SGK1 in the kidney: disrupted sodium transport in diabetes and beyond

Renal complications of diabetes can be severe; however, the mechanisms that underlie the development and progression of diabetic nephropathy are poorly understood. Recent evidence suggests that the serum and glucocorticoid induced kinase-1 (SGK1) may be key to this process. SGK1 expression and function are increased in models of diabetes and polymorphisms of the SGK1 gene are associated with type 2 diabetes mellitus. A key regulator of sodium transport within the renal epithelium of the distal nephron, SGK1 was originally isolated as a glucocorticoid-sensitive gene that regulated the epithelial sodium channel (ENaC; known also as the sodium channel, nonvoltage-gated 1, SCNN1). It is now apparent that SGK1 modulates sodium re-absorption by a number of sodium transporters/channels throughout the length of the nephron including; the Na+/H+ exchange isoform 3 (NHE3), the Na+Cl- co-transporter (NCC) and the Na+/K+-ATPase. In addition, SGK1 is regulated by a diverse range of factors including; insulin, glucose, intracellular calcium, transforming growth factor-beta1, flow rate and osmolality. This brief review examines the evidence supporting an involvement of SGK1 in diabetic nephropathy and discusses how dysregulated sodium transport may account for the development of secondary hypertension associated with the condition. Furthermore, the article examines how aberrant SGK1 expression and activity may be responsible for the cellular changes seen in the damaged nephron.</p

Serum and glucocorticoid regulated kinase and disturbed renal sodium transport in diabetes

Diabetes is associated with a number of side effects including retinopathy, neuropathy, nephropathy and hypertension. Recent evidence has shown that serum and glucocorticoid regulated kinase-1 (SGK1) is increased in models of diabetic nephropathy. While clearly identified as glucocorticoid responsive, SGK1 has also been shown to be acutely regulated by a variety of other factors. These include insulin, hypertonicity, glucose, increased intracellular calcium and transforming growth factor-beta, all of which have been shown to be increased in type II diabetes. The principal role of SGK1 is to mediate sodium reabsorption via its actions on the epithelial sodium channel (now known is sodium channel, nonvoltage-gated 1). Small alterations in the sodium resorptive capacity of the renal epithelia may have dramatic consequences for fluid volume regulation, and SGK1 maybe responsible for the development of hypertension associated with diabetes. This short commentary considers the evidence that Supports the involvement of SGK1 in diabetic hypertension, but also discusses how aberrant sodium reabsorption may account for the cellular changes seen in the nephron.</p

Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy

Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48?hrs, high glucose and mannitol (25?mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy.</p

SGK1 in the kidney: disrupted sodium transport in diabetes and beyond

Renal complications of diabetes can be severe; however, the mechanisms that underlie the development and progression of diabetic nephropathy are poorly understood. Recent evidence suggests that the serum and glucocorticoid induced kinase-1 (SGK1) may be key to this process. SGK1 expression and function are increased in models of diabetes and polymorphisms of the SGK1 gene are associated with type 2 diabetes mellitus. A key regulator of sodium transport within the renal epithelium of the distal nephron, SGK1 was originally isolated as a glucocorticoid-sensitive gene that regulated the epithelial sodium channel (ENaC; known also as the sodium channel, nonvoltage-gated 1, SCNN1). It is now apparent that SGK1 modulates sodium re-absorption by a number of sodium transporters/channels throughout the length of the nephron including; the Na+/H+ exchange isoform 3 (NHE3), the Na+Cl- co-transporter (NCC) and the Na+/K+-ATPase. In addition, SGK1 is regulated by a diverse range of factors including; insulin, glucose, intracellular calcium, transforming growth factor-beta1, flow rate and osmolality. This brief review examines the evidence supporting an involvement of SGK1 in diabetic nephropathy and discusses how dysregulated sodium transport may account for the development of secondary hypertension associated with the condition. Furthermore, the article examines how aberrant SGK1 expression and activity may be responsible for the cellular changes seen in the damaged nephron.</p

Functional expression of TRPV4 channels in human collecting duct cells: implications for secondary hypertension in diabetic nephropathy

Background. The Vanilloid subfamily of transient receptor potential (TRPV) ion channels has been widely implicated in detecting osmotic and mechanical stress. In the current study, we examine the functional expression of TRPV4 channels in cell volume regulation in cells of the human collecting duct. Methods. Western blot analysis, siRNA knockdown, and microfluorimetry were used to assess the expression and function of TRPV4 in mediating Ca2+-dependent mechanical stimulation within a novel system of the human collecting duct (HCD). Results. Native and siRNA knockdown of TRPV4 protein expression was confirmed by western blot analysis. Touch was used as a cell-directed surrogate for osmotic stress. Mechanical stimulation of HCD cells evoked a transient increase in [Ca2+]i that was dependent upon thapsigargin-sensitive store release and Ca2+ influx. At 48?hrs, high glucose and mannitol (25?mM) reduced TRPV4 expression by 54% and 24%, respectively. Similar treatment doubled SGK1 expression. Touch-evoked changes were negated following TRPV4 knockdown. Conclusion. Our data confirm expression of Ca2+-dependent TRPV4 channels in HCD cells and suggest that a loss of expression in response to high glucose attenuates the ability of the collecting duct to exhibit regulatory volume decreases, an effect that may contribute to the pathology of fluid and electrolyte imbalance as observed in diabetic nephropathy.</p

Serum and glucocorticoid regulated kinase and disturbed renal sodium transport in diabetes

Diabetes is associated with a number of side effects including retinopathy, neuropathy, nephropathy and hypertension. Recent evidence has shown that serum and glucocorticoid regulated kinase-1 (SGK1) is increased in models of diabetic nephropathy. While clearly identified as glucocorticoid responsive, SGK1 has also been shown to be acutely regulated by a variety of other factors. These include insulin, hypertonicity, glucose, increased intracellular calcium and transforming growth factor-beta, all of which have been shown to be increased in type II diabetes. The principal role of SGK1 is to mediate sodium reabsorption via its actions on the epithelial sodium channel (now known is sodium channel, nonvoltage-gated 1). Small alterations in the sodium resorptive capacity of the renal epithelia may have dramatic consequences for fluid volume regulation, and SGK1 maybe responsible for the development of hypertension associated with diabetes. This short commentary considers the evidence that Supports the involvement of SGK1 in diabetic hypertension, but also discusses how aberrant sodium reabsorption may account for the cellular changes seen in the nephron.</p

Functional expression of TRPV4 in human collecting duct (HCD) cells: a role in diabetic nephropathy

Background and aims: According to the World Health Organisation (WHOFact sheet 312, Sept 2006) diabetes is among the leading causes of kidney failurewith a10-20% mortality. When hyperglycaemia spills over into the urine(glucosuria), loss of glucose causes osmotic drag and diuresis and exposesthe kidney to various physical and nutrient stresses. In collecting duct epithelium,sodium absorption is linked to cell volume regulation and dependsupon detection of these mechanical stimuli. Prolonged exposure of renalepithelial to osmotic changes and increased fluid flow has led to a numberof suggestions concerning the mechanisms involved in stimulus-responsecoupling. One mechanism involves the calcium permeable transient receptorpotential-vanilloid-4 (TRPV4) channel. Gated by changes in osmolarity ormembrane stretch, these mechano-sensitive channels reputedly regulate cellvolume via a Ca2+ dependent mechanism. In the present study, mechanicalstimulation was employed as a surrogate form of osmotic stress to elucidatea role for TRPV4 in cell volume regulation in a novel model system of thehuman collecting duct.Materials and methods: RT-PCR and western blot analysis were used todetermine mRNA and protein expression for TRPV4 in a human collectingduct (HCD) cell line. Fura-2-microfluorimetry was used to assess generationof touch evoked calcium transients. Knockdown of TRPV4 expression wasachieved using a TRPV4 specific siRNA and the siLentGene U6 Casette RNAInterferance System different siRNA protocols.Results: Bands corresponding to TRPV4 were identified from HCD mRNAand protein. Mechanical stimulation of a single HCD cell evoked a transientincrease in cytosolic calcium ([Ca2+]i) that propagated between neighbouringcells within a cluster (5 separate experiments). Touch evoked changes in[Ca2+]i were still observed under Ca2+-free conditions, however the basal topeak amplitude of the response was only 35% of that obtained in the presenceof extracellular calcium (P<0.05 n=6). Pre-incubation with the Ca2+-ATPaseinhibitor thapsigargin (1?M) was used to assess contribution of intracellularCa2+-stores on touch-evoked changes in [Ca2+]i. Whilst removal of extracellularcalcium alone, failed to negate the generation of touch evoked Ca2+transients, the chelation of extracellular calcium in conjunction with the depletionof Ca2+-stores completely prevented touch-evoked changes in [Ca2+]i(P<0.05; n=6). Confirmation of a role for TRPV4 in the generation of touchevoked Ca2+-transients was assessed by siRNA knockdown. Transient transfectionof HCD cells with TRPV4 siRNA reduced TRPV4 protein expressionby approximately 40% (n=4; P<0.01 compared to control), whilst mechanicalstimulation of individual RFP-tagged anti-TRPV4 cells failed to significantlyelevate [Ca2+]i, compared to control cells observed in the same field of viewunder identical culture conditions.Conclusion: These data confirm a functional role for TRPV4 in generationof touch evoked Ca2+-signals between coupled cells of the human collectingduct and suggest that knockdown of TRPV4 expression inhibit the cells abilityto both detect and respond to osmotic signals via a Ca2+-dependent mechanism.These data have implications for altered fluid and electrolyte handlingin the diabetic kidney.</p

Deranged transcriptional regulation of SGK and ENaC: a link between hyperglycaemia and diabetic nephropathy

Background and Aims: Alterations in the resorptivecapacity of renal epithelia to sodium can have severeimplications for the normal functioning of the nephron andare most likely to be prominent in the development andpathogenesis of secondary hypertension, a condition associatedwith a number of renal diseases including diabeticnephropathy and glomerulonephritis. One of the majorregulators of sodium reabsorption in the nephron is theserum and glucocorticoid induced kinase (SGK), analdosterone regulated gene, which mediates sodium reabsorptionvia its actions on the epithelial sodium channel(ENaC). Recent reports have identified SGK as a keysignaling element whose level of expression appears to beupregulated in kidneys of diabetic humans and diabeticnephropathy. Hyperglycamia is believed to be the keypathophysiological component in promoting intercellularchanges ultimately leading to the deranged transcriptionalregulation of SGK.Materials and Methods: RT-PCR, immunocytochemistryand western blot analysis confirmed that human corticalcollecting duct (HCD) cells express SGK and alpha ENaC.For glucose, experiments, HCD cells were treated with(25 mM) glucose for 24 and 48 hours. For TGF-beta (2 nm)and Ionomycin (1 2m) experiments, HCD cells were treatedfor 4,6,8,12 and 24 hrs. A functional correlate for theseincreased levels of expression was provided by sodiummicrofluorimetry.Results: Incubation of HCD cells with high glucose(25 mM) for 24 and 48 hours increased expression ofSGK and Alpha ENaC protein (SGK, 261.7T14.9% ofcontrol (5 mM) at 48 hours; n=3, p<0.01%; alpha ENaC,316.5%T9.9% of control (5 mM) at 48 hours n=6, p<0.01).Hyperglycaemia has been linked to both raised calcium andTGF-beta levels. Application of TGF-beta or Ionomycinalone induced a significant increase in SGK proteinexpression (TGF-beta, 125.5%T7.5% of control at 8 hours;n=3 p<0.001%; Ionomycin 263.7%T8.6% of control at8 hours; n=3 p<0.001) suggesting a possible role for bothTGF-beta and calcium in mediating downstream effects ofhyperglycaemia. Intracellular sodium levels were found tobe significantly elevated following 24 (107%T0.88%) and48 (114%T0.93%) hours exposure to 25 mM glucose, ascompared to cells cultured in low (5 mM) glucose.Conclusion: This data taken in conjunction with thatdescribed in previous literature suggests that increases inintracellular sodium in response to high glucose after 48hours may be mediated via the increased expression of bothSGK and alpha-ENaC. This data highlights a possible linkbetween hyperglycaemia and the deranged sodium reabsorptionobserved in cases of diabetic nephropathy, andmay lead to identification of potential therapeutic targets.</p

Functional expression of TRPV4 in human collecting duct (HCD) cells: a role in diabetic nephropathy

Background and aims: According to the World Health Organisation (WHOFact sheet 312, Sept 2006) diabetes is among the leading causes of kidney failurewith a10-20% mortality. When hyperglycaemia spills over into the urine(glucosuria), loss of glucose causes osmotic drag and diuresis and exposesthe kidney to various physical and nutrient stresses. In collecting duct epithelium,sodium absorption is linked to cell volume regulation and dependsupon detection of these mechanical stimuli. Prolonged exposure of renalepithelial to osmotic changes and increased fluid flow has led to a numberof suggestions concerning the mechanisms involved in stimulus-responsecoupling. One mechanism involves the calcium permeable transient receptorpotential-vanilloid-4 (TRPV4) channel. Gated by changes in osmolarity ormembrane stretch, these mechano-sensitive channels reputedly regulate cellvolume via a Ca2+ dependent mechanism. In the present study, mechanicalstimulation was employed as a surrogate form of osmotic stress to elucidatea role for TRPV4 in cell volume regulation in a novel model system of thehuman collecting duct.Materials and methods: RT-PCR and western blot analysis were used todetermine mRNA and protein expression for TRPV4 in a human collectingduct (HCD) cell line. Fura-2-microfluorimetry was used to assess generationof touch evoked calcium transients. Knockdown of TRPV4 expression wasachieved using a TRPV4 specific siRNA and the siLentGene U6 Casette RNAInterferance System different siRNA protocols.Results: Bands corresponding to TRPV4 were identified from HCD mRNAand protein. Mechanical stimulation of a single HCD cell evoked a transientincrease in cytosolic calcium ([Ca2+]i) that propagated between neighbouringcells within a cluster (5 separate experiments). Touch evoked changes in[Ca2+]i were still observed under Ca2+-free conditions, however the basal topeak amplitude of the response was only 35% of that obtained in the presenceof extracellular calcium (P<0.05 n=6). Pre-incubation with the Ca2+-ATPaseinhibitor thapsigargin (1?M) was used to assess contribution of intracellularCa2+-stores on touch-evoked changes in [Ca2+]i. Whilst removal of extracellularcalcium alone, failed to negate the generation of touch evoked Ca2+transients, the chelation of extracellular calcium in conjunction with the depletionof Ca2+-stores completely prevented touch-evoked changes in [Ca2+]i(P<0.05; n=6). Confirmation of a role for TRPV4 in the generation of touchevoked Ca2+-transients was assessed by siRNA knockdown. Transient transfectionof HCD cells with TRPV4 siRNA reduced TRPV4 protein expressionby approximately 40% (n=4; P<0.01 compared to control), whilst mechanicalstimulation of individual RFP-tagged anti-TRPV4 cells failed to significantlyelevate [Ca2+]i, compared to control cells observed in the same field of viewunder identical culture conditions.Conclusion: These data confirm a functional role for TRPV4 in generationof touch evoked Ca2+-signals between coupled cells of the human collectingduct and suggest that knockdown of TRPV4 expression inhibit the cells abilityto both detect and respond to osmotic signals via a Ca2+-dependent mechanism.These data have implications for altered fluid and electrolyte handlingin the diabetic kidney.</p

Deranged transcriptional regulation of SGK and ENaC: a link between hyperglycaemia and diabetic nephropathy

Background and Aims: Alterations in the resorptivecapacity of renal epithelia to sodium can have severeimplications for the normal functioning of the nephron andare most likely to be prominent in the development andpathogenesis of secondary hypertension, a condition associatedwith a number of renal diseases including diabeticnephropathy and glomerulonephritis. One of the majorregulators of sodium reabsorption in the nephron is theserum and glucocorticoid induced kinase (SGK), analdosterone regulated gene, which mediates sodium reabsorptionvia its actions on the epithelial sodium channel(ENaC). Recent reports have identified SGK as a keysignaling element whose level of expression appears to beupregulated in kidneys of diabetic humans and diabeticnephropathy. Hyperglycamia is believed to be the keypathophysiological component in promoting intercellularchanges ultimately leading to the deranged transcriptionalregulation of SGK.Materials and Methods: RT-PCR, immunocytochemistryand western blot analysis confirmed that human corticalcollecting duct (HCD) cells express SGK and alpha ENaC.For glucose, experiments, HCD cells were treated with(25 mM) glucose for 24 and 48 hours. For TGF-beta (2 nm)and Ionomycin (1 2m) experiments, HCD cells were treatedfor 4,6,8,12 and 24 hrs. A functional correlate for theseincreased levels of expression was provided by sodiummicrofluorimetry.Results: Incubation of HCD cells with high glucose(25 mM) for 24 and 48 hours increased expression ofSGK and Alpha ENaC protein (SGK, 261.7T14.9% ofcontrol (5 mM) at 48 hours; n=3, p<0.01%; alpha ENaC,316.5%T9.9% of control (5 mM) at 48 hours n=6, p<0.01).Hyperglycaemia has been linked to both raised calcium andTGF-beta levels. Application of TGF-beta or Ionomycinalone induced a significant increase in SGK proteinexpression (TGF-beta, 125.5%T7.5% of control at 8 hours;n=3 p<0.001%; Ionomycin 263.7%T8.6% of control at8 hours; n=3 p<0.001) suggesting a possible role for bothTGF-beta and calcium in mediating downstream effects ofhyperglycaemia. Intracellular sodium levels were found tobe significantly elevated following 24 (107%T0.88%) and48 (114%T0.93%) hours exposure to 25 mM glucose, ascompared to cells cultured in low (5 mM) glucose.Conclusion: This data taken in conjunction with thatdescribed in previous literature suggests that increases inintracellular sodium in response to high glucose after 48hours may be mediated via the increased expression of bothSGK and alpha-ENaC. This data highlights a possible linkbetween hyperglycaemia and the deranged sodium reabsorptionobserved in cases of diabetic nephropathy, andmay lead to identification of potential therapeutic targets.</p