17 ␣-Endosulfine in Diabetic Nephropathy Jerry Yee, MD, and Balazs Szamosfalvi, MD CONTENTS EFFECTS OF SULFONYLUREAS ON CULTURED MESANGIAL CELLS SULFONYLUREA AGENTS REGULATE MESANGIAL ATP-SENSITIVE K CHANNELS KIDNEY SUR NDOGENOUS IGANDS E KAT P L α NDOSULFINE EGULATION OF -E R KAT P GLOMERULAR EFFECTS OF SULF IN INSULIN-DEFICIENT DIABETES MELLITUS α-ENDOSULFINE EXPRESSION ALTERS MATRIX METABOLISM CONCLUSIONS REFERENCES EFFECTS OF SULFONYLUREAS ON CULTURED MESANGIAL CELLS The sulfonylureas (SULF) have long been utilized as oral agents in the treatment of type 2 diabetes mellitus (1). The primary effect of SULF is the stimulation of insulin secretion following binding to specific SULF receptors (SUR) on pancreatic β-cells. However, SUR have extensive representation in a multitude of extrapancreatic tissues. Therefore, it is not unanticipated that SULF may induce metabolic changes aside from that of insulin secretion. These drugs have been shown to increase glucose uptake and glucose transporter (GLUT) expression in myocytes, adipocytes, and skeletal muscle cells (2–5). Moreover, we have documented significant SULF-induced metabolic effects in cultured rat mesangial cells (MCs), including alterations in mesangial matrix metabolism and MC contractility, independent of their effect on the ambient level of glycemia. The latter effect mimicked that provided by other known MC effectors of contractility, for example, atrial natriuretic peptide and angiotensin II. In short-term (acute) experiments of rat MC, the exposure to a first-generation SULF, tolazamide (1.5 mM), augmented mesangial glucose uptake. This effect was attributed to an elevated rate of cytosol-to-membrane translocation of GLUT1. This direct effect subsequently stimulated MC extracellular matrix (ECM) synthesis, driven by transforming β growth factor (TGF)- 1, which was demonstrated to accumulate in the conditioned From: Contemporary Diabetes: The Diabetic Kidney Edited by: P. Cortes and C. E. Mogensen © Humana Press Inc., Totowa, NJ 305 306 Yee and Szamosfalvi media (6). By contrast, chronic exposure of MC to glibenclamide (10 nM), a more potent, second-generation SULF, did not enhance MC glucose uptake, yet produced an intense inhibition of high glucose concentration-induced ECM accumulation (7). Taken collectively, SULF, in addition to their action as insulin secretagogues, exert important metabolic changes through affecting MC matrix metabolism to the extent that the devel- opment and evolution of diabetic glomerulosclerosis may be altered by them. Furthermore, it is highly probable that the aforementioned effects are mediated via membrane-bound and/or intracellular SUR. SULFONYLUREA AGENTS REGULATE MESANGIAL ATP-SENSITIVE K CHANNELS Several SUR have been identified and cloned from diverse species and tissues (8–10). SUR provide the regulatory subunits of adenosine triphosphate (ATP)-sensitive K channels (KAT P ). Classical KAT P consist of two subunits, a potassium ion pore and a SUR. These structurally unrelated subunits complex as four heterodimers to comprise a single functional KAT P . The ion pore belongs to the Kir6.x subfamily of weak inwardly rectifying K+ channels and is represented as either Kir6.1 or Kir6.2. SUR are members of the cystic fibrosis transmembrane regulator/multidrug resistance protein subfamily of the ATP-binding cassette protein (ABC) superfamily (11,12). The pancreatic β-cell SUR is a high-affinity receptor and is designated SUR1. This SUR is encoded by the gene ABCC8, whereas SUR2, the lower affinity receptor, is encoded by the gene ABCC9. The heterogeneous properties and functional diversity of KAT P are based on differing complexations of Kir6.x and SUR isoforms and cellular distribution. Nearly 90% of KAT P are not localized to the plasmalemma, but to endoplasmic reticulum, mitochondria, and secretory granules (13–16). Consequently, nearly 70% of all SULF binding is cytosolic (17), and this intracellular site of action for KAT P represents an important determinant of SULF action. For example, in β-cells, chronic glibenclamide exposure induces translo- cation of membranous SUR to the cytoplasm, thereby reducing insulin secretion (18). β Consistent with the above, the SULF binding site of the -cell KAT P is on its cytosolic aspect (19). These observations reconcile the greater potency of the more highly lipophilic SULF compounds: they more easily permeate the plasma membrane and have greater affinity for SUR (20,21). Overall, regardless of their location, all SUR ligands act intra- cellularly, consequently, candidate endogenous SUR ligands would be anticipated to exert their effects intracellularly as well. Although K were first described in cardiac myocytes, the membrane-bound pancreatic β AT P -cell KAT P , a tetradimer of SUR1/Kir6.2, represents the most extensively studied of these channels, and it is regarded as the “classical” KAT P (22,23). In the functional channel, Kir6.2 confers KAT P inhibition by ATP, whereas SUR increases pore sensitivity to ATP and regulates channel activation by magnesium-bound adenosine diphosphate (MgADP) and closure by SULF (24,25). Finally, SUR respond variably to KAT P channel openers (KCO), for example, diazoxide, cromakalim, or pinacidil. The prevailing axiom defines KAT P as molecular switches that link the cell’s metabolic state to calcium-dependent signaling (26). In the β-cell, SULF and/or elevations of the cytosolic ATP/adenosine diphosphate (ADP) ratio inhibit KAT P leading to a chain of events: channel closure, membrane depolarization, Ca2+ influx, and insulin secretion (25). The opposite series of events are observed with declines in the cytosolic ATP/ADP ratio or after exposure to KCO. Currently, the roles of KAT P are broadening with recent α-Endosulfine in Diabetic Nephropathy 307 evidence reinforcing the diversity of KAT P and the void of knowledge regarding their functions (9,10,27). SUR2 is the more ubiquitous extrapancreatic KAT P subunit, and it is found as two major low-affinity splice variants, SUR2A and SUR2B. These isoforms respectively reconstitute the cardiac-type KAT P (SUR2A/Kir6.2), predominantly found in heart and skeletal muscle, and the more ubiquitous vascular smooth muscle-type KAT P (SUR2B/Kir6.1 or Kir6.2) found in brain, heart, liver, kidney, intestine, bladder, vascular smooth muscle, and uterus (9,10,28–30). KAT P , in these tissues regulate a myriad functions, including cell survival, differentiation, and responses to ischemic injury, neurotransmitter release, and vascular smooth muscle cell contraction. The latter has been extensively studied in coronary vessels where KAT P control arteriolar tone (31,32). Not unexpectedly, pharmacological evaluations have also documented the diversity of KAT P among various tissues, with respect to their SULF affinities and sensitivity to KCO (33). Finally, an intact system of actin filaments is critical to extrapancreatic KAT P activity (34–37). Disruption of filamentous actin reduces the sensitivity of cardiac and smooth muscle KAT P for ATP, SULF, and presumably, for any endogenous SUR ligand(s). In the context of diabetic kidney disease, the dependence of KAT P on normal actin assembly becomes highly relevant because high-glucose concentrations induce MC actin fiber disassembly (38). Finally, the discrete localization and control of protein kinase A (PKA) requires actin cytoskeleton targeting by specific proteins, for example, gravin and Wiskott- Aldrich syndrome protein (WAVE), that dually anchor actin and PKA (39). KIDNEY SUR The SUR2B splice variant is widely expressed in the kidney, including the distal nephron where it presumably mediates, in part, potassium transport (40). However, in the proximal tubule, the combination of Kir6.1 with SUR2A and/or SUR2B forms a taurine-sensitive KAT P (41). SUR2B may also couple to murine ROMK2, a Kir that resides in the cortical ascending limb and cortical collecting duct of the distal nephron (42). We hypothesized that the observed MC effects of SULF agents were mediated by specific MC KAT P channels. Subsequently, using membrane preparations from rat MC, we demonstrated specific [3H]glibenclamide binding to low-affinity SUR (8). A func- tional KAT P was subsequently demonstrated in MC. Cultured cells, following a single exposure to glibenclamide (5 μM), initiated prolonged cycles of oscillatory cytoplas- mic Ca2+ transients that were coupled to the enhancement of MC contractility (8). These observations were in alignment with results of other investigators who demon- strated similar Ca2+ oscillations in MC exposed to angiotensin II. We subsequently cloned two SUR2 cDNAs from rat MC, a 6.7 kbp smooth muscle- type rSUR2B that had been previously described and a unique 4.8 kbp serum-regulatable MC-specific splice variant, mcSUR2B. This variant was homologous, in large part, with the larger splice variant, rSUR2B. Our findings additionally revealed expression of Kir 6.1 but not of Kir6.2 in MC (43). These studies suggest that the KAT P of MC and also of isolated glomeruli are comprised of (rSUR2B/Kir6.1)4 and possibly, (mcSUR2B/Kir6.1)4 (8,43). In this context, the marked inhibition of established high-glucose concentration- fostered ECM accumulation by glibenclamide at 10 nM is a highly relevant observation because the experimental concentrations are within the clinically relevant range for this compound (peak plasma concentration: 50–60 nM after a 5-mg dose) (44). In addition, 308 Yee and Szamosfalvi the KD of 6 nM for glibenclamide, as determined by complexation of SUR2B to Kir6.1 in intact cells, is consonant with this hypothesis (45). Finally, immunoreactivity for rSUR2B and mcSUR2 in primary and cloned MC (16KC2) lines as delineated by a spe- cific antibody directed against the common C-terminal epitope of SUR2A and SUR2B further substantiates this argument (43). Thus, it is plausible that the metabolic actions of low-concentration glibenclamide on MC are mediated through KAT P comprised of SUR2B/Kir6.1. ENDOGENOUS KATP LIGANDS β The central role that pancreatic -cell KAT P (SUR1/Kir6.2)4 plays in regulating insulin secretion and the ubiquitous nature of the SUR2-based KAT P led to a search for endogenous ligand(s) of these channels.