Aminoglycoside antibiotics reduce glucose reabsorption in kidney through down-regulation of SGLT1

Nephrotoxicity is known to be a major clinical side effect of aminoglycoside antibiotics. Aminoglycosides cause damage to proximal tubular cells in kidney, however the mechanism of toxicity is still unclear. In order to elucidate the mechanism of nephrotoxicity, we studied the effect of aminoglycoside antibiotics on glucose transport systems in vitro and in vivo. As a result, we found that the aminoglycosides significantly reduced Na(+)/glucose cotransporter (SGLT1)-dependent glucose transport and also down-regulated mRNA and protein levels of the SGLT1 in pig proximal tubular LLC-PK(1) cells. To obtain evidence about SGLT1 down-regulation in vivo, we studied the mRNA expression of SGLT1 using gentamicin C-treated murine kidney and found that gentamicin C down-regulated SGLT1 in vivo as well as in vitro. Furthermore, the gentamicin C-treated mice showed significant rise in urinary glucose excretion. These results indicate that one of the mechanisms of aminoglycoside nephrotoxicity is the down-regulation of SGLT1, which causes reduction in glucose reabsorption in kidney.     

Phosphatidylinositol 3-kinase mediates inhibitory effect of angiotensin II on sodium/glucose cotransporter in renal epithelial cells

Effects of angiotensin II (ANGII) on regulation of sodium/glucose cotransporter (SGLT1) activity were investigated in LLC-PK(1) cells, renal proximal epithelial cell line. ANGII inhibited alpha-[14C] methyl-D-glucopyranoside (AMG) uptake into LLC-PK(1) cells in a dose-dependent manner. This inhibition was based on a decrease in maximal transport rate (Vmax) of AMG from 2.20 nmol/mg protein/15 min to 1.19 nmol/mg protein/15 min, although apparent affinity constant (Km) did not alter. In western blot analysis, protein level of SGLT1 in brush border membrane (BBM) was decreased by ANGII, although total SGLT1 was not altered. In the aspect of intracellular signal transduction, ANGII blocked the formation of cAMP. Pertussis toxin, an inactivator of Gi protein that control intracellular cAMP level, completely prevented the decrease of AMG uptake caused by ANGII. 8-Br-cAMP, a cell membrane permeable cAMP analogue, increased AMG uptake and protein level of SGLT1 in BBM. Both wortmannin and LY294002 that are phosphatidylinositol (PI) 3-kinase inhibitors, inhibited the SGLT1 activity, and also attenuated the effect of 8-Br-cAMP on SGLT1 activity. Those inhibitors prevented the 8-Br-cAMP-induced expression of SGLT1 in plasma membrane. We conclude that ANGII plays an important role in post-translational regulation in SGLT1. Inhibition of SGLT1 translocation is suggested to be caused by inactivation of protein kinase A and decrease of PI 3-kinase activity.     

Megalin deficiency offers protection from renal aminoglycoside accumulation.

Aminoglycosides are antibiotics commonly used to treat life-threatening Gram-negative bacterial infections. However, their use is hampered by their severe nephrotoxicity due to accumulation in renal proximal tubules. Several pathways have been implicated in the renal uptake of aminoglycosides including megalin, an endocytic receptor in proximal tubular cells. Here, we have used mouse models with genetic or functional megalin deficiency to explore the contribution of megalin and other pathways to renal aminoglycoside uptake in vivo. We demonstrate that the uptake of aminoglycosides into the kidney directly correlates with renal megalin activity and is completely eliminated in mice lacking the receptor. Thus, our studies provide unequivocal evidence that megalin is the only major pathway responsible for renal aminoglycoside accumulation and that the receptor represents a unique drug target to prevent aminoglycoside-induced nephrotoxicity in patients.     

Endocytosis of gentamicin in a proximal tubular renal cell line.

The mechanisms by which aminoglycosides are accumulated in renal proximal tubular cells remain unclear. Adsorptive mediated endocytosis, via a common pathway for cationic proteins, or receptor endocytosis, mediated by the glycoprotein 330/megalin, have been proposed to be involved in gentamicin transport in renal cells. We used the LLC-PK1 cell line, derived from the pig proximal tubule, to explore further the regulation of gentamicin endocytosis in these cells and to determine the role of clathrin mediated endocytosis and G proteins in this function. Gentamicin endocytosis was strictly temperature dependent, whereas total uptake (endocytosis plus binding) did not significantly differ at 4 or 37 degrees C. Substances that suppress receptor mediated, clathrin dependent endocytosis, such as monensin, phenylarsine oxide and dansylcadaverine, or inhibit caveolae mediated endocytosis, such as nystatin, did not affect gentamicin entrance in LLC-PK1 cells. Among substances that disrupt the actin cytoskeleton, only cytochalasin D, that is active also on fluid phase endocytosis, significantly reduced the intracellular concentrations of the aminoglycoside. Other maneuvers that perturb clathrin dependent endocytosis without affecting clathrin independent pathway, such as acidification of cytosol or incubation in hypertonic medium, were also without effect. Mastoparan, a well known stimulator of heterotrimeric G proteins, strongly increased endocytosis of gentamicin, and the same effect was evident with two other G protein stimulators, aluminum fluoride and fluoride alone; however the effect seems not to be mediated by an activation of adenylyl cyclase. In conclusion, gentamicin endocytosis in LLC-PK1 cells is probably clathrin independent, limited by cytochalasin D, which interacts with cytoskeleton, and increased by substances like mastoparan and aluminum fluoride, which activate heterotrimeric G proteins.     

SARS-CoV-2 spike protein inhibits megalin-mediated albumin endocytosis in proximal tubule epithelial cells

Patients with COVID-19 have high prevalence of albuminuria which is used as a marker of progression of renal disease and is associated with severe COVID-19. We hypothesized that SARS-CoV-2 spike protein (S protein) could modulate albumin handling in proximal tubule epithelial cells (PTECs) and, consequently contribute to the albuminuria observed in patients with COVID-19. In this context, the possible effect of S protein on albumin endocytosis in PTECs was investigated. Two PTEC lines were used: HEK-293A and LLC-PK1. Incubation of both cell types with S protein for 16 h inhibited albumin uptake at the same magnitude. This effect was associated with canonical megalin-mediated albumin endocytosis because: (1) DQ-albumin uptake, a marker of the lysosomal degradation pathway, was reduced at a similar level compared with fluorescein isothiocyanate (FITC)-albumin uptake; (2) dextran-FITC uptake, a marker of fluid-phase endocytosis, was not changed; (3) cell viability and proliferation were not changed. The inhibitory effect of S protein on albumin uptake was only observed when it was added at the luminal membrane, and it did not involve the ACE2/Ang II/AT1R axis. Although both cells uptake S protein, it does not seem to be required for modulation of albumin endocytosis. The mechanism underlying the inhibition of albumin uptake by S protein encompasses a decrease in megalin expression without changes in megalin trafficking and stability. These results reveal a possible mechanism to explain the albuminuria observed in patients with COVID-19.     

Megalin-mediated endocytosis of cystatin C in proximal tubule cells

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.     

Induction of nephrotoxicity by high doses of gentamicin in diabetic rats

Rats with streptozotocin-induced diabetes mellitus (DM) are resistant to aminoglycoside (AG) nephrotoxicity presumably because of defective transport and accumulation of drug by proximal tubular cells. To test this hypothesis we injected DM rats with saline or with gentamicin, 100, 200, and 400 mg/kg per day for 6 days, to determine if the renal cortical concentration of gentamicin could be raised to toxic levels. Nephrotoxicity was assessed by monitoring for evidence of accelerated lipid peroxidation in the renal cortex, for elevation of the serum creatinine concentration, and for evidence of proximal tubular cell injury and necrosis by light and electron microscopy. At 100 mg/kg per day renal cortical gentamicin was 454 +/- 85 micrograms/g. Except for an increase in renal cortical phospholipids these rats manifested no evidence of accelerated lipid peroxidation or elevation of serum creatinine. At 200 mg/kg per day renal cortical gentamicin rose to 636 +/- 20 micrograms/g. These rats manifested mild functional and morphological evidence of toxicity. At 400 mg/kg renal cortical gentamicin rose to 741 +/- 43 micrograms/g. These rats developed severe nephrotoxic injury as manifested by a marked increase of lipid peroxidation evident by an increase of malondialdehyde from a control level of 0.48 +/- 0.02 to 1.72 +/- 0.12 nmole/mg protein, a shift from unsaturated to saturated fatty acids esterified in renal cortical phospholipids, depression of superoxide dismutase and catalase, and a shift from reduced to oxidized glutathione. The serum creatinine rose from a baseline level of 0.24 +/- 0.01 to 0.46 +/- 0.05 mg/dl. Light and electron microscopy revealed enlarged lysosomes distended with typical myeloid bodies and extensive proximal tubular cell necrosis. These observations provide compelling evidence in support of the view that the resistance of DM rats to AG nephrotoxicity is causally linked to the low rate of drug uptake by renal proximal tubular cells. When the renal cortical concentration reaches a critical level, it elicits a pattern of toxic injury indistinguishable from that of nondiabetic rats. Thus, there is nothing inherent to the diabetic state that prevents AGs from causing their usual adverse effects on the metabolism of renal proximal tubular cells once they gain access in sufficient quantity into these cells.     

A two-receptor pathway for catabolism of Clara cell secretory protein in the kidney

Clara cell secretory protein (CCSP) is a transport protein for lipophilic substances in bronchio-alveolar fluid, plasma, and uterine secretion. It acts as a carrier for steroid hormones and polychlorinated biphenyl metabolites. Previously, the existence of receptors for uptake of CCSP.ligand complexes into the renal proximal tubules had been suggested. Using surface plasmon resonance analysis, we demonstrate that CCSP binds to cubilin, a peripheral membrane protein on the surface of proximal tubular cells. Binding to cubilin results in uptake and lysosomal degradation of CCSP in cultured cells. Surprisingly, internalization of CCSP is blocked not only by cubilin antagonists but also by antibodies directed against megalin, an endocytic receptor that does not bind CCSP but associates with cubilin. Consistent with a role of both receptors in renal uptake of CCSP in vivo, patients deficient for cubilin or mice lacking megalin exhibit a defect in tubular uptake of the protein and excrete CCSP into the urine. These findings identify a cellular pathway consisting of a CCSP-binding protein (cubilin) and an endocytic coreceptor (megalin) responsible for tissue-specific uptake of CCSP and associated ligands.     

Expression of the Na+-d-Glucose Cotransporter SGLT1 in Neurons

Abstract: In brains of the rabbit, pig, and human, expression of the high-affinity Na⁺-d -glucose cotransporter SGLT1 and of the protein RS1, which alters the activity of SGLT1, was demonstrated. In situ hybridization showed that SGLT1 and RS1 are transcribed in pyramidal cells of brain cortex and hippocampus and in Purkinje cells of cerebellum. In neurons of pig brain SGLT1 protein was demonstrated by western blotting with synaptosomal membranes and by immunohistochemistry, which showed SGLT1 in pyramidal and Purkinje cells. To test whether SGLT1 in neurons may be activated during increased d -glucose consumption, an epileptic seizure was induced in rat brain, and the uptake of specific nonmetabolized substrates of SGLT1 {[¹⁴C]methyl-α-d -glucopyranoside ([¹⁴C]AMG)} and of Na⁺-independent transporters {2-deoxy-d -[¹⁴C]glucose([¹⁴C]2-DG)} was analyzed by autoradiography. During the seizure the uptake of AMG and 2-DG was increased in the focus. Within two hours after the seizure 2-DG uptake in the focus returned to normal. In contrast, the AMG uptake in the focus area was still increased 1 day later. The data show that the high-affinity Na⁺-d -glucose cotransporter SGLT1 is expressed in neurons and can be up-regulated.     

Nucleoside transport in cultured LLC-PK1 epithelia.

The transport of nucleosides by LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, was characterised. Uridine influx was saturable (apparent Km approximately 34 microM at 22 degrees C) and inhibited by greater than 95% by nitrobenzylthioinosine (NBMPR), dilazep and a variety of purine and pyrimidine nucleosides. In contrast to other cultured animal cells, the NBMPR-sensitive nucleoside transporter in LLC-PK1 cells exhibited both a high affinity for cytidine (apparent Ki approximately 65 microM for influx) and differential 'mobility' of the carrier (the kinetic parameters of equilibrium exchange of formycin B are greater than those for formycin B influx). An additional minor component of sodium-dependent uridine influx in LLC-PK1 cells became detectable when the NBMPR-sensitive nucleoside transporter was blocked by the presence of 10 microM NBMPR. This active transport system was inhibited by adenosine, inosine and guanosine but thymidine and cytidine were without effect, inhibition properties identical to the N1 sodium-dependent nucleoside carrier in bovine renal outer cortical brush-border membrane vesicles (Williams and Jarvis (1991) Biochem. J. 274, 27-33). Late proximal tubule brush-border membrane vesicles of porcine kidney were shown to have a much reduced Na(+)-dependent uridine uptake activity compared to early proximal tubule porcine brush-border membrane vesicles. These results, together with the recent suggestion of the late proximal tubular origin of LLC-PK1 cells, suggest that in vivo nucleoside transport across the late proximal tubule cell may proceed mainly via a facilitated-diffusion process.     

SLC5A9/SGLT4, a new Na+-dependent glucose transporter, is an essential transporter for mannose, 1,5-anhydro-D-glucitol, and fructose

We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) > D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).     

Sodium-Glucose Transporter-2 (SGLT2; SLC5A2) Enhances Cellular Uptake of Aminoglycosides

Aminoglycoside antibiotics, like gentamicin, continue to be clinically essential worldwide to treat life-threatening bacterial infections. Yet, the ototoxic and nephrotoxic side-effects of these drugs remain serious complications. A major site of gentamicin uptake and toxicity resides within kidney proximal tubules that also heavily express electrogenic sodium-glucose transporter-2 (SGLT2; SLC5A2) in vivo. We hypothesized that SGLT2 traffics gentamicin, and promotes cellular toxicity. We confirmed in vitro expression of SGLT2 in proximal tubule-derived KPT2 cells, and absence in distal tubule-derived KDT3 cells. D-glucose competitively decreased the uptake of 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), a fluorescent analog of glucose, and fluorescently-tagged gentamicin (GTTR) by KPT2 cells. Phlorizin, an SGLT2 antagonist, strongly inhibited uptake of 2-NBDG and GTTR by KPT2 cells in a dose- and time-dependent manner. GTTR uptake was elevated in KDT3 cells transfected with SGLT2 (compared to controls); and this enhanced uptake was attenuated by phlorizin. Knock-down of SGLT2 expression by siRNA reduced gentamicin-induced cytotoxicity. In vivo, SGLT2 was robustly expressed in kidney proximal tubule cells of heterozygous, but not null, mice. Phlorizin decreased GTTR uptake by kidney proximal tubule cells in Sglt2^+/− mice, but not in Sglt2^−/− mice. However, serum GTTR levels were elevated in Sglt2^−/− mice compared to Sglt2^+/− mice, and in phlorizin-treated Sglt2^+/− mice compared to vehicle-treated Sglt2^+/− mice. Loss of SGLT2 function by antagonism or by gene deletion did not affect gentamicin cochlear loading or auditory function. Phlorizin did not protect wild-type mice from kanamycin-induced ototoxicity. We conclude that SGLT2 can traffic gentamicin and contribute to gentamicin-induced cytotoxicity.     

Megalin antagonizes activation of the parathyroid hormone receptor

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (Kd 0.5 microM). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.     

Nephrotoxic cell death by diclofenac and meloxicam

The nephrotoxicity of diclofenac, a non-steroidal anti-inflammatory drug that inhibits both isoforms of cyclooxygenase (COX) has been reported to be fatal to vultures but this was not so with meloxicam which is COX-2 selective. Our study showed that diclofenac was more toxic than meloxicam to both the proximal tubular LLC-PK1 cells and the distal tubular Madin-Darby canine kidney type II (MDCKII) cells, and that LLC-PK1 cells were more susceptible. Exposure of MDCKII cells to meloxicam caused activation of caspase-9/-3 and release of cytochrome c. These observations together with a positive annexin V-FITC staining implicate an intrinsic mitochondrial cell death pathway by apoptosis. Diclofenac-treated MDCKII cells on the other hand showed extensive propidium iodide staining, suggestive of cell death by necrosis. The mode of cell death in LLC-PK1 cells was however less well-defined with positive annexin V-FITC staining but minimal increase in caspase-3 activity alluding to a caspase-independent pathway.     

Calcitriol mediates the activity of SGLT1 through an extranuclear initiated mechanism that involves intracellular signaling pathways

The present study explored whether calcitriol plays a role in the regulation of sodium-dependent glucose transporter protein 1 (SGLT1) activity. For this purpose, alpha-methyl glucoside (AMG) uptake in stable transfected Chinese hamster ovary (CHO-G6D3) cells expressing rabbit SGLT1 (rbSGLT1) was used. The involvement of second messengers, intracellular signaling pathways, and pro-inflammatory cytokines were examined using specific inhibitors before incubation with calcitriol for 15 min. The present study demonstrated the involvement of second messengers produced by phospholipase A₂, phospholipase C, calmodulin, diacylglycerol kinase, and phosphoinositide 3 kinase on calcitriol-regulated AMG uptake. Pretreatment with inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway increased calcitriol-induced AMG uptake. In contrast, inhibition of the phosphoinositide 3-kinase PI3K/Akt/mTOR signaling pathway decreased the effect of calcitriol on AMG uptake. These findings suggest that calcitriol regulates rbSGLT1 activity through a rapid, extranuclear initiated mechanism of action stimulated by MAPK and inhibited by PI3K/Akt/mTOR. Another important finding was the effect of pro-inflammatory cytokines on calcitriol-induced AMG uptake. Interleukin-6 increased while tumor necrosis factor-α decreased calcitriol-induced AMG uptake. In conclusion, the present study demonstrates the involvement of calcitriol in the regulation of rbSGLT1 activity. This is due to the activation of intracellular signaling pathways triggered by second messenger molecules and cytokines after a short time (15 min) exposure to calcitriol.     

Depletion of renal cortical glutathione and nephrotoxicity by cephaloridine,cephalothin and gentamicin in male sprague-dawley rats

Cephaloridine and gentamicin are selectively accumulated in renal cortex and produce necrosis of proximal tubular cells. However, the mechanisms responsible for renal cortical accumulation of these two antibiotics are quite different; therefore the early pathogenetic processes may not be the same. In the present study, effects of two cephalosporins (cephaloridine and cephalothin) and an aminoglycoside (gentamicin) on rat renal cortical glutathione were determined. Cephaloridine produced a dose-related depletion of renal cortical glutathione one hour following a single administration of the drug. In contrast, cephalothin in equivalent doses did not reduce renal cortical glutathione. Gentamicin had no effect on renal cortical glutathione, even when an acutely lethal dose (1000 mg/kg) was used. Pretreatment of rats with diethyl maleate (0.4 ml/kg) markedly depleted renal cortical glutathione and this pretreatment also potentiated cephaloridine nephrotoxicity. These results suggest that glutathione may play a protective role against cephaloridine but not gentamicin nephrotoxicity.     

Calcium-sensing receptor antagonism or lithium treatment ameliorates aminoglycoside-induced cell death in renal epithelial cells

The aminoglycoside antibiotic gentamicin elicits proximal tubular toxicity and cell death. In calcium-sensing receptor (CaR)-transfected HEK-293 (CaR-HEK) cells and CaR-expressing proximal tubule-derived opossum kidney (OK) cells, chronic gentamicin treatment elicits dose-dependent, caspase-mediated apoptotic cell death. Here we investigated whether the renal cell toxicity of the CaR agonist gentamicin could be prevented by CaR antagonism or by lithium cotreatment which may interfere with receptor-mediated signalling. Chronic treatment of OK and CaR-HEK cells with low concentrations of gentamicin elicited cell death, an effect that was ameliorated by cotreatment with the CaR negative allosteric modulator (calcilytic) NPS-89636. This calcilytic also attenuated CaR agonist-induced ERK activation in these cells. In addition, 1 mM LiCl, equivalent to its therapeutic plasma concentration, also inhibited gentamicin-induced toxicity in both cell types. This protective effect of lithium was not due to the disruption of phosphatidylinositol-mediated gentamicin uptake as the cellular entry of Texas red-conjugated gentamicin into OK and CaR-HEK cells was unchanged by lithium treatment. However, the protective effect of lithium was mimicked by glycogen synthase 3beta inhibition. Together, these data implicate CaR activation and a lithium-inhibitable signalling pathway in the induction of cell death by gentamicin in renal epithelial cells in culture.     

Light chains are a ligand for megalin.

Cubilin and megalin are giant glycoprotein receptors abundant on the luminal surface of proximal tubular cells of the kidney. We showed previously that light chains are a ligand for cubilin. As cubilin and megalin share a number of common ligands, we further investigated the ligand specificity of these receptors. Three lines of evidence suggest that light chains can also bind megalin: 1) anti-megalin antiserum largely displaces brush-border light chain binding and megalin-expressing BN-16 cell uptake more than anti-cubilin antiserum, 2) direct binding studies on isolated proteins using surface plasmon resonance techniques confirm that megalin binds light chains, and 3) light chains compete with known megalin ligands for brush-border membrane binding and BN-16 cell uptake. The megalin-light chain interaction is divalent ion dependent and similar for both kappa- and lambda-light chains. A fit of the data on light chain binding to megalin over a concentration range 0.078-2.5 mg/ml leads to an estimated dissociation constant of 6 x 10(-5) M, corresponding approximately to one light chain-binding site per megalin and in the same range for dissociation constants for cubilin binding. These data suggest that light chains bind the tandem megalin-cubilin complex. Megalin is the major mediator of light chain entry into megalin-expressing membrane such as the apical surface of proximal tubular epithelial cells.