Compensatory kidney hypertrophy/hyperplasia after nephrectomy in mice: alterations of connexin 43 (Cx43) phosphorylated isoforms (original) (raw)
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Kidney International, 2014
Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43 þ / À mice. The renal cortex of 5-month-old RenTgCx43 þ / À mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43 þ / À mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43 þ / À or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.
Cell Communication and Signaling
Background: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannelmediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Methods: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43 +/− mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Results: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43 +/− mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Conclusion: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD.
Nephrology Dialysis Transplantation, 2006
Background. Significance of podocyte injury in the progression of diabetic nephropathy is not wellunderstood. In this study, we examined whether alteration of gap junction protein connexin43 (Cx43) expression in podocytes is associated with the progression of overt diabetic nephropathy. Methods. We recruited 29 type 2 diabetic patients with overt nephropathy who underwent renal biopsy. Nephrectomized kidney samples obtained from seven subjects with localized neoplasm and biopsy specimens from five patients diagnosed as minor glomerular abnormalities were used as controls. Cx43 staining on paraffin-embedded kidney sections were studied by immunohistochemistry. Results. In controls, Cx43 was expressed at podocytes in a linear pattern along the glomerular basement membrane. In contrast, downregulation and loss of uniformly linear staining of Cx43 (Cx43 heterogeneity) in podocytes were observed in diabetic nephropathy. Cx43 intensity correlated with current renal function (R ¼ 0.647, P < 0.005), whereas the magnitude of Cx43 heterogeneity correlated well with the degree of future decline in renal function (R ¼ À0.705, P < 0.001). Conclusions. Alteration of Cx43 expression in podocytes was closely associated with the progression of overt diabetic nephropathy. These results indicate that change in Cx43 expression at podocytes represents a progressive stage in overt diabetic nephropathy and that it may be a convenient way to predict future decline in renal function.
Up-Regulation of Connexin43 in Glomerular Podocytes in Response to Injury
The American Journal of Pathology, 2002
Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of focal segmental glomerulosclerosis. However, it is poorly understood how podocytes respond to injury. In this study, glomerular expression of connexin43 (Cx43) gap junction protein was examined at both protein and transcript levels in an experimental model of podocyte injury, puromycin aminonucleoside (PAN) nephrosis. A striking increase in the number of immunoreactive dots with anti-Cx43 antibody was demonstrated along the glomerular capillary wall in the early to nephrotic stage of PAN nephrosis. The conspicuous change was not detected in the other areas including the mesangium and Bowman's capsule. Immunoelectron microscopy showed that the immunogold particles for Cx43 along the capillary wall were localized predominantly at the cell-cell contact sites of podocytes. Consistently, Western blotting and ribonuclease protection assay revealed a distinct increase of Cx43 protein, phosphorylation, and transcript in glomeruli during PAN nephrosis. The changes were detected by 6 hours after PAN injection. These findings indicate that the increase of Cx43 expression is one of the earliest responses that have ever been reported in podocyte injury. To show the presence of functional gap junctional intercellular communication (GJIC) in podocytes, GJIC was assessed in podocytes in the primary culture by transfer of fluorescent dye, Lucifer yellow, after a single-cell microinjection. Diffusion of the dye into adjacent cells was observed frequently in the cultured podocytes, but scarcely in cultured parietal epithelial cells of Bowman's capsule, which was compatible with their Cx43 staining. Thus, it is concluded that Cx43-mediated GJIC is present between podocytes, suggesting that podocytes may respond to injury as an integrated epithelium on a glomerulus rather than individually as a separate cell. (Am J
Alteration of connexin expression is an early signal for chronic kidney disease
AJP: Renal Physiology, 2011
Chronic kidney disease is promoted by a variety of factors that induce chronic inflammation and fibrosis. Inflammation and excessive scaring have been recently associated with disruptions of the gap junction-mediated intercellular communication. Nevertheless, little is known about alterations of the expression of gap junction proteins such as connexin (Cx) 43 and 37 in chronic renal disease. In this study, we investigated the expression of these two Cxs in the hypertensive RenTg mice, the anti-glomerular basement membrane glomerulonephritis, and the unilateral ureteral obstruction models, all leading to the development of chronic kidney disease in mice. Expression of Cx43 was almost negligible in the renal cortex of control mice. In contrast, Cx43 was markedly increased in the endothelium of peritubular and glomerular capillaries of the 3-mo-old RenTg mice, in the glomeruli of mice suffering from glomerulonephritis, and in the tubules after obstructive nephropathy. The Cx43 expressi...
Screening for genes up-regulated in 5/6 nephrectomized mouse kidney
Kidney …, 1999
Screening for genes up-regulated in 5/6 nephrectomized mouse glomerulosclerosis in immunological and nonimmunokidney. logical human glomerular diseases. Subtotal renal abla-Background. In diabetic and nondiabetic renal diseases, glotion (5/6 nephrectomy) of rat has been used as a longmerular hyperfiltration is believed to play a central role in the standing and extensively investigated animal model to subsequent progression of glomerulosclerosis and interstitial explore the mechanism of glomerular hyperfiltration, alrenal scarring. To identify genes involved in the process of hyperfiltration and hypertrophy, a polymerase chain reaction though the susceptibility to the glomerulosclerosis is (PCR)-based subtraction method, that is, representational difquite different among various strains [1]. In Wistarference analysis of cDNA (cDNA-RDA), was employed. Kyoto and Sprague-Dawley rats, partial ablation of renal Methods. Ten-week-old ICR mice were 5/6 nephrectomized mass initiates the cycle of progressive renal injury in the and sham operated. After two weeks, mRNAs were isolated from control and remnant kidneys and were subjected to the remnant kidney associated with glomerular hypertrophy, cDNA-RDA procedure. hyperfiltration, and systemic hypertension [2, 3]. The Results. We identified 10 known and 9 novel genes. Among histopathological studies of nephrectomized renal tissue 19 clones, 12 clones (8 known and 4 novel) showed 1.5-to revealed that a complex response mainly consists of three 6-fold up-regulation by Northern blot analyses. The remaining seven clones were rarely expressed genes and were barely steps: (a) the rapid hypertrophic phase (2 to 4 weeks detected by Northern blot analyses, and their up-regulated after ablation), (b) the quiescence phase with minimal expression was confirmed by Southern blot analysis using the histological alterations (4 to 10 weeks), and (c) the devel-PCR-amplified representative amplicons. The known genes inopment of segmental glomerular sclerosis and tubuloincluded kidney androgen-regulated protein, major urinary protein, lysozyme M, metalloproteinase-3 tissue inhibitor, chaperterstitial fibrosis (after 10 weeks) [4, 5]. Many researchers onin 10, cytochrome oxidase I, ⑀-sarcoglycan, ribosomal protein have reported that various molecules are up-regulated S3a, G-protein␥ 10 subunit, and splicing factor 9G8. All of the during different periods of nephrectomized kidneys. isolated known genes have not been reported to be up-regu-mRNA and protein expression studies of diverse genes, lated in the nephrectomized mouse kidney and suggest the possible role of androgen action, mitochondrial functions, masuch as of hormones [6], cytokines, and growth factors trix metabolism, cell-matrix interactions, and intracellular sig-[7-10], growth factor receptors [8, 11], transcription facnaling events in the initiation of the progressive renal injury tors [12], cell cycle regulators [13], proto-oncogenes [14], of the remnant kidney. Furthermore, cDNA-RDA facilitates vasoactive peptides [15-18], adhesion molecules [19], exthe discovery of novel genes, including two kidney-specific genes. Conclusions. The isolated known and novel genes may be tracellular matrix (ECM) glycoproteins [20], ECM deinvolved in the pathobiological process of initial hyperfiltration grading proteases and its inhibitors [21], have been reand hypertrophy of remnant kidney. ported. In the sclerotic phase, mRNA expression of ECM glycoproteins, including type I, III, and IV collagen, fibronectin, laminin, and proteoglycans, was up-reg-Glomerular hyperfiltration has been considered to be ulated, and the expansion of mesangial and interstitial the initial process for the subsequent development of matrix and the accumulation of ECM glycoproteins were observed by immunohistochemistry [20]. Prior to the
In various models of chronic kidney disease, the amount and localization of Cx43 in the nephron is known to increase, but the intracellular pathways that regulate these changes have not been identified. Therefore, we proposed that: "In the model of renal damage induced by infusion of angiotensin II (AngII), a RhoA/ROCK-dependent pathway, is activated and regulates the abundance of renal Cx43”. In rats, we evaluated: 1) the time-point where the renal damage induced by AngII is no longer reversible; and 2) the involvement of a RhoA/ROCK-dependent pathway and its relationship with the amount of Cx43 in this irreversible stage. Systolic blood pressure (SBP) and renal function (urinary protein/urinary creatinine: Uprot/UCrea) were evaluated as systemic and organ outcomes, respectively. In kidney tissue, we also evaluated: 1) oxidative stress (amount of thiobarbituric acid reactive species), 2) inflammation (immunoperoxidase detection of the inflammatory markers ED-1 and IL-1β), 3) f...
Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells
Cell and Tissue Research, 2003
We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation.
Evidence for the Presence of a Free C-Terminal Fragment of Cx43 in Cultured Cells
Cell Communication and Adhesion, 2007
Migration of the gap junction protein connexin 43 (Cx43) in SDS-PAGE yields 2 to 4 distinct bands, detectable in the 40-47 kDa range. Here, we show that antibodies against the carboxyterminal domain of Cx43 recognized an additional 20-kDa product. This protein was detected in some culture cell lysates. The presence of the 20-kDa band was not prevented by the use of protease inhibitors (Complete R and phenylmethylsulfonyl fluoride (PMSF), 1-5 mM). The band was absent from cells treated with Cx43-specific RNAi, and from those derived from Cx43-deficient mice, indicating that this Cx43-immunoreactive protein is a product of the Cx43 gene. Treatment of CHO cells with cyclosporin A caused a reduction in the amount of full-length Cx43 and a concomitant increase in the amount of the 20-kDa band. Overall, our data show that a fraction of the Cx43-immunoreactive protein pool within a given cell may correspond to a C-terminal fragment of the protein.
International Journal of Molecular Sciences
Numerous evidence corroborates roles of gap junctions/hemichannels in proper kidney development. We analyzed how Dab1 gene functional silencing influences expression and localization of Cx37, Cx40, Cx43, Cx45, Panx1 and renin in postnatal kidneys of yotari mice, by using immunohistochemistry and electron microscopy. Dab1 Δ102/221 might lead to the activation of c-Src tyrosine kinase, causing the upregulation of Cx43 in the medulla of yotari mice. The expression of renin was more prominent in yotari mice (p < 0.001). Renin granules were unusually present inside the vascular walls of glomeruli capillaries, in proximal and distal convoluted tubules and in the medulla. Disfunction of Cx40 is likely responsible for increased atypically positioned renin cells which release renin in an uncontrolled fashion, but this doesn’t rule out simultaneous involvement of other Cxs, such as Cx45 which was significantly increased in the yotari cortex. The decreased Cx37 expression in yotari medulla ...