The Role of the NLRP3 Inflammasome in Mediating Glomerular and Tubular Injury in Diabetic Nephropathy.

The NOD-like receptor protein 3 (NLRP3) inflammasome is a multi-protein signalling complex integral to the chronic inflammatory response, activated in response to sterile and non-sterile cellular damage. The assembly and activation of the NLRP3 inflammasome comprise a two-step process involving nuclear factor kappa B (NFkB)-mediated priming, followed by canonical, non-canonical or alternative signalling pathways. These result in the maturation and release of inflammatory cytokines interleukin 1 beta (IL1ß) and interleukin-18 (IL18), which are associated with chronic inflammatory conditions including diabetic kidney disease. Diabetic nephropathy is a condition affecting ∼40% of people with diabetes, the key underlying pathology of which is tubulointerstitial inflammation and fibrosis. There is growing evidence to suggest the involvement of the NLRP3 inflammasome in this chronic inflammation. Early deterioration of kidney function begins in the glomerulus, with tubular inflammation dictating the progression of late-stage disease. Priming and activation of the NLRP3 inflammasome have been linked to several clinical markers of nephropathy including proteinuria and albuminuria, in addition to morphological changes including mesangial expansion. Treatment options for diabetic nephropathy are limited, and research that examines the impact of directly targeting the NLRP3 inflammasome, or associated downstream components are beginning to gain favour, with several agents currently in clinical trials. This review will explore a role for NLRP3 inflammasome activation and signalling in mediating inflammation in diabetic nephropathy, specifically in the glomerulus and proximal tubule, before briefly describing the current position of therapeutic research in this field.

TGFß modulates cell-to-cell communication in early epithelial-to-mesenchymal transition.

AIMS/HYPOTHESIS: A key pathology in diabetic nephropathy is tubulointerstitial fibrosis. The condition is characterised by increased deposition of the extracellular matrix, fibrotic scar formation and declining renal function, with the prosclerotic cytokine TGF-ß1 mediating many of these catastrophic changes. Here we investigated whether TGF-ß1-induced epithelial-to-mesenchymal transition (EMT) plays a role in alterations in cell adhesion, cell coupling and cell communication in the human renal proximal tubule. METHODS: Whole-cell and cell compartment abundance of E-cadherin, N-cadherin, snail, vimentin, ß-catenin and connexin-43 was determined in human kidney cell line (HK)2 and human proximal tubule cells with or without TGF-ß1, using western blotting and immunocytochemistry, followed by quantification by densitometry. The contribution of connexin-43 in proximal tubule cell communication was quantified using small interfering RNA knockdown, while dye-transfer was used to assess gap junctional intercellular communication (GJIC). Functional tethering was assessed by single-cell force spectroscopy with or without TGF-ß1, or by immunoneutralisation of cadherin ligation. RESULTS: High glucose (25 mmol/l) increased the secretion of TGF-ß1 from HK2 cells. Analysis confirmed early TGF-ß1-induced morphological and phenotypical changes of EMT, with altered levels of adhesion and adherens junction proteins. These changes correlated with impaired cell adhesion and decreased tethering between coupled cells. Impaired E-cadherin-mediated adhesion reduced connexin-43 production and GJIC, these effects being mimicked by neutralisation of E-cadherin ligation. Upregulation of N-cadherin failed to restore adhesion or connexin-43-mediated GJIC. CONCLUSIONS/INTERPRETATION: We provide compelling evidence that TGF-ß1-induced EMT instigates a loss of E-cadherin, cell adhesion and ultimately of connexin-mediated cell communication in the proximal tubule under diabetic conditions; these changes occur ahead of overt signs of renal damage.

The calcium-sensing receptor and insulin secretion: a role outside systemic control 15 years on.

In the 15 years since the identification and characterisation of the extracellular calcium-sensing receptor (CaR), it has become increasingly apparent that this cationic binding receptor is found in many tissues, not associated with the control of plasma calcium. One of these tissues is the pancreatic islet where insulin secretion provides the basis of energy regulation. It seems inherently unlikely that the islet responds to alterations in systemic calcium and a more plausible and intriguing possibility is that the CaR mediates cell-to-cell communication through local increases in the concentration of extracellular Ca(2+), co-released with insulin. This short article explores this possibility and suggests that this novel mechanism of cell communication, along with direct coupling via gap junctions and other local paracrine regulators helps explain why the glucose responsiveness of the intact islet is greater than the sum of the composite parts in isolation.

Calcium-dependent translocation of cytosolic phospholipase A2 in pancreatic beta-cells.

Cytosolic phospholipase A(2)(cPLA(2)), an enzyme responsible for the generation of arachidonic acid, is located in the cytosolic compartment in most tissues and it translocates to membrane compartments when activated. We found that cPLA(2) distribution in pancreatic beta-cells is different from that of most other mammalian cells: it is evenly distributed throughout the beta-cell, in both cytoplasmic and nuclear compartments. Agents that increased intracellular Ca(2+) in the MIN6 beta-cell line also stimulated a redistribution of cPLA(2) immunoreactivity such that the majority of the enzyme moved from the nucleus to the cytoplasm. The time course of events was compatible with the elevation in Ca(2+) being responsible for translocation of cPLA(2). These observations suggest that cPLA(2) may be compartmentalised in unstimulated beta-cells, perhaps to limit its access to substrate prior to elevations in intracellular Ca(2+).

Co-ordinated Ca(2+)-signalling within pancreatic islets: does beta-cell entrainment require a secreted messenger.

Isolated beta-cells are heterogeneous in sensory, biosynthetic and secretory capabilities, however, to enable efficient and appropriate secretion, cellular activity within the intact islet is synchronised. Historically, the entrainment of activity to a common pattern has been attributed to gap-junction mediated cell-to-cell communication. Although clearly influential, the possibility remains for other local synchronising mechanisms. In this study, we have used small clusters of insulin-secreting MIN6 cells to assess how contact-dependent, homotypic interactions between cells influences nutrient- and non-nutrient- evoked Ca(2+)-handling and insulin secretion, and to determine whether a secreted product plays a role in the synchronisation of oscillatory activity. Tolbutamide evoked a concentration-dependent recruitment of active cells within cell clusters, both in terms of numbers of cells and amplitude of the evoked Ca(2+)-response. The change in [Ca(2+)](i) was characteristically oscillatory above a mean elevated plateau, and was in phase between member cells of an individual cluster. Even at maximal concentrations (100 microM) some cells within a cluster responded before their immediate neighbours. Subsequent oscillatory behaviour then became entrained between member cells within that cluster. Inhibiting exocytosis using the microtubule inhibitors vincristine and nocodazole, or the adrenergic agent noradrenaline, did not prevent tolbutamide-evoked oscillatory changes in [Ca(2+)](i) but did reduce the probability of obtaining synchronous activity within an individual cluster. Above a threshold glucose concentration, the number of cells secreting insulin increased, without a commensurate change in secretory efficiency. This recruitment of cells secreting insulin mirrored Ca(2+) data that showed a glucose-dependent increase in cell number, without a change in the mean basal-to-peak change in [Ca(2+)](i). Together these data suggest that synchronised behaviour in MIN6 cells is dependent, in part, on a secreted factor that acts in a local paracrine fashion to recruit heterogeneous individual cellular activity into an organised group response.

Role of adenine nucleotides in insulin secretion from MIN6 pseudoislets.

Insulin secretion from MIN6 cells configured as cell aggregates by culture on a gelatin substrate (pseudoislets) is enhanced compared to that of MIN6 cells grown as monolayers on tissue culture plastic, indicating the importance of beta-cell-to-beta-cell proximity for insulin release. In this study we have shown that glucose induced a biphasic release of insulin from pseudoislets, whereas the amplitude and duration of the responses of equivalent monolayer cells were much reduced. Purinergic aqonists have been implicated in intercellular communication between beta-cells, so we investigated whether adenine nucleotides co-released with insulin are responsible for the enhanced secretory responses of pseudoislets. We have demonstrated that MIN6 cells express purinergic A(1) and P2Y receptors, and that adenine nucleotides increased [Ca(2+)](i) with an efficacy of agonists being ATP > ADP > AMP. However, neither suramin nor the more selective A(1) antagonist 1,3-dipropyl-8-cyclopentylxanthine reduced glucose-induced insulin secretion from pseudoislets, and stimulation of monolayer cells with a range of adenine nucleotides did not enhance glucose-induced secretion. These results suggest that enhanced secretion from MIN6 pseudoislets is not due to increased paracrine/autocrine action of adenine nucleotides.

Interdependence of steroidogenesis and shape changes in Y1 adrenocortical cells: studies with inhibitors of phosphoprotein phosphatases.

Y1 adrenocortical cells respond to activators of the cyclic AMP-dependent protein kinase (PKA) signalling pathway not only with increases in steroid secretion but also with a characteristic change in cell morphology from flat and adherent to round and loosely attached. This change of shape, which may facilitate cholesterol transport to the mitochondrion, requires tyrosine dephosphorylation of the focal adhesion protein, paxillin, and can be blocked by inhibitors of phosphotyrosine phosphatase (PTP) activity. In a previous study we demonstrated that inhibition of phosphoserine/threonine phosphatase 1 and 2A (PP1/2A) activities caused a similar morphological response to PKA activation whilst opposing the effects on steroid production. We have now investigated the responses to PKA activation and inhibition of PP1/2A and used PTP inhibitors to examine the relationship between the morphological changes and enhanced steroid production. Both forskolin (FSK) and the PP1/2A inhibitor, calyculin A (CA), caused rapid and extensive rounding of Y1 cells. FSK-induced cell rounding was reversible and accompanied by a reduction in the tyrosine phosphorylation of paxillin. Rounding was prevented by the PTP inhibitors pervanadate (PV) and calpeptin (CP) and was associated with the maintained tyrosine phosphorylation of paxillin. In contrast, CA-induced cell rounding was not reversible over a 2-h period and was not affected by the presence of PTP inhibitors, and CA had no effect on the tyrosine phosphorylation of paxillin. Although neither CA nor FSK produced any gross changes in cell viability as judged by Trypan Blue exclusion or mitochondrial activity, CA-treated cells showed a marked reduction in total protein synthesis assessed by (35)S-incorporation. The effects of FSK and the PTP inhibitors on cell rounding were reflected in their effects on steroid production since PV and CP also inhibited FSK-stimulated steroid production. These results suggest that the mechanism through which inhibition of PP1/2A activities induces morphological changes in Y1 cells is fundamentally different from that seen in response to activation of PKA. They are consistent with PKA-induced shape changes in adrenocortical cells being mediated through increased PTP activity and the dephosphorylation of paxillin, and support the view that the morphological and functional responses to PKA activation in steroidogenic cells are intimately linked.

Human granulosa-lutein cells express functional EP1 and EP2 prostaglandin receptors.

Prostaglandin E(2) (PGE(2)) exerts mainly luteotrophic effects in the corpus luteum. In other tissues, PGE(2) acts via specific PGE(2) receptor subtypes including EP1, which modulates intracellular calcium ([Ca(2+)](i)) and EP2, which is coupled to cyclic AMP (cAMP) generation. We have therefore investigated the presence of functional EP1 and EP2 receptors using human granulosa-lutein (GL) cells. Reverse-transcription PCR revealed that GL cells expressed mRNA transcripts encoding both EP1 and EP2 receptors. When GL cells were challenged with ligands that can bind to both receptor subtypes (PGE(2) and 16,16 dimethyl PGE(2)) or exclusively to EP2 (butaprost), both cAMP formation and progesterone synthesis were stimulated. Furthermore, the cAMP response to these agonists could be significantly blocked by an EP1/2 antagonist AH6809 but not by an EP1-selective antagonist SC19220. Exposure of GL cells to 16,16-dm PGE(2) transiently raised [Ca(2+)](i) levels, which could be prevented by both AH6809 and SC19220. We therefore conclude that human GL cells express functional EP1 and EP2 receptors.

Mechanism of action of the calcium-sensing receptor in human antral gastrin cells.

BACKGROUND AND AIMS: Human G cells express the calcium-sensing receptor and respond to extracellular calcium by releasing gastrin. However, the receptor on G cells is insensitive to serum calcium levels. We investigated whether this is a result of differential regulation of signaling pathways compared with parathyroid or calcitonin cells. METHODS: Gastrin release from primary cultures of human antral epithelial cells enriched for G cells (35%) was measured by radioimmunoassay. G cells were stimulated by increasing extracellular calcium concentration for 1 hour in the presence or absence of antagonists of specific intracellular signaling pathways. Intracellular calcium levels were monitored to evaluate the effect of the antagonists on calcium influx. RESULTS: Inhibition of phospholipase C decreased calcium-stimulated gastrin release, but blockers of adenylate cyclase, phospholipase A(2), or mitogen-activated protein kinase had no effect. Inhibition of protein kinase C, nonselective cation channels, and phosphodiesterase increased basal and calcium-stimulated gastrin release while decreasing calcium influx. These data were consistent with basally active phosphodiesterase. CONCLUSIONS: The calcium-sensing receptor on the G cell activates phospholipase C and opens nonselective cation channels, resulting in an influx of extracellular calcium. Protein kinase C isozymes expressed by the G cells play multiple roles regulating both gastrin secretion and phosphodiesterase activity.

Synchronization of Ca(2+)-signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers.

The secretory response of the intact islet is greater than the response of individual beta-cells in isolation, and functional coupling between cells is critical in insulin release. The changes in intracellular Ca(2+)([Ca(2+)](i)) which initiate insulin secretory responses are synchronized between groups of cells within the islet, and gap-junctions are thought to play a central role in coordinating signalling events. We have used the MIN6 insulin-secreting cell line, to examine whether uncoupling gap-junctions alters the synchronicity of nutrient- and non-nutrient-evoked Ca(2+)oscillations, or affects insulin secretion. MIN6 cells express mRNA species that can be amplified using PCR primers for connexin 36. A commonly used gap-junctional inhibitor, heptanol, inhibited glucose- and tolbutamide-induced Ca(2+)-oscillations to basal levels in MIN6 cell clusters at concentrations of 0.5 mM and greater, and it had similar effects in pseudoislets when used at 2.5 mM. Lower heptanol concentrations altered the frequency of Ca(2+)transients without affecting their synchronicity, in both monolayers and pseudoislets. Heptanol also had effects on insulin secretion from MIN6 pseudoislets such that 1 mM enhanced secretion while 2.5 mM was inhibitory. These data suggest that heptanol has multiple effects in pancreatic beta-cells, none of which appears to be related to uncoupling of synchronicity of Ca(2+)signalling between cells. A second gap-junction uncoupler, 18 alpha-glycyrrhetinic acid, also failed to uncouple synchronized Ca(2+)-oscillations, and it had no effect on insulin secretion. These data provide evidence that Ca(2+)signalling events occur simultaneously across the bulk mass of the pseudoislet, and suggest that gap-junctions are not required to coordinate the synchronicity of these events, nor is communication via gap junctions essential for integrated insulin secretory responses.

The extracellular calcium-sensing receptor on human beta-cells negatively modulates insulin secretion.

The presence and functional significance of the extracellular calcium-sensing receptor (CaR) on human pancreatic beta-cells were investigated. Reverse transcriptase-polymerase chain reaction with primers for the extracellular domain of the CaR expressed in human parathyroid-secreting cells identified a product of the expected size in human pancreatic mRNA. Immunocytochemistry using an antibody against the extracellular region of CaR showed extensive immunoreactivity in insulin- and glucagon-containing cells but not in somatostatin-containing cells. In perifusion experiments, elevations in extracellular Ca2+ produced initial transient increases in insulin secretion, followed by a concentration-dependent and prolonged, but reversible, inhibition of secretion. Microfluorometric measurements of intracellular Ca2+ ([Ca2+]i) in isolated human beta-cells demonstrated that elevations in extracellular Ca2+ (0.5-10 mmol/l) caused rapid elevations in [Ca2+]i. Increases in extracellular Ca2+ caused small increases in the cyclic AMP content of whole human islets. These studies demonstrated that human beta-cells express an extracellular CaR and that activation of the receptor inhibits basal and nutrient-stimulated insulin secretion. The transduction mechanism that mediates this inhibitory effect is unknown, but our results suggest that it is unlikely to be through the adenylate cyclase-cyclic AMP pathway or through the phospholipase C-IP3 pathway. This CaR-mediated inhibitory mechanism may be an important autoregulatory mechanism in the control of insulin secretion.

Non-Ca2+-homeostatic functions of the extracellular Ca2+-sensing receptor (CaR) in endocrine tissues.

The extracellular Ca(2+)-sensing receptor (CaR) links changes in the concentration of extracellular Ca(2+) to changes in cell function. For cells involved in the control of systemic Ca(2+) concentration, this provides an efficient receptor-mediated mechanism to rapidly counteract slight fluctuations in the circulating concentration of Ca(2+). However, all cells that express the CaR are not necessarily involved in Ca(2+) homeostasis. The recent localisation of CaR expression on a variety of cell types more usually associated with non-Ca(2+)-homeostatic endocrine function may have serious repercussions for the interpretation of data in those systems which routinely culture cells under standard hypercalcaemic conditions. This short commentary considers the literature surrounding the identification of the CaR and the potential effects of its localisation on endocrine cells not directly involved in the control of systemic Ca(2+ )homeostasis.

Depolarizing stimuli reduce Ca(2+)/calmodulin-dependent protein kinase II activity in islets of Langerhans.

Elevations in intracellular Ca(2+) ([Ca(2+)](i)) initiate insulin secretion from pancreatic beta-cells, but the secretory responses become rapidly desensitised to maintained elevations in [Ca(2+)](i). We have investigated the mechanisms underlying the Ca(2+) desensitization of insulin secretion using electrically permeabilized rat islets of Langerhans. Measurements of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) enzyme activity and immunoreactivity in permeabilized islets demonstrated Ca(2+)-induced reductions in enzyme activity which could not be attributed to reductions in CaMK II immunoreactive protein. Measurements in intact islets demonstrated that the Ca(2+)-induced reduction of CaMK II activity was also operative in intact cells, suggesting that this mechanism may have pathophysiological implications for beta-cell function.

Signaling through the p38 and p42/44 mitogen-activated families of protein kinases in pancreatic beta-cell proliferation.

The present study has focused on the role of the 42- and 44-kDa mitogen-activated protein kinases (p42/44 MAPKs) and the 38-kDa mitogen-activated protein kinase (p38 MAPK) in the proliferation of the pancreatic beta-cell line MIN6. MIN6 beta-cell proliferation was assessed by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into cellular DNA. Inhibition of both the p42/44 MAPK pathway using the MEK inhibitor PD098059 (PD) and the p38 MAPK pathway using the p38 inhibitor SB203580 (SB) caused a marked, concentration-dependent reduction in the BrdU immunostaining observed in the presence of 15% FCS when assessed using fluorescence immunocytochemistry. These data provide direct evidence of a role for p42/44 MAPKs in the mitogenic response of MIN6 beta-cells to FCS. Furthermore, these data also suggest a novel role for the p38 MAPK pathway in MIN6 beta-cell proliferation.

Pancreatic beta-cell-to-beta-cell interactions are required for integrated responses to nutrient stimuli: enhanced Ca2+ and insulin secretory responses of MIN6 pseudoislets.

The effect of cell-to-cell contact on Ca2+ influx and secretory responses in the beta-cell line MIN6 was studied using MIN6 pseudoislets, which are three-dimensional islet-like cell aggregates that develop when MIN6 cells are cultured for 6-8 days on gelatin. The formation of pseudoislets is dependent on the Ca2+-dependent adhesion molecule E-cadherin (E-CAD), since the process can be inhibited by incubation in the absence of Ca2+ or in the presence of an anti-E-CAD antibody. Glucose and alpha-ketoisocaproic acid (KIC) evoked a Ca2+ influx in only a small fraction of the MIN6 monolayer cells, whereas >80% of cell groups within the pseudoislets responded to both nutrients. In contrast, changes in the intracellular free Ca2+ concentration ([Ca2+]i) were observed in all or most monolayer cells or pseudoislet cell groups in response to physical or pharmacological depolarizing stimuli. No significant increase in insulin release was observed from MIN6 monolayer cells in response to nutrient or nonnutrient insulin secretagogues. Conversely, pseudoislets were found to respond significantly to both nutrients and nonnutrients. These results suggest that close cell-to-cell contact improves the functional responsiveness of MIN6 cells and that pseudoislets may therefore serve as a useful research model in the study of beta-cell function.

Bombesin-evoked gastrin release and calcium signaling in human antral G cells in culture.

Amplification of mRNA from a human antral cell culture preparation demonstrated the presence of two receptors of the bombesin and gastrin-releasing peptide family, GRPR-1 and BRS-3. Single cell microfluorometry demonstrated that most cells that exhibited bombesin-evoked changes in intracellular Ca2+ concentration were gastrin immunoreactive, indicating that antral G cells express the GRPR subtype. There were two components to the intracellular Ca2+ response: an initial nitrendipine-insensitive mobilization followed by a sustained phase that was inhibited by removal of extracellular Ca2+ and 20 mM caffeine and was partially inhibited by 10 microM nitrendipine. Preexposure of cells to thapsigargin and caffeine prevented the response to bombesin, indicating activation of inositol 1,4,5-trisphosphate (IP3)-sensitive stores. Gastrin release could be partially reversed by removal of extracellular Ca2+ and blockade of L-type voltage-dependent Ca2+ channels, indicating that a component of the secretory response to bombesin was dependent on Ca2+ influx. These data demonstrated that bombesin-stimulated gastrin release from human antral G cells resulted from activation of GRPRs and involved both release of intracellular Ca2+ and influx of extracellular Ca2+ through a combination of L-type voltage-gated and IP3-gated Ca2+ channels.

L-type calcium channels regulate gastrin release from human antral G cells.

In mRNA samples isolated from a gastrin (G) cell-enriched human antral cell preparation, reverse transcription-polymerase chain reaction identified products encoding part of the alpha 1-subunit of class C and D L-type voltage-dependent Ca2+ channels (VDCCs). Analysis of the polymerase chain reaction products demonstrated a 100% homology with the known human gene sequences. An antibody to the class D alpha 1-subunit immunostained 30-40% of the cultured cells; of these 90% were gastrin immunoreactive. Gastrin release stimulated by terbutaline (beta 2-agonist) and forskolin was abolished by blockade of L-type VDCCs; the effect of 3.6 mM extracellular Ca2+ was only partially reversed. In G cells the rise in intracellular Ca2+ observed in response to increasing extracellular Ca2+ from 0.5 to 3.6 mM was reduced by nitrendipine. These results indicated that human antral cells expressed class C and D L-type VDCCs. Activation of G cells with beta-adrenergic agonists required an influx of extracellular Ca2+ through these channels to stimulate gastrin release. However, activation of L-type channels was not the only mechanism underlying Ca(2+)-stimulated gastrin release.