Control of fluid absorption in the renal proximal tubule

Glomerulotubular balance was investigated in isolated, perfused rabbit proximal tubules in vitro in order to evaluate some of the mechanisms proposed to account for the proportionate relationship between glomerular filtration rate and fluid absorption generally observed in vivo. The rate of fluid transport from lumen to bath in proximal convoluted tubules in vitro was approximately equal to the estimated normal rate in vivo. The absorption rate in proximal straight tubules however was approximately one-half as great. If the mechanism responsible for maintenance of glomerulotubular balance is intrinsic to the proximal tubule, as has been proposed on the basis of micropuncture studies, the rate of fluid absorption in vitro should be directly related to the perfusion rate and/or tubule volume. In the present studies absorption rate was only minimally affected when perfusion rate was increased or the tubule distended. Thus, glomerulotubular balance is not mediated by changes in velocity of flow of the tubular fluid or tubular diameter and therefore is not an intrinsic property of the proximal tubule. It has also been proposed that glomerulotubular balance results from a humoral feedback mechanism in which angiotensin directly inhibits fluid absorption by the proximal convoluted tubule. In the present experiments, angiotensin was found to have no significant effect on absorption rate.     

Challenges to potassium metabolism: internal distribution and external balance

A complex pump-leak system involving both active and passive transport mechanisms is responsible for the appropriate distribution of potassium (K) between the intra- and extracellular fluid compartments. In addition, the kidneys, and to a lesser extent the colon, safeguard maintenance of the narrow range of low K concentrations in the extracellular fluid. Early renal clearance studies showed that K is normally both reabsorbed and secreted by renal tubules, and that regulated secretion is the major source of K excretion. Net K secretion occurs mainly in principal cells while K absorption takes place in intercalated cells. Studies on single tubules and principal and intercalated cells have defined the determinants of K secretion and reabsorption including the electrochemical driving forces, specific carriers, ATPases, and K channels. Recent studies on the properties and molecular identity of renal K channels have also contributed significantly to understanding the renal mechanisms that transport and regulate K excretion.     

The renal sodium-calcium exchanger.

The functional expression of the renal sodium-calcium exchanger has been amply documented in studies on renal cortical basolateral membranes. In perfused renal tubules, other investigators have shown sodium-calcium exchange activity in the proximal convolution of the rat and in the distal convolution, the connecting tubule, and the collecting tubule of the rabbit. In rat proximal tubules, we found that the sodium-calcium exchanger is an important determinant of cytosolic calcium homeostasis, since inhibition of sodium-dependent calcium efflux mode caused a large accumulation of tubular calcium. In membranes from rat proximal tubules sodium-calcium activity was high, and in intact proximal tubules, the tubular sodium-calcium exchanger exhibited a high affinity for cytosolic calcium and had a substantial transport capacity, which may be absolute requirements for the maintenance of stable cytosolic calcium in proximal tubules.     

Renal cystic diseases as a ciliopathy

Renal cystic disease is characterized by expansion of renal tubules. Abnormal cell proliferation and randomly oriented cell division angle are thought to induce cystic changes in renal tubules. Recent advancements have identified many of causative genes. Interestingly, those gene products are localized in cilia or centriole, suggesting that cilia have some role to control tubular diameter. Several systemic syndromes accompany renal cystic diseases, particularly nephronophthisis. To identify nephronophthisis related pathway will be a clue to understand mechanisms to develop not only renal cysts but also phenotypes associated with ciliopathies.     

Calciuretic renal function in patients with essential hypertension

Under the conditions of standard and customary calcium use, patients suffering from essential hypertension (EH) do not manifest any changes in calciuresis either at the expense of the glomerular or tubular mechanisms. After intravenous hypercalcemic injections EH patients demonstrate well-defined disorders in calciuretic renal function, caused by inadequate suppression of tubular reabsorption of calcium by parathyroid hormone (PTH). The hormonal-renal correlations in EH patients differ from those in normals. More pronounced alterations in the concentration of radioimmune PTH and calcitonin under acute hypercalcemia are not associated with an adequate increment of fractional excretion of calcium whereas the calciuretic effect of exogenous calcium-regulating hormones (CRH) realized at the tubular level is less remarkable. Therefore EH patients manifest changes not only in CRH secretion but also in the sensitivity to them of the renal tubules. White changing parathyroid regulation of calcium metabolism prolonged administration of calcium to EH patients enhances body capabilities of resisting acute alterations in calcemia because of normalization of calciuretic renal function, especially tubular calcium transport. In addition, it lowers arterial pressure and enables reduction of the dose of calcium antagonists used in the treatment of EH.     

Altered distribution pattern of Na+-K+-ATPase and succinate dehydrogenase activities along the nephron in human acute post-transplant renal failure

The catalytic activities of Na+-K+-ATPase and succinate dehydrogenase, marker enzymes for active salt reabsorptive capacity of renal basolateral plasma membranes and for respiratory capacity of mitochondrial cristae membranes, were studied in the maintenance phase of human acute post-transplant renal failure. Biopsies of 4 kidney-allografts taken at transplantation operation and additionally at different post-transplantation periods, either with good function or in various stages of dysfunction, were compared with the unaffected part of a human kidney nephrectomized due to hypernephroma. In single nephron segments, Na+-K+-ATPase activity was determined after microdissection by microfluorometry, and succinate dehydrogenase activity was determined by a microphotometric procedure in stained cryosections. In intraoperative and postoperative biopsies of a well-functioning allograft, both Na+-K+-ATPase and succinate dehydrogenase activities did not differ from those of normal renal tissue. In contrast, the catalytic activities were found to be decreased in the distal tubules of 2 anuric allografts when compared with their intraoperative controls. In addition, succinate dehydrogenase activity was reduced in distal tubules of a recovering allograft. Catalytic activities appeared to be unaffected in glomeruli, proximal tubules, and collecting ducts. It is suggested that the predominant distal tubular alterations with regard to these parameters are a consequence of increased distal tubular vulnerability due to circulatory and metabolic conditions.     

Catalytic activities of alkaline phosphatase and N-acetyl-beta-D-glucosaminidase in human cortical nephron segments: heterogeneous changes in acute renal failure and acute rejection following kidney allotransplantation

The catalytic activities of alkaline phosphatase and N-acetyl-beta-D-glucosaminidase, constituents of luminal brush-border membranes and lysosomes of kidney tubular cells, were measured in human kidney allografts in the maintenance and recovery phases of acute renal failure and in acute rejection crisis. The enzyme activities were fluorometrically determined in single microdissected cortical nephron segments of biopsies from 4 kidney allografts taken intraoperatively and postoperatively at different periods, which exhibited either good function or dysfunction. For comparison, the unaffected part of a human kidney nephrectomized due to hypernephroma as well as a biopsy of a morphologically normal human kidney were examined. Both enzymes displayed highest activities in the proximal part of the human nephron. In some intraoperative and postoperative biopsies with acute renal failure, alkaline phosphatase activity was reduced in proximal tubules, predominantly in the straight portion. This reduction could not be correlated with function. In acute rejection, very low alkaline phosphatase activities were uniformly found in proximal convoluted and straight tubules. Furthermore, intraoperative biopsies and biopsies of the functioning allograft have only approximately 50% of normal N-acetyl-beta-D-glucosaminidase activity in proximal convoluted tubules, but generally normal values in the straight portion. However, in acute renal failure, this enzyme activity was several-fold enhanced along the whole nephron, when compared with intraoperative values. In acute rejection, N-acetyl-beta-D-glucosaminidase activity was slightly reduced in proximal convoluted tubules, when compared with biopsies showing good function. It is suggested that the decrease of proximal tubular enzyme activities is the consequence of increased enzymuria and inadequate enzyme regeneration. On the other hand, the overshoot of N-acetyl-beta-D-glucosaminidase activity in the maintenance phase of acute renal failure appears to indicate increased degradative capacity, associated with cellular regeneration along the whole nephron.     

Profound pharmacologic inhibition of renal tubule sodium and water reabsorption in rats

Vanadate, furosemide, chlorothiazide, acetazolamide, and amiloride were infused concomitantly into conscious rats to determine the extent to which renal sodium reabsorption can be inhibited. The animals were maintained in exact fluid balance despite exorbitant urine flow rates by intravenous infusion of an isotonic salt solution sufficient to keep body weight constant. In maximally effective doses vanadate, furosemide, chlorothiazide, acetazolamide, and amiloride increased sodium excretion to an amount equal to 69% of the filtered load. Together with isotonic extracellular fluid expansion equal to 7% of body weight, these pharmacologic agents increased sodium excretion to an amount equal to 83.7% of the filtered load, a level of sustained sodium excretion that has not been reported previously in mammals. These studies demonstrate that tubular sodium and water reabsorption can be profoundly inhibited in vivo by drugs that diminish sodium and water transport in proximal as well as in distal tubules.     

Direct determination of PCO2 in the rat renal cortex.

The mechanism by which the kidney reabsorbs sodium bicarbonate could be a result of (a) H+ secretion, (b) direct HCO3- reabsorption, or (c) a combination of both processes. Most of the studies which have supported the H+ secretory theory have involved the assumption that tubular fluid and arterial PCO2 were equal. We have utilized a new PCO2 microelectrode to directly determine in situ PCO2 of tubular fluid and stellate vessel blood in the cortex of the rat kidney during control conditions and after alterations in acid-base status. In 21 control rats, proximal tubular fluid PCO2 exceeded systemic arterial PCO2 (deltaCO2) by 25.9 +/- 0.92 mm Hg (P less than 0.001). The values obtained for both distal tubular fluid and stellate vessel blood were not significantly different from proximal tubular PCO2. Evaluation of PCO2 in the proximal tubules of Munich-Wistar rats did not reveal evidence for a declining profile for PCO2 along the length of the nephron. When proximal bicarbonate reabsorption was increased or decreased acutely by alterations in acid-base status, deltaPCO2 changed in paralle. Furthermore, benzolamide administration significantly reduced deltaPCO2. We conclude: (a) that the PCO2 in tubular fluid is significantly greater than systemic arterial PCO2, (b) that there is no tendency for the observed PCO2 to fall along the proximal tubule, (c) the mean PCO2 in the proximal and distal tubules as well as the stellate vessle is not significantly different, thereby rendering the concept of a "diffusion barrier" for CO2 in the proximal tubule unlikely, and (d) the level of renal cortical PCO2 appears to vary directly with the magnitude of bicarbonate reabsorption.     

Inhibitory effects of volume expansion performed in vivo on transport in the isolated rabbit proximal tubule perfused in vitro.

To examine the renal tubular sites and mechanisms involved in the effects of hypooncotic volume expansion (VE) on renal electrolyte excretion, we performed clearance and isolated tubular perfusion studies using intact and thyroparathyroidectomized (TPTX) rabbits. We also examined the effect of VE on luminal brush border transport. In the microperfusion studies, proximal convoluted (PCT) and straight (PST) tubules were taken from rabbits without prior VE or after 30 min of 6% (body wt) VE. Acute VE increased the percentage excretion of Na, Ca, and P in TPTX animals and the percentage and absolute excretions of these ions in intact rabbits. In PST from VE animals, fluid flux (Jv) was depressed compared with Jv in PST from nonVE rabbits: Jv = 0.18 +/- 0.03, (VE) vs. 0.31 +/- 0.03 nl/mm.min, (nonVE) P less than 0.02. Phosphate transport (Jp) in the PST from VE animals was also depressed: JP = 1.58 +/- 0.10 (VE) vs. 2.62 +/- 0.47 pmol/mm.min, (nonVE) P less than 0.05. Similar results were obtained with TPTX animals. In the PCT from VE animals, Jv was decreased (0.49 +/- 0.10 (VE) vs. 0.97 +/- 0.14 nl/mm.min, (nonVE) P less than 0.02), but JP was not affected significantly. Transport inhibition was stable over approximately 90 min of perfusion. In the brush border vesicle studies, sodium-dependent phosphate transport was inhibited compared with that in control animals, at the 9-, 30-, and 60-s time points. These findings indicate that the inhibition of renal ionic transport by VE occurs in both PCT and PST and is, in part, the result of a direct effect of VE on tubular transport mechanisms.     

A question of balance--dehydration in the elderly

The maintenance of optimal water balance is a priority for homeostasis; unfortunately, the aging process adversely affects the mechanisms of water balance, making it more difficult for the body to adequately defend itself against water loss. Four major age-related changes predispose the elderly to dehydration and hypernatremia: a decrease in total body water, an altered sense of thirst, a decrease in the renal urine concentrating ability, and a decrease in the effectiveness of ADH. In the case of fluid balance, careful assessment cannot be overemphasized due to the fact that there are few symptoms unique to dehydration that readily allow a nurse to know that fluid imbalances are involved.     

Effect of Intraluminal Flow on Proximal Tubular Reabsorption

Micropuncture techniques in the rat were used to reinvestigate the possibility that intraluminal flow rate per se may influence net volume reabsorption by the proximal tubule. An experimental design was devised which lowered intraluminal flow without affecting filtration rate of the nephron under study or without directly affecting other renal hemodynamics. In 11 rats flow of tubular fluid between early and late proximal tubular sites was reduced by partially collecting tubular fluid at the early puncture site. In 42 nephrons the rate of flow of tubular fluid was reduced an average of 45% without changing nephron filtration rate and there was an associated reduction in reabsorption between the two sites which averaged 29%. This indicated 63% balance between delivery of tubular fluid and the rate of reabsorption between two sites along proximal tubules. The results of these studies indicate that a reduction in delivery of normal filtrate along the proximal tubule is associated with a concordant reduction in the absolute rate of reabsorption. Since this relationship occurred in the absence of changes in renal hemodynamics or even a change in filtration rate of the nephron under study it is concluded that changes in intraluminal load per se play an important role in the phenomenon of glomerulotubular balance.     

Glomerular and tubular adaptive responses to acute nephron loss in the rat. Effect of prostaglandin synthesis inhibition.

These studies, using in vivo micropuncture techniques in the Munich-Wistar rat, document the magnitude of changes in glomerular and tubular function and structure 24 h after approximately 75% nephron loss (Nx) and compared these results with those obtained in sham-operated rats. The contribution of either nephron hypertrophy or renal prostaglandin to these adjustments in nephron function was also explored. After acute Nx, single nephron GFR (SNGFR) was increased, on average by approximately 30%, due primarily to glomerular hyperperfusion and hypertension. The approximately 45% reduction in preglomerular and the constancy in postglomerular vascular resistances was entirely responsible for these adaptations. Although increases in fluid reabsorption in proximal convoluted tubules correlated closely with increase in SNGFR, the fractional fluid reabsorption between late proximal and early distal tubular segments was depressed. Nephron hypertrophy could not be substantiated based on either measurements of protein content in renal tissue homogenates or morphometric analysis of proximal convoluted tubules. However, acute Nx was associated with increased urinary excretory rates per functional nephron for 6-keto-PGF1 alpha and TXB2. Prostaglandin synthesis inhibition did not affect function in control nephrons, but this maneuver was associated with normalization of glomerular and tubular function in remnant nephrons. The results suggest that enhanced synthesis of cyclooxygenase-dependent products is one of the earliest responses to Nx, and even before hypertrophy the pathophysiologic effects of prostaglandin may be important contributors to the adaptations in remnant nephron function.     

Ion channels in the kidney: molecular biological aspects]

Ion channels in renal tubules are involved in maintenance of the volume and ion composition of the body. In the kidney these channels are expressed at the apical and/or basolateral cell membranes and are responsible for vectorial ion transport. Structure and function of the renal ion channels differs from those of previous channels which were cloned in excitable tissues. In this review I outline the various types of ion channels in renal and non-renal cells and give examples that emphasize the physiological roles of these channels in the nephron.     

Augmented bicarbonate reabsorption by both the proximal and distal nephron maintains chloride-deplete metabolic alkalosis in rats.

Whether augmented bicarbonate reabsorption by renal tubular epithelium contributes to the maintenance of chloride-deplete metabolic alkalosis is not clear. This study used free-flow micropuncture to investigate bicarbonate reabsorption by surface nephron segments in a rat model of diuretic-induced alkalosis compared to control. The proximal and distal nephron of the alkalotic animals had higher values for both delivered load to and absolute reabsorption from these segments. The proximal tubules of alkalotic and control animals had similar values for the slopes of the linear regression of delivered load vs. reabsorption and for the bicarbonate tubular fluid to plasma (TF/P) ratio at the late proximal tubule. By contrast, the corresponding analysis for the distal segment of alkalotic animals revealed a greater slope (0.98 vs. 0.81, P less than 0.003) and a smaller bicarbonate TF/P ratio at the late distal tubule (0.10 vs. 0.16, P less than 0.006). The data indicate that augmented bicarbonate reabsorption by both the proximal and distal nephron contributes to maintaining the alkalosis of this model. The data suggest primary stimulation of bicarbonate reabsorption in the distal nephron and load-dependent reabsorption in the proximal tubule.     

Intrarenal cells, not bone marrow-derived cells, are the major source for regeneration in postischemic kidney

Ischemic injury to the kidney produces acute tubular necrosis and apoptosis followed by tubular regeneration and recovery of renal function. Although mitotic cells are present in the tubules of postischemic kidneys, the origins of the proliferating cells are not known. Bone marrow cells (BMCs) can differentiate across lineages to repair injured organs, including the kidney. However, the relative contribution of intrarenal cells and extrarenal cells to kidney regeneration is not clear. We produced transgenic mice that expressed enhanced GFP (EGFP) specifically and permanently in mature renal tubular epithelial cells. Following ischemia/reperfusion injury (IRI), EGFP-positive cells showed incorporation of BrdU and expression of vimentin, which provides direct evidence that the cells composing regenerating tubules are derived from renal tubular epithelial cells. In BMC-transplanted mice, 89% of proliferating epithelial cells originated from host cells, and 11% originated from donor BMCs. Twenty-eight days after IRI, the kidneys contained 8% donor-derived cells, of which 8.4% were epithelial cells, 10.6% were glomerular cells, and 81% were interstitial cells. No renal functional improvement was observed in mice that were transplanted with exogenous BMCs. These results show that intrarenal cells are the main source of renal repair, and a single injection of BMCs does not make a significant contribution to renal functional or structural recovery.     

A micropuncture study of renal phosphate transport in rats with chronic renal failure and secondary hyperparathyroidism.

Micropuncture studies were carried out in rats to determine changes in tubular transport of phosphate which occur in chronic renal failure and secondary hyperparathyroidism. Rats underwent subtotal nephrectomy (NX) and were fed a low calcium, high phosphorus diet for 3--4 wk. Other groups consisted of normal control animals, normal rats infused with sodium phosphate to raise filtered load of phosphate, subtotal NX rats parathyroidectomized (PTX) on the day of experiment, and normal PTX rats infused with sodium phosphate. It was found that filtered phosphate/nephron is markedly increased in subtotal NX rats due to high single nephron filtration rates, proximal tubular fluid plasma phosphate ratios are less than 1.0, and fractional reabsorption of phosphate is decreased in the proximal tubule. More phosphate was present in the final urine than in surface distal convoluted tubules. Acute PTX in subtotal NX rats resulted in a striking increase in proximal phosphate reabsorption, and urinary phosphate became approximately equal to that remaining in surface distal tubules. Phosphate loading in normal rats reduced fractional reabsorption in the proximal tubule, but urinary phosphate was not greater than that at the end of surface distal tubules. Acute PTX in normal phosphate-loaded animals had no significant effect on proximal tubular phosphate reabsorption. These observations suggest that phosphate homeostasis in chronic renal failure is acheived by inhibition of proximal phosphate reabsorption, counteracting a greatly enhanced intrinsic capacity for reabsorption. In addition, the large amount of urinary phosphate is consistent either with secretion by the collecting ducts or with a disproportionately high contribution by deep nephrons. The changes in phosphate transport are mediated by parathyroid hormone and are completely abolished by acute removal of the hormone.     

Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney.

Kidneys have long been recognized as a major source of plasminogen activators (PAs). However, neither the sites of synthesis of the enzymes nor their role in renal function have been elucidated. By the combined use of zymographies on tissue sections and in situ hybridizations, we have explored the cellular distribution of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators and of their mRNAs in developing and adult mouse kidneys. In 17.5-d old embryos, renal tubules synthesize u-PA, while S-shaped bodies produce t-PA. In the adult kidney, u-PA is synthesized and released in urine by the epithelial cells lining the straight parts of both proximal and distal tubules. In contrast, t-PA is produced by glomerular cells and by epithelial cells lining the distal part of collecting ducts. The precise segmental distribution of PAs suggests that both enzymes may be implicated in the maintenance of tubular patency, by catalyzing extracellular proteolysis to prevent or circumvent protein precipitation.     

Metabolic Requirement for Inorganic Phosphate by the Rabbit Proximal Tubule: EVIDENCE FOR A CRABTREE EFFECT

These studies examine the effects of acute changes in the availability of inorganic phosphate on the function of isolated proximal renal tubules from rabbit kidney. We removed phosphate from the extracellular fluids and measured fluid absorption rates in isolated perfused tubules and oxygen consumption rates in suspensions of cortical tubules. In proximal convoluted tubules, the selective removal of phosphate from the luminal fluid reduced fluid absorption rates from 1.11±0.12 to −0.01±0.08 nl/mm · min. This effect on fluid absorption was dependent on the presence of glucose transport and metabolism. The addition of phlorizin to the phosphate-free luminal fluid preserved fluid absorption rates (1.12±0.12 nl/mm · min) as did the substitution of nonmetabolized α-methyl d-glucopyranoside for glucose (1.05±0.21 nl/mm · min) or the addition of 2-deoxyglucose, an inhibitor of glycolysis, to the bathing medium (1.01±0.15 nl/mm · min). There was no effect on fluid absorption if phosphate was removed from the bath only. Additionally, removal of phosphate from the luminal fluid of proximal straight rather than convoluted tubules had no effect on fluid absorption rates. Oxygen consumption rates in suspensions of cortical tubules were reduced from 18.9±0.6 to 10.6±0.6 nmol O₂/mg tubular protein · min by the removal of phosphate from the medium. This inhibition was prevented by the substitution of α-methyl d-glucopyranoside for glucose in the phosphate-free medium. The data indicate that under certain conditions, proximal convoluted tubules require the presence of phosphate in the luminal fluid to preserve tubular function. In the absence of intraluminal phosphate, glucose metabolism causes a reduction in both oxidative metabolism and fluid absorption. This response is analogous to the Crabtree effect and suggests limitations on the intracellular availability of inorganic phosphate.     

Intrarenal effects of ecadotril during acute volume expansion in dogs with congestive heart failure

Neutral endopeptidase 24.11 (NEP) inhibitors are known to have vascular, diuretic, and natriuretic effects that may be helpful in the treatment of congestive heart failure (CHF). Most NEP inhibitors may act principally through intrarenal mechanisms, which are not completely understood. The purpose of this study was to determine the principal renal effects of the NEP inhibitor ecadotril in dogs with progressive CHF induced by rapid ventricular pacing. Renal function was measured before, during, and after acute i.v. infusion of normal saline in a total of six dogs during normal cardiac function, early left ventricular dysfunction, and overt CHF. During overt CHF, each dog was treated with either ecadotril or placebo orally for 1 week. Parameters measured included glomerular filtration rate, renal blood flow, urine output, sodium clearance, sodium fractional excretion, and proximal and distal sodium reabsorption. Ecadotril treatment resulted in increased urine output, sodium clearance, and renal sodium excretion relative to placebo-treated controls. The principal intrarenal effect of ecadotril was decreased distal renal tubular sodium reabsorption. Both glomerular filtration rate and renal blood flow declined during overt CHF and were unaffected by ecadotril treatment. The results of this study are consistent with the principal action of ecadotril occurring by way of intrarenal events as opposed to changes in renal hemodynamics. The principal effect of ecadotril on distal tubular sodium reabsorption suggests that inhibition of NEP activity in the proximal renal tubules may allow increased binding of filtered atrial natriuretic peptide to natriuretic peptide receptor sites in the distal renal tubules and collecting ducts.