Endosulfine in Diabetic Nephropathy

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 development 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 translocation 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 intracellularly, 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 functional KAT P was subsequently demonstrated in MC. Cultured cells, following a single exposure to glibenclamide (5 μM), initiated prolonged cycles of oscillatory cytoplasmic Ca2+ transients that were coupled to the enhancement of MC contractility (8). These observations were in alignment with results of other investigators who demonstrated 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 specific 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. Subsequently, a putative endogenous polypep- tide ligand of K , α-endosulfine was isolated from ovine and porcine brain. Purified α AT P -endosulfine displaced prebound glibenclamide from these KAT P -rich substrates. Because of this initial characterization, human α-endosulfine (ARPP-19e) has been cloned and proposed as an endogenous ligand for pancreatic KAT P (46). Consonant with this hypothesis are electrophysiological observations that demonstrate KAT P inhibition by recombinant α-endosulfine in Syrian hamster insulinoma tumor cells and the induction of insulin secretion from MIN6 and RINm5F pancreatic β-cells via a Ca2+- dependent mechanism. α-Endosulfine and its splice variant β-endosulfine reveal similarity to a group of mammalian neuronal phosphoproteins, which are phosphorylated by PKA. Of these cyclic AMP-regulated phosphoproteins (ARPPs), two closely related isoforms have been characterized by apparent molecular mass, ARPP-16 and ARPP-19. The former is only detected in brain, with enrichment in the neostriatum, whereas the latter is ubiqui- tously expressed in neurons and non-neuronal tissues, including fibroblasts and renal tubular epithelial cells (47). In vertebrates, the ARPP protein family is characterized by a core of 82 identical amino acids that encompasses an absolutely conserved serine residue, which is phosphorylated, by the catalytic subunit of PKA (47). The conserva- tion of this amino acid contends that ARPP regulation via PKA-dependent phosphorylation represents a fundamental biological process. Although ARPP are without known enzymatic activity, they possess characteristics common to intracellular regulatory proteins that alter the activities of enzymes involved in signal transduction, for example, calmodulin and the PKA inhibitor (Walsh inhibitor, PKI) (47). Presently, little is known regarding the physiological roles of ARPP 16/19, but ARPP-19 mediates nerve growth factor signaling through posttranscriptional control of gene expression via binding to and stabilizing GAP-43 mRNA, which results in amplified protein expression (48). The human gene encoding α-endosulfine, ENSA, had initially been reported on chro- mosome 14 and colocalized with a known insulin-dependent diabetes mellitus (IDDM) susceptibility locus (49). Subsequently, based on the results derived from the human genome project, ENSA was remapped to locus 1q23.1, relegating the previously reported locus to the status of a pseudogene. In functional experiments, recombinant α-endosulfine at micromolar concentrations competitively inhibits [3H]glibenclamide binding to SUR. Moreover, there is also diminution of KAT P conductance in oocytes transduced to express the recombinant channel. The inhibition of current is most evident when α- endosulfine is delivered intracellularly where it can rapidly appose itself to its cytoplasmic locus of action (50). α-Endosulfine in Diabetic Nephropathy 309

Although α-endosulfine is expressed in a large number of tissues, including heart, lung, spleen, skeletal muscle, pancreatic somatostatin δ-cells, liver, and kidney (51), its various tissue-specific roles remain at present undetermined. However, it has been recently shown that brain α-endosulfine from Alzheimer’s disease patients was greatly reduced (52). Recently, it was posited that α-endosulfine did not mediate its actions in an autocrine or paracrine manner (51). This assumption appears likely given that treatment of pancreatic α cells with somatostatin secretagogues fails to induce α-endosulfine α release, KAT P inhibition only occurs in high extracellular -endosulfine concentrations, plasma α-endosulfine levels are in the picomolar range, and because α-endosulfine is a cytosolic protein that associates with the particulate fraction. In the aggregate, these data favor α-endosulfine as an intracellular regulator (51). α -ENDOSULFINE REGULATION OF KATP α As -endosulfine represents a putative ligand of KAT P and KAT P are present in MC, we postulated that α-endosulfine was expressed in the rat kidney in vivo and in cultured rat MC in vitro. We determined that ENSA was expressed abundantly in rat kidney by in situ hybridization. Further observations, utilizing Northern blotting and RT-PCR, established α-endosulfine message in cultured MC in vitro and in whole rat kidney in vivo, with expression predominantly localized to the glomeruli. Further studies of α-endosulfine expression by immunoblotting and confocal microscopy confirmed its presence in vitro and in vivo. ENSA gene and protein expression in MC is altered in response to a hyperglycemic milieu. Cultured cells increased ENSA gene expression by twofold following exposure to 30 mM glucose within 24–48 h, and this effect persisted for at least 10 d, even after withdrawal of the inciting stimulus (53). Thus, MC expression of SUR2B, Kir 6.1 and α-endosulfine lays the foundation for the existence of a regulatable intracellular

KAT P /endosulfine receptor/ligand system, which could influence MC contractility and matrix metabolism (54). GLOMERULAR EFFECTS OF SULF IN INSULIN-DEFICIENT DIABETES MELLITUS SULF have been extensively used in the treatment of type 2 diabetes. However, the renal effects of SULF in diabetes have not been determined. A study of complications in patients with type 2 diabetes investigated the effects of intensive glycemic control with insulin or SULF (UK Prospective Diabetes Study) (55). These results revealed a reduction in proteinuria and renal failure, in association with improved glycemic control. However, owing to either the relatively few number of patients who developed renal disease or the confounding effects of combinations of sequential therapies in individual patients, no conclusive differences were detected between insulin- and SULF-treated groups. Since this trial, other studies have proven inconclusive (56,57). Therefore, the current status of clinical knowledge does not render definitive conclusions regarding any beneficial or deleterious renal effects by exogenous SULF or an endogenous SULF- like ligand. In an attempt to extend prior observations of altered matrix metabolism and increased cellular contractility in MCs after SULF exposure, the effect of chronic administration of various SULF on the progression of diabetic nephropathy was evaluated in animal models of types 1 and 2 diabetes mellitus. Our observations revealed an attenuation of proteinuria 310 Yee and Szamosfalvi

Fig. 1. Mesangial KAT P are formed as tetradimers of Kir6.1 and SUR2B. KAT P mediate changes in extracellular matrix (ECM) metabolism following binding of either sulfonylurea (SULF) or α-endosulfine. α Low-concentration SULF inhibits -endosulfine action at the KAT P site and limits ECM formation. However, high concentrations of SULF increase ECM accumulation as a consequence of the increased expression of GLUT1 and enhanced GLUT1-mediated intracellular glucose transport with β the consequent upregulation of TGF- 1. This growth factor, in turn upregulates membrane-associated GLUT1 expression. Heightened mesangial contractility represents a Ca2+-mediated event in response to exposure to SULF and possibly to α-endosulfine. and morphological glomerular alterations after glibenclamide or tolazamide administration to streptozotocin-treated rats (58). Notably, these salutary effects proceeded in the absence of changes in glycemia or (as studied in the case of glibenclamide) in glomerular filtration rate or renal hypertrophy. In separate experiments with insulin-resistant db/db mice, SULF treatment did not impact the course of glomerulosclerosis in functional or morphological assays. In toto, these observations imply that the direct glomerular effects of SULF at low concentrations (and by extrapolation, of their endogenous counterpart, α β -endosulfine) may suppress or retard the TGF- 1-mediated accrual of glomerular extracellular matrix (ECM), the hallmark of diabetic glomerulosclerosis and may signifi- cantly alter the course of insulin-deficient diabetic kidney disease. α-ENDOSULFINE EXPRESSION ALTERS MATRIX METABOLISM To more fully understand the role of ENSA expression in MCs with regard to ECM formation, a stably overexpressing α-endosulfine cell line was produced by retroviral transduction. The resulting cell line reliably upregulated ENSA by eightfold. In cells, exposed to a high-glucose environment (25 mM) for 2 wk, there was increased accumulation of collagen type I in the conditioned medium, compared with control cells transduced by a nonvirus-containing empty vector. A similar finding was noted for type-4 collagen. Contrastingly, ENSA downregulation by RNA interference during tran- sient transfection experiments with three different siRNAs led to decrements of types I and IV collagen accumulation in the conditioned media of cells incubated for 2 wk in 5 mM glucose. We conclude that, although modifications of ECM gene expression via ENSA may not be translated directly to changes in ECM protein expression, these results imply that α-endosulfine enhances ECM accumulation, an effect that is particularly invoked by a high-glucose environment. In addition, in other preliminary data, these effects occur downstream of the perinuclear and nuclear translocation of α-endosulfine in MCs following their cAMP-mediated PKA phosphorylation. α-Endosulfine in Diabetic Nephropathy 311

CONCLUSIONS

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