Tubular proteinuria in patients with HNF1α mutations: HNF1α drives endocytosis in the proximal tubule.

Hepatocyte nuclear factor 1α (HNF1α) is a transcription factor expressed in the liver, pancreas, and proximal tubule of the kidney. Mutations of HNF1α cause an autosomal dominant form of diabetes mellitus (MODY-HNF1A) and tubular dysfunction. To gain insights into the role of HNF1α in the proximal tubule, we analyzed Hnf1a-deficient mice. Compared with wild-type littermates, Hnf1a knockout mice showed low-molecular-weight proteinuria and a 70% decrease in the uptake of ß2-microglobulin, indicating a major endocytic defect due to decreased expression of megalin/cubilin receptors. We identified several binding sites for HNF1α in promoters of Lrp2 and Cubn genes encoding megalin and cubilin, respectively. The functional interaction of HNF1α with these promoters was shown in C33 epithelial cells lacking endogenous HNF1α. Defective receptor-mediated endocytosis was confirmed in proximal tubule cells from these knockout mice and could be rescued by transfection of wild-type but not mutant HNF1α. Transfection of human proximal tubule HK2 cells with HNF1α was able to upregulate megalin and cubilin expression and to increase endocytosis of albumin. Low-molecular-weight proteinuria was consistently detected in individuals with HNF1A mutations compared with healthy controls and patients with non-MODY-HNF1A diabetes mellitus. Thus, HNF1α plays a key role in the constitutive expression of megalin and cubilin, hence regulating endocytosis in the proximal tubule of the kidney. These findings provide new insight into the renal phenotype of individuals with mutations of HNF1A.

Impaired Lysosomal Function Underlies Monoclonal Light Chain-Associated Renal Fanconi Syndrome.

Monoclonal gammopathies are frequently complicated by kidney lesions that increase the disease morbidity and mortality. In particular, abnormal Ig free light chains (LCs) may accumulate within epithelial cells, causing proximal tubule (PT) dysfunction and renal Fanconi syndrome (RFS). To investigate the mechanisms linking LC accumulation and PT dysfunction, we used transgenic mice overexpressing human control or RFS-associated κLCs (RFS-κLCs) and primary cultures of mouse PT cells exposed to low doses of corresponding human κLCs (25 µg/ml). Before the onset of renal failure, mice overexpressing RFS-κLCs showed PT dysfunction related to loss of apical transporters and receptors and increased PT cell proliferation rates associated with lysosomal accumulation of κLCs. Exposure of PT cells to RFS-κLCs resulted in κLC accumulation within enlarged and dysfunctional lysosomes, alteration of cellular dynamics, defective proteolysis and hydrolase maturation, and impaired lysosomal acidification. These changes were specific to the RFS-κLC variable (V) sequence, because they did not occur with control LCs or the same RFS-κLC carrying a single substitution (Ala30→Ser) in the V domain. The lysosomal alterations induced by RFS-κLCs were reflected in increased cell proliferation, decreased apical expression of endocytic receptors, and defective endocytosis. These results reveal that specific κLCs accumulate within lysosomes, altering lysosome dynamics and proteolytic function through defective acidification, thereby causing dedifferentiation and loss of reabsorptive capacity of PT cells. The characterization of these early events, which are similar to those encountered in congenital lysosomal disorders, provides a basis for the reported differential LC toxicity and new perspectives on LC-induced RFS.

Cystic gene dosage influences kidney lesions after nephron reduction.

Cystic kidney disease is characterized by the progressive development of multiple fluid-filled cysts. Cysts can be acquired, or they may appear during development or in postnatal life due to specific gene defects and lead to renal failure. The most frequent form of this disease is the inherited polycystic kidney disease (PKD). Experimental models of PKD showed that an increase of cellular proliferation and apoptosis as well as defects in apico-basal and planar cell polarity or cilia play a critical role in cyst development. However, little is known about the mechanisms and the mediators involved in acquired cystic kidney diseases (ACKD). In this study, we used the nephron reduction as a model to study the mechanisms underlying cyst development in ACKD. We found that tubular dilations after nephron reduction recapitulated most of the morphological features of ACKD. The development of tubular dilations was associated with a dramatic increase of cell proliferation. In contrast, the apico-basal polarity and cilia did not seem to be affected. Interestingly, polycystin 1 and fibrocystin were markedly increased and polycystin 2 was decreased in cells lining the dilated tubules, whereas the expression of several other cystic genes did not change. More importantly, Pkd1 haploinsufficiency accelerated the development of tubular dilations after nephron reduction, a phenotype that was associated to a further increase of cell proliferation. These data were relevant to humans ACKD, as cystic genes expression and the rate of cell proliferation were also increased. In conclusion, our study suggests that the nephron reduction can be considered a suitable model to study ACKD and that dosage of genes involved in PKD is also important in ACKD.

Dedifferentiation and aberrations of the endolysosomal compartment characterize the early stage of nephropathic cystinosis.

Nephropathic cystinosis, a lysosomal storage disease caused by mutations in the CTNS gene encoding the lysosomal cystine transporter cystinosin, is characterized by generalized proximal tubule (PT) dysfunction that progresses, if untreated, to end-stage renal disease. The pathogenesis of defective PT cellular transport in nephropathic cystinosis remains unclear. We characterized a recently generated line of C57BL/6 Ctns mice and analyzed endocytic uptake, lysosome function, and dedifferentiation and proliferation markers using primary cultures of PT epithelial cells derived from Ctns(-/-) and Ctns(+/+) littermates. Metabolic studies revealed that Ctns(-/-) mice show a progressive PT dysfunction characterized by low-molecular-weight (LMW) proteinuria, glucosuria and phosphaturia, before structural damage and in the absence of renal failure. These changes are related to decreased expression of the multi-ligand receptors megalin and cubilin and to increased dedifferentiation (ZONAB transcription factor) and proliferation (PCNA and Cyclin D1) rates. Studies on PT cells derived from Ctns(-/-) kidneys confirmed cystine overload, with accumulation of enlarged, dysfunctional lysosomes and reduced expression of endocytic receptors reflected by decreased uptake of specific ligands. These changes were related to a loss of integrity of tight junctions with a nuclear translocation of ZONAB and increased proliferation, as observed in Ctns(-/-) kidneys. These data reveal that the absence of cystinosin in PT cells triggers aberrations of the endolysosomal compartment, transport defects and an abnormal transcription program in the early stage of nephropathic cystinosis. Insights into the early manifestations of cystinosis may offer new targets for intervention, before irreversible renal damage.

A primary culture system of mouse thick ascending limb cells with preserved function and uromodulin processing.

The epithelial cells lining the thick ascending limb (TAL) of the loop of Henle perform essential transport processes and secrete uromodulin, the most abundant protein in normal urine. The lack of differentiated cell culture systems has hampered studies of TAL functions. Here, we report a method to generate differentiated primary cultures of TAL cells, developed from microdissected tubules obtained in mouse kidneys. The TAL tubules cultured on permeable filters formed polarized confluent monolayers in ∼12 days. The TAL cells remain differentiated and express functional markers such as uromodulin, NKCC2, and ROMK at the apical membrane. Electrophysiological measurements on primary TAL monolayers showed a lumen-positive transepithelial potential (+9.4 ± 0.8 mV/cm(2)) and transepithelial resistance similar to that recorded in vivo. The transepithelial potential is abolished by apical bumetanide and in primary cultures obtained from ROMK knockout mice. The processing, maturation and apical secretion of uromodulin by primary TAL cells is identical to that observed in vivo. The primary TAL cells respond appropriately to hypoxia, hypertonicity, and stimulation by desmopressin, and they can be transfected. The establishment of this primary culture system will allow the investigation of TAL cells obtained from genetically modified mouse models, providing a critical tool for understanding the role of that segment in health and disease.

Decreased renal accumulation of aminoglycoside reflects defective receptor-mediated endocytosis in cystic fibrosis and Dent's disease.

The clinical use of aminoglycoside (AG) antibiotics is limited by their renal toxicity, which is caused by drug accumulation in proximal tubule (PT) cells. Clinical studies reported that renal clearance of AG is enhanced in cystic fibrosis (CF) patients, which might reflect the role of CFTR in PT cell endocytosis. In order to assess the role of chloride transporters on the renal handling of AG, we investigated gentamicin uptake and renal accumulation in mice lacking functional CFTR (Cftr ( ∆F/∆F)) or knock-out for the Cl(-)/H(+) exchanger ClC-5 (Clcn5 ( Y/- )). The latter represent a paradigm of PT dysfunction and defective receptor-mediated endocytosis. As compared with controls, Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mice showed a 15% to 85% decrease in gentamicin accumulation in the kidney, respectively, in absence of renal failure. Studies on primary cultures of Cftr ( ∆F/∆F) and Clcn5 ( Y/- ) mouse PT cells confirmed the reduction in gentamicin uptake, although colocalization with endosomes and lysosomes was maintained. Quantification of endocytosis in PT cells revealed that gentamicin, similar to albumin, preferentially binds to megalin. The functional loss of ClC-5 or CFTR was reflected by a decrease of the endocytic uptake of gentamicin, with a more pronounced effect in cells lacking ClC-5. These results support the concept that CFTR, as well as ClC-5, plays a relevant role in PT cell endocytosis. They also demonstrate that the functional loss of these two chloride transporters is associated with impaired uptake of AG in PT cells, reflected by a decreased renal accumulation of the drug.

The inositol Inpp5k 5-phosphatase affects osmoregulation through the vasopressin-aquaporin 2 pathway in the collecting system.

Inositol Inpp5k (or Pps, SKIP) is a member of the inositol polyphosphate 5-phosphatases family with a poorly characterized function in vivo. In this study, we explored the function of this inositol 5-phosphatase in mice and cells overexpressing the 42-kDa mouse Inpp5k protein. Inpp5k transgenic mice present defects in water metabolism characterized by a reduced plasma osmolality at baseline, a delayed urinary water excretion following a water load, and an increased acute response to vasopressin. These defects are associated with the expression of the Inpp5k transgene in renal collecting ducts and with alterations in the arginine vasopressin/aquaporin-2 signalling pathway in this tubular segment. Analysis in a mouse collecting duct mCCD cell line revealed that Inpp5k overexpression leads to increased expression of the arginine vasopressin receptor type 2 and increased cAMP response to arginine vasopressin, providing a basis for increased aquaporin-2 expression and plasma membrane localization with increased osmotically induced water transport. Altogether, our results indicate that Inpp5k 5-phosphatase is important for the control of the arginine vasopressin/aquaporin-2 signalling pathway and water transport in kidney collecting ducts.

High-dose steroid treatment increases free water transport in peritoneal dialysis patients.

The water channel aquaporin-1 (AQP1) is the molecular counterpart of the ultrasmall pore that mediates free water transport during peritoneal dialysis (PD). Proof-of-principle studies performed in rats have shown that treatment with corticosteroids upregulates the expression of AQP1 in the peritoneal capillaries, causing a significant increase in free water transport. Whether such a beneficial effect could be observed in end-stage renal disease patients treated by PD remains unknown. Peritoneal transport parameters were evaluated in three patients on PD, shortly before and after living-donor renal transplantation and treatment with high-dose methylprednisolone (1.0-1.2 g/m(2)). As compared with pre-transplantation values, the post-transplantation test revealed an ∼2-fold increase in the sodium sieving and ultrasmall pore ultrafiltration volume, suggesting an effect on AQP1 water channels. In contrast, there was no change in the parameters of small solute transport. The direct involvement of AQP1 in these changes is suggested by the expression of glucocorticoid receptors in the human peritoneum and the presence of conserved glucocorticoid response elements in the promoter of the human AQP1 gene.

CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia.

Familial hypomagnesemia is a rare human disorder caused by renal or intestinal magnesium (Mg(2+)) wasting, which may lead to symptoms of Mg(2+) depletion such as tetany, seizures, and cardiac arrhythmias. Our knowledge of the physiology of Mg(2+) (re)absorption, particularly the luminal uptake of Mg(2+) along the nephron, has benefitted from positional cloning approaches in families with Mg(2+) reabsorption disorders; however, basolateral Mg(2+) transport and its regulation are still poorly understood. Here, by using a candidate screening approach, we identified CNNM2 as a gene involved in renal Mg(2+) handling in patients of two unrelated families with unexplained dominant hypomagnesemia. In the kidney, CNNM2 was predominantly found along the basolateral membrane of distal tubular segments involved in Mg(2+) reabsorption. The basolateral localization of endogenous and recombinant CNNM2 was confirmed in epithelial kidney cell lines. Electrophysiological analysis showed that CNNM2 mediated Mg(2+)-sensitive Na(+) currents that were significantly diminished in mutant protein and were blocked by increased extracellular Mg(2+) concentrations. Our data support the findings of a recent genome-wide association study showing the CNNM2 locus to be associated with serum Mg(2+) concentrations. The mutations found in CNNM2, its observed sensitivity to extracellular Mg(2+), and its basolateral localization signify a critical role for CNNM2 in epithelial Mg(2+) transport.

Fluid transport and cystogenesis in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most frequent inherited nephropathy. The development and enlargement of cysts in ADPKD requires tubular cell proliferation, abnormalities in the extracellular matrix and transepithelial fluid secretion. Multiple studies have suggested that fluid secretion across ADPKD cyst-lining cells is driven by the transepithelial secretion of chloride, mediated by the apical CFTR channel and specific basolateral transporters. The whole secretory process is stimulated by increased levels of cAMP in the cells, probably reflecting modifications in the intracellular calcium homeostasis and abnormal stimulation of the vasopressin V2 receptor. This review will focus on the pathophysiology of fluid secretion in ADPKD cysts, starting with classic, morphological and physiological studies that were followed by investigations of the molecular mechanisms involved and therapeutic trials targeting these pathways in cellular and animal models and ADPKD patients. This article is part of a Special Issue entitled: Polycystic Kidney Disease.

The transcription factor HNF1α regulates expression of chloride-proton exchanger ClC-5 in the renal proximal tubule.

The Cl(-)/H(+) exchanger ClC-5 is essential for the endocytic activity of the proximal tubule cells and the tubular clearance of proteins filtered in the glomeruli. The mechanisms that regulate the expression of ClC-5 in general and its specific expression in the proximal tubule are unknown. In this study, we investigated the hypothesis that the hepatocyte nuclear transcription factor HNF1α, which is predominantly expressed in proximal tubule segments, may directly regulate the expression of ClC-5. In situ hybridization demonstrated that the expression of Clcn5 overlaps with that of Hnf1α in the developing kidney as well as in absorptive epithelia, including the digestive tract and yolk sac. Multiple binding sites for HNF1 were mapped in the 5'-regulatory sequences of the mouse and human Clcn5/CLCN5 genes. The transactivation of the Clcn5/CLCN5 promoter by HNF1α was verified in vitro, and the binding of HNF1α to the Clcn5 promoter in vivo was confirmed by chromatin immunoprecipitation in mouse kidney. The expression of Clcn5 was reduced in the proximal tubule segments of HNF1α-null kidneys, and it was rescued upon transfection of HNF1α-null cells with wild-type but not with mutant HNF1α. These data demonstrate that HNF1α directly regulates the expression of ClC-5 in the renal proximal tubule and yield insights into the mechanisms governing epithelial differentiation and specialized transport activities in the kidney.

A homozygous mutation in INVS causing juvenile nephronophthisis with abnormal reactivity of the Wnt/beta-catenin pathway.

Mutations in the INVS gene coding for inversin have been identified in patients with nephronophthisis type 2 (NPHP2), typically causing infantile onset of ESRD and potentially associated with situs inversus. We report a novel family with a homozygous INVS mutation (c.2695 C > T; p.Arg899X) deleting the C-terminus of inversin. Both affected patients had juvenile ESRD and were discordant for situs inversus. The end-stage kidneys showed chronic interstitial nephritis with cysts and abnormal expression of ß-catenin and Dishevelled-1 supporting up-regulated canonical Wnt pathway in tubular cells. This case shows that INVS mutation can cause juvenile nephronophthisis with abnormal reactivity of the Wnt/ß-catenin pathway.

The soluble intracellular domain of megalin does not affect renal proximal tubular function in vivo.

Megalin-mediated endocytic uptake constitutes the main pathway for clearance of plasma proteins from the glomerular filtrate in proximal tubules. Little is known, however, about mechanisms that control megalin expression and activity in the kidney. A widely discussed hypothesis states that upon ligand binding a regulated intramembrane proteolysis releases the cytosolic domain of megalin and this fragment subsequently modulates megalin gene transcription. Here, we tested this by generating a mouse model that co-expressed both the soluble intracellular domain and full-length megalin. Despite pronounced synthesis in the proximal tubules, the soluble intracellular domain failed to exert distinct effects on renal proximal tubular function, including megalin expression, endocytic retrieval of proteins, or global renal gene transcription. Hence, our study argues that the soluble intracellular domain does not have a role in regulating the activity of megalin in the kidney.

Cell therapy for cystinosis.

In the September 2009 issue of Blood, Syres et al. [1] report on syngeneic bone marrow cell (BMC) and haematopoietic stem cell (HSC) therapy as a successful treatment in a mouse model of cystinosis, an autosomal recessive metabolic disease caused by a defect in the transport of cystine across the lysosomal membrane. The accumulation of cystine crystals in lysosomes leads to a multi-organ dysfunction including proximal tubulopathy and renal failure, corneal deposits, myopathy and central nervous system defects. By using Ctns knock-out (Ctns(-/-)) mice as a model for cystinosis, Syres et al. show that BMC transplantation leads to a major reduction of cystine content in all tissues tested, reflected by a significant attenuation of the development and progression of kidney injury and reduction in the number of mice with corneal cystine crystals. These changes were correlated with the engraftment of donor BMC producing a functional cystine transporter in the tissues tested. The transplantation of mouse HSC had the same therapeutic effect than whole BMC in this model, which is important as such HSC can readily be isolated from peripheral blood in humans. This work suggests that BMC or HSC transplantation is a potential treatment for cystinosis and other renal tubular disorders.

CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease.

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

PKD1 haploinsufficiency causes a syndrome of inappropriate antidiuresis in mice.

Mutations in PKD1 are associated with autosomal dominant polycystic kidney disease. Studies in mouse models suggest that the vasopressin (AVP) V2 receptor (V2R) pathway is involved in renal cyst progression, but potential changes before cystogenesis are unknown. This study used a noncystic mouse model to investigate the effect of Pkd1 haploinsufficiency on water handling and AVP signaling in the collecting duct (CD). In comparison with wild-type littermates, Pkd1(+/-) mice showed inappropriate antidiuresis with higher urine osmolality and lower plasma osmolality at baseline, despite similar renal function and water intake. The Pkd1(+/-) mice had a decreased aquaretic response to both a water load and a selective V2R antagonist, despite similar V2R distribution and affinity. They showed an inappropriate expression of AVP in brain, irrespective of the hypo-osmolality. The cAMP levels in kidney and urine were unchanged, as were the mRNA levels of aquaporin-2 (AQP2), V2R, and cAMP-dependent mediators in kidney. However, the (Ser256) phosphorylated AQP2 was upregulated in Pkd1(+/-) kidneys, with AQP2 recruitment to the apical plasma membrane of CD principal cells. The basal intracellular Ca(2+) concentration was significantly lower in isolated Pkd1(+/-) CD, with downregulated phosphorylated extracellular signal-regulated kinase 1/2 and decreased RhoA activity. Thus, in absence of cystic changes, reduced Pkd1 gene dosage is associated with a syndrome of inappropriate antidiuresis (positive water balance) reflecting decreased intracellular Ca(2+) concentration, decreased activity of RhoA, recruitment of AQP2 in the CD, and inappropriate expression of AVP in the brain. These data give new insights in the potential roles of polycystin-1 in the AVP and Ca(2+) signaling and the trafficking of AQP2 in the CD.

A primary culture of mouse proximal tubular cells, established on collagen-coated membranes.

A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and gamma-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na(+) similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures.

Cystic fibrosis is associated with a defect in apical receptor-mediated endocytosis in mouse and human kidney.

Inactivation of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) causes cystic fibrosis (CF). Although CFTR is expressed in the kidney, no overwhelming renal phenotype has been documented in patients with CF. This study investigated the expression, subcellular distribution, and processing of CFTR in the kidney; used various mouse models to assess the role of CFTR in proximal tubule (PT) endocytosis; and tested the relevance of these findings in patients with CF. The level of CFTR mRNA in mouse kidney approached that found in lung. CFTR was located in the apical area of PT cells, with a maximal intensity in the straight part (S3) of the PT. Fractionation showed that CFTR co-distributed with the chloride/proton exchanger ClC-5 in PT endosomes. Cftr(-/-) mice showed impaired (125)I-beta(2)-microglobulin uptake, together with a decreased amount of the multiligand receptor cubilin in the S3 segment and a significant loss of cubilin and its low molecular weight (LMW) ligands into the urine. Defective receptor-mediated endocytosis was found less consistently in Cftr(DeltaF/DeltaF) mice, characterized by a large phenotypic heterogeneity and moderate versus mice that lacked ClC-5. A significant LMW proteinuria (and particularly transferrinuria) also was documented in a cohort of patients with CF but not in patients with asthma and chronic lung inflammation. In conclusion, CFTR inactivation leads to a moderate defect in receptor-mediated PT endocytosis, associated with a cubilin defect and a significant LMW proteinuria in mouse and human. The magnitude of the endocytosis defect that is caused by CFTR versus ClC-5 loss likely reflects functional heterogeneity along the PT.

An octamer motif is required for activation of the inducible nitric oxide synthase promoter in pancreatic beta-cells.

Nitric oxide, generated by the inducible form of nitric oxide synthase (iNOS), is a potential mediator of cytokine-induced beta-cell dysfunction in type 1 diabetes mellitus. We have previously shown that cytokine-induced iNOS expression is cycloheximide (CHX) sensitive and requires nuclear factor-kappa B (NF-kappa B) activation. In the present study, we show that an octamer motif located 20 bp downstream of the proximal NF-kappa B binding site in the rat iNOS promoter is critical for IL-1 beta and interferon-gamma induction of promoter activity in rat primary beta-cells and insulin-producing RINm5F cells. In gel shift assays, the octamer motif bound constitutively the transcription factor Oct1. Neither Oct1 nor NF-kappa B binding activities were blocked by CHX, suggesting that other factor(s) synthesized in response to IL-1 beta contribute to iNOS promoter induction. The high mobility group (HMG)-I(Y) protein also bound the proximal iNOS promoter region. HMG-I(Y) binding was decreased in cells treated with CHX and HMG-I(Y) silencing by RNA interference reduced IL-1 beta-induced iNOS promoter activity. These results suggest that Oct1, NF-kappa B, and HMG-I(Y) cooperate for transactivation of the iNOS promoter in pancreatic beta-cells.