (GFR). Objective: To develop an equation to predict GFR from serum creatinine concentration and other factors. Design: Cross-sectional study of GFR, creatinine clearance, serum creatinine concentration, and demographic and clinical characteristics in patients with chronic renal disease. Patients: 1628 patients enrolled in the baseline period of the Modification of Diet in Renal Disease (MDRD) Study, of whom 1070 were randomly selected as the training sample ; the remaining 558 patients constituted the validation sample. Methods: The prediction equation was developed by stepwise regression applied to the training sample. The equation was then tested and compared with other prediction equations in the validation sample. Results: To simplify prediction of GFR, the equation included only demographic and serum variables. Independent factors associated with a lower GFR included a higher serum creatinine concentration, older age, female sex, nonblack ethnicity, higher serum urea nitrogen levels, and lower serum albumin levels (P < 0.001 for all factors). The multiple regression model explained 90.3% of the variance in the logarithm of GFR in the validation sample. Measured creatinine clearance overestimated GFR by 19%, and creatinine clearance predicted by the Cockcroft-Gault formula overestimated GFR by 16%. After adjustment for this overestimation, the percentage of variance of the logarithm of GFR predicted by measured creatinine clearance or the Cockcroft-Gault formula was 86.6% and 84.2%, respectively. Conclusion: The equation developed from the MDRD Study provided a more accurate estimate of GFR in our study group than measured creatinine clearance or other commonly used equations.
By using the in vitro microperfusion technique, were examined functions of the descending limbs of Henle's loop obtained from either the short-loop nephron (SDL) or the upper portion of the long-loop nephron of hamsters (LDLu). Morphological distinctions between these segments were confirmed by light and electron microscopic observations. Both segments were highly permeable to water. The LDLu was highly permeable to sodium and to chloride: efflux coefficients (10−7 cm2·s−1) for22Na and36Cl were 41.0±5.4 and 3.8±0.6, respectively. The SDL were less permeable to sodium and to chloride: efflux coefficient for22Na and36Cl were 2.9±1.4 and 0.9±0.2, respectively. In contrast, the SDL was more permeable to urea as compared to the LDL, efflux coefficients for urea being 5.1±1.4 vs 1.4±0.3, respectively. When composition of the perfusate was identical to that of the bathing fluid, no transepithelial voltage was demonstrated. The volume flux was very small or undetectable. From these observations, we propose that the internephron heterogencity must be taken into consideration for constructing a model of countercurrent system in the renal medulla.
Specific antibodies against human erythrocyte carbonic anhydrase isozyme C were used to determine the ultrastructural localization of this enzyme in the collecting ducts of rat kidney. Using a pre-embedding labeling technique, carbonic anhydrase C was found in the cytoplasm of intercalated cells, whereas the principal cells were negative.
In order to investigate the mechanism of Na and K transport, rabbit cortical collecting tubules were perfused in vitro and the concentrations of Na and K in lumen and bathing fluid and the transtubular electrical potential difference (PD) were measured. When the perfusate and external bath contained 150 Na-5 K (mEq liter(-1)), the sodium concentration decreased and the potassium concentration increased by an approximately equal amount in collected tubular fluid. The transtubular electrical potential was equal at both ends of the tubule in the steady state and ranged between 21 and 67 mv, lumen negative. In all tubules perfused at rates less than 0.5 nl min(-1), the K concentration of the collected fluid was higher and the Na concentration lower than that predicted for electrochemical equilibrium between lumen fluid and external bath, evidence for active transtubular transport of both cations. These results differ from those observed in rat distal tubule in which potassium secretion is passive. Active Na and K transport and the transtubular PD were decreased by (a) ouabain, (b) removal of sodium from the perfusate, or (c) removal of potassium from the external bath, evidence of interdependence of Na and K transport. The dependence of active K secretion on intraluminal Na concentration accounts for the phenomenon of "distal" Na-K exchange noted previously in clearance and stop-flow studies. The mechanism of Na transport may in part be electrogenic since the rate of decline of the transtubular PD in low K media was faster than could be accounted for on the basis of a reduction in cell potassium concentration.
The hamster renal pelvis has been studied by means of low-power light microscopy, scanning electron microscopy and morphometric analyses. The results of this study are highly suggestive that the contact of pelvic urine with the other medulla as well as with the inner medulla may be an important aspect of final urine formation. The outer medulla constituted nearly 50% of the total pelvic surface area, with the inner stripe of the outer medulla more than twice the pelvic surface area of the outer stripe of the outer medulla. The large outer medullary pelvic surface area was accounted for by the elaboration of the upper pelvic walls into peripelvic columns, opercula ("secondary pyramids"), fornices and secondary pouches. A thin simple-squamous to low cuboidal pelvic epithelium separated pelvic urine from outer medullary parenchyma. The inner medulla which constituted about one quarter of the total pelvic surface area was covered by a cuboidal to columnar pelvic epithelium which appeared morphologically similar to the papillary collecting duct epithelium. Tubules and capillaries of the inner medulla did not appear as closely juxtaposed to the pelvic epithelium as did those of the outer medulla. Cortical tissue comprised only 11.7% of the total pelvic surface area and was covered by transitional epithelium similar to that of ureter and bladder. The previously reported impermeability of this epithelium suggests that pelvic urine contact with the cortex is unimportant in final urine formation. The rich layer of smooth muscle under the transitional epithelium probably functions to move urine into and out of the pelvis during pelvic peristalsis, which has been observed in vivo.
Studies on adenylate cyclase response of the nephron fragments to hormones and drugs have suggested that there is a functionally distinct segment tentatively called the connecting tubule (CNT), which is located between the distal (DT) and the cortical collecting tubule (CCT). The functional significance of these biochemical findings was examined with isolated rabbit renal tubules perfused in vitro. The transepithelial voltage (PDt) of the DT, CNT, and CCT were, respectively, -28.7 +/- 3.24 mV (24), -27.0 +/- 2.69 mV (24), and -3.5 +/- 2.14 mV (11) in the normal rabbits. The PDt of the CCT increased to -32.2 +/- 2.02 mV (33) when rabbits were pretreated for at least 3 days with deoxycorticosterone acetate, DOCA (1 mg/kg/day, i.m.), whereas the PDt of the DT and the CNT remained unchanged. The PDt of the CCT obtained from deoxycorticosterone acetate- (DOCA) treated animals decreased after addition of antidiuretic hormone (ADH) or isoproterenol (ISO) to the bath. The PDt of the CNT also responded to these agents, but the dose required to obtain the same response was quite different: The CNT was 100-fold more sensitive to ISO as compared to the CCT, whereas the CCT was 10-fold more sensitive to ADH than was the CNT. In contrast, the PDt of the DT did not respond to any of these agents, even at a higher concentration. After addition of ADH (200 microU/ml) to the bath, the osmotic water permeability (10(-8) cm2 . sec-1 . atm-1) of the CCT increased from 1.13 +/- 0.83 to 7.46 +/- 2.36, but that of the CNT remained low (0.36 +/- 0.78 in control vs. 0.48 + 0.64 after ADH). These observations support the view that the CNT is functionally distinct from either the DT or the CCT.
Rat thin limbs of Henle were studied by freeze-fracture electron microscopy. Thin limb segments in both short- and long-looped nephrons were identified by previously developed ultrastructural criteria, continuity with known thick segments, and architectural relationships in the outer medulla. Intramembrane particle (IMP) density and the number of intramembrane fibrils comprising the zonula occludens were determined for each morphologically identifiable thin limb segment. The IMP density on the protoplasmic faces of both the luminal and abluminal membranes of the upper portion of the descending thin limb (DTL) of the long-looped nephron is quantitatively greater than in the short-looped thin limb, lower portion of the long-looped DTL, and in the ascending thin limb. The zonulae occludens in the long-looped upper DTL consists of a single fibril; the long-looped lower DTL contains 3.13 +/- 0.14 fibrils; the ascending thin limb contains 1.31 +/- 0.09 fibrils; and the short-looped DTL contains 3.75 +/- 0.19 fibrils. These studies further support the contention that there is anatomic heterogeneity among the thin limb segments. Because direct physiologic studies in the thin limbs are incomplete and conflicting, the need for correlative physiologic studies on anatomically characterized structures is indicated.
Single segments of the loop of Henle, the cortical thick ascending limb (CTAL), were dissected from fresh renal slices (rabbit) and individually perfused in vitro. Direct analysis of transepithelial NaCl and water movement was carried out at two functional states of epithelial transport. I. Differentiating thick ascending loop of Henle (d-CTAL, day 2–10 postnatal,n
t=31); II. Mature thick ascending loop of Henle (m-CTAL, day 25–30,n
t=23). Nephron segments were dissected without collagenase. Perfusion rates were similar per unit surface area in both functional states. Perfusate was ultrafiltrate of rabbit serum or artificial solution identical to bath medium, except when osmotic hydraulic permeability or steady-state ion gradients were analyzed. Results: Osmotic hydraulic permeability (bath medium hyperosmotic) was 4.72 cm3 cm–2 min–1 kPa–1 10–8 [or 4.78(S.D.) 1.15 cm3 cm–2 min–1 atm–1 10–6] in the d-CTAL, similar to the m-CTAL (5.191.21). Solute transport (ratio of collected to perfused osmolarity) was hyperosmotic in the m-CTAL (0.620.13), but only 0.910.07 in the d-CTAL. Net sodium transport across the d-CTAL was 2.130.37 mol cm–1s–110–12, and 11.61.3 in the m-CTAL. Steady-state sodium concentration in the single loop lumen was 658 mmoll–1 in the m-CTAL, and 972.7 in the d-CTAL (bath medium: 148 mmoll–1). Conclusions: Differentiation of the thick ascending loop of Henle is characterized by constant osmotic hydraulic permeability. Sodium absorption, transepithelial steady-state sodium concentration gradient, and transtubular osmotic disequilibrium suggest that the electrolyte transport capacity of the single cell of the diluting segment is the major determinant of renal medullary countercurrent differentiation.
Descending limbs of Henle's loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional “Sylgard seal” pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H2O) even when the bath was hyperosmotic: ΔJ
v
=−0.06 ±0.18 nl/min (n=7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16±8 mM (n=7) and 254±38 mOsm (n=7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea.
When the “Sylgard seal” collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H2O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 ±5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153±8 mmole/l; 6 tubules).
The14C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 ×10−7 cm2·s−1±0.23×10−7 (11 tubules). When the bath was made hyperosmotic using urea or raffinose the14C urea permeability increased significantly.
Recent micropuncture studies have suggested that the collecting tubule may be involved in the regulation of extracellular fluid volume. The present studies were designed to evaluate chloride transport across the in vitro-perfused rabbit cortical collecting tubule inasmuch as chloride ion would ultimately affect extracellular fluid volume. The tubules were perfused and bathed with artificial solutions simulating ultrafiltrate. Four groups of studies were conducted. In groups one and two, tubules from rabbits not receiving desoxycorticosterone (DOCA) were compared to tubules from rabbits which had received DOCA (5 mg/day) for 1 wk. In groups three and four, tubules were obtained only from rabbits not receiving DOCA. In group one, sequential bidirectional chloride fluxes were measured. The ratio of chloride efflux to influx was 0.99+/-0.04 in tubules obtained from rabbits not receiving DOCA whereas it was 1.28+/-0.09 in tubules obtained from rabbits receiving DOCA, suggesting stimulation of net chloride flux under these conditions. In group 2, chemical chloride concentration and osmolality of the collected fluid were measured. Neither the chemical chloride concentration nor the osmolality of the collected fluid decreased significantly below their respective perfusion fluid values in tubules from non-DOCA-treated rabbits but there was a significant decrease in the chemical chloride concentration (10-42 meq/liter) and osmolality (10-42 mosmol/kg H(2)O of the collected fluid in tubules from DOCA-treated rabbits. In group three, unidirectional chloride permeabilities from lumen-to-bath were determined during the passage of current down the perfusion pipette. The alterations of the average lumen potential, -35+/-4 and +28+/-2 mV, did not influence unidirectional chloride movement suggesting that the cortical collecting tubule is quite impermeable to chloride. In group four, unidirectional chloride permeability from lumen-to-bath was measured before and after substitution of NaCH(3)SO(4) for sodium chloride in the bath. Replacement of chloride by CH(3)SO(4) reversibly decreased the apparent chloride permeability from 2.41+/-0.50 to 0.69+/-0.08 (x10(-5) cm/s) demonstrating that (36)Cl permeability is dependent on the chemical concentration of chloride.THE CURRENT STUDIES DEMONSTRATE THAT: (a) the cortical collecting tubule is able to reabsorb salt under the modulation of circulating mineralocorticoids and, thus, may participate in overall volume homeostasis; (b) the chloride permeability and the major portion of isotopic chloride flux across the cortical collecting tubule is via exchange diffusion; and (c) under certain circumstances the cortical collecting tubule may act as a diluting segment.
Previous studies in the mammalian proximal tubule have suggested that para-aminohippurate (PAH) secretion is approximately threefold greater in the straight segment, or pars recta, than in the convoluted segment, or pars convoluta. However, the possibility that the site of maximal PAH secretion might be related better to particular tubule segments as identified by cell type had not been explored. In addition, the presence or absence of differences in PAH secretion between morphologically identical regions of superficial (SF) vs. juxtamedullary (JM) proximal tubules has not been examined. These issues were studied using a combination of histologic methods and measurement of [(3)H]PAH secretion in isolated perfused tubules. Measurements of microdissected SF and JM proximal tubules from young and adult rabbits revealed that SF proximal tubules were slightly but significantly longer than JM tubules ([young rabbits: SF, 8.69+/-SE 0.14 mm vs. JM, 7.97+/-SE 0.13 mm; P < 0.01] [adult rabbits: SF, 10.61+/-SE 0.28 mm; JM, 9.17+/-SE 0.19 mm; P < 0.001]). Light and electron microscopy revealed three sequential segments (S(1), S(2), and S(3)) along the length of SF and JM proximal tubules as defined by cell type. PAH secretion was measured in each of these three segments by the isolated perfused tubule technique. Net PAH secretion in fmol/mm per min in SF proximal tubules was: S(1), 281+/-SE 21; S(2), 1,508+/-SE 104; S(3), 318+/-SE 46. Corresponding values in JM proximal tubules were 353+/-SE 31, 1,391+/-SE 72, and 188+/-SE 23. Net PAH secretion did not differ between comparable segments of SF and JM proximal tubules. It is concluded that differences in PAH secretion along the proximal tubule correlate best with cell type rather than the arbitrary division of the proximal tubule into pars convoluta and pars recta according to its external configuration. Evidence of functional heterogeneity between comparable segments of SF and JM proximal tubules was not observed.
A circuit is described that can produce a high output voltage to automatically short-circuit an epithelial tissue mounted in an Ussing chamber. Because of its high output voltage it can short-circuit preparations requiring over 500 muA even in conjunction with high-resistance agar bridges. The circuit, which is easy to build, uses an inexpensive, low-voltage, integrated circuit operational amplifier, which is electrically isolated from the high-voltage part of the circuit by an optical isolator. The device can also be modified for use as a high impedance preamplifier for monitoring the spontaneous membrane potential.
The possibility that the rabbit collecting tubule is heterogeneous with respect to electrical and morphologic properties was examined in isolated segments. Collecting tubules from rabbits treated with desoxycorticosterone acetate were dissected from various locations within the cortex and outer medulla, and the transepithelial potential was measured. The cortical collecting tubule always displayed a negative potential. In contrast, collecting tubules dissected from the inner stripe of the outer medulla displayed a positive potential. That this change is a function of position within the kidney is supported by the observation that some tubules generated a negative potential at the superficial (cortical) end and a positive potential at the deep (medullary) end of the same tubule. Histologic evaluation of tubules after perfusion resulted in a striking correlation between the existence of a negative potential and the presence of dark cells. The results of this study provide evidence that the collecting tubule is heterogeneous with respect to active transport processes and suggest that these functional differences may correlate with morphologic differences between the cortical segment and the medullary segment of the collecting tubule.
Studies utilizing in vitro microperfusion were designed to examine whether urea is actively or passively transported across superficial and juxtamedullary straight segments of rabbit proximal tubules. With perfusate and bath solutions containing 1 mM urea and electrolytes similar to normal plasma, the efflux (lumen-to-bath) isotopic permeability (X 10(-5) cm s-1) of superficial segments was 1.37 +/- 0.16 and of juxtamedullary segments was 2.14 +/- 0.20. In the same tubules, the influx (bath-to-lumen) isotopic permeability was 3.70 +/- 0.35 in superficial segments and 4.75 +/- 0.37 in juxtamedullary segments. Despite net water movement in the opposite direction (0.5 nl mm-1 min-1), the influx rate was significantly higher than the efflux rate of urea in both groups. With a low perfusion rate (2 nl/min) and equivalent specific activities of [14C]urea in bath and perfusate, the collected-to-perfused ratio of [14C]urea, corrected for volume marker change, was 1.07 +/- 0.01 in superficial and 1.09 +/- 0.01 in juxtamedullary nephrons, thus indicating net secretion in both segments. In separate studies urea influx was inhibited by hypothermia (decrease from 37 degrees to 28 degrees C), by phloretin (0.1 mM in bath), by cyanide (1 mM), but not by probenecid (0.2 mM). In each case the inhibition was highly significant and reversible. These data suggest that urea is actively secreted by the straight segments of both the superficial and juxtamedullary proximal tubules. These segments may, therefore, contribute significantly to the high urea concentration found at the bend of Henle's loop by micropuncture.
Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although
selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane
protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions.
Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited
by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane
water channels.
The thin limb segments of the long loop of Henle are thought to play important roles in the urinary concentrating mechanism. In this study, we present new approaches to the identification, dissection, and in vitro perfusion of individual thin limb segments from all levels of the chinchilla renal medulla, including the deepest portions of the papilla. We have applied these techniques to the investigation of the osmotic water permeability along the chinchilla long loop of Henle. The results demonstrate that the osmotic water permeability of the thin descending limb is not uniformly high along its length, as previously thought, but that the distal 20% of the long-loop descending limb has a very low water permeability (approximately 50 microns/s). The transition to the low water permeability region of the thin descending limb is accompanied by a relatively abrupt change in morphology (increased cellularity and decreased diameter) that is readily perceptible in the perfused segments and even in the dissection dish. In contrast, the upper part of the chinchilla long-loop thin descending limb had an extremely high osmotic water permeability (greater than 2,000 microns/s) as observed in other species. Thin ascending limbs from deep in the inner medulla had water permeabilities that were indistinguishable from zero, as previously found in thin ascending limbs from near the inner-outer medullary junction. The presence of a low-water-permeability portion of the long-loop thin descending limb in chinchilla may have important implications with regard to the inner medullary concentrating process. A relatively low osmotic water permeability (397 microns/s) was also found in the deep inner medullary portion of the thin descending limb from the rat.
We have shown that urea transport across the terminal inner medullary collecting duct (terminal IMCD) is mediated by a vasopressin-stimulated, facilitated diffusion process exhibiting properties consistent with a transporter. To investigate whether hypertonic NaCl, as exists in vivo in the inner medulla, affects urea permeability, we studied isolated perfused rat terminal IMCD segments. Perfusate and bath osmolality were varied symmetrically by adding or removing NaCl or mannitol. Urea permeability rose progressively when osmolality was increased with NaCl or mannitol from 290 to 690 mOsm/kg H2O in the absence of vasopressin; there was no further increase at 890 mOsm/kg H2O. In the presence of 10(-8) M arginine vasopressin, urea permeability increased when NaCl was added to raise osmolality from 290 to 490 mOsm/kg H2O but there was no further increase at 690 mOsm/kg H2O. When 1 mM 8-bromo cyclic AMP was added to the bath, raising NaCl still increased urea permeability. These results suggest that urea transport across the rat terminal IMCD is regulated both by vasopressin and by osmolality at values present in the renal inner medulla. Osmolality seems to activate urea transport across the rat terminal IMCD by mechanisms distinct from those of vasopressin or cyclic AMP.
Currents through individual Na channels in the apical membrane of the rat cortical collecting tubule were resolved by using the patch-clamp technique. In cell-attached patches, the channels had a conductance of 5 pS with 140 mM NaCl in the pipet. The conductance was a saturable function of external Na, with a maximal value of about 8 pS and a half saturation at about 75 mM Na. In excised inside-out patches, the selectivity of the channels for Na over K was estimated from reversal potentials to be at least 10:1. The channels underwent spontaneous transitions between open and closed states. Both states had mean lifetimes of 3-4 sec. Amiloride (0.5 microM) added to the pipet induced more frequent closures and openings of the channels and a reduction in the mean open time. These channels are presumed to mediate Na reabsorption by this nephron segment in vivo.
We examined the hypothesis that proton-potassium-activated adenosine triphosphatase (H-K-ATPase) mediates K absorption and acidification in the inner stripe of the outer medullary collecting duct (OMCDi). Rabbits were fed a low-K diet (0.55% K) for 7-14 d because we have demonstrated previously that this low-K diet stimulates K-absorptive flux by the OMCDi. Proton secretion was measured as net total CO2 flux (JTCO2) by microcalorimetry. After basal collections, either vehicle or an inhibitor of gastric H-K-ATPase, omeprazole (0.1 mM), was added to the perfusate during the second period. Addition of vehicle to the perfusate changed neither the transepithelial voltage (VT, in millivolts) nor the JTCO2. In contrast, the addition of omeprazole (0.1 mM) to the perfusate abolished JTCO2 (from 14.5 +/- 5.6 to -0.1 +/- 3.1 pmol.mm-1.min-1) without significantly affecting VT. In additional experiments, in 16 tubules there was significant net K absorption (JK) of 5.0 +/- 1.0 pmol.mm-1.min-1 during the basal period, which exceeded the rate of K absorption that could be attributed to a paracellular voltage-mediated pathway (JKP = 1.0 +/- 0.4 pmol.mm-1.min-1, P less than 0.01). Administration of vehicle did not significantly affect either VT or JK. However, omeprazole abolished JK (from 5.1 +/- 1.0 to 0.1 +/- 2.5 pmol.mm-1.min-1) without affecting VT or JNa. The present results demonstrate that the OMCDi possesses an active, omeprazole-sensitive acidification and K-absorptive mechanism. These findings are consistent with the presence of H-K-ATPase activity in this nephron segment.
Synthesis of the sodium pump, Na+-K+-ATPase, is regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine if triiodothyronine (T3) regulates the concentration of the mRNAs coding for the two enzyme subunits, alpha and beta, and the time course of the response. A single dose of T3 (250 micrograms/100 g body wt) was administered to hypothyroid rats that were killed at various times after injection. In the kidney cortexes of the T3-injected animals, as well as hypothyroid and euthyroid rats, alpha- and beta-mRNA concentrations were measured by dot blot using cDNAs corresponding to the two mRNAs; alpha-subunit abundance was measured by Western blot using antibodies to the enzyme, and Na+-K+-ATPase activity was measured enzymatically. alpha- and beta-mRNAs increased coordinately, after a 6-h time lag to 1.6-fold over hypothyroid levels by 12 h after T3. alpha-Subunit abundance increased significantly by 48 h and to 1.4-fold over hypothyroid by 72 h after T3. Na+-K+-ATPase activity increased with the same time course as the increase in alpha-subunit abundance to 1.3-fold over hypothyroid by 72 h after T3. We conclude that T3 regulates Na+-K+-ATPase synthesis and activity by coordinately increasing the mRNAs of both the alpha- and beta-subunits of the enzyme.
To determine the exact site and mechanism of action of thiazide diuretics, effects of 10(-4) M trichlormethiazide (TCM) on NaCl transport were examined in the distal convoluted tubule (DCT), the connecting tubule (CNT) and the cortical collecting duct (CCD) of rabbit kidney by the in vitro microperfusion technique. TCM added to the lumen decreased lumen-to-bath 36Cl flux (JCl(LB)) only in the CNT without changing the transmural voltage (VT). In the DCT, 10(-4) M furosemide did not change JCl(LB) even if it was added to the lumen with 10(-4) M TCM, whereas 10(-5) M amiloride in the lumen decreased the lumen-to-bath 22Na flux (JNa(LB)) and VT. In the CNT, TCM added to the lumen did not affect the bath-to-lumen 36Cl flux. Addition of TCM to the bath slightly decreased JCl(LB). Luminal addition of 10(-4) M TCM also decreased JNa(LB). Amiloride at 10(-5) M in the lumen decreased both JNa(LB) and VT. Addition of TCM with 10(-5) M amiloride further decreased JNa(LB) without affecting VT, indicating that TCM affects the electroneutral Na+ transport, which is distinct from the amiloride-sensitive conductive Na+ pathway. When Na+ was removed from the lumen, JCl(LB) was markedly decreased, but addition of TCM did not cause further decrease in JCl(LB). Furosemide did not affect JCl(LB), but addition of both 10(-4) M TCM and furosemide decreased JCl(LB), indicating that Na+-K+-2Cl- cotransport is not involved in the action of TCM. Removal of HCO3- slightly decreased JCl(LB), and TCM caused further decrease in JCl(LB). Amiloride at 10(-3) M, a concentration supposed to inhibit the Na+/H+ antiport, slightly decreased JCl(LB), and addition of TCM caused a further marked decrease in JJl(LB). The similar results were also obtained when the combined effects of 10(-3) M 4,4'-diisothiocyano-stilben-2,2'-disulfonate(DIDS) and 10(-4) M TCM were examined. These findings suggest that the parallel antiport of Na+/H+ and Cl-/HCO3- is not involved in the action of TCM. By excluding other possible mechanisms involving neutral Na+-dependent Cl- transport, we conclude that TCM inhibits Na+-Cl- cotransport in the luminal membrane of the rabbit CNT.
In a review published in 1977, Sachs [1] concluded that “biochemical studies on renal tissue have so far not shown the presence of any active pump mechanism other than Na,K-ATPase,” and pointed out that transport of Na, K, H, Cl, and HCO3can be explained by various modes of direct or indirect coupling to Na,K-ATPase. In the intervening years a large body of information further established the crucial role of Na,K-ATPase in tubular electrolyte transport, while at the same time providing evidence for the presence of other transport ATPases in the kidney. In parallel, microdissection and microanalysis techniques have made possible the examination of metabolic and enzymatic processes in discrete tubule segments that revealed the remarkable biochemical heterogeneity of the nephron [2], much as in the recent past in vitro tubule microperfusion has demonstrated the axial heterogeneity of its transport processes [3, 4].
Given the functional specialization of different nephron segments, one can easily appreciate why studies of renal biochemistry with classical methods using tissue slices or homogenates are subject to the same kind of limitations as, for example, the analysis of kidney function with clearance techniques: Both provide information about the kidney as a whole, rather than about its component units. To understand nephron function, one has to study it in its subdivisions of interest; to evaluate the regulation and the role of ATPases in tubular transport, it is essential to examine the behavior of these enzymes in individual nephron segments, and first to delineate their location along the nephron. This paper reviews the nephron distribution and function of several classes of ATPase, with emphasis on the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase), and including the calcium- and proton-ATPases, about which much less is known at this time.
Mineralocorticoid hormones stimulate Na+ absorption and K+ secretion by the cortical collecting tubule. There is good evidence that this stimulation involves increasing luminal membrane Na+ permeability and the turnover rate (or number) of the Na+-K+ pumps. These experiments were designed to examine whether mineralocorticoid hormones also increase cell K+ permeability. Using 42K tracer measurements in tubules treated with amiloride to inhibit active Na+ and K+ transport, passive K+ permeation increased with increasing mineralocorticoid effect. Net Na+ absorption and the (passive) K+ efflux rate coefficient (KK) showed a linear relationship. The stimulatory effect was evident in vitro since 0.2 microM aldosterone added to the bath of tubules harvested from NaCl-loaded rabbits increased KK at 3 hrs while time controls showed no change. Since these tubules were also treated with amiloride, this increase in KK was not dependent on increasing Na+ absorption. The results indicate that in addition to the well-described effects of aldosterone on Na+ permeability and cell metabolism, the mineralcorticoid effect includes an increase in cellular K+ permeability.
To test prolactin (PRL) action on osmoregulation in mammals, we evaluated in the rat the effect of this hormone on a major enzyme in renal regulation of water and electrolyte: renal Na-K-ATPase. Enzyme activity was determined by cytochemistry in medullary ascending limb (MAL) and distal convoluted tubule (DCT) from rats treated either by bromocriptine, or by PRL. Three hours after a bromocriptine injection (0.1 mg/100 g IP) a significant decrease of Na-K-ATPase activity is observed in both MAL (80% of control values, p less than 0.001) and DCT (78% p less than 0.01). Reciprocally, a significant (p less than 0.001) increase in enzyme activity is induced 3 h after a single PRL injection (140 micrograms/100 g IM), in both segments (MAL: 165%, DCT: 172% of control activities) and persists 6 h after the injection (MAL: 130%, DCT: 118%). Na-K-ATPase activity was correlated to plasma PRL levels (r = 0.78 in DCT, r = 0.89 in MAL). A direct effect of PRL on the tubule is suggested by results from experiments in which PRL, at various concentrations, is added in vitro on renal slices before Na-K-ATPase activity measurements. The increase in Na-K-ATPase activity exhibits a log-dose dependency with PRL concentration (p less than 0.01) and is still observed when AVP antagonist is added before PRL incubation, ruling out the possible role of AVP contamination of PRL. These results suggest a direct effect of PRL on renal Na-K-ATPase in MAL and DCT.
Our previous studies in cortical collecting ducts isolated from rat kidneys have shown that vasopressin increases both sodium absorption and potassium secretion, while bradykinin inhibits sodium absorption without affecting potassium transport. To determine which anions are affected by these agents, we perfused cortical collecting ducts from rats treated with deoxycorticosterone and measured net chloride flux, net bicarbonate flux (measured as total CO2), transepithelial voltage, and the rate of fluid absorption. Arginine vasopressin (10(-10) M in the peritubular bath) caused a sustained sixfold increase in net chloride absorption and a two- to threefold increase in the magnitude of the lumen negative transepithelial voltage. Before addition of vasopressin, the tubules secreted bicarbonate. Vasopressin abolished the bicarbonate secretion, resulting in net bicarbonate absorption (presumably due to proton secretion) in many tubules. Bradykinin (10(-9) M added to the peritubular bath) caused a reversible 40% inhibition of net chloride absorption, but did not affect the transepithelial voltage or the bicarbonate flux. We concluded: (a) that arginine vasopressin stimulates absorption of chloride and inhibits bicarbonate secretion (or stimulates proton secretion) in the rat cortical collecting duct; and (b) that bradykinin inhibits net chloride absorption in the rat cortical collecting duct without affecting transepithelial voltage or bicarbonate flux. Combining these results with the previous observations on cation fluxes described above, we conclude that bradykinin inhibits electroneutral NaCl absorption (or stimulates electroneutral NaCl secretion) in the rat cortical collecting duct.
In order to further characterize Cl- transport of the thin ascending limb of Henle's loop (TAL), we observed the effects of glutaraldehyde on Na+ and Cl- transport in hamster TAL perfused in vitro. We found that 0.1 mol/l glutaraldehyde added either to the lumen or to the bath caused a rapid irreversible reversal of the NaCl diffusion potential. This was mainly accounted for by an inhibition of Cl- permeability (10(-7) cm2 s-1) from 93.51 +/- 8.39 to 14.89 +/- 3.91 (P less than 0.01, n = 9). By contrast, Na+ permeability changed little from 34.18 +/- 3.27 to 26.56 +/- 2.74 (P less than 0.01, n = 6). Glutaraldehyde treatment abolished the halogen-permselectivity of the TAL as determined by the voltage deflection seen upon ionic substitution. Permeabilities for Cl-, Br-, I-, and SCN- relative to Na+ were changed from 3.16 +/- 0.20, 3.22 +/- 0.19, 2.97 +/- 0.26 and 4.36 +/- 0.36 to 0.38 +/- 0.07, 0.35 +/- 0.06, 0.36 +/- 0.07 and 0.58 +/- 0.05, respectively. The effect of glutaraldehyde on the NaCl diffusion potential was dose-dependent in the range from 10(-5) to 10(-1) M. The effect was reversible at concentrations lower than 10(-3) M. Glutaraldehyde did not affect the NaCl diffusion potential of the long-loop descending limb. These observations constitute additional evidence that the mechanism of Cl- transport across the TAL is different from that of Na+ transport. Glutaraldehyde might inhibit Cl- transport in the TAL by cross-linking amino acid residues of the proteins essential for halogen transport across this segment.
The stromal-to-tear transport of Cl- by the corneal epithelium of the frog is increased by pharmacological effectors (secretagogues) that are known to raise the intracellular levels of cyclic AMP or Ca2+. It has been shown in the past that the Cl- secretagogues increase the apical membrane permeability to Cl- and thus facilitate the cell-to-tear flux of the anion. In this report, we combine transepithelial and microelectrode studies to show that three of these secretagogues, epinephrine, the Ca2+ ionophore A23187, and forskolin, also increase the K+ conductance of the basolateral membrane by two- to threefold. The increase in the K+ conductance is not dependent on membrane potential, since this increase occurred equally when the basolateral membrane potential either exceeded 60 mV, as measured with microelectrodes, or was depolarized by voltage clamping after apical permeabilization with amphotericin B. It is proposed that both Cl- and K+ conductances are under the control of intracellular mediators that act independently on each pathway. The increase in basolateral K+ conductance favors the Cl- secretory process.
The possible role of protein kinase c in regulating the electrical events in the B-cell plasma membrane was examined by using the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a known activator of this enzyme. TPA has been found to enhance glucose- and sulfonylurea-induced insulin secretion with little or no effect on the fluxes of 86Rb+ or 45Ca2+ across the plasma membrane. TPA, 0.2 microM, did not influence the membrane potential from 0 to 5.6 mM glucose but increased by two- to threefold the fraction of the plateau phase of the oscillatory electrical activity induced by 7.0-11.1 mM glucose. This effect of TPA was completely blocked by 0.5 mM spermidine, an inhibitor of protein kinase c. However, spermidine had no influence on the electrical activity elicited by glucose alone. Glyburide, 10 nM, initiated slow depolarization and constant spike activity after about 18 and 25 min, respectively. TPA or 2.8 mM glucose reduced the lag period for glyburide to elicit an electrical response by about 75%. The duration of the spikes was increased two- to threefold by the presence of glucose or TPA with glyburide. There were also characteristic differences in the shape of the spikes under each experimental condition. Spermidine inhibited the influence of glucose, but not TPA, on the glyburide-induced electrical response. These results indicate that TPA may influence stimulant-induced electrical events via protein kinase c or by directly altering the ionic permeability of the plasma membrane.
To demonstrate that osmotic work can be accomplished across the inner medullary collecting duct (IMCD) by the difference in reflection coefficients for urea and NaCl, phenomenological coefficients for urea and NaCl transport were determined in isolated segments of the hamster IMCD perfused in vitro. Arginine vasopressin at 100 microU/ml increased urea permeability from 11.5 +/- 2.9 to 31.7 +/- 4.2 x 10(-7) cm2 s-1 in the middle IMCD but not in the upper IMCD. Urea transport in the middle IMCD consisted of two components, transport with saturable kinetics and simple passive diffusion. Permeability to Na+ was very low (2 x 10(-7) cm2 s-1). Reflection coefficients as measured by the equiosmolality method, with raffinose being a reference solute, were 0.87 +/- 0.05 and 0.71 +/- 0.04 for urea and 1.03 +/- 0.07 and 0.91 +/- 0.04 for NaCl in the upper and the middle IMCD, respectively. Reflection coefficient for urea in the middle IMCD was 0.68 when determined by the zero volume flux method. When the middle IMCD was perfused with bicarbonate Krebs-Ringer (BKR) solution containing 200 mmol/l urea, the replacement of urea in the bathing fluid with equisomolal NaCl caused large volume flux (3.81 +/- 0.45 nl mm-1 min-1) associated with dilatation of intercellular space. The existence of vasopressin in the bath was essential for this phenomenon. This effect was inhibited by 5 x 10(-4) M phloretin in the bath, suggesting that the vasoressin-stimulated urea transport is responsible for this phenomenon. From these observations, we conclude that transport parameters of the middle IMCD are appropriate for accomplishment of osmotic work across this segment in the absence of physicochemical osmotic gradients.
The interaction of 12-HETE with MDCK cells was investigated to determine whether this lipoxygenase product might influence tubular epithelial function. MDCK cells took up 12-HETE from both the luminal and interstitial surfaces. Much of the 12-HETE was incorporated into phospholipids but, unlike arachidonic acid, there was no specificity for the sn-2 position. 12-HETE was incorporated without chemical modification, suggesting that it might perturb tight packing in the lipid bilayer. 12-HETE uptake was reduced by the presence of arachidonic acid, but not by oleic acid. 12-HETE was removed from the cell lipids more rapidly than arachidonic acid and it was released into the medium in the form of both more and less polar metabolites. When MDCK cells were incubated with 12-HETE, their capacity to produce prostaglandin E2 was reduced. Since 12-HETE enters epithelial phospholipids and is converted to metabolic products and reduces prostaglandin production, it apparently has the capacity to modulate renal function if it is released in the proximity of the tubular epithelium.
The existence of a steep osmotic gradient in the renal medullary interstitium is the most critical in the formation of concentrated urine [1]. The architectural organization of the renal tubules and blood vessels in the medulla constitutes counterflow systems which are essential for both generating and maintaining high osmotic pressure of the renal medulla [2–4]. While it has been generally accepted that active NaCl transport in the thick ascending limb of Henle's loop plays the most fundamental role in the operation of the countercurrent multiplication system in the renal medulla, it is still a matter of considerable dispute whether the thin ascending limb (tAL) also has an active salt transport system to provide a “single effect” necessary for the operation of the countercurrent multiplication system.
We have analyzed the histotopography of the renal medulla of the rat in terms of cycles and separations. Cycles are pathways by which solute leaving the medulla in an ascending structure (AVR, AHL) is returned to a deeper medullary level. Separations are based on spatial incontiguity and special characteristics of the interstitium and blood supply. The two concepts are complementary: the compartmentalization resulting from separations imparts specificity to the cycles. Structural lateral heterogeneity, consisting in distinct domains organized around vascular bundles, is present in one form or another in all three medullary zones. Such compartmentalization probably leads to heterogeneity in interstitial solute concentrations, a state of affairs inconsistent with the requirements of a "central core". In all such considerations of exchanges between compartments, the lack of a unitary interstitium must be borne in mind. Instead, three general types of interstitium may be distinguished: corresponding roughly to those of the OS and VB, the interbundle region of the IS, and the IM. Among the histotopographic features of the renal medulla not usually included in models of the urinary concentrating mechanism but likely to have functional significance are the association of CD with completely distinct populations of AVR and AHL in the OM and IM; a clear-cut separation throughout the medulla between cycles involving long loops and those involving short loops; the lack of an effective countercurrent association between ascending and descending limbs of short loops in the IS; and a pronounced separation of the venous drainage of the IM from that of the OM.
There is increasing evidence of acidification along the entire mammalian collecting duct including the inner medullary collecting duct (IMCD). Recent studies have provided morphologic evidence that the intercalated cells are involved in hydrogen ion secretion in the cortical and outer medullary collecting duct of the rat. In the present study we performed a quantitative and qualitative morphologic examination of the intercalated cells in the IMCD of the rat and compared the results to observations obtained from intercalated cells in the collecting duct in the inner stripe of the outer medulla (OMCDi). Kidneys of male rats were preserved by in vivo perfusion with glutaraldehyde and processed for morphologic evaluation. With light microscopy and scanning electron microscopy intercalated cells were found in the outer third of the IMCD (IMCD1) and accounted for 10% of the total cell population. They were absent in the terminal two-thirds of the IMCD. Examination of the intercalated cells using transmission electron microscopy revealed striking similarities between the cells of the IMCD1 and those in the OMCDi. In addition, no differences were found in the surface densities of the apical or basolateral plasma membranes or the volume densities of the mitochondria of the intercalated cells in the two regions. In light of the morphologic similarity with the intercalated cells of the OMCDi that are believed to be involved in hydrogen ion secretion, it is likely that the intercalated cells of the IMCD1 are also involved in the acidification of tubular fluid.
To compare passive urea transport across the inner medullary collecting ducts (IMCDs) and the papillary surface epithelium (PSE) of the kidney, two determinants of passive transport were measured, namely permeability coefficient and surface area. Urea permeability was measured in isolated perfused IMCDs dissected from carefully localized sites along the inner medullas of rats and rabbits. Mean permeability coefficients (X 10(-5) cm/s) in rat IMCDs were: outer third of inner medulla (IMCD1), 1.6 +/- 0.5; middle third (IMCD2), 46.6 +/- 10.5; and inner third (IMCD3), 39.1 +/- 3.6. Mean permeability coefficients in rabbit IMCDs were: IMCD1, 1.2 +/- 0.1; IMCD2, 11.6 +/- 2.8; and IMCD3, 13.1 +/- 1.8. The rabbit PSE was dissected free from the underlying renal inner medulla and was mounted in a specially designed chamber to measure its permeability to urea. The mean value was 1 X 10(-5) cm/s both in the absence and presence of vasopressin (10 nM). Morphometry of renal papillary cross sections revealed that the total surface area of IMCDs exceeds the total area of the PSE by 10-fold in the rat and threefold in the rabbit. We conclude: the IMCD displays axial heterogeneity with respect to urea permeability, with a high permeability only in its distal two-thirds; and because the urea permeability and surface area of the PSE are relatively small, passive transport across it is unlikely to be a major source of urea to the inner medullary interstitium.
The protein A-gold technique was used to localize carbonic anhydrase isozymes on ultrathin frozen sections of kidney collecting duct epithelial cells and erythrocytes. The particulate nature of the gold marker gives a more precise appreciation of the intracellular distribution of this enzyme than has been previously possible, and allows the intensity of the labeling to be quantified. Intercalated cells showed four times more labeling over the cytosol than adjacent principal cells in collecting ducts from the inner stripe of the outer medulla: by double-labeling using protein A-gold particles of different sizes, carbonic anhydrase isozymes B and C were simultaneously localized in erythrocytes.
Several factors interact to maintain precise control of electrolyte transport in the mammalian cortical collecting duct. We have studied the effects of deoxycorticosterone, arginine vasopressin, and bradykinin on net transepithelial sodium and potassium transport in isolated, perfused rat cortical collecting ducts. Chronic administration of deoxycorticosterone to rats increased both sodium absorption and potassium secretion above very low basal levels. Consequently, deoxycorticosterone-treated rats were used for all remaining studies. Arginine vasopressin (10(-10) M in the bath) caused a sustained fourfold increase in net sodium absorption and a sustained threefold increase in net potassium secretion. Bradykinin (10(-9) M in the bath) caused a reversible 40-50% inhibition of net sodium absorption without affecting net potassium transport or the transepithelial potential difference. In the perfusate, up to 10(-6) M bradykinin had no effect. We conclude: As in rabbits, chronic deoxycorticosterone administration to rats increases sodium absorption and potassium secretion in cortical collecting ducts perfused in vitro. Arginine vasopressin causes a reversible increase in net potassium secretion and net sodium absorption. Bradykinin in the peritubular bathing solution reversibly inhibits net sodium absorption, possibly by affecting an electroneutral sodium transport pathway.
Structural differences in thin limbs of Henle: Physiological implications. Rat thin limbs of Henle (TL) were studied by electron microscopy. Descending and ascending thin limbs (DTL, ATL) were identified by tracing their continuity with known structures and by histotopography. Two types of TL were identified. Type I showed cellular interdigitation and very shallow intercellular occluding junctions. Junctions between noninterdigitating cells in type II had longer apical-basal depths. In short loops, DTL were type II. In long loops, DTL were type 1 in the outer medulla and possessed “complex” features such as microvilli and basal cytoplasmic invaginations. “Complex” type I DTL extended to varying distances in the inner medulla, where they underwent transition to type II limbs. ATL were type I, but appeared “simple” compared to the upper portions of DTL (no microvilli or basal invaginations). Transition from type II to “simple” type I epithelium occurred in the terminal portions of DTL at short distances (56 to 133 µ) before their junctions with ATL at the bends. Counts showed that profiles of “complex” type I DTL were outnumbered by type II DTL in the upper one-half, and virtually absent in the lower one-half of the inner medulla. Ultrastructural studies suggest that the degree of passive ion permeation across epithelia may be determined by the depth of their occluding junctions. Thus, type I cell junctions may be more permeable than type II. Cellular interdigitation provides for a larger paracellular pathway in type I limbs, and thus may enhance their permeability. These considerations suggest a role for TL structural differences in medullary counter-current mechanisms.
Permeability of medullary nephron segments to urea and water: Effect of vasopressin. High papillary urea concentrations are necessary for the formation of maximally concentrated urine, while low papillary urea concentrations are associated with less concentrated urine. In the present studies we examined the membrane characteristics that are important in determining the medullary urea concentration profiles. Using isolated segments of rabbit nephrons perfused in vitro, we measured the diffusional urea and water permeability in the medullary thick ascending limb of Henle (MTALH), the outer medullary collecting duct (MCD) and the papillary collecting duct (PCD), in the presence and absence of antidiuretic hormone (ADH). Segments of MTALH and MCD were essentially impermeant to 14C-urea. This degree of impermeability was maintained both with or without ADH. The urea permeability (× 10−5 cm sec−1) of the PCD was moderate (2.2± 0.3 without ADH, 2.4± 0.6 with ADH). Diffusional water permeability (× 10−5 cm sec−1) of the MTALH was low both with and without ADH; but the diffusional water permeability of the PCD increased from 39.9 ± 7.6 to 56.9 ± 8.6 (P < 0.002) with the addition of 200 µU/ml of ADH to the bath. In summary: 1) ADH has no influence on urea permeability across the nephron segments studied; 2) urea permeability of the PCD is higher than that of the MCD or the MTALH; and 3) ADH increases diffusional water permeability of the PCD but not of the MTALH. We conclude that pathways of urea movement are not the same as the principal pathways of water movement across the papillary collecting duct epithelium.
Ultrastructure of the renal pelvic epithelium of the rat. The ultrastructure of the renal pelvis of six rats was examined. Transitional epithelium, similar to that found in the bladder, was limited to the contrapapillary surface and overlay cortex and outer medulla. Like the bladder, the luminal plasma membrane of these cells measured 125Å and its outer leaflet was thicker than the cytoplasmic. The papillary epithelium was comprised of simple cuboidal cells, whose luminal membrane was 75Å in thickness. In the fornix, groups of cells intermediate in appearance were observed with regions of both 75Å and 125Å membrane present along the luminal surface. A correlation was observed between the urea permeability of each pelvic cell type and the concentration of urea in the underlying renal parenchyma reported by other investigators ; that is, the urea-permeable papillary epithelial cells invest the inner medulla which is a region of high urea concentration, whereas the transitional cells believed to be impermeable overlie the urea-poor cortex and outer medulla. It is suggested that the pelvic epithelial cells may participate in the regulation of urea reabsorption from the pelvic urine.
Countercurrent multiplication system without active transport in inner medulla. The present study reports a model for countercurrent multiplication in which both the descending limb of Henle (DLH) and thin ascending limb of Henle (ALH) operate as purely passive equilibrating segments. This model is based largely on transport characteristics obtained by perfusing isolated segments of rabbit nephrons in vitro. For this passive equilibration model to be operative the following segmental membrane characteristics should be present: thin DLH, relatively impermeant to solute but having high osmotic water permeability (osmotic water equilibration raises intraluminal Na concentration to higher levels than adjacent interstitium); thin ALH, more permeant to NaCl than urea and relatively impermeant to osmotic flow of water (hypoosmolal fluid can be generated passively by allowing more net NaCl to diffuse out than influx of urea); thick ALH, relativly water and solute impermeable with capacity of active outward NaCl transport (hypotonic fluid with a low NaCl concentration is developed); distal tubule, cortical and outer medullary collecting duct, impermeant to urea both in presence and absence of vasopressin (intraluminal urea is raised to high concentrations by virtue of water abstraction); inner medulla and papillary collecting duct, urea permeant (allows for generation of high interstitial urea concentration by passive diffusion down chemical concentration gradient). The salient feature of the proposed model is that the energy generated by active outward NaCl transport by thick ALH (expressed as high urea concentration in outer medullary CD by virtue of water abstraction) is transmitted to the papilla (by way of urea diffusing down its concentration gradient). In turn, papillary interstitial urea abstracts water out of DLH generating high intraluminal NaCl concentrations which allows the entire system to operate by passive diffusion of NaCl out of the thin ALH. By this model, the observed medullary concentration gradients are developed without invalidating the mass balance equations.
Long loops of Henle in rat kidneys fixed during an osmotic diuresis were marked by microinjection with a silicone rubber. In histological serial sections corresponding descending and ascending thin limbs in the inner medullary zone were identified and their luminal diameters measured.
At any level of the inner medulary zone, the luminal diameter of descending limbs is considerably smaller than that of corresponding ascending limbs. This diameter difference is most drastic at the border between inner and outer zones and diminishes towards the bend of the loops. Starting with a pre-bend enlargement the ascending thin limbs increase in diameter towards the outer medullary zone.
Concentration of urine in a central core model of the renal counterflow system. In this model descending Henle's limbs (DHL), ascending Henle's limbs (AHL) and collecting ducts (CD) exchange with a central vascular core (VC) formed by vasa recta loops—assumed so highly permeable that the core functions as a single tube, open at the cortical end and closed at the papillary end. Solute supplied to the VC primarily by AHL increases VC osmolality and so extracts water from DHL and CD, increasing their osmolality while diluting AHL fluid. This single effect multiplied by the counterflow arrangement leads to a high papillary osmolality in all structures. Some of the solute may enter DHL to be recycled. In single solute system energy requirements for transport out of AHL decrease from outer to inner medulla. In two solute systems (e.g. salt and urea) mixing in the central core can supply part of the energy for the final concentration of urine. Urea cycling, regulated by ADH, allows active Na+ transport in the outer medulla and cortex to be used for concentration in the inner medulla.