Epigenetics refers to a heritable change in the pattern of gene expression that is mediated by a mechanism specifically not due to alterations in the primary nucleotide sequence. Well known epigenetic mechanisms encompass DNA methylation, chromatin remodeling (histone modifications) and RNA interference. Functionally, epigenetics provides an extra layer of transcriptional control and plays a crucial role in normal physiological development, as well as in pathological conditions. Aberrant DNA methylation is implicated in immune dysfunction, inflammation and insulin resistance. Epigenetic changes may be responsible for “metabolic memory” and development of micro- and macrovascular complications of diabetes. MicroRNAs are critical in the maintenance of glomerular homeostasis and hence RNA interference may be important in the progression of renal disease. Recent studies have shown that epigenetic modifications orchestrate the epithelial-mesenchymal transition and eventually fibrosis of the renal tissue. Oxidative stress, inflammation, hyperhomocysteinemia and uremic toxins could induce epimutations in chronic kidney disease. Epigenetic alterations are associated with inflammation and cardiovascular disease in patients with chronic kidney disease. Reversible nature of the epigenetic changes gives an unique opportunity to halt or even reverse the disease process through targeted therapeutic strategies.
Diabetic nephropathy is a common complex disease with a clear genetic predisposition. Human gene association studies are beginning to bear fruit by identifying gene loci that increase diabetic nephropathy risk. Chua et al. report a similar study in diabetic mice that reveals a major nephropathy locus on chromosome 8. Could this be a human nephropathy gene? Time will tell, but such findings will at least improve the use of mouse models of human kidney disease.
Frequent hemodialysis can alter volume status, blood pressure and the concentration of osmotically active solutes, each of which might affect residual kidney function (RKF). In the Frequent Hemodialysis Network Daily and Nocturnal Trials, we examined the effects of assignment to 6 compared to 3 times per week hemodialysis on follow up RKF. In both trials, baseline RKF was inversely correlated with number of years since onset of ESRD. In the Nocturnal Trial, 63 participants had non-zero RKF at baseline (mean urine volume 0.76 l/d, urea clearance 2.3 ml/min, and creatinine clearance 4.7 ml/min). In those assigned to frequent nocturnal dialysis, these indices were all significantly lower at month 4 and were mostly so at month 12 compared to controls. In the frequent dialysis group, urine volume had declined to zero in 52% and 67% of patients at months 4 and 12, respectively, compared to 18% and 36% in controls. In the Daily Trial, 83 patients had non-zero RKF at baseline (mean urine volume 0.43 l/d, urea clearance 1.2 ml/min, and creatinine clearance 2.7 ml/min). Here, treatment assignment did not significantly influence follow-up levels of the measured indices, although the range in baseline RKF was narrower, potentially limiting power to detect differences. Thus, frequent nocturnal hemodialysis appears to promote a more rapid loss of RKF, the mechanism of which remains to be determined. Whether RKF also declines with frequent daily treatment could not be determined.
Oxidative stress and inflammation are mediators in the development and progression of chronic kidney disease (CKD) and its complications, and they are inseparably linked as each begets and amplifies the other. CKD-associated oxidative stress is due to increased production of reactive oxygen species (ROS) and diminished antioxidant capacity. The latter is largely caused by impaired activation of Nrf2, the transcription factor that regulates genes encoding antioxidant and detoxifying molecules. Protective effects of Nrf2 are evidenced by amelioration of oxidative stress, inflammation, and kidney disease in response to natural Nrf2 activators in animal models, while Nrf2 deletion amplifies these pathogenic pathways and leads to autoimmune nephritis. Given the role of impaired Nrf2 activity in CKD-induced oxidative stress and inflammation, interventions aimed at restoring Nrf2 may be effective in retarding CKD progression. Clinical trials of the potent Nrf2 activator bardoxolone methyl showed significant improvement in renal function in CKD patients with type 2 diabetes. Results of the ongoing BEACON trial investigating the effect of this drug on time to end-stage renal disease or cardiovascular death will help further characterize the efficacy of Nrf2 pharmacological modulation in CKD. This article provides an overview of the role of impaired Nrf2 activity in the pathogenesis of CKD-associated oxidative stress and inflammation and the potential utility of targeting Nrf2 in the treatment of CKD.
Oxidative stress; Inflammation; CKD progression; Cardiovascular disease; ESRD; Antioxidant therapy; bardoxolone methyl
Experimental and clinical evidence suggests that long chain n-3 fatty acids may protect against sudden cardiac death, the leading cause of mortality in hemodialysis patients. Here we investigated whether long chain n-3 fatty acids have a protective relationship with sudden cardiac death in 100 patients who died of sudden cardiac death during the first year of starting hemodialysis and 300 patients who survived. Individuals were selected from a nationally representative cohort of over 1000 U.S. hemodialysis units in 2004–2005. The odds of sudden cardiac death were calculated by quartile of long chain n-3 fatty acids levels over the first year. There was a significant inverse relationship between long chain n-3 fatty acids and the risk of sudden cardiac death even after adjusting for relevant co-morbid conditions, biochemical values, and dietary fats. The odds of sudden cardiac death at 1 year for the second, third, and fourth quartile groups of long chain n-3 fatty acids were 0.37, 0.22, and 0.20, respectively, compared to the lowest quartile. This significant inverse relationship was maintained even during the highest-risk first few months on hemodialysis. Thus, long chain n-3 fatty acids are strongly and independently associated with a lower risk of sudden cardiac death in hemodialysis patients throughout the first year of hemodialysis.
sudden cardiac death; hemodialysis; omega-3; n-3; fatty acids
Using large-scale small molecule screening techniques, Li et al. have identified a compound that inhibits the UT-B urea channel. They propose that this or similar compounds could be used as aquaretic agents to increase water excretion without changes in electrolyte excretion. Such compounds would potentially be useful in treatment of hyponatremic disorders. Here we review the physiological basis for the action of urea channel inhibitors in the kidney and assess their clinical potential.
Podocyte (glomerular epithelial cell; GEC) dysfunction and loss are the hallmarks of focal segmental glomerulosclerosis (FSGS). Over the recent years, changes, including activation and proliferation of parietal epithelial cells (PEC) have been increasingly appreciated in FSGS. The functional role of PECs in FSGS is still a hotly debated issue. Here Ueno et al. report that Notch signaling plays a role in orchestrating PEC cell phenotypic changes in FSGS.
Chronic kidney disease risk factors may associate with the estimated glomerular filtration rate (eGFR) differently than with the measured GFR. To examine this, we evaluated 1150 patients (mean age 65) in two community cohorts for risk factors, measured GFR by iothalamate clearance, and eGFR based on creatinine (Cr), cystatin C (CysC), or both. The interaction between each risk factor and eGFR (relative to measured GFR) identified risk factor associations with eGFR along non-GFR pathways. In a subset of 40 patients with two visits, the mean coefficient of variation was 8.2% for measured GFR, 6.4% for eGFRCr, 8.2% for eGFRCr-CysC, and 10.7% for eGFRCysC. The measured GFR was better correlated with eGFRCr-CysC (r, 0.74) than eGFRCr (r, 0.70) or eGFRCysC (r, 0.68). Lower measured GFR associated with lower 24-hour urine creatinine, albuminuria, hypertension, diabetes, higher triglycerides, and higher uric acid. Lower eGFRCr had these same associations except for an association with higher 24-hour urine creatinine along a non-GFR pathway. Lower eGFRCysC and eGFRCr-CysC also had these same associations but also associated with obesity, albuminuria, hypertension, diabetes, higher triglycerides, higher C-reactive protein, and higher uric acid along non-GFR pathways. Thus, cystatin C improves estimation of GFR over creatinine alone; however, the association between most of the risk factors and GFR was more accurate by eGFR based on creatinine alone. This is explained by the association of these risk factors with the non-GFR determinants of cystatin C.
In order to define the intensity of immunosuppression, we examined risk factors for acute rejection in desensitization protocols that use baseline donor specific antibody levels measured as mean fluorescence intensity (MFImax). The study included 146 patients transplanted with a negative flow crossmatch and a mean follow-up of 18 months with the majority (83%) followed for at least 1 year. At the time of transplant, mean calculated panel reactive antibody and MFImax ranged from 10.3% to 57.2%, and 262 to 1691, respectively, between low and high-risk protocols. Mean MFImax increased significantly from transplant to one-week and one-year. The incidence of acute rejection (mean 1.65 months) as a combination of clinical and subclinical rejection was 32% including 14% cellular, 12% antibody-mediated and 6% mixed rejection. In regression analyses, only C4d staining in post-reperfusion biopsies (hazard ratio 3.3, confidence interval 1.71 to 6.45) and increased donor specific antibodies at 1 week post-transplant were significant predictors of rejection. A rise in MFImax by 500 was associated with a 2.8-fold risk of rejection. Thus, C4d staining in post-reperfusion biopsies and an early rise in donor specific antibodies after transplantation are risk factors for rejection in moderately sensitized patients.
Elevated fibroblast growth factor 23 (FGF23) is associated with cardiovascular disease in patients with chronic kidney disease. As a potential mediating mechanism, FGF23 induces left ventricular hypertrophy; however, its role in arterial calcification is less clear. In order to study this we quantified coronary artery and thoracic aorta calcium by computed tomography in 1501 patients from the Chronic Renal Insufficiency Cohort (CRIC) study within a median of 376 days (interquartile range 331 to 420 days) of baseline. Baseline plasma FGF23 was not associated with prevalence or severity of coronary artery calcium after multivariable adjustment. In contrast, higher serum phosphate levels were associated with prevalence and severity of coronary artery calcium, even after adjustment for FGF23. Neither FGF23 nor serum phosphate were consistently associated with thoracic aorta calcium. We could not detect mRNA expression of FGF23 or its co-receptor, klotho, in human or mouse vascular smooth muscle cells, or normal or calcified mouse aorta. Whereas elevated phosphate concentrations induced calcification in vitro, FGF23 had no effect on phosphate uptake or phosphate-induced calcification regardless of phosphate concentration or even in the presence of soluble klotho. Thus, in contrast to serum phosphate, FGF23 is not associated with arterial calcification and does not promote calcification experimentally. Hence, phosphate and FGF23 promote cardiovascular disease through distinct mechanisms.
phosphate; fibroblast growth factor 23; vascular calcification; vascular smooth muscle; chronic kidney disease
Netrin-1 regulates inflammation but the mechanism by which this occurs is unknown. Here we explore the role of netrin-1 in regulating the production of the prostanoid metabolite PGE2 from neutrophils in in vitro and in vivo disease models. Ischemia reperfusion in wild-type and RAG-1 knockout mice induced severe kidney injury that was associated with a large increase in neutrophil infiltration and COX-2 expression in the infiltrating leukocytes. Administration of netrin-1 suppressed COX-2 expression, PGE2 and thromboxane production, and neutrophil infiltration into the kidney. This was associated with reduced apoptosis, inflammatory cytokine and chemokine expression, and improved kidney function. Treatment with the PGE2 receptor EP4 agonist enhanced neutrophil infiltration and renal injury which was not inhibited by netrin-1. Consistent with in vivo data, both LPS and IFNγ-induced inflammatory cytokine production in macrophages and IL-17-induced IFNγ production in neutrophils were suppressed by netrin-1 in vitro by suppression of COX-2 expression. Moreover, netrin-1 regulates COX-2 expression at the transcriptional level through the regulation of NFκB activation. Thus, netrin-1 regulates the inflammatory response of neutrophils and macrophages through suppression of COX-2 mediated PGE2 production. This could be a potential drug for treating many inflammatory immune disorders.
Netrin-1; inflammation; cyclooxygenase; PGE2
Podocytes are specialized cells that contribute critically to the normal structure and function of the glomerular filtration barrier. Their depletion plays an important role in the pathogenesis of glomerulosclerosis. Here, we report generation of a genetic model of conditional podocyte ablation and regeneration in zebrafish using a bacterial nitroreductase strategy to convert a prodrug, Metronidazole, into a cytotoxic metabolite. A transgenic zebrafish line was generated that expresses a green fluorescence protein (GFP) and the nitroreductase fusion protein under the control of the podocin promoter Tg(podocin:nitroreductase-GFP). Treatment of these transgenic zebrafish with Metronidazole results in podocyte apoptosis, a loss of nephrin and podocin expression, foot process effacement, and a leaky glomerular filtration barrier. Following Metronidazole washout, proliferating cells were detected in the glomeruli of recovering transgenic fish with a restoration of nitroreductase-GFP fluorescence, nephrin and podocin expression, a reestablishment of normal foot process architecture and glomerular barrier function. Thus, our studies show that zebrafish podocytes are capable of regenerating following depletion and establish the Tg(podocin:NTR-GFP) fish as a new model to study podocyte injury and repair.
Podocyte; nitroreductase; zebrafish; ablation; kidney regeneration
Diabetic nephropathy is the leading cause of end stage renal disease. The urinary albumin to creatinine ratio is used as a predictor for the development of nephropathy but it is neither sensitive nor specific. Here we used liquid chromatography/mass spectrometry on urine of eight normoalbuminuric patients with type 2 diabetes from the VA Diabetes Trial to identify candidate markers for loss of renal function. Initial verification of 7 markers (agrin, haptoglobin, mannan-binding lectin serine protease 2, LAMP-2, angiotensinogen, NGAL and uromodulin) in the urine of an additional 30 patients showed that haptoglobin was the best predictor of early renal functional decline. We then measured this in the urine of 204 patients with type 2 diabetes who did not yet have significant kidney disease (eGFR stage 2 or better and an albumin to creatinine ratio less than 300 mg/g). In comparing the highest to lowest tertile, the odds ratio for having early renal function decline was 2.70 (CI 1.15, 6.32) using the haptoglobin to creatinine ratio compared to 2.50 (CI 1.14, 5.48) using the albumin to creatinine ratio after adjusting for treatment group and use of ACE inhibitors. Addition of the haptoglobin to creatinine ratio to a model using the albumin to creatinine ratio to predict early renal function decline resulted in improved predictive performance. Thus, the haptoglobin to creatinine ratio may be useful to predict patients with type 2 diabetes at risk of nephropathy prior to the development of macroalbuminuria or reduced GFR.
Diabetes; diabetic nephropathy; type 2 diabetes; urine; biological markers; chronic kidney disease
Chronic kidney disease (CKD) impairs muscle protein metabolism leading to muscle atrophy, and exercise can counteract this muscle wasting. Here we evaluated how resistance exercise (muscle overload) and endurance training (treadmill running) affect CKD-induced abnormalities in muscle protein metabolism and progenitor cell function using mouse plantaris muscle. Both exercise models blunted the increase in disease-induced muscle proteolysis and improved phosphorylation of Akt and the forkhead transcription factor FoxO1. Muscle overloading, but not treadmill running, corrected protein synthesis and levels of mediators of protein synthesis such as phosphorylated mTOR and p70S6K in the muscles of mice with CKD. In these mice, muscle overload, but not treadmill, running, increased muscle progenitor cell number and activity as measured by the amounts of MyoD, myogenin, and eMyHC mRNAs. Muscle overload not only increased plantaris weight and reduced muscle proteolysis but also corrected intracellular signals regulating protein and progenitor cell function in mice with CKD. Treadmill running corrects muscle proteolysis but not protein synthesis or progenitor cell function. Our results provide a basis for evaluating different types of exercise on muscle atrophy in patients with chronic kidney disease.
chronic kidney disease; exercise; metabolism; nutrition; signaling
Congenital abnormalities of the kidney and urinary tract (CAKUT) constitute the most frequent cause of chronic kidney disease in children, accounting for ~50% of all cases. Although many forms of CAKUT are likely caused by single-gene defects, only few causative genes have been identified. To identify new causative genes many candidate genes need to be analyzed due to the broad genetic locus heterogeneity of CAKUT. We therefore applied our newly developed approach of DNA pooling with consecutive massively parallel exon resequencing to overcome this problem. We pooled DNA of 20 individuals and amplified by PCR all 313 exons of 30 CAKUT candidate genes. PCR products were then subjected to massively parallel exon resequencing. Mutation carriers were identified using Sanger sequencing. We repeated the experiment to cover 40 patients in total (29 with unilateral renal agenesis and 11 with other CAKUT phenotypes). We detected 5 heterozygous missense mutations in 2 candidate genes that were not previously implicated in non-syndromic CAKUT in humans, 4 mutations in the FRAS1 gene and 1 in FREM2. All mutations were absent from 96 healthy control individuals and had a PolyPhen score of >1.4 (“possibly damaging”). Recessive truncating mutations in FRAS1 and FREM2 were known to cause Fraser syndrome in humans and mice, whereas a phenotype in heterozygous carriers has not been described. We hereby identify heterozygous missense mutations in FRAS1 and FREM2 as a new cause of non-syndromic CAKUT in human.
Steroid-free immunosuppression in kidney transplantation has been gaining popularity over the past decade, as documented by a continuous and steady rise in the number of kidney transplant patients discharged on steroid-free regimens. This increased interest in steroid-free immunosuppression is fueled by the recognition that half of transplant loss is related to patient death due to cardiovascular disease and/or infectious complications and that the long-term use of steroids contributes to such elevated cardiovascular morbidity and mortality. The availability of newer and more potent immunosuppressive agents has furthered such interest. Many clinical trials over the past two decades have demonstrated the feasibility of steroid-free regimens, at the expense of a slight increase in the rate of acute rejection, which is an important end point in any clinical trial of relatively short duration. The largest epidemiological study to date has reassured the transplant community that the selective use of steroid-free immunosuppression in kidney transplant patients provides no inferior outcome in patient and graft survival at intermediate term. Steroid-free regimens have the potential to improve cardiovascular risk profile. The challenges that remain are to identify the subset of kidney transplant patients who may not benefit from steroid-free immunosuppression and to demonstrate the survival advantage of steroid-free immunosuppresion in suitable kidney transplant candidates.
cardiovascular risk; graft survival; kidney transplant; new-onset diabetes; patient survival; steroid-free immunosuppression
Renal cysts, pain and hematuria are common presentations of autosomal dominant polycystic kidney disease (ADPKD) in children. Renal function, however, is typically preserved in these patients despite increased renal volume. Since angiogenesis has been implicated in promotion of renal cyst growth in ADPKD we measured the serum level of various angiogenic factors and early renal structural changes and cardiovascular parameters in 71 patients with ADPKD with a mean age of 16 years. Renal structure and left ventricular mass index were measured by magnetic resonance imaging or by echocardiogram. Renal function was assessed by creatinine clearance, and urinary protein excretion. Serum growth factor levels were measured by enzyme-linked immunosorbent assay. Because of skewed distributions, the various parameters are reported as log10. Serum Log10 vascular endothelial growth factor was positively correlated with renal and cardiac structure, but negatively correlated with creatinine clearance. Serum angiopoietin 1 levels significantly correlated with structural change in both the kidney and the heart and with urinary protein. Thus, the correlation between angiogenic growth factors with both renal and cardiac disease severity is compatible with a possible role for angiogenesis in the early progression of disease in ADPKD.
In children with idiopathic nephrotic syndrome rituximab can maintain short-term remission with withdrawal of prednisone and calcineurin-inhibitors. Long-term effects including number of repeated infusions to maintain remission are unknown.
We treated with rituximab 46 consecutive children with idiopathic nephrotic syndrome lasting for at least one year (6.3±4.1 years), who were maintained in remission with oral prednisone and calcineurin inhibitors. They received 1–5 rituximab courses during a median follow-up of three years (range 1–5). Oral agents were tapered after each infusion, and completely withdrawn within 45 days.
Rituximab was well tolerated. Six-month probabilities of remission were 48% after the first infusion and 37% after subsequent infusions. One- and two-year-remission probabilities were respectively 20% and 10%. Median time intervals between complete oral-agent withdrawal and relapse were 5.6 and 8.5 months respectively following the first and subsequent courses. Time to reconstitution of CD20 cells correlated with the duration of remission, but was not associated with variation in FcyR, CD20 or SMPDL-3B polymorphisms. Podocyte Src phosphorylation was normal.
Rituximab can be safely and repeatedly used as prednisone and calcineurin-inhibitor-sparing therapy in a considerable proportion of children with dependent forms of idiopathic nephrotic syndrome. Further research is needed to identify patients who will benefit most from rituximab therapy.
Bardoxolone methyl, a synthetic triterpenoid, improves the estimated glomerular filtration rate (GFR) in patients with chornic kidney disease and type 2 diabetes. Since the contractile activity of mesangial cells may influence glomerular filtration, we evaluated the effect of the synthetic triterpenoid RTA405 with structural similarity to bardoxolone methyl, on GFR in rats and on mesangial cell contractility in freshly isolated glomeruli. In rats, RTA 405 increased basal GFR, assessed by inulin clearance, and attenuated the angiotensin II-induced decline in GFR. RTA 405 increased the filtration fraction, but did not affect arterial blood pressure or renal plasma flow. Glomeruli from RTA 405-treated rats were resistant to angiotensin II-induced volume reduction ex vivo. In cultured mesangial cells, angiotensin II-stimulated contraction was attenuated by RTA 405, in a dose- and time-dependent fashion. Further, Nrf2 targeted gene transcription (regulates antioxidant, anti-inflammatory, and cytoprotective responses) in mesangial cells was associated with decreased basal and reduced angiotensin II-stimulated hydrogen peroxide and calcium ion levels. These mechanisms contribute to the GFR increase that occurs following treatment with RTA 405 in rats and may underlie the effect of bardoxolone methyl on the estimated GFR in patients.
Novel biomarkers may improve our ability to predict which patients with chronic kidney disease (CKD) are at higher risk for progressive loss of renal function. Here we assessed the performance of urine neutrophil gelatinase-associated lipocalin (NGAL) for outcome prediction in a diverse cohort of 3386 patients with CKD in the CRIC study. In this cohort, the baseline mean estimated glomerular filtration rate (eGFR) was 42.4 ml/min/1.73m2; the median 24-hour urine protein was 0.2 gm/day; and the median urine NGAL concentration was 17.2 ng/mL. Over an average follow-up of 3.2 years, there were 689 cases in which the eGFR was decreased by half or incident end-stage renal disease developed. Even after accounting for eGFR, proteinuria and other known CKD progression risk factors, urine NGAL remained a significant independent risk factor (Cox model hazard ratio 1.70 highest to lowest quartile). The association between baseline urine NGAL levels and risk of CKD progression was strongest in the first two years of biomarker measurement. Within this time frame, adding urine NGAL to a model which included eGFR, proteinuria and other CKD progression risk factors led to net reclassification improvement of 24.7%; but the C-statistic remained nearly identical. Thus, while urine NGAL was an independent risk factor of progression among patients with established CKD of diverse etiology, it did not substantially improve prediction of outcome events.
Renal K+ excretion is increased rapidly following dietary K+ intake, but the underlying molecular mechanisms are largely unknown. Sorensen and colleagues show that K+ intake in mice provoked rapid and near complete dephosphorylation of the renal distal convoluted tubule NaCl cotransporter, temporally associated with increases in both Na+ and K+ excretion. This response was independent of aldosterone and may be a crucial component of the acute homeostatic adaptation of the kidney to K+ intake.
20-Hydroxyeicosatetraenoic acid (20-HETE) production is increased in ischemic kidney tissue and may contribute to ischemia/reperfusion (I/R) injury by mediating vasoconstriction and inflammation. To test this hypothesis, uninephrectomized male Lewis rats were exposed to warm ischemia following pretreatment with either an inhibitor of 20-HETE synthesis (HET0016), an antagonist (20-hydroxyeicosa-6(Z),15(Z)-dienoic acid), an agonist (20-hydroxyeicosa-5(Z),14(Z)-dienoic acid), or vehicle via the renal artery and the kidneys were examined 2 days after reperfusion. Pretreatment with either the inhibitor or the antagonist attenuated I/R-induced renal dysfunction as shown by improved creatinine clearance and decreased plasma urea levels, compared to controls. The inhibitor and antagonist also markedly reduced tubular lesion scores, inflammatory cell infiltration, and tubular epithelial cell apoptosis. Administering the antagonist accelerated the recovery of medullary perfusion, as well as renal medullary and cortical re-oxygenation, during the early reperfusion phase. In contrast, the agonist did not improve renal injury and reversed the beneficial effect of the inhibitor. Thus, 20-HETE generation and its action mediated kidney injury due to I/R. Whether or not these effects are clinically important will need to be tested in appropriate human studies.
acute kidney injury; eicosanoids; ischemia/reperfusion
The guanine nucleotide exchange factor C3G, in complex with the adaptor protein CrkII, mediates GTP loading of small GTPases Rap1 and R-Ras. Hence, C3G facilitates the activation of downstream signaling pathways, shown to be important in glomerulonephritis (GN). We evaluated glomerular expression of C3G in an experimental model of accelerated anti-GBM antibody induced GN. C3G expression (assessed by western blotting) was upregulated in glomeruli after induction of GN and was prominent in glomerular epithelial cells (assessed by immunostaining). In order to examine the consequences of upregulation of C3G expression in glomerular epithelial cells (GEC), we used adenovirus mediated gene transfer of C3G into cultured GEC and analyzed GTP-loading (activation) of Rap1 and R-Ras. Whereas activation of Rap1 was not affected by C3G, overexpression of C3G in GEC decreased the basal level of GTP-bound R-Ras and enhanced activation of R-Ras in response to endothelin. Furthermore C3G overexpression led to significant reduction in cultured GEC spreading and augmented cell migration accompanied by decreased E-cadherin and podocine expression. Taken together, these data represent the first report of pathologic renal C3G overexpression and suggest that it is involved in modulation of GEC morphology and behavior.
C3G; glomerular epithelial cells; endothelin-1; small GTPases; cell spreading; migration; E-cadherin; glomerulonephritis