Outer Medullary Tubules Research Articles

Acute hydroxyurea treatment reduces tubular damage following bilateral ischemia-reperfusion injury in a mouse model of sickle cell disease

Ischemic injury is a primary contributor to the initiation of renal tubular epithelial cell damage in sickle cell disease (SCD). In this study, we investigated the effects of bilateral ischemia-reperfusion injury, which is a common type of acute kidney injury (AKI), in male and female genetic mouse model of SCD. Bilateral occlusion of both renal hila for 21 min led to a significantly higher detection of established serum markers of AKI (creatinine, KIM-1 and NGAL) compared to sham-operated male SCD mice. Severe damage to the outer medullary tubules was determined in the ischemia-reperfision injury (IRI)-treated SCD male mice. In female SCD mice with a longer ischemic time (23 min), the serum markers of AKI were not as highly elevated compared to their male counterparts, and the extent of outer medullary tubular injury was less severe. To assess the potential benefit in the use of hydroxyurea (50 mg/kg IP) following bilateral renal IRI, we observed that the serum markers of AKI and the outer medullary tubular damage were markedly improved compared to male SCD mice that were not treated with hydroxyurea. In this study, we confirmed that male SCD mice were more susceptible to increased tubular damage and a loss in renal function compared to female SCD mice, and that hydroxyurea may partially prevent the extent of tubular injury following severe ischemia-reperfusion injury in SCD.

Renal response to L-arginine in diabetic rats. A possible link between nitric oxide system and aquaporin-2.

The aim of this study was to evaluate whether L-Arginine (L-Arg) supplementation modifies nitric oxide (NO) system and consequently aquaporin-2 (AQP2) expression in the renal outer medulla of streptozotocin-diabetic rats at an early time point after induction of diabetes. Male Wistar rats were divided in four groups: Control, Diabetic, Diabetic treated with L-Arginine and Control treated with L-Arginine. Nitric oxide synthase (NOS) activity was estimated by [14C] L-citrulline production in homogenates of the renal outer medulla and by NADPH-diaphorase staining in renal outer medullary tubules. Western blot was used to detect the expression of AQP2 and NOS types I and III; real time PCR was used to quantify AQP2 mRNA. The expression of both NOS isoforms, NOS I and NOS III, was decreased in the renal outer medulla of diabetic rats and L-Arg failed to prevent these decreases. However, L-Arg improved NO production, NADPH-diaphorase activity in collecting ducts and other tubular structures, and NOS activity in renal homogenates from diabetic rats. AQP2 protein and mRNA were decreased in the renal outer medulla of diabetic rats and L-Arg administration prevented these decreases. These results suggest that the decreased NOS activity in collecting ducts of the renal outer medulla may cause, at least in part, the decreased expression of AQP2 in this model of diabetes and constitute additional evidence supporting a role for NO in contributing to renal water reabsorption through the modulation of AQP2 expression in this pathological condition. However, we cannot discard that another pathway different from NOS also exists that links L-Arg to AQP2 expression.

257 KIDNEY INNERVATION BY ANGIOTENSINERGIC FIBERS AND THEIR FATE AFTER UNILATERAL RENAL DENERVATION

Objective: The renal nerves release neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and substance P (SP) as co-transmitters to modulate kidney function via specific postjunctional target cell receptors. We hypothesized, that they also express and release Ang II which was not known. Methods: Cryosections of rat, pig and human kidneys were stained immunocytochemically with a monoclonal antibody against angiotensin (Ang) II (4B3) and co-stained for tyrosine-3 or dopamine β-hydroxylase (TH, DBH), CGRP, SP, synaptophysin and renin. Detection was by confocal light or laser scanning microscopy. Left kidneys of 8 normotensive Wistar-Kyoto rats were surgically denervated and 8 rats sham operated before kineys were investigated 6 days later. Results: Ang II-positive nerve fibers were present in the renal nerves, the pelvic region, within the arterial nerve plexus up to the periglomerular arterioles, in the cortical interstitium and outer medulla. Also, juxtaglomerular cells, central veins and the renal capsule were innervated but not glomerula or the papilla. The fibers classified as distinct subpopulations of postganglionic (TH-, DBH-positive) or sensory fibers (CGRP-, SP-positive) with different morphology and distribution vs. Ang II-negative fibers. Varicose angiotensinergic postganglionic fibers preferentially innervated the periglomerular arterioles and outer medullary tubules. Neurons in microganglia were angiotensinergic, catecholaminergic or both. After kidney denervation, all ipsilateral fibers disappeared except for rare Ang II-positive fibers from microganglia and the ureter. Conclusions: The kidney receives an important angiotensinergic sympathetic and sensory-afferent innervation that provides an autonomous renal Ang II source and may play an independent role in the pathogenesis of hypertension.

Renal actions of dendroaspis natriuretic peptide in rabbits

Dendroaspis natriuretic peptide (DNP) is one of four members of the natriuretic peptide family sharing functional and structural properties. The purpose of the present study was to elucidate the physiological role of DNP on renal functions and its cellular mechanism in the rabbit kidney. DNP (5μg/kg/min) infused intravenously increased urine volume and urinary excretion of electrolytes. These renal actions induced by DNP were more pronounced than those caused by atrial natriuretic peptide (ANP). We compared profiles of 125I-ANP and 125I-DNP by reverse-phase HPLC during incubation in rabbit plasma at 37°C for 1, 2, and 4h. While 125I-ANP was quickly degraded within 1h, 125I-DNP was still stable in plasma for 4h. DNP induced the greatest cyclic guanosine monophosphate (cGMP) production in the glomeruli in a dose-dependent manner, when compared to other renal structures including cortical tubules, outer medullary tubules, and inner medullary tubules. Affinity cross-linking analysis revealed NPR-A is selective receptor for DNP in glomeruli. Forskolin, a stimulator of adenylyl cyclase, significantly decreased cGMP production in the renal glomeruli but not in the renal medulla. In summary, DNP is a more effective activator of renal functions than ANP, possibly because of the degradation resistance of DNP against the endogenous peptidases in plasma or tissues. These findings suggest that DNP plays a pivotal role as a renal regulating peptide via specific natriuretic peptide receptors with a guanylyl cyclase domain.

Renal-specific oxidoreductase biphasic expression under high glucose ambience during fetal versus neonatal development

Renal-specific oxidoreductase (RSOR) has been recently identified in mice kidneys of diabetic animals, and it is developmentally regulated. Its expression during fetal, neonatal, and postnatal periods was assessed under high glucose ambience. Whole-mount immunofluorescence and confocal microscopy were performed to assess the effect of high glucose on the morphogenesis of mice fetal kidneys. RSOR mRNA and protein expression was assessed by competitive polymerase chain reaction (PCR) and immunoprecipitation methods in embryonic kidneys (day E13 to E17) subjected to high glucose ambience and by Northern and Western blot analyses of kidneys of newborn and 1-week-old mice with hyperglycemia. The spatiotemporal changes in the RSOR expression were assessed by in situ hybridization analyses and immunofluorescence microscopy. In addition, the extent of apoptosis in the kidneys was determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Whole-mount microscopy of the embryonic metanephroi revealed a dose-dependent disruption in the ureteric bud iterations with reduced population of the nascent nephrons. Both gene and protein expressions were reduced in day E13 to E17 metanephroi, while increased in kidneys of newborn and 1-week-old mice. In day E13 and day E15 kidneys, the RSOR was expressed in the ureteric bud branches and some of the immature tubules, and its expression was reduced with high glucose treatment. In day E17 kidneys the RSOR was expressed in the tubules of the deeper cortex, and its expression was marginally decreased. In newborn kidneys, this enzyme was expressed in the subcortical tubules and it spread to the entire width of the renal cortex in hyperglycemic state. In 1-week-old mice kidneys, the RSOR was localized to the entire cortex, and in animals with blood glucose above 300 mg/dL, its intensity increased with extension of expression into the outer medullary tubules. A dose-dependent fulminant apoptosis was observed in day E13 to E17 kidneys subjected to high glucose ambience. In newborn and 1-week-old mice control kidneys, the apoptosis was minimal although slightly increased during hyperglycemia. High glucose has a differential effect on the RSOR expression in kidneys during the embryonic versus neonatal/postnatal period. This may partly be related to the differential degree of apoptosis, a process reflective of oxidant stress that is seen in diabetic milieu, which as previously has been shown to adversely effect the modulators of fetal development and thereby the morphogenesis of the kidney and RSOR expression.

Influence of dehydration on glycerophosphorylcholine and choline distribution along the rat nephron.

Glycerophosphorylcholine is one of the four major organic osmolytes in renal medullary cells, changing their intracellular osmolyte concentration in parallel with extracellular tonicity during cellular osmoadaptation. In this study, the tubular content of glycerophosphorylcholine was quantified in untreated and 48-h-dehydrated male rats. A chemiluminescence ultra-micromethod was developed to measure choline at the picomolar level in single tubules microdissected from collagenase-treated kidneys. The glycerophosphorylcholine level was calculated as the difference between total choline after acid hydrolysis and the free tubular choline content. In accordance with the glycerophosphorylcholine distribution pattern in different renal zones of untreated rats, low amounts of glycerophosphorylcholine were found in all cortical and outer medullary structures (< 35 pmol/mm), whereas increasing amounts were detected towards the papillary tip (163 pmol/mm). As a percentage of total choline, the level of free tubular choline varied from 4.2% in outer medullary proximal tubules to 30.3% in the inner medullary collecting ducts adjacent to the outer medulla (IMCD1). Antidiuresis led to a nearly twofold increase in glycerophosphorylcholine content in papillary collecting ducts. The osmolality-dependent regulation of organic osmolytes in single microdissected tubules has been demonstrated for the first time. Furthermore, the high tubular glycerophosphorylcholine concentration compared to sorbitol and myo-inositol emphasizes the predominance of glycerophosphorylcholine in the inner medulla and papilla of the rat kidney.

Expression of angiopoietin-1, angiopoietin-2, and the Tie-2 receptor tyrosine kinase during mouse kidney maturation.

The Tie-2 receptor tyrosine kinase transduces embryonic endothelial differentiation, with Angiopoietin-1 (Ang-1) acting as a stimulatory ligand and Ang-2 postulated to be a naturally occurring inhibitor. Expression of these genes was sought during mouse kidney maturation from the onset of glomerulogenesis (embryonic day 14 [E14]) to the end of nephron formation (2 wk postnatal [P2]), and during medullary maturation into adulthood (P8). Using Northern and slot blotting of RNA extracted from whole organs, these three genes were expressed throughout the experimental period with peak levels at P2 to P3. By in situ hybridization analysis at E18, P1, and P3, Ang-1 mRNA was found to localize to condensing renal mesenchymal cells, proximal tubules, and glomeruli in addition to maturing tubules of the outer medulla. In contrast, Ang-2 transcripts were more spatially restricted, being detected only in differentiating outer medullary tubules and the vasa recta bundle area. Using in situ hybridization and immunohistochemistry, Tie-2 was detected in capillaries of the nephrogenic cortex, glomerular tufts, cortical interstitium, and medulla including vessels in the vasa recta. Using Western blotting of protein extracted from whole organs, Tie-2 protein was detected between E14 and P8 with tyrosine phosphorylated Tie-2 evident from E18. These data are consistent with the hypothesis that Tie-2 has roles in maturation of both glomeruli and vasa rectae.

Effect of cAMP on the activity and the phosphorylation of Na+,K+-ATPase in rat thick ascending limb of Henle

In rat kidney medullary thick ascending limb of Henle's loop (MTAL), activation of protein kinase A (PKA) was previously reported to inhibit Na+,K(+)-ATPase activity. This is paradoxical with the known stimulatory effect of cAMP on sodium reabsorption. Because this inhibition was mediated by phospholipase A2 (PLA2) activation, a pathway stimulated by hypoxia, we evaluated the influence of oxygen supply on cAMP action on Na+,K(+)-ATPase in MTAL. Ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity were measured in isolated MTALs. Cellular ATP content and the phosphorylation level of Na+,K(+)-ATPase were determined in suspensions of outer medullary tubules. Experiments were carried out under nonoxygenated or oxygenated conditions in the absence or presence of PKA activators. cAMP analogues or forskolin associated with 3-isobutyl-1-methylxanthine (IBMX) inhibited ouabain-sensitive 86Rb uptake in nonoxygenated MTALs. In contrast, when oxygen supply was increased, cAMP stimulated ouabain-sensitive 86Rb uptake and Na+,K(+)-ATPase activity. Improved oxygen supply was associated with increased intracellular ATP content. The phosphorylation level of the Na+,K(+)-ATPase alpha subunit was increased by cAMP analogues or forskolin associated with IBMX in oxygenated as well as in nonoxygenated tubules. Under nonoxygenated conditions, the inhibition of Na+,K(+)-ATPase was dissociated from its cAMP-dependent phosphorylation, whereas under oxygenated conditions, the stimulatory effect of cAMP analogues on ouabain-sensitive 86Rb uptake was linearly related and cosaturated with the level of phosphorylation of the Na+,K(+)-ATPase alpha subunit. In oxygenated MTALs, PKA-mediated stimulation of Na+,K(+)-ATPase likely participates in the cAMP-dependent stimulation of sodium reabsorption. Under nonoxygenated conditions, this stimulatory pathway is likely overridden by the PLA2-mediated inhibitory pathway, a possible adaptation to protect the cells against hypoxic damage.

Intrarenal and subcellular localization of rat CLC5.

Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Dysregulation of cytokine expression in tubulointerstitial nephritis associated with murine malaria

Renal tubule regeneration after ischemic injury is coupled to the up-regulation and activation of cyclins and cyclin dependent kinases

Proliferation of renal tubules after acute injury is a reactive process of renal regeneration for recovery of renal function. Molecular and cellular mechanisms of the re-entrance of renal cells into the cell cycle after injury remain largely unknown. We have measured the correlations among the extent of proliferative activity and expression of cyclins and CDKs, and activity of each CDK during the regeneration period in the outer medullae of kidneys after ischemic injury in rats. The ratio of proliferating cell nuclear antigen (PCNA) positively immuno-stained nuclei to total nuclei per each section of the outer medulla of kidney indicated the proliferative index (PI) for this study. PI in the control period was 0.1%. The PI was increased at day 1 (13.4%), remained at a plateau at days 3 and 5 (30.5 and 32.3%), and decreased at day 7 and day 14 (17.3 and 12.2%) after ischemic injury. Proliferative activity was readily detectable in renal tubules, but was hardly detectable in glomeruli or blood vessels. As the PI increased, the mRNA and protein levels of cyclins D1, D3 and B, the mRNA levels of cyclin A, the protein levels of CDK4 and CDK2, and the activities of CDKs (CDK4, CDK2 and cdc2) increased in the outer medullae of kidneys after ischemic injury. These findings suggest that the temporal induction of proliferative activity in outer medullary tubules was closely linked with the cyclin/CDK system for regeneration of kidney after ischemic injury.

Kinetics and osmoregulation of Na(+)-and Cl(-)-dependent betaine transporter in rat renal medulla.

Betaine is one of the major organic osmolytes that accumulate in the renal medulla in response to high extracellular tonicity. Recent studies in MDCK cells have shown that betaine is taken up by an Na(+)- and Cl(-)-dependent transporter located on the basolateral membrane. We demonstrate here the presence of Na(+)-Cl(-)-dependent betaine transporter(s) in tubule suspensions prepared from the rat outer and inner medulla. The betaine transport activity was two to three times higher in the inner medulla compared with the outer medulla. The removal of Na+ and Cl- reduced betaine uptake in the outer medullary tubules by 86% and 82%, respectively. The betaine uptake was decreased by 39% in hypotonic buffer (189 mosmol/ kgH2O) and increased by 82% in hypertonic buffer (545 mosmol/kgH2O), compared with isotonic buffer (308 mosmol/ kgH2O). Kinetic studies of Na(+)-dependent betaine uptake in the outer medullary tubules revealed both a low- and a high-affinity component as follows: low-affinity and high volume component with Michaelis constant (K(m)1) of 8.6 mM and maximal uptake rate (Vmax1) of 112 pmol.microgram protein-1.h-1; and a low-volume and high-affinity component with K(m)2 of 0.141 mM and Vmax2 of 10 pmol. microgram protein-1.h-1. To investigate whether the Na(+)-Cl(-)-dependent betaine transporter is regulated by tonicity in vivo, we quantitated its mRNA in rat renal cortex and outer and inner medulla using both canine and rat Na(+)-Cl(-)-dependent betaine transporter cDNA probes. A single band of 3.0 kb was seen in the Northern blots prepared from both outer and inner medulla, but not in the cortex. Water deprivation for 3 days increased the abundance of this mRNA in the outer and inner medulla by 140% and 170%, respectively, but did not affect its expression in the cortex. In conclusion, Na(+)-Cl(-)-dependent betaine transporter(s) is present in rat outer and inner medullary tubules, and betaine transporter mRNA abundance is regulated by the hydration state in vivo.

Characterization of G proteins in obstructed kidneys.

Urinary tract obstruction has a marked effect on renal function. Activation of phospholipases which results in incremental production of vasoactive eicosanoids may contribute to the hemodynamic changes characteristic of an obstructed kidney. G proteins play an important role in transmembrane signal transduction, which control phospholipase activities and eicosanoid production. The present study was designed to determine the presence of G proteins in obstructed kidneys in rats, and to characterize the differences between unilateral ureteral obstruction (UUO) and bilateral ureteral obstruction (BUO). Several G-protein alpha subunits (G alpha s, G alpha i1,2, and G alpha i3) and the beta subunit (G beta) were determined by immunoblotting and immunocytochemical techniques using specific antibodies against these G proteins. Immunoblots demonstrated a decreased G alpha i3 content in the outer medullary tubules and a significantly lower G beta level in the glomeruli of UUO. In BUO, there was an increased level of G beta in the cortical tubules, and the G alpha s level was markedly reduced in the inner medullary tubules. Immunocytochemical studies revealed that these G proteins were predominantly localized in the brush border side of the cortical tubules. However, we could not demonstrate staining differences between UUO and BUO. These results indicate that a modulation of G-protein-coupled transmembrane signal transduction may contribute to the renal functional changes in an obstructed kidney. A different level of expression of G-protein subunits between UUO and BUO may be a factor in the differences of hemodynamics and renal tubular damage between UUO and BUO.

Cellular and sub‐cellular location of kallikrein gene products induced in outer medullary tubules by furosemide treatment

Summary: Renal kallikrein is normally synthesized and secreted by connecting tubule cells of the renal cortex. Treatment of mice with furosemide induces an expansion of the kallikrein synthesizing tubule cell population from the cortex to the inner stripe of the outer medulla. There is no induction of kallikrein gene expression in the medulla in sodium restricted mice or in untreated controls. We used hybridization histochemistry on semi‐thin plastic sections to identify the medullary segment recruited to express the renal kallikrein gene after furosemide treatment. In the outer medulla, the thick ascending limb (TAL) was the only tubule segment in which kallikrein gene expression was identified. Immunogold cytochemistry revealed that kallikrein is in granules located in the cytoplasm of medullary TAL cells from furosemide‐treated mice, confirming that furosemide‐induced kallikrein mRNA is translated in these cells. There was no immunoreactive kallikrein found in other tubule segments of the outer medulla, nor in TAL cells from sodium or untreated mice. Granules containing immunoreactive kallikrein were secreted from both poles of the cells into the tubule lumen from the apical surface and into the interstitial space from the basolateral surface. Secretion of kallikrein from the basolateral pole of TAL cells into the interstitial space enables kinins to be generated in close apposition to the vasculature underlying renal tubules, promoting local vasodilatation. the expansion of kallikrein synthesis and secretion to the medulla after furosemide treatment may contribute to the vasodilatation which follows furosemide‐induced diuresis.

Effect of increased distal sodium delivery on organic osmolytes and cell electrolytes in the renal outer medulla.

Sodium absorption in distal tubule segments was stimulated by increasing the distal delivery via infusion of hypertonic saline. In these animals, and in control rats, electrolyte concentrations in thick ascending limb cells, light and dark cells of the collecting duct in the outer and inner stripe of the outer medulla and in cells of the proximal straight tubule (outer stripe only) were studied. The measurements were performed by electron microprobe analysis of freeze-dried cryosections of the outer medulla. In addition, organic osmolytes (glycerophosphorylcholine, betaine and myo-inositol) were measured by high performance liquid chromatography in cortex and outer medulla. Augmented delivery of sodium chloride to the distal tubule was associated with increased sodium concentrations of thick ascending limb cells both in the outer and inner stripe and of medullary collecting duct light and dark cells in the outer stripe. While the sum of organic osmolyte concentrations was 28% higher in the outer medulla of the salt-loaded animals compared with controls, this value was unchanged in the renal cortex. These findings indicate that the primary event underlying stimulation of sodium absorption along the thick ascending limb during increased distal sodium delivery is enhanced entry of sodium across the apical cell membrane. This would be expected to lead to higher cell sodium concentrations and stimulation of basolateral active Na-K-exchange. The enhanced transport activity of outer medullary tubules may be associated with increased interstitial tonicities and intracellular retention of organic osmolytes.

Messenger RNA expression and synthesis of endothelin-1 along rat nephron segments.

The kidney both produces and responds to endothelin. We examined the production and the expression of mRNA of endothelin-1 (ET-1) in tubule suspensions and microdissected nephron segments. ET-1 production was measured by RIA using an ET-1-specific antibody. We applied the reverse transcription and polymerase chain reaction (PCR) technique to detect ET-1 mRNA along the nephron segments. Stimulation of ET-1 production was observed in the presence of FCS and transforming growth factor-beta (TGF-beta) in inner medullary tubules but not in cortical or outer medullary tubule suspensions. Among dissected nephron segments, ET-1 production was observed in glomeruli and inner medullary collecting ducts (IMCD), whereas it was negligible in proximal convoluted tubules (PCT) and medullary thick ascending limbs (MAL). In addition, the PCR product of ET-1 mRNA was also higher in glomeruli and IMCD, whereas it was undetectable in PCT and MAL. Furthermore, FCS and TGF-beta increased ET-1 mRNA in microdissected glomeruli and IMCD. These data clearly demonstrated that the production sites of ET-1 are glomeruli and IMCD among the nephron segments. ET-1 is an autocrine factor in these sites.

Impaired cyclic AMP generation in outer medullary tubules of gentamicin-treated rats

We have examined the effects of chronic gentamicin treatment on arginine 8-vasopressin (AVP)-dependent cyclic AMP (cAMP) metabolism in rat medullary collecting tubules (oMCT) and medullary thick ascending limbs of Henle's loop (mTALH). Gentamicin attenuated AVP-stimulated cAMP accumulation to a greater extent in the mTALH (Δ −51%) than in the oMCT (Δ −25%). The mechanism of attenuation differed between segments, and could not be attributed to either direct inhibition of adenylate cyclase activity nor direct potentiation of cAMP-phosphodiesterase activity. These data suggest that the gentamicin-induced decrease in renal concentrating ability may be due at least in part to reduced AVP-dependent cAMP accumulation in the oMCT and mTALH.

Metabolic Regulation of Organic Osmolytes in Tubules from Rat Renal Inner and Outer Medulla

Glycerophosphorylcholine (GPC), sorbitol and inositol were quantitated in renal tubule suspensions from inner and outer medulla of untreated Sprague-Dawley rats to study the regulation of organic osmolyte concentrations under different metabolic conditions and varying extracellular osmolalities in vitro. Inner medullary tubules prepared in hypertonic saline (550 mosm/kg) contained osmolyte concentrations comparable to those found in the kidney in vivo. Incubation for up to 8 h at 5 mmol/l glucose increased sorbitol in the inner medullary tubules and medium in an osmolality-dependent fashion, whereas GPC and inositol remained constant. At a given glucose concentration the rate of sorbitol formation decreased linearly with increasing tubular sorbitol concentration, which was regulated by an osmolality-dependent export mechanism. Perturbation of tubular mechanisms by inhibition of glycolysis or oxidative phosphorylation did not change the tubular osmolyte content. In contrast to papilla outer medullary tubules contained only inositol. Lactate added as a metabolic substrate to the outer medullary tubules did not change the cellular inositol levels. In outer medullary tubules osmolality changes (320-710 mosm/kg) had no effect on tubular inositol. Addition of furosemide was without effect, when added in vitro. The results indicate that tubular sorbitol formation is regulated by glucose concentration, the level of tubular sorbitol, and an osmolality-dependent export mechanism. In contrast, cellular inositol and GPC levels seem to be independent of acute changes in tubular metabolism.