Role of megalin, a proximal tubular endocytic receptor, in the pathogenesis of diabetic and metabolic syndrome-related nephropathies: protein metabolic overload hypothesis

SUMMARY:   Megalin is an endocytic receptor on the apical membranes of proximal tubule cells (PTC) in the kidney, and is involved in the reabsorption and metabolism of various proteins that have been filtered by glomeruli. Patients with diabetes, especially type 2 diabetes, or metabolic syndrome are likely to have elevated serum levels of advanced glycation end products, liver-type fatty acid binding protein, angiotensin II, insulin and leptin, and renal metabolism of these proteins is potentially overloaded. Some of these proteins are themselves nephrotoxic, while others are carriers of nephrotoxic molecules. Megalin is involved in the proximal tubular uptake of these proteins. We hypothesize that megalin-mediated metabolic overload in PTC leads to compensatory cellular hypertrophy and sustained Na⁺ reabsorption, causing systemic hypertension and glomerular hyperfiltration via tubuloglomerular feedback, and named this as 'protein metabolic overload hypothesis'. Impaired metabolism of bioactive proteins such as angiotensin II and insulin in PTC may enhance hypertrophy of PTC and/or Na⁺ reabsorption. Sleep apnoea syndrome, a frequent complication of diabetes and metabolic syndrome, may cause renal hypoxia and result in relative overload of protein metabolism in the kidneys. The development of strategies to identify patients with diabetes or metabolic syndrome who are at high risk for renal metabolic overload would allow intensive treatment of these patients in an effort to prevent the development of nephropathy. Further studies on the intracellular molecular signalling associated with megalin-mediated metabolic pathways may lead to the development of novel strategies for the treatment of nephropathies related to diabetes and metabolic syndrome.     

Segmental reabsorption measured by micropuncture and clearance methods during hypertonic sodium infusion in the rat

BACKGROUND: We wanted to validate by direct measurements in rat tubulesa technique used to calculate segmental volume absorption byeach segment of the human nephron. METHODS: Experiments were performed on 17 rats during hypertonic Na infusionprior to and after frusemide administration. Tubular sampleswere taken from early distal and last proximal sites. The rateof filtration of single nephrons (SNGFR) was calculated by thetechnique of total collection of tubular fluid using labelledinulin as a marker. Reabsorption was computed by the tubularfluid to plasma (TF/P) inulin concentration ratio. RESULTS: SNGFR was 50±4 nl/min at the distal (n=82), 51±3nl/min at the proximal sampling site (n=112, P>0.65) duringbaseline conditions. Percent reabsorptions were 85±1and 69±2% respectively (P<0.0001). During frusemidethese values were 52±6 nl/min and 76±2% at thedistal, 49±5 nl/min and 66±2% at the proximalsite. In 83 paired proximal collections, fractional (68±1versus 67±1%, P>0.32), absolute reabsorption (34±2versus 33±2, P>0.50) and SNGFR (50±2 nl/minversus 50±3 nl/min, P>0.99) were not different betweenbaseline and frusemide. In 25 re-collections from the distaltubule these same values were 83±2% versus 76±2%,and 48±4 nl/min versus 55±6 nl/min respectively.Very similar results were obtained in 55 paired distalproximalcollections during baseline, and 42 such pairs during frusemide. In the presence of the diuretic the fractional urine excretionwas significantly correlated (R=0.83, P< 0.0001) with fractionalproximal delivery. Na^± resorption by Henle's loop was22±2% calculated from clearance data and 23±1%of GFR from micropuncture data respectively. They were not significantlydifferent (P>0.70) and were significantly correlated (R=0.57,P<0.02). CONCLUSIONS: These data demonstrate that frusemide does not act proximallyand that delivery beyond the proximal tubule approximates urineflow rate during the action of the drug. The values of segmentalreabsorption along the nephron computed on clearance measurementsare superimposable upon those obtained directly by micropuncture.     

Kinetics and characterization of initially regenerating proximal tubules in S3 segment in response to various degrees of acute tubular injury.

BACKGROUND: We examined kinetics and characterization of regenerating proximal tubule (PT) cells after various degrees of tubular injury in S3 segments of PT and assessed label-retaining slow cycling cells in S3. METHODS: PT injury was induced by different doses of uranyl acetate (UA) injection into rats, and initially regenerating PTs were identified by in vivo bromodeoxyuridine (BrdU)-labelling before sacrifice or were examined on vimentin positivity. Next, the 3H-thymidine pulse/chase approach was applied to the early regenerating PTs identified by BrdU-labelling after UA injection. RESULTS: Low-dose UA induced focal PT depletion and initial BrdU positivity in the proximal three-quarters of the S3 segment of PT. Autoradiography showed the increased number of label-retaining and label-diluted cells in the proximal three-quarters of S3 in rats treated with low-dose UA compared to normal rats. High-dose UA induced almost complete PT depletion in the proximal three-quarters of S3 and less PT depletion in the distal quarter of S3 and initial BrdU+ cells were restrictedly found in the distal quarter of S3. Label-retaining and label-diluted cells were increasingly found in the entire S3 at day 7, but only label-retaining cells remained in similar numbers in the distal quarter of S3 until day 42. Initially regenerating PT cells with any doses of UA expressed vimentin, suggesting dedifferentiated PT cells. CONCLUSIONS: Initially regenerating cells after PT injury in S3 are dedifferentiated pre-existing PT cells, which may scatter throughout S3 and be responsible for focal repair of S3. Some initially regenerating PT cells in the distal S3 showed persistent label-retaining cells at day 42 after high-dose UA insult and contributed to renewal of the entire S3, thus they might be slow cycling cells with responsibility for S3 repair.     

Micropuncture and clearance measurements of segmental reabsorption by the rat nephron

BACKGROUND.: We wanted to verify whether the calculations of segmental tubularreabsorption obtained during water diuresis were supported bydirect micropuncture measurements. METHODS.: Experiments were performed on 18 rats during baseline waterdiuresis (B) and after the administration of frusemide (F),10 mg/kg, by whole-kidney clearance measurements and micropuncturecollections from early distal (ED) and last proximal (LP) tubularsegments. RESULTS.: GFR was 957±79 in B, 1053±77 µl/min in F,P>0±13. SNGFR was 38±1 in 166 and 38±1nl/min in 165 tubules respectively, P>0.77. In LP collectionsthe percentage reabsorption was 71±2 in B and 76±2%during F (P> 0.07)in 99 and 95 samples respectively. Theabsolute proximal reabsorption was not changed by F (27.6±1.5versus 27.7±1·3 nl/min, P>0.96). The data weresuperimposable when the analysis was restricted to paired data.The difference between ED and LP resorption was 17±3during B and fell significantly (P<0.008) to 5±3%during F. The percentage of GFR excreted during F, measuredby clearance techniques, and the percentage delivery of filtratebeyond the proximal tubule, measured independently by micropuncture,were not different (27±2 versus 24±2%, P>0.10),while they were significantly correlated (P<0.04). The calculationsof segmental Na reabsorption along the different nephron segmentsby clearance techniques were not statistically different fromand were significantly correlated with the reabsorptions measureddirectly by micropuncture. CONCLUSIONS.: The present experiments validate the calculations of reabsorptionby techniques applicable to human studies of clinical physiology.     

Early proximal tubule injury in experimental aristolochic acid nephropathy: functional and histological studies.

BACKGROUND: Aristolochic acid (AA), the plant extract of Aristolochia species, is involved in the onset of progressive tubulointerstitial renal fibrosis in humans. Clinical and in vitro findings have previously suggested that the proximal tubule was the target of AA. METHODS: Using a rat model of AA nephropathy, the proximal tubular lesions induced by daily subcutaneous injections of AA for 35 or 5 days were characterized biochemically and histologically. Urinary excretion of proteins, albumin, low molecular weight proteins, N-acetyl-beta-d-glucosaminidase, alpha-glutathione S-transferase, leucine aminopeptidase and neutral endopeptidase (NEP) was determined and related to histological conventional findings and immunostainings of NEP and megalin. RESULTS: In both protocols, an acute phase of release of urinary markers was observed within the first 3 days of AA treatment in parallel with a significant increase of specific AA-related DNA adducts reflecting early tubular intoxication. A dramatic loss of the proximal tubule brush border was histologically confirmed, while the expression of megalin decreased at the damaged apical epithelium (mainly of the S3 segment). CONCLUSION: Proximal tubule injury occurs early after AA intoxication in rats, with a link between specific AA-DNA adduct formation, decreased megalin expression and inhibition of receptor-mediated endocytosis of low molecular weight proteins, bringing in vivo confirmation of previous in vitro studies.     

Role of protein kinase C and oxidative stress in interleukin-1beta-induced human proximal tubule cell injury and fibrogenesis

BACKGROUND: Interleukin (IL)-1beta, a pro-inflammatory macrophage-derived cytokine, is implicated as a key mediator of interstitial fibrosis and tubular loss or injury in progressive renal insufficiency. This study investigates some of the mechanisms of action of IL-1beta on the proximal tubule. METHODS: Confluent cultures of primary human proximal tubule cells (PTC) were incubated in serum-free media supplemented with either IL-1beta (0-4 ng/mL), phorbol-12-myristate 13-acetate (PMA, protein kinase C activator) (6.25-100 nmol/L), or vehicle (control), together with a non-specific protein kinase C inhibitor (H7), a specific protein kinase C inhibitor (BIM-1), an anti-oxidant (NAC) or a NADPH oxidase inhibitor (AEBSF). RESULTS: Interleukin-1beta-treated PTC exhibited time-dependent increases in fibronectin secretion (ELISA), cell injury (LDH release) and reactive nitrogen species (RNS) release (Griess assay). Proximal tubule cell DNA synthesis (thymidine incorporation) was also significantly suppressed. The effects of IL-1beta, which were reproduced by incubation of PTC with PMA (6.25-100 nmol/L), were blocked by H7 but not by BIM-1. The anti-oxidant (4 mmol/L) partially blocked IL-1beta-induced fibronectin secretion by PTC, but did not affect IL-1beta-induced LDH release, RNS release or growth inhibition. The NADPH oxidase inhibitor (AEBSF) significantly attenuated all observed deleterious effects of IL-1beta on PTC. CONCLUSION: Interleukin-1beta directly induces proximal tubule injury, extracellular matrix production and impaired growth. The anti-oxidant, NAC, appears to ameliorate part of the fibrogenic effect of IL-1beta on PTC through mechanisms that do not significantly involve protein kinase C activation or nitric oxide release.     

Recreating the size-dependent reabsorption function of proximal convoluted tubule towards artificial kidney applications: Structural analysis and computational study

Human kidneys tend to be affected adversely and fail to function more often than any other organ in the body because of diet, heredity, and lifestyle of a person. Dialysis is the technique presently in use for replacing the failed kidney function but it is packed with painfulness, bulkiness, and is costly also. There is a growing need for development of an artificial kidney that eradicates the problems associated with dialysis. This article proposes a structure that mimics the most important aspect of the human kidney: the size-dependent reabsorption of endothelial cells in the proximal convoluted tubule (PCT). The proposed structure consists of transporting channels connecting blood tubules surrounded on both sides of a main tubule. Geometries of the channels are analyzed for optimum flow by varying angles with respect to the main tubule. The analytical formulae have been developed by considering proper boundary conditions governing the flow in the structure, which makes the model as robust, concise, and realistic as the actual PCT. The mathematical model is validated against the benchmark FEM tool COMSOL Multiphysics and the results seem to be satisfactory. This article concludes, that slant channels possess a considerably higher average flow velocity of 5.39 × 10^–5 m/s (≈52% reabsorption rate) than straight channels with 4.77 × 10^–5 m/s (≈46% reabsorption rate) which is closer to the actual PCT reabsorption rate of 60%. The proposed model is first of its kind in nature among the reported works which creates and exhibits simulation environment of PCT reabsorption function supported by mathematical formulation and also can be useful to study and develop artificial kidney in near future.     

Dynamics of renal glucose reabsorption in rat

Summary: Glucose filtered load (FL), glomerular filtration rate (GFR) and tubular glucose reabsorption (TG) were simultaneously measured in the right, control kidney and in the left kidney with cortical necrosis (CN kidney) in rats with increasing plasma glucose concentrations (PG). Urinary glucose excretion starts when plasma concentrations (threshold value) averages 2.69 ± 0.20 mg/mL in the control kidney and 2.78 ± 0.16 mg/mL in the CN kidney (non significant; NS). For PG above the threshold value, increases in glucose FL were associated with increases in TG reabsorption. Maximal glucose reabsorption was not reached even when PG levels were 10 mg/mL. Moreover, there is a linear correlationship between TG reabsorbed and glucose FL in this range of PG. Among the two factors determining FL, our results support a linear correlationship between glucose reabsorption and GFR. Finally, no significant differences in tubular glucose reabsorption dynamics appears between control and CN kidney suggesting similar dynamics for glucose reabsorption in cortical and juxtamedullary nephrons.     

Proximal tubule endocytic apparatus as the specific renal uptake mechanism for vitamin D-binding protein/25-(OH)D3 complex

The renal proximal tubule exhibits a very extensive apical endocytic apparatus that is involved in the reabsorption of molecules filtered in the glomeruli. Several key receptors appear to be involved in this function, which serves not only to conserve protein but also to reabsorb different vitamins in complex with their binding proteins. Recent research has established megalin as probably the most important receptor in this endocytosis process. Cubilin is another receptor identified in the proximal tubule endocytic apparatus. Because cubilin lacks transmembrane or cytoplasmic domains required for endocytosis, this receptor associates with megalin to recycle and internalize its ligands. Recent studies have shown that vitamin D-binding protein (DBP)/25-(OH)D3 complex is one of the megalin/cubilin ligands. Megalin knockout mice develop vitamin D deficiency and bone disease owing to an inability of the proximal tubules to capture the DBP/25-(OH)D3 complexes from the glomerular filtrate. In the same way, kidney-specific megalin knockout mice have severe plasma vitamin D deficiency, hypocalcaemia and serious bone disease, like the complete megalin knockout mice. Anti-cubilin antibodies inhibit cellular uptake of DBP/25-(OH)D3 by up to 70%. Anti-megalin antibodies produced a similar reduction in DBP/25-(OH)D3 endocytosis. When both antibodies were applied, impairment of DBP/25-(OH)D3 was only slightly more impaired (around 80%), suggesting that cubilin and megalin function through the same endocytic pathway. Specific forms of renal Fanconi syndrome are associated with endocytic pathway dysfunction with disruption of megalin-mediated uptake DBP/25-(OH)D3 complex, producing metabolic bone disease in affected individuals as a prominent clinical finding.     

Aldosterone induces collagen synthesis via activation of extracellular signal-regulated kinase 1 and 2 in renal proximal tubules

Aim: Aldosterone plays a crucial role in renal fibrosis by inducing mesangial cell proliferation and promoting collagen synthesis in renal fibroblasts. However, renal proximal tubule involvement in aldosterone-induced collagen synthesis has not yet been identified. The aim of this study was to examine the potential role of aldosterone in collagen expression and its possible mineralocorticoid receptor (MR)-dependent pathway, mediated by activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2) in cultured human renal proximal tubular epithelial (HKC) cells. Methods: After HKC cells were stimulated by aldosterone with different concentrations for various time and periods, the gene expression and protein synthesis of collagen I, II, III and IV were measured by real-time polymerase chain reaction and western blot, respectively. ERK1/2 activation, α-smooth muscle actin (α-SMA), and E-cadherin were also detected by western blot. Results: Aldosterone can increase ERK1/2 phosphorylation of human renal proximal tubular epithelial cells in a time- and dose-dependent manner. Although aldosterone had no effect on collagen I and II expression, it increased expression of α-SMA and collagen III and IV and decreased that of E-cadherin in HKC cells after 48 h. These effects could be prevented by a ERK pathway inhibitor, U0126, or by a selective MR antagonist, spironolactone. Conclusion: The results suggest that aldosterone plays a pivotal role in tubulointerstitial fibrosis by promoting tubular epithelial–mesenchymal transition and collagen synthesis in proximal tubular cells. The process is MR-dependent, and mediated by ERK1/2 mitogen-activated protein kinase pathway.     

Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro.

BACKGROUND:Mycophenolic acid has been shown to be effective for the prevention and treatment of renal allograft rejection. Rejection episodes were found to be associated with an infiltration of lymphocytes and macrophages/monocytes into the diseased kidney. Expression of RANTES, HLA-DR and ICAM-1 may be important for the pathogenesis of this leukocyte infiltration. Therefore the aim of this study was to evaluate the effect of the antiproliferative and immunosuppressive agent mycophenolic acid (MPA) on cell growth and cytokine-induced expression of RANTES, HLA-DR and ICAM-1 of highly purified proximal (PTC) and distal tubular cells (DTC) from human kidney. METHODS:Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.25-50 microM) or MPA plus guanosine (100 microM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1beta+gammaIFN+TNFalpha=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis. RESULTS:MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression. CONCLUSIONS:We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.     

Dissociation of gluconeogenesis from fluid and phosphate reabsorption in isolated rabbit proximal tubules

Gluconeogenesis in the kidney is a metabolic function characteristic of proximal tubules. Recent studies suggest that renal gluconeogenesis (GNG) may in some way be coupled to fluid and phosphate reabsorption in the proximal tubule. Therefore, the present studies examined more directly the relationship between GNG and transport of fluid and phosphate using isolated proximal straight tubules of rabbit kidney. Glucose production rates were determined in isolated tubules with a glucose microassay while both phosphate (Jp) and net fluid fluxes (Jv) were measured by the in vitro isolated tubule perfusion technique. Glucose production rates from individual substrates, including pyruvate, lactate, glutamate and alpha-ketoglutarate, differed significantly, but neither Jv nor Jp was altered when different substrates in the bath medium were used. Inhibition of GNG by 3-mercaptopicolinate did not alter the Jv or Jp. Acid pH (7.0) stimulated GNG and suppressed both Jv and Jp. The addition of 3-mercaptopicolinate at acid pH abolished the stimulatory effect of acid pH on GNG but had no effect on Jv or Jp. These data thus indicate that, in the isolated rabbit renal proximal tubule, GNG rates can be dissociated from both the Jv and Jp and suggest that renal GNG may not directly regulate the fluid and phosphate transport rates in the proximal straight tubule.     

Inhibitors of arachidonic acid metabolism modulate the insulin-like growth factor-1-induced growth of proximal tubular cells in primary culture

Summary: Insulin-like growth factor 1 (IGF-1) is involved in the regulation of kidney growth during maturation as well as during regenerative processes. Some observations point to eicosanoids as possible intracellular mediators. In rat proximal tubular cells in primary culture we found that IGF-1 (100 ng/mL) stimulated the release of arachidonic acid. We therefore investigated the effects of different inhibitors of arachidonic acid handling on IGF-1-induced hypertrophy (increase in cell protein) and hyperplasia (increase in cellular thymidine incorporation and cell number). the hypertrophic response was abolished by dibucaine (inhibitor of phospholipase A₂) or proadifen (inhibitor of cytochrome P-450 enzymes). Clotrimaole (inhibitor of epoxygenase) reduced the hypertrophic response significantly. Indomethacin (inhibitor of cyclooxygenase) or NDGA (nordihydroguaiaretic acid; inhibitor of lipoxygenase) did not affect IGF-1- induced hypertrophy. the hyperplastic response was abolished completely in the presence of dibucaine, and reduced significantly in the presence of proadifen or clotrimazole. Indomethacin did not affect the hyperplastic response, whereas NDGA reduced it slightly under certain conditions. From our results we conclude that the action of IGF-1 on growth of proximal tubular cells is, at least in part, mediated by arachidonic acid or its cytochrome P-450 metabolites. the underlying mechanism of action might be either stimulated metabolism or enhanced release of arachidonic acid.     

The effect of cholesterol overload on mouse kidney and kidney-derived cells

Introduction: Dyslipidemia is one of the onset and risk factors of chronic kidney disease and renal function drop is seen in lipoprotein abnormal animal models. However, the detailed molecular mechanism of renal lipotoxicity has not been clarified. Therefore, the present study aimed to investigate the influence of cholesterol overload using mouse kidney tissue and kidney-derived cultured cells.

Methods: C57BL/6 mice were fed normal diet (ND) or 1.25% cholesterol-containing high-cholesterol diet (HCD) for 11?weeks, and we used megalin as a proximal tubule marker for immunohistology. We added beta-very low density lipoprotein (βVLDL) to kidney-derived cells and examined the effect of cholesterol overload on megalin protein and mRNA expression level, cell proliferation and cholesterol content in cells.

Results: In the kidney of HCD mice, the gap between glomerulus and the surrounding Bowman’s capsule decreased and the expression level of megalin decreased. After βVLDL treatment to the cells, the protein expression and mRNA expression level of megalin decreased and cell proliferation was restrained. We also observed an increase in cholesterol accumulation in the cell and free cholesterol/phospholipid ratios increased.

Conclusions: These findings suggest that the increased cholesterol load on kidney contribute to the decrease of megalin and the overloaded cholesterol is taken into the renal tubule epithelial cells, causing suppression on cell proliferation, which may be the cause of kidney damage.     

Effects of mycophenolic acid on IL-6 expression of human renal proximal and distal tubular cells in vitro.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine which regulates immune responses and host defence mechanisms. IL-6 has been found to be increased in certain inflammatory conditions of the kidney, in which tubular epithelial cells play a pivotal role. Human renal tubular cells express IL-6. Until now no data about the effect of the immunosuppressant drug mycophenolic acid (MPA) on IL-6 expression were available. METHODS: Proximal and distal tubular epithelial cells (PTC/DTC) have been isolated immunomagnetically. Confluent monolayers were stimulated with interleukin-1beta (IL-1beta; 25 U/ml), IL-1beta+ MPA (0.25-50 micro M) or MPA alone for 48 h. Release of IL-6 protein into the supernatant was evaluated with an enzyme immunoassay, IL-6 mRNA expression was evaluated using the Quantikine mRNA kit. RESULTS: After IL-1beta stimulation, a highly significant 2.6- (PTC) and 3.8-fold (DTC) upregulation of IL-6 expression was detectable. IL-6 mRNA was upregulated by IL-1beta [1.57- (PTC) and 2.03-fold (DTC)]. MPA inhibited this cytokine-induced IL-6 expression in a dose-dependent manner. Incubation with the lowest MPA concentration had no effect on the stimulated upregulation, whereas all higher doses significantly decreased IL-6 expression. Dexamethasone significantly inhibited the cytokine-induced IL-6 protein release in PTC, but not in DTC. CONCLUSIONS: In this study we demonstrated for the first time an inhibitory effect of MPA on the stimulated IL-6 expression of renal tubular epithelial cells. In contrast to older data, which showed a synergistic upregulation of the expression of a CC-chemokine by a combination of cytokines and MPA, in the present study we could demonstrate an immunosuppressive effect of MPA on the expression of an important cytokine.     

A hypothesis linking sodium and lithium reabsorption in the distal nephron.

BACKGROUND: A hypothesis is proposed linking Na(+) and Li(+) reabsorption in the distal nephron. The handling of these two ions in the distal nephron is related because they share the same apical membrane entry mechanism: the amiloride-sensitive Na(+) channel (ENaC). However, the two ions exit the cell through different transport mechanisms: Na(+) via the Na(+)-K(+)-ATPase and Li(+) via the Na(+)/H(+) exchanger. Studies in rats have shown that under normal circumstances hardly any Li(+) is reabsorbed in the distal nephron, so that the urinary excretion of Li(+), expressed as a fraction of the delivery to the early distal tubule (FE(Li dist)), amounts to approximately 0.97. In contrast, during severe dietary Na(+) restriction, FE(Li dist) decreases to 0.50-0.60. Our hypothesis is that the absence of distal Li(+) reabsorption during intake of a normal diet can be explained by a negative driving force for Li(+) entrance across the apical membrane in those segments in which ENaC is active. METHOD: We propose a model that incorporates this concept. RESULTS: The model indicates that the lowering of FE(Li dist) during dietary Na(+) restriction can be explained by activation of apical ENaC in extra sub-segments further downstream. In these extra sub-segments the driving force for Li(+) reabsorption is positive, leading to significant Li(+) reabsorption. During dietary K(+) restriction, FE(Li dist) is reduced to 0.35-0.55. The model shows that this reduction in FE(Li dist) can be explained by hyperpolarization of the apical membrane in ENaC-containing sub-segments, which is known to occur in this condition. CONCLUSION: We conclude that the model may improve current understanding of both Na(+) and Li(+) handling in the distal nephron.     

Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo

In vivo microperifbsion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo . Somniferous and caput and cauda epididymal tubules were perihsed for 3 h. with [³⁵S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed.
Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules.
Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and path physiological effects on this important epithelial hnction in vim