Are filtered plasma proteins processed in the same way by the kidney?

In order to understand the mechanism of albuminuria we have explored how other plasma proteins are processed by the kidney as compared to inert molecules like Ficolls. When fractional clearances are plotted versus protein radius there is a remarkable parallelism between protein (molecular weight range 30-150kDa) clearance in healthy controls, in Dent's disease, in nephrotic states and the clearance of Ficolls. Although there are significant differences in the levels of fractional clearances in these states. Dent's disease results in a 2-fold increase in the fractional clearance of proteins as compared to healthy controls whereas in nephrotic states there is a further 3-fold increase in fractional clearance. Previous thinking that albumin uptake was controlled primarily by the megalin/cubilin receptor does not explain the albumin urinary excretion data and is therefore an incorrect concept. Protein clearance in nephrotic states approach the fractional clearance of inert Ficolls for a given radius. It therefore appears that there are two pathways processing these proteins. A low capacity pathway associated with megalin/cubilin that degrades filtered protein (that is inhibited in Dent's disease) and a high capacity pathway that retrieves the filtered protein and returns it to the blood supply (without retrieval nephrotic protein excretion will occur and this will account for hypoproteinemia). On the other hand low molecular weight proteins (<20kDa) are processed entirely differently by the kidney. They are not retrieved but are comprehensively degraded in the kidney with the degradation products predominantly returned to the blood supply.

Oral intoxication of mice with Shiga toxin type 2a (Stx2a) and protection by anti-Stx2a monoclonal antibody 11E10.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains cause food-borne outbreaks of hemorrhagic colitis and, less commonly, a serious kidney-damaging sequela called the hemolytic uremic syndrome (HUS). Stx, the primary virulence factor expressed by STEC, is an AB5 toxin with two antigenically distinct forms, Stx1a and Stx2a. Although both toxins have similar biological activities, Stx2a is more frequently produced by STEC strains that cause HUS than is Stx1a. Here we asked whether Stx1a and Stx2a act differently when delivered orally by gavage. We found that Stx2a had a 50% lethal dose (LD50) of 2.9 µg, but no morbidity occurred after oral intoxication with up to 157 µg of Stx1a. We also compared several biochemical and histological parameters in mice intoxicated orally versus intraperitoneally with Stx2a. We discovered that both intoxication routes caused similar increases in serum creatinine and blood urea nitrogen, indicative of kidney damage, as well as electrolyte imbalances and weight loss in the animals. Furthermore, kidney sections from Stx2a-intoxicated mice revealed multifocal, acute tubular necrosis (ATN). Of particular note, we detected Stx2a in kidney sections from orally intoxicated mice in the same region as the epithelial cell type in which ATN was detected. Lastly, we showed reduced renal damage, as determined by renal biomarkers and histopathology, and full protection of orally intoxicated mice with monoclonal antibody (MAb) 11E10 directed against the toxin A subunit; conversely, an irrelevant MAb had no therapeutic effect. Orally intoxicated mice could be rescued by MAb 11E10 6 h but not 24 h after Stx2a delivery.

Sodium-independent carrier-mediated inositol transport in cultured renal epithelial (LLC-PK1) cells.

In addition to the concentrative, Na(+)-dependent inositol transport system demonstrated in many cell types, carrier-mediated, Na(+)-independent inositol transport is also shown to exist in LLC-PK1 renal epithelia. Inhibition of inositol uptake in Na(+)-free saline by 0.1 mM phloretin, and self-inhibition by net concentrations of inositol exceeding 10 mM, demonstrate the carrier-mediation of the Na(+)-independent uptake and distinguish it from flux through anion channels. The Na(+)-dependent uptake exhibits higher affinity for inositol, as seen by the stronger self-inhibition at lower inositol concentrations in Na+ saline. Kinetic analyses indicate a Km of 178 microM and a Vmax of 2447 pmol/min per microgram DNA for the Na(+)-dependent system, whereas the lower affinity, lower capacity Na(+)-independent system manifests a Km of 5.2 mM and a Vmax of 249 pmol/min per microgram DNA. the Na(+)-independent uptake further differs from the Na(+)-dependent transport by the lack of inhibitory effect of 10 microM glucose, and the greater relative inhibition of phloretin compared to that of phlorizin. Both types of uptake appear to localize predominantly to the basal-lateral cell surface. The Na(+)-independent transport is bidirectional, functioning in efflux as well as influx of inositol.

Prevention of albuminuria by aminoguanidine or ramipril in streptozotocin-induced diabetic rats is associated with the normalization of glomerular protein kinase C.

This study examined whether the prevention of diabetes-related albuminuria by aminoguanidine (AG) or ramipril (RAM) may be mediated by a common post-glomerular basement membrane renal intracellular mechanism involving protein kinase C (PKC). The renal handling of albumin was examined over 24 weeks in control and streptozotocin (STZ)-induced diabetic rats. A radioimmunoassay (RIA) that measures intact albumin, and intravenously injected tritium-labeled rat serum albumin, was used to assess the proportion of intact albumin and albumin fragments in urine. Diabetes was induced in male Sprague-Dawley rats by the intravenous administration of STZ at a dose of 50 mg/kg. Age-matched control rats received buffer alone. Diabetes was characterized by an increase in blood glucose (>15 mmol/l), an increase in GHb (means at 24 weeks 29.3+/-1.1%; control 6.1+/-0.1%, P<0.005), an increase in glomerular filtration rate (GFR) (4.13+/-0.15 ml/min; control 3.54+/-0.19 ml/min, P<0.005), an increase in intact albumin excretion rate (expressed as geometric mean 11.64 times/divided by 2.11 mg/24 h; control 0.74 times/divided by 1.57 mg/24 h, P<0.005) as measured by RIA, and an increase in glomerular PKC activity (26.83+/-2.38 pmol x mg(-1) x min(-1); control 14.6+/-2.99 pmol x mg(-1) x min(-1), P<0.005). Treatment of diabetic rats with either AG or RAM prevented the rise in intact albuminuria and glomerular PKC activity. Renal lysosomal cathepsin activity decreased in diabetic rats and this was not prevented by AG or RAM. Neither drug affected glycemic control or GFR, but RAM reduced systolic blood pressure (BP), whereas AG did not. These data indicate that urinary excretion of intact albumin and albumin-derived fragments in diabetes may be modulated independently of glycemic control (AG and RAM) and systolic BP (RAM). While both drugs are known for their different mechanisms of action, the fact that both prevent diabetes-related increases in glomerular PKC activity and albuminuria supports the hypothesis that PKC plays a central role in the development of diabetic nephropathy.

The TGV transgenic vectors for single-copy gene expression from the Escherichia coli chromosome.

Plasmid-based cloning and expression of genes in Escherichia coli can have several problems: plasmid destabilization; toxicity of gene products; inability to achieve complete repression of gene expression; non-physiological overexpression of the cloned gene; titration of regulatory proteins; and the requirement for antibiotic selection. We describe a simple system for cloning and expression of genes in single copy in the E. coli chromosome, using a non-antibiotic selection for transgene insertion. The transgene is inserted into a vector containing homology to the chromosomal region flanking the attachment site for phage lambda. This vector is then linearized and introduced into a recombination-proficient E. coli strain carrying a temperature-sensitive lambda prophage. Selection for replacement of the prophage with the transgene is performed at high temperature. Once in the chromosome, transgenes can be moved into other lysogenic E. coli strains using standard phage-mediated transduction techniques, selecting against a resident prophage. Additional vector constructs provide an arabinose-inducible promoter (P(BAD)), P(BAD) plus a translation-initiation sequence, and optional chloramphenicol-, tetracycline-, or kanamycin-resistance cassettes. These Transgenic E. coli Vectors (TGV) allow drug-free, single-copy expression of genes from the E. coli chromosome, and are useful for genetic studies of gene function.

Controversies in nephrology: response to 'renal albumin handling, facts, and artifacts'.

For 40 years indirect measurements of the glomerular sieving coefficient of albumin yielded very low values. The first direct measurement by 2-photon microscopy by Russo et al (Kidney Int (2007) 71, 504-513) gives values 50-times higher. This demonstrated that relatively large quantities of albumin are normally filtered based on size selectivity alone. Most of this albumin is retrieved and returned to the blood supply. These new discoveries represent a paradigm shift in our understanding of albumin processing by the kidney. They also serve to explain several anomalous aspects of previous studies on glomerular filtration and mechanism of albuminuria and support the fact that glomerular charge selectivity is not a major factor controlling glomerular permselectivity.

The normal kidney filters nephrotic levels of albumin retrieved by proximal tubule cells: retrieval is disrupted in nephrotic states.

The origin of albuminuria remains controversial owing to difficulties in quantifying the actual amount of albumin filtered by the kidney. Here we use fluorescently labeled albumin, together with the powerful technique of intravital 2-photon microscopy to show that renal albumin filtration in non-proteinuric rats is approximately 50 times greater than previously measured and is followed by rapid endocytosis into proximal tubule cells (PTCs). The endocytosed albumin appears to undergo transcytosis in large vesicles (500 nm in diameter), identified by immunogold staining of endogenous albumin by electron microscopy, to the basolateral membrane where the albumin is disgorged back to the peritubular blood supply. In nephrotic rats, the rate of uptake of albumin by the proximal tubule (PT) is decreased. This is consistent with reduced expression of clathrin, megalin, and vacuolar H(+)-ATPase A subunit, proteins that are critical components of the PT endocytotic machinery. These findings strongly support the paradigm-shifting concept that the glomerular filter normally leaks albumin at nephrotic levels. Albuminuria does not occur as this filtered albumin load is avidly bound and retrieved by PTCs. Dysfunction of this retrieval pathway leads to albuminuria. Thus, restoration of the defective endocytotic and processing function of PT epithelial cells might represent an effective strategy to limit urinary albumin loss, at least in some types of nephrotic syndrome.

Idiopathic restrictive cardiomyopathy in children.

OBJECTIVE: To define the natural history of idiopathic restrictive cardiomyopathy in a paediatric population and to identify any factors predictive of outcome. DESIGN: Retrospective analysis of patients born between 1970 and 2002 were identified from the Children's Hospital of Pittsburgh cardiology database. Demographic data, mode of presentation, echocardiographic and haemodynamic findings at diagnosis, survival time, and manner of death were evaluated. SETTING: Tertiary referral and transplant centre for paediatric patients with cardiac disease. PATIENTS: All local and referred patients with idiopathic restrictive cardiomyopathy born after 1970 and under 21 years of age at time of diagnosis. RESULTS: 21 patients were identified. Probability of survival at 1, 5, and 10 years was 80.5% (95% confidence interval (CI) 58 to 100), 39% (95% CI 17 to 61), and 20% (95% CI 0 to 42), respectively. Median age of presentation was 3.8 years (mean (SD) 5.7 (6.1) years). Median survival without transplantation was 2.2 years (mean (SD) 4.6 (5.4) years). Age at presentation, sex, and presence or absence of heart failure symptoms at presentation were not associated with clinical course. Right (p = 0.05) and left ventricular end diastolic pressures (p = 0.04) and ratio of left atrial to aortic root dimensions (LA:Ao) (p = 0.03) at presentation had a significantly negative correlation with survival time after diagnosis. CONCLUSIONS: Without transplantation, most children with restrictive cardiomyopathy have a very poor prognosis. Longer survival from diagnosis was correlated with lower LA:Ao and cardiac filling pressures at diagnosis. Survival time was not influenced by the symptoms present at diagnosis.

The protein kinase C inhibitor, bisindolylmaleimide, inhibits the TPA-induced but not the TNF-induced increase in LLC-PK1 transepithelial permeability.

The transepithelial paracellular permeability of an epithelium formed by LLC-PK1 cells increases upon activation of protein kinase C (PKC) by the phorbol ester tumor promoter, TPA, or in response to the cytokine tumor necrosis factor-alpha (TNF). Until recently, however, we have not been able to inhibit the permeability effects of TPA or TNF using any of the currently available serine-threonine kinase inhibitors. In this study we report the treatment of epithelial cell sheets with the selective PKC inhibitor bisindolylmaleimide, GF109203X, completely prevents the TPA-induced but not the TNF-alpha induced increase in tight junction permeability. While PKC-alpha still translocates from the cytosol to the membrane of TPA-stimulated epithelial cells overall PKC activity in the membrane fraction is markedly reduced in the presence of GFX.

Long-term effects of tumor necrosis factor on LLC-PK1 transepithelial resistance.

Renal epithelial LLC-PK1 cell sheets incubated with tumor necrosis factor (TNF) undergo an acute, spontaneous, and rapidly reversible decrease in transepithelial resistance (TER). (Mullin et al., 1992). However, 24 to 72 h following TNF exposure, TER across the cell sheet increases 2-fold. This later effect of TNF is also reversible, albeit slowly. The TER of TNF-treated cell sheets then declines toward initial levels between 72 and 144 h following exposure to the cytokine. Whereas the long-term increase in TER following TNF exposure is not associated with a decreased transepithelial 14C-mannitol flux (size selectivity), the charge (anionic) selectivity of the LLC-PK1 tight junction is decreased. Basal-lateral (ouabain and bumetanide-insensitive) Rb+ and apical Na+-dependent alpha-methylglucoside (AMG) uptake into the cell are both reduced in cultures exposed to TNF 24 h earlier. Correspondingly, this long-term effect on TER is accompanied by a 30% decrease in short circuit current (iscc). Along with an observed increase in basal-lateral methylamino-isobutyric acid (MeAIB) influx into the cells, an increased incorporation of [3H]-thymidine into DNA indicates increased cell cycling after exposure to TNF. While the increase in cell cycling is not sustained for the duration of the elevation in TER, it does appear to initiate a sequence of events that lead to the sustained increase in TER. A decrease in the lateral intercellular space, observed between these epithelial cells after long-term TNF exposure, may be a mechanism for the elevated TER following from the mitogenesis and/or transport changes. This overall long-term tightening of an epithelium in response to TNF may function, in part, as a compensatory action of the epithelium to reestablish its effectiveness as a physiological barrier, following the acute effect of TNF.

Basolateral 3-O-methylglucose transport by cultured kidney (LLC-PK1) epithelial cells.

In previous work we demonstrated the similarity of basolateral sugar transport of LLC-PK1 renal epithelia to basolateral kidney sugar transport using 2-deoxy-D-glucose as a substrate. In this study we first examine a central limitation to use of 2-deoxyglucose for basolateral sugar transport study in LLC-PK1 epithelia, namely, a shift of the rate-limiting step in uptake from transport to phosphorylation. Use of 3-O-methylglucose avoids this complication because it is not phosphorylated. However, use of 3-O-methylglucose requires much shorter incubation periods to examine linear rates of uptake (steady state is reached by 60 s at 22 degrees C for 0.1 mM 3-O-methylglucose). As was true for 2-deoxyglucose, apical uptake of 3-O-methylglucose was only a fraction of total uptake. Basolateral uptake was characteristically more sensitive to phloretin and cytochalasin B inhibition, relative to phlorizin. Inhibition studies indicate a requirement for a free hydroxyl on C-1 carbon of the pyranose ring, as is characteristic for renal basolateral sugar transport. Kinetic analysis indicates a single transport system with a Km of 10.9 mM and Vmax of 17.2 pmol.micrograms DNA-1.15 s-1. Subconfluent, undifferentiated LLC-PK1 cells show a similar Km (12.7 mM) but a ninefold higher Vmax (166.2 pmol.micrograms DNA-1.15 s-1). Stimulation of 3-O-methylglucose transport rate in confluent cultures by phorbol ester is relatively small (less than 100%) compared with effects on other somatic cells. The uptake rate of 3-O-methylglucose is not affected by glucose starvation, but subsequent refeeding with glucose-containing medium does significantly stimulate uptake.

Modulation of tumor necrosis factor-induced increase in renal (LLC-PK1) transepithelial permeability.

Tumor necrosis factor-alpha (TNF) causes a spontaneously reversible increase in tight junction permeability. TNF was the only cytokine tested that produced this effect. The effect on transepithelial permeability proceeds in four distinct phases: 1) a 60- to 90-min delay from time of application of TNF, 2) a rapid decrease in transepithelial resistance, 3) a recovery of transepithelial resistance to control level within 1 h, and 4) a further increase of transepithelial resistance above control levels. The recovery of transepithelial resistance occurs with or without TNF in the culture medium. Different protein kinase inhibitors affected different phases of this overall process. The tyrosine kinase inhibitor genistein significantly blocked the TNF effect. Neither transcription nor protein synthesis was required for transepithelial permeability to increase, but were required for the recovery. After the tight junctions have opened at 2 h in response to TNF, a second application of TNF will not produce the effect again for at least 12 h. The tight junctions will, however, open in response to phorbol esters during this time frame. Electron microscopy studies using apically applied ruthenium red suggest that TNF action results in < 10% of the junctions having increased permeability at any given time during the resistance decrease. The role of epithelial barrier permeability changes in TNF action in vivo is discussed.

cAMP modulates transepithelial resistance response of LLC-PK1 renal epithelia to tumor necrosis factor.

For "leaky" epithelia the transepithelial resistance (Rt) is an electrophysiological measure of the paracellular pathway within the epithelial barrier. The Rt across a monolayer of LLC-PK1 porcine renal epithelial cells is specifically an inverse measure of paracellular transepithelial permeability and displays a multiphasic and reversible response to the cytokine tumor necrosis factor-alpha (TNF). The Rt response to TNF can be inhibited by the nonhydrolyzable adenosine 3',5'-cyclic monophosphate (cAMP) analogue, dibutyryl-cAMP. In addition, activation of adenylate cyclase (forskolin) or inhibition of phosphodiesterase (3-isobutyl-1-methylxanthine, Ro-20-1724, and pentoxifylline), each of which have been reported to elevate cellular cAMP levels, also inhibited the Rt response to TNF. Incubation of the LLC-PK1 cell sheet with N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cAMP-dependent protein kinase (PKA), potentiated the Rt response to TNF. The Rt response to TNF was completely prevented by preincubation of the cultures with cholera toxin, whereas pertussis toxin pretreatment had a slight but significant potentiating effect on the response. Pretreatment with cholera toxin was associated with an approximately 18-fold elevation in cAMP levels in both control and TNF-treated cultures. Measurements of cellular cAMP content at selected intervals after TNF administration showed a significant elevation (P < 0.01) of 140% above time-matched controls at 1 h after the administration of TNF to the cell sheet. The level of cAMP then declined to approximate control level within 2.5 h of TNF administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic exposure of LLC-PK1 epithelia to the phorbol ester TPA produces polyp-like foci with leaky tight junctions and altered protein kinase C-alpha expression and localization.

Acute exposure (up to 4 h) of LLC-PK1 epithelial cell sheets to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) causes a rapid decrease of transepithelial electrical resistance (TER) to less than 15% of initial value. As the TPA exposure period is continued by chronically passaging cells in the presence of TPA, TER begins to recover. By 6 weeks of exposure, TER recovers to almost 50% of its initial value, suggesting that tight junctions (TJs) are recovering barrier function even in the continued presence of TPA. Between 6 and 8 weeks, TER then decreases a second time to approximately 20 to 40% of initial values, and TER values remain at this level for at least 18 weeks of exposure. Transepithelial (paracellular) fluxes of D-mannitol inversely correspond with TER changes. Across chronically treated cell sheets, rates are higher than those across control cell sheets, but lower than those across acutely treated cell sheets. The decrease of TER at 6-8 weeks coincides with the appearance of multilayered, polyp-like foci (PLFs) on the otherwise one cell layer thick epithelium. Electron microscopy shows that the electron-dense dye ruthenium red cannot penetrate across TJs of control cells but passes across all of the TJs of a cell sheet treated acutely with TPA. In chronically treated cultures, ruthenium red penetrates TJs between most cells of PLFs, but not TJs of adjacent morphologically normal epithelium. A clonal subline derived from cells of a PLF (clone PLF-A) is multilayered almost throughout and exhibits ruthenium red penetration across nearly all of its tight junctions, monolayer or multilayer. Acute exposure of control cell sheets to TPA induces activation, translocation, and down-regulation of protein kinase C-alpha (PKC-alpha). In chronically TPA-treated and clone PLF-A cells, total PKC-alpha levels are reduced even further and almost all remaining PKC-alpha is found in the membrane-associated and Triton-insoluble fractions. Immunofluorescence shows that PKC-alpha expression is restricted to the PLFs in chronically TPA-treated cells and is more homogeneously distributed in clone PLF-A cultures. In summary, the data show that chronic treatment of epithelial cells with a tumor promoter induces the formation of abnormal cell architecture (PLFs) associated with increased leakiness of TJs and membrane translocation of PKC-alpha. Recovery of barrier function in portions of chronically TPA-treated cultures does not correlate with up-regulation of PKC-alpha nor translocation back to the cytosolic compartment.

Allergic skin rash with lamotrigine and concomitant valproate therapy

Cutaneous rash is one of the commonest adverse events associated with lamotrigine. We assessed whether the risk is increased in patients receiving concomitant valproate therapy in a population of 103 adult patients with intractable epilepsy, who had lamotrigine added to their treatment. Of the 33 patients taking valproate, 10 (30 per cent) developed a rash, whilst of the 70 not taking valproate, only 6 (8 per cent) developed a rash. This suggests a significantly higher risk of cutaneous rash when starting lamotrigine in patients already taking valproate (p