High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal--regulated kinase and protein kinase Cdelta activities in human mesangial cells

High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-beta1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-beta1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-beta1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-beta1. Neither TGF-beta1 bioactivity nor TGF-beta receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-beta1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-beta expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCdelta-specific inhibitor rottlerin, whereas G?6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCbeta and delta kinase assay. High glucose increased expression of several PKC isozymes, but only PKCdelta showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCdelta inhibition during glucose conditioning abrogated enhanced alpha1(I) collagen mRNA and promoter induction by TGF-beta1. Taken together, these data strongly suggest that heightened ERK and PKCdelta activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-beta1 and increased extracellular matrix production in mesangial cells.     

Glomerular expression of CTGF, TGF-beta 1 and type IV collagen in diabetic nephropathy

BACKGROUND: In development of progressive extracellular matrix accumulation, connective tissue growth factor (CTGF) may act as a downstream mediator of transforming growth factor-beta 1 (TGF-beta 1). However, the association and the correlation of these cytokines and extracellular matrix accumulation in human diabetic nephropathy (DN) is not fully understood. METHODS: To explore the possible involvement of TGF-beta 1 and CTGF in extracellular matrix accumulation in DN, high-resolution in situ hybridization with digoxigenin-labeled antisense oligonucleotides to CTGF, TGF-beta 1 and type IV collagen mRNAs were performed in DN and in histologically normal human kidney (NHK). To quantify expression of each mRNA, the fraction of all nuclear cells that were positively stained in the cytoplasm was determined in at least 10 randomly selected cross-sections of nonsclerotic glomeruli. RESULTS: Both in DN and in NHK, CTGF, TGF-beta 1 and type IV collagen mRNAs were mainly expressed by glomerular mesangial, visceral epithelial and parietal epithelial cells. The percentages of positive glomerular resident cells were significantly higher for each mRNA in DN compared with NHK. Especially, the expression of CTGF mRNA was also notably increased in case of DN with only mild histopathologic lesions. The extent of expression of each mRNA was significantly correlated to that of each other mRNA examined. CONCLUSION: Our study indicated that CTGF and TGF-beta may play an important role in glomerular histopathologic change in DN.     

CTGF expression in mesangial cells: involvement of SMADs,MAP kinase,and PKC

BACKGROUND: The induction of excess matrix in renal fibrosis seems to be mediated, at least in part, by the transforming growth factor-beta (TGF-beta)-mediated induction of connective tissue growth factor (CTGF) in mesangial cells. METHODS: By examining CTGF protein and mRNA expression and promoter activity in the presence or absence of TGF-beta or inhibitors, the signaling pathways controlling basal and TGF-beta-induced CTGF expression in mesangial cells were investigated. RESULTS: TGF-beta enhances CTGF mRNA and protein expression in mesangial cells. Mutation of a consensus SMAD binding element in the CTGF promoter completely abolished TGF-beta-induced CTGF expression and reduced basal CTGF expression. The previously identified basal control element-1 (BCE-1) site, but not Sp1 contributes to basal CTGF promoter activity. Ras/MEK/ERK, protein kinase C (PKC) and tyrosine kinase activity also contribute to basal and TGF-beta-induced CTGF promoter activity in cultured mesangial cells. CONCLUSIONS: The TGF-beta-induction of CTGF in mesangial cells requires SMADs and PKC/ras/MEK/ERK pathways. SMADs are involved in basal CTGF expression, which presumably reflects the fact that mesangial cells express TGF-beta endogenously. TGF-beta also induces CTGF through ras/MEK/ERK. Inhibiting ras/MEK/ERK seems not to reduce phosphorylation (that is, activation) of SMADs, suggesting that SMADs, although necessary, are insufficient for the TGF-beta-stimulation of the CTGF promoter through ras/MEK/ERK. Thus, maximal TGF-beta induction of CTGF requires synergy between SMAD and ras/MEK/ERK signaling.     

In-situ hybridization studies of protein kinase C α and β I isoforms in human diabetic nephropathy

Background. Hyperglycemia is the most important contributor to the development of diabetic nephropathy (DN). The activation of protein kinase C (PKC) caused by hyperglycemia is implicated in the pathogenesis of DN. PKC, which comprises a family of enzymes, plays a role in many signal transduction pathways and is involved in the regulation of cell growth and differentiation. However, the precise role of PKC in the progression of human DN is not fully understood. Methods. To evaluate the pathological role of PKC in DN, we examined the mRNA expression levels of PKC α and PKC β I isoforms in normal renal tissues and in renal tissues affected by DN. Tissues from open renal biopsies were obtained from 20 type 2 diabetic patients with DN. The expression levels of PKC α and PKC β I mRNA in kidneys with DN and in normal human kidney (NHK) were evaluated by in-situ hybridization, using digoxigenin-labeled oligonucleotide probes. Results. Cells positive for PKC α and β I mRNA were mainly observed in glomeruli, and some mRNA was also detected in the tubulointerstitium. The percentages of glomerular cells positive for PKC α and β I mRNA were higher in kidneys with DN than in NHK. In the glomeruli of kidneys with DN, the percentage of cells positive for PKC β I mRNA, but not the percentage of cells positive for PKC α mRNA, tended to be decreased with the degree of mesangial expansion. Conclusions. Our results suggest that the expression of mRNA for PKC α and/or β I may be associated with the progression of DN. Received: March 26, 2001 / Accepted: September 17, 2001     

BMK1 is activated in glomeruli of diabetic rats and in mesangial cells by high glucose conditions

BACKGROUND: High glucose causes renal cell injury through various signal transduction pathways, including mitogen-activated protein (MAP) kinases cascades. Big MAP kinase 1 (BMK1), also known as extracellular signal-regulated kinase 5 (ERK5), is a recently identified MAP kinase family member and was reported to be sensitive to osmotic and oxidative stress. However, the role of BMK1 in diabetic nephropathy has not been elucidated yet. METHODS: We investigated whether BMK1 is activated in the glomeruli of Otsuka Long Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus in comparison with the control Long Evans Tokushima Otsuka (LETO) rats. We also examined the effect of high glucose on BMK1 activity in cultured rat mesangial cells. RESULTS: BMK1 and ERK1/2 but not p38 were activated in the glomeruli of OLETF rats, which showed diabetic nephropathy at 52 weeks of age. High glucose, in addition to a high concentration of raffinose, caused rapid and significant activation of BMK1 in rat mesangial cells. MAP kinase/ERK kinase (MEK) inhibitors, U0126 and PD98059, both inhibited BMK1 activation by high glucose in a concentration-dependent manner. Protein kinase C (PKC) inhibition by GF109203X and PKC down-regulation with long-time phorbol myristate acetate (PMA) treatment both inhibited BMK1 and Src kinase activation. Src kinase inhibitors, herbimycin A and PP2, also inhibited high glucose-induced BMK1 activation. PKC inhibitors, Src inhibitors and MEK inhibitors, all inhibited cell proliferation by high glucose. Finally, transfection of dominant-negative MEK5, which is an upstream regulator of BMK1, abolished the BMK1-mediated rat mesangial cell proliferation stimulated by high glucose. CONCLUSION: In the present study, we demonstrated that high glucose activates BMK1 both in vivo and in vitro. It was suggested that high glucose induces PKC- and c-Src-dependent BMK1 activation. It could not be denied that BMK1 activation is induced through an osmotic stress-sensitive mechanism. BMK1-mediated mesangial cell growth may be involved in the pathogenesis of diabetic nephropathy.     

High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha1(IV) collagen expression in response to endothelin-1: role of specific protein kinase C isozymes

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal-regulated protein kinase (ERK1/2) signaling and alpha1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1-stimulated alpha1(IV) collagen mRNA expression from 1.2 +/- 0.1-fold to 1.9 +/- 0.2-fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)-PKC-delta, -epsilon, or -zeta inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1-driven GAL4 luciferase activity to 11 +/- 1-fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC-delta, -epsilon, or -zeta attenuated this response to NG levels. HG enhanced ET-1-stimulated intracellular alpha1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN-PKC-delta, -epsilon, -zeta, as well as DN-PKC-alpha and -beta, attenuated the response. Thus, HG-enhanced ET-1 stimulation of alpha1(IV) collagen expression requires PKC-delta, -epsilon, and -zeta to act through an ERK1/2-dependent pathway and via PKC-alpha and -beta, which are independent of ERK1/2.     

Thiazolidinedione compounds ameliorate glomerular dysfunction independent of their insulin-sensitizing action in diabetic rats

Thiazolidinedione (TZD) compounds are widely used as oral hypoglycemic agents. Herein, we provide evidence showing that troglitazone, one of the TZD compounds, is able to prevent glomerular dysfunction in diabetic rats through a novel mechanism independent of its insulin-sensitizing action. We examined the effect of troglitazone on functional and biochemical parameters of glomeruli in streptozotocin-induced diabetic rats. Troglitazone was able to prevent not only diabetic glomerular hyperfiltration and albuminuria, but an increase in mRNA expression of extracellular matrix proteins and transforming growth factor-beta1 in glomeruli of diabetic rats, without changing blood glucose levels. Biochemically, an increase in diacylglycerol (DAG) contents and the activation of the protein kinase C (PKC)-extracellular signal-regulated kinase (ERK) pathway in glomeruli of diabetic rats were abrogated by troglitazone. The activation of DAG-PKC-ERK pathways in vitro in mesangial cells cultured under high glucose conditions was also inhibited by troglitazone. Troglitazone enhanced the activities of DAG kinase, which could metabolize DAG to phosphatidic acid, in both glomeruli of diabetic rats and mesangial cells cultured under high glucose conditions. Surprisingly, pioglitazone, another TZD compound without alpha-tocopherol moiety in its structure, also prevented the activation of the DAG-PKC pathway and activated DAG kinase in mesangial cells cultured under high glucose conditions. These results may identify the TZDs as possible new therapeutic agents for diabetic nephropathy that prevent glomerular dysfunction through the inhibition of the DAG-PKC-ERK pathway.     

Glomerular expression of platelet-derived growth factor (PDGF)-A, -B chain and PDGF receptor-Α, -Β in human diabetic nephropathy

Background Mesangial expansion is thought to be a major cause of diabetic nephropathy (DN). Platelet-derived growth factor (PDGF) plays an important role in the production of extracellular matrix proteins in renal diseases. The present study was designed to determine the expression of PDGF and PDGF receptor (PDGFR) mRNA in the renal tissues of type 2 diabetic patients with DN.Methods We examined open renal biopsies of 20 type 2 diabetic patients with DN, and 10 normal human kidneys (NHK). Histopathologically, the severity of DN was classified as grade I (DN I, n = 10; mild mesangial expansion) or grade II (DN II, n = 10; moderate mesangial expansion). We evaluated the expression and localization of PDGF-A, -B, and PDGFR-, - using in situ hybridization, and quantified PDGF and PDGFR mRNA expression by counting all nuclei, and nuclei surrounded by PDGF-positive cytoplasm and PDGFR-positive cytoplasm, in at least ten randomly selected cross-sections of nonsclerotic glomeruli.Results In all glomeruli, PDGF and PDGFR mRNAs were expressed mainly in glomerular resident cells, predominantly glomerular mesangial and epithelial cells. The percentages of cells positive for PDGF-A and PDGFR- mRNA in DN were similar to those in NHK. In contrast, the percentages of PDGF-B and PDGFR- mRNA-positive cells in DN were significantly higher than those in NHK, and were significantly higher in DN I than in DN II. The percentages of cells positive for PDGF-B correlated with the PDGFR- mRNA level.Conclusions Our results suggest that the expression of PDGF-B and PDGFR- is an important factor in histologically early glomerular lesions of DN.     

Expression of megsin mRNA, a novel mesangium-predominant gene, in the renal tissues of various glomerular diseases

Mesangial cells play an important role in maintaining a structure and function of the glomerulus and in the pathogenesis of glomerular diseases. Recently, we discovered a new mesangium-predominant gene termed "megsin." Megsin is a novel protein that belongs to the serine protease inhibitor (serpin) superfamily. To elucidate the pathophysiologic role of megsin in the kidney, the expression and localization of megsin mRNA in renal tissues of patients with IgA nephropathy (IgA-N), diabetic nephropathy (DN), minimal change nephrotic syndrome (MCNS), membranous nephropathy (MN), and normal human kidney (NHK) was evaluated by in situ hybridization using digoxigenin-labeled oligonucleotide. Individual cells positive for megsin mRNA were observed only in glomeruli in all renal tissues. Their localization coincided with those of mesangial cells. The percentage of positive cells for megsin mRNA in total glomerular cells was significantly greater in IgA-N than in MCNS, MN, and NHK. It was also significantly greater in DN than in MCNS and NHK. In IgA-N, the percentage of megsin mRNA-positive cells was greater in tissues from those with mesangial cell proliferation and slightly mesangial matrix expansion (periodic acid-Schiff-positive area in the total glomerulus area, <30%; cell number in mesangial matrix area, >30; assessed in cross-sections through their vascular poles) than in tissues from those with severe mesangial matrix expansion (periodic acid-Schiff-positive area in total glomerulus area, >30%; cell number in mesangial matrix area, <30). In conclusion, megsin mRNA was predominantly expressed in glomerular mesangial cells in all renal tissues. The expression of megsin mRNA was upregulated in IgA-N and DN, both of which are diseases accompanied with mesangial cell proliferation and/or mesangial matrix expansion. These data suggest a link of megsin expression to the pathogenesis of IgA-N and DN, two major causes of end-stage renal failure.     

Extracellular signal-regulated kinase mediates stimulation of TGF-beta1 and matrix by high glucose in mesangial cells

High ambient glucose exerts its injurious effects on renal cells through nonenzymatic and enzymatic pathways, including altered signal transduction and upregulation of the transforming growth factor-beta (TGF-beta) system. Extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) cascade, is activated in mesangial cells cultured in high glucose and in glomeruli of diabetic rats. However, the biologic consequences of ERK activation in the kidney have not been investigated. To clarify the role of ERK activation, mouse mesangial cells were exposed to normal (5.5 mM) or high (25 mM) glucose with or without addition of PD98059, a specific inhibitor of MAPK/ERK kinase (MEK), an upstream kinase activator of ERK. Cells that were exposed to high glucose exhibited significant increases in ERK activity, TGF-beta1 expression (total protein, mRNA levels, and promoter activity), [(3)H]-proline uptake, and alpha1(I) collagen and fibronectin mRNA levels. Treatment with PD98059 (up to 25 microM) significantly inhibited these parameters. In contrast, 25 microM PD98059 had no significant effect on any of the parameters measured in cells that were exposed to normal glucose. Overexpression of MAPK phosphatase CL 100 prevented TGF-beta1 promoter activation by high glucose, confirming the involvement of the MEK-ERK pathway in response to high glucose. The conclusion is that activation of ERK in mesangial cells is responsible for high-glucose-induced stimulation of TGF-beta1 and contributes to the increased extracellular matrix expression.     

Glomerular expression of connective tissue growth factor mRNA in various renal diseases

SUMMARY:   Connective tissue growth factor (CTGF) is a cysteine-rich member of a new family of growth regulators. It is an important factor in the pathogenesis of mesangial matrix accumulation and progressive glomerulosclerosis. The present study was designed to elucidate the role of CTGF in diabetic nephropathy (DN), immunoglobulin A nephropathy (IgA-N), membranous nephropathy (MN), and minimal change nephrotic syndrome (MCNS). We evaluated the expression and localization of CTGF mRNA in surgically excised renal tissue samples from 10 patients with DN, 10 with IgA-N, 10 with MN, 10 with MCNS, and 10 normal human kidney (NHK) tissue samples, by using high-resolution in situ hybridization with digoxigenin-labelled oligonucleotide. To quantify CTGF mRNA expression, we counted all nuclei, and nuclei surrounded by CTGF-positive cytoplasm, in at least 10 randomly selected cross-sections of non-sclerotic glomeruli, and expressed the results as a percentage of total glomerular cells. In all glomeruli, CTGF mRNA was expressed mainly in glomerular intrinsic cells, including glomerular mesangial and epithelial cells and some cells of Bowman's capsule. The percentage of cells positive for CTGF mRNA was significantly higher in DN and IgA-N than in MN, MCNS and NHK. However, there was no significant difference in the percentage of CTGF mRNA-positive cells between DN and IgA-N. Our study indicates that CTGF may play an important role in the development and progression of glomerulosclerosis in DN and IgA-N, which are both accompanied by mesangial matrix expansion and comprise two major causes of end-stage renal failure.     

Localization of connective tissue growth factor mRNA in human diabetic nephropathy by in situ hybridization

Background. Progressive expansion of the mesangial matrix is one of the most characteristic histological features of diabetic nephropathy (DN). Connective tissue growth factor (CTGF) is an important factor in the pathogenesis of mesangial matrix expansion and progressive glomerulosclerosis. Methods. To evaluate the expression and localization of CTGF mRNA in the renal tissues of 23 patients with DN and in normal human kidney (NHK), high-resolution in situ hybridization, using a digoxigenin-labeled oligonucleotide, was performed. The patients with DN were classified into three groups based on the histopathological severity of the DN: mild (grade I; n = 9), moderate (grade II; n = 10), and severe (grade III; n = 4). Mesangial expansion and tubulointerstitial injury were evaluated histologically. To quantitate the expression of CTGF mRNA, all nuclei as well as nuclei surrounded by CTGF-positive cytoplasm, in at least ten randomly selected cross-sections of nonsclerotic glomeruli were counted, and the results were expressed as a percentage of the total number of glomerular cells. Results. In both DN and NHK, CTGF mRNA was expressed mainly in intrinsic glomerular cells, including glomerular mesangial cells, epithelial cells and cells of Bowman's capsule. In the tubulointerstitial area, some tubules, particularly atrophic tubules, and some infiltrating cells in DN, were positively stained for CTGF mRNA, especially in DN grade III. The percentage of CTGF mRNA-positive cells was significantly higher in DN than in NHK, and the percentage of these cells was higher in grades I and II DN than in grade III DN. Conclusions. Our results suggest that the expression of CTGF mRNA may be associated with the development and progression of human diabetic nephropathy. Received: April 3, 2001 / Accepted: October 20, 2001     

Expression of mitogen-activated protein kinases in human renal dysplasia

BACKGROUND: We previously reported that the expression of mitogen-activated protein kinases (MAPKs) is developmentally regulated. Dysregulation of MAPKs may lead to kidney malformation. Thus, we investigated the expression of MAPKs in human renal dysplasia, one of the most common kidney malformations. METHODS: Prenatal (gestational ages 20 to 36 weeks, N = 6) and postnatal (2 years old, N = 1) dysplastic kidneys, and normal kidneys (gestational ages 19 to 34 weeks, N = 4) were examined. Immunohistochemical studies were performed using antibodies against extracellular signal-regulated kinase (ERK), p38 MAPK (p38), c-Jun N-terminal kinase (JNK), phospho-MAPKs (P-MAPKs), and proliferating cell nuclear antigen (PCNA). Apoptosis was detected by the TUNEL method. RESULTS: In dysplastic kidneys, proliferation was prominent in dysplastic tubules and also found in cyst epithelia. TUNEL staining was detected in dysplastic tubules and cysts, and occasionally in undifferentiated cells. p38 and anti-phospho-p38 (P-p38) were strongly expressed in dysplastic epithelia, but not detected in normal kidneys at any stage examined. On the other hand, JNK and P-JNK were positive in tubular epithelia of normal kidneys, whereas their expression was barely detectable in dysplastic tubules and cysts. ERK was expressed in all tubular segments, and P-ERK was detected in distal tubules and collecting ducts of normal kidneys. Dysplastic kidney epithelia stained exclusively positive for ERK and P-ERK. CONCLUSIONS: p38 is ectopically expressed, and JNK is down-regulated in dysplastic kidney epithelia. Furthermore, dysplastic epithelia are exclusively positive for ERK and P-ERK. Activated p38 and ERK may mediate hyperproliferation of dysplastic tubules resulting in cyst formation, whereas down-regulated JNK expression may be the cause or the result of an undifferentiated state of dysplastic epithelia.     

Renal activation of extracellular signal-regulated kinase in rats with autosomal-dominant polycystic kidney disease

BACKGROUND: Abnormal proliferation of renal tubule epithelial cells is a central factor in the biogenesis and sustained expansion of cysts in autosomal-dominant polycystic kidney disease (ADPKD). Recent evidence from in vitro studies of human cyst wall epithelial cells has implicated a role for the mitogen-activated protein (MAP) kinase pathway in this aberrant proliferation. To determine the extent to which this signaling pathway is involved in cyst pathogenesis in vivo, we measured the expression of select components of the MAP kinase cascade in Han:SPRD rats with ADPKD at an early stage of the disease. METHODS: Kidneys of 8-week-old normal Han:SPRD rats (+/+) or rats heterozygous (Cy/+) for ADPKD were examined by Western blot analysis and immunohistochemistry to determine the expression of extracellular-regulated kinase (ERK), phosphorylated ERK (P-ERK), Raf-1 (MAPKKK), phosphorylated Raf-1 (P-Raf-1), B-Raf, Rap-1 and phosphorylated protein kinase A (P-PKA). RESULTS: P-ERK was expressed to a greater extent in Cy/+ kidneys (3.74 +/- 1.07 fold) than in normal kidneys, whereas ERK abundance was not different. P-Raf-1 levels were higher in Cy/+ than in +/+ kidneys (1.53 +/- 0.08 fold) consistent with upstream stimulation of receptor tyrosine kinase. B-Raf and Raf-1 abundances were greater in Cy/+ than in +/+ (1.74 +/- 0.25 and 1.27 +/- 0.08 fold, respectively). In Cy/+, immunohistochemistry showed increased P-ERK and B-Raf expression in the abnormal mural epithelial cells within cysts. These findings, together with the detection of P-PKA and the small G protein, Rap-1, in cyst epithelial cells, implicate a potential role for cyclic adenosine monophosphate (AMP) in the activation of ERK in ADPKD cells. CONCLUSIONS: We conclude that the MAP kinase pathway is activated to the level of ERK in the abnormal mural epithelial cells lining cysts in animals with a dominantly inherited type of polycystic kidney disease. We suggest that cAMP, acting through PKA, Rap-1 and B-Raf, may contribute to the activation of ERK in a way that complements receptor tyrosine kinase-mediated agonists in the promotion of cyst enlargement.     

Ouabain binds with high affinity to the Na,K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.     

Calcium restores a normal proliferation phenotype in human polycystic kidney disease epithelial cells

Polycystic kidney disease (PKD) is a lethal disorder characterized by progressive expansion of renal cysts. Genetic mutations associated with PKD are thought to disrupt intracellular Ca2+ regulation, leading to abnormal proliferation of tubule epithelial cells. cAMP stimulates the B-Raf/MEK/extracellular signal-regulated kinase (B-Raf/MEK/ERK) pathway and accelerates the proliferation of cells that are cultured from PKD cysts. By contrast, cAMP inhibits the proliferation of cells from normal human kidneys (NHK) and M-1 mouse collecting duct cells. Previously, it was found that a sustained reduction of intracellular Ca2+ levels in NHK and M-1 cells that were treated with Ca2+ entry blockers allowed cAMP activation of the B-Raf/MEK/ERK pathway, switching the cells to a cAMP-growth stimulated phenotype. In this study, primary cultures of cyst epithelial cells from autosomal dominant (ADPKD) and recessive (ARPKD) PKD kidneys were used to determine whether controlled addition of Ca2+ could reverse the aberrant mitogenic response to cAMP. Steady-state intracellular Ca2+ levels were found to be 20 nM lower in cyst-derived ADPKD cells (57 +/- 2 nM) compared with NHK cells (77 +/- 2 nM). Treatment of ADPKD cells or ARPKD cells with either Bay K8644, a Ca2+ channel activator, or A23187, a Ca2+ ionophore, caused sustained increases in intracellular Ca2+ levels and completely reversed the mitogenic response to cAMP. Elevation of intracellular Ca2+ levels in ADPKD cells increased Akt activity and blocked cAMP-dependent B-Raf and ERK activation. Thus, increases in [Ca2+]i are able to restore the normal anti-mitogenic response to cAMP in cells that are derived from two genetically distinct forms of PKD.