Michal Herman-Edelstein

E-Cadherin Expression Is Regulated by miR-192/215 by a Mechanism That Is Independent of the Profibrotic Effects of Transforming Growth Factor-β

OBJECTIVE Increased deposition of extracellular matrix (ECM) within the kidney is driven by profibrotic mediators including transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF). We investigated whether some of their effects may be mediated through changes in expression of certain microRNAs (miRNAs). RESEARCH DESIGN AND METHODS Proximal tubular cells, primary rat mesangial cells, and human podocytes were analyzed for changes in the expression of key genes, ECM proteins, and miRNA after exposure to TGF-β (1–10 ng/μl). Tubular cells were also infected with CTGF-adenovirus. Kidneys from diabetic apoE mice were also analyzed for changes in gene expression and miRNA levels. RESULTS TGF-β treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells. TGF-β treatment also modulated the expression of certain miRNAs, including decreased expression of miR-192/215 in tubular cells, mesangial cells, which are also decreased in diabetic kidney. Ectopic expression of miR-192/215 increased E-cadherin levels via repressed translation of ZEB2 mRNA, in the presence and absence of TGF-β, as demonstrated by a ZEB2 3′-untranslated region luciferase reporter assay. However, ectopic expression of miR-192/215 did not affect the expression of matrix proteins or their induction by TGF-β. In contrast, CTGF increased miR-192/215 levels, causing a decrease in ZEB2, and consequently increased E-cadherin mRNA. CONCLUSIONS These data demonstrate the linking role of miRNA-192/215 and ZEB2 in TGF-β/CTGF–mediated changes in E-cadherin expression. These changes appear to occur independently of augmentation of matrix protein synthesis, suggesting that a multistep EMT program is not necessary for fibrogenesis to occur.

Altered Renal Lipid Metabolism and Renal Lipid Accumulation in Human Diabetic Nephropathy

Animal models link ectopic lipid accumulation to renal dysfunction, but whether this process occurs in the human kidney is uncertain. To this end, we investigated whether altered renal TG and cholesterol metabolism results in lipid accumulation in human diabetic nephropathy (DN). Lipid staining and the expression of lipid metabolism genes were studied in kidney biopsies of patients with diagnosed DN (n = 34), and compared with normal kidneys (n = 12). We observed heavy lipid deposition and increased intracellular lipid droplets. Lipid deposition was associated with dysregulation of lipid metabolism genes. Fatty acid β-oxidation pathways including PPAR-α, carnitine palmitoyltransferase 1, acyl-CoA oxidase, and L-FABP were downregulated. Downregulation of renal lipoprotein lipase, which hydrolyzes circulating TGs, was associated with increased expression of angiopoietin-like protein 4. Cholesterol uptake receptor expression, including LDL receptors, oxidized LDL receptors, and acetylated LDL receptors, was significantly increased, while there was downregulation of genes effecting cholesterol efflux, including ABCA1, ABCG1, and apoE. There was a highly significant correlation between glomerular filtration rate, inflammation, and lipid metabolism genes, supporting a possible role of abnormal lipid metabolism in the pathogenesis of DN. These data suggest that renal lipid metabolism may serve as a target for specific therapies aimed at slowing the progression of glomerulosclerosis.

Dedifferentiation of Immortalized Human Podocytes in Response to Transforming Growth Factor-β: A Model for Diabetic Podocytopathy

OBJECTIVE Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis. RESEARCH DESIGN AND METHODS Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes. RESULTS Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α−smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes. CONCLUSIONS In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature podocytes into the cell cycle, which results in enhanced proliferation and apoptosis. These “pathoadaptive” changes are seen early in the diabetic glomerulus and ultimately contribute to albuminuria, glomerulosclerosis, and podocytopenia.

Obesity-related glomerulopathy: clinical and pathologic characteristics and pathogenesis

The prevalence of obesity-related glomerulopathy is increasing in parallel with the worldwide obesity epidemic. Glomerular hypertrophy and adaptive focal segmental glomerulosclerosis define the condition pathologically. The glomerulus enlarges in response to obesity-induced increases in glomerular filtration rate, renal plasma flow, filtration fraction and tubular sodium reabsorption. Normal insulin/phosphatidylinositol 3-kinase/Akt and mTOR signalling are critical for podocyte hypertrophy and adaptation. Adipokines and ectopic lipid accumulation in the kidney promote insulin resistance of podocytes and maladaptive responses to cope with the mechanical forces of renal hyperfiltration. Although most patients have stable or slowly progressive proteinuria, up to one-third develop progressive renal failure and end-stage renal disease. Renin–angiotensin–aldosterone blockade is effective in the short-term but weight loss by hypocaloric diet or bariatric surgery has induced more consistent and dramatic antiproteinuric effects and reversal of hyperfiltration. Altered fatty acid and cholesterol metabolism are increasingly recognized as key mediators of renal lipid accumulation, inflammation, oxidative stress and fibrosis. Newer therapies directed to lipid metabolism, including SREBP antagonists, PPARα agonists, FXR and TGR5 agonists, and LXR agonists, hold therapeutic promise.

miR-21 promotes renal fibrosis in diabetic nephropathy by targeting PTEN and SMAD7.

The cytokine transforming growth factor (TGF)-β1 plays a central role in diabetic nephropathy (DN) with data implicating the miRNA (miR) miR-21 as a key modulator of its prosclerotic actions. In the present study, we demonstrate data indicating that miR-21 up-regulation positively correlates with the severity of fibrosis and rate of decline in renal function in human DN. Furthermore, concomitant analyses of various models of fibrotic renal disease and experimental DN, confirm tubular miR-21 up-regulation. The fibrotic changes associated with increased miR-21 levels are proposed to include the regulation of TGF-β1-mediated mothers against decapentaplegic homolog 3 (SMAD3)- and phosphoinositide 3-kinase (PI3K)-dependent signalling pathways via co-ordinated repression of mothers against decapentaplegic homolog 7 (SMAD7) and phosphatase and tensin homologue (PTEN) respectively. This represents a previously uncharacterized interaction axis between miR-21 and PTEN-SMAD7. Targeting of these proteins by miR-21 resulted in de-repression of the respective pathways as reflected by increases in SMAD3 and V-Akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation. Many of the changes typically induced by TGF-β1, including phosphorylation of signalling mediators, were further enhanced by miR-21. Collectively, these data present a unified model for a key role for miR-21 in the regulation of renal tubular extracellular matrix (ECM) synthesis and accumulation and provide important insights into the molecular pathways implicated in the progression of DN.

SGLT2 Protein Expression Is Increased in Human Diabetic Nephropathy SGLT2 PROTEIN INHIBITION DECREASES RENAL LIPID ACCUMULATION, INFLAMMATION, AND THE DEVELOPMENT OF NEPHROPATHY IN DIABETIC MICE

There is very limited human renal sodium gradient-dependent glucose transporter protein (SGLT2) mRNA and protein expression data reported in the literature. The first aim of this study was to determine SGLT2 mRNA and protein levels in human and animal models of diabetic nephropathy. We have found that the expression of SGLT2 mRNA and protein is increased in renal biopsies from human subjects with diabetic nephropathy. This is in contrast to db-db mice that had no changes in renal SGLT2 protein expression. Furthermore, the effect of SGLT2 inhibition on renal lipid content and inflammation is not known. The second aim of this study was to determine the potential mechanisms of beneficial effects of SGLT2 inhibition in the progression of diabetic renal disease. We treated db/db mice with a selective SGLT2 inhibitor JNJ 39933673. We found that SGLT2 inhibition caused marked decreases in systolic blood pressure, kidney weight/body weight ratio, urinary albumin, and urinary thiobarbituric acid-reacting substances. SGLT2 inhibition prevented renal lipid accumulation via inhibition of carbohydrate-responsive element-binding protein-β, pyruvate kinase L, SCD-1, and DGAT1, key transcriptional factors and enzymes that mediate fatty acid and triglyceride synthesis. SGLT2 inhibition also prevented inflammation via inhibition of CD68 macrophage accumulation and expression of p65, TLR4, MCP-1, and osteopontin. These effects were associated with reduced mesangial expansion, accumulation of the extracellular matrix proteins fibronectin and type IV collagen, and loss of podocyte markers WT1 and synaptopodin, as determined by immunofluorescence microscopy. In summary, our study showed that SGLT2 inhibition modulates renal lipid metabolism and inflammation and prevents the development of nephropathy in db/db mice.

Proximal Tubular Hypertrophy and Enlarged Glomerular and Proximal Tubular Urinary Space in Obese Subjects with Proteinuria

Background Obesity is associated with glomerular hyperfiltration, increased proximal tubular sodium reabsorption, glomerular enlargement and renal hypertrophy. A single experimental study reported an increased glomerular urinary space in obese dogs. Whether proximal tubular volume is increased in obese subjects and whether their glomerular and tubular urinary spaces are enlarged is unknown. Objective To determine whether proximal tubules and glomerular and tubular urinary space are enlarged in obese subjects with proteinuria and glomerular hyperfiltration. Methods Kidney biopsies from 11 non-diabetic obese with proteinuria and 14 non-diabetic lean patients with a creatinine clearance above 50 ml/min and with mild or no interstitial fibrosis were retrospectively analyzed using morphometric methods. The cross-sectional area of the proximal tubular epithelium and lumen, the volume of the glomerular tuft and of Bowman’s space and the nuclei number per tubular profile were estimated. Results Creatinine clearance was higher in the obese than in the lean group (P=0.03). Proteinuria was similarly increased in both groups. Compared to the lean group, the obese group displayed a 104% higher glomerular tuft volume (P=0.001), a 94% higher Bowman’s space volume (P=0.003), a 33% higher cross-sectional area of the proximal tubular epithelium (P=0.02) and a 54% higher cross-sectional area of the proximal tubular lumen (P=0.01). The nuclei number per proximal tubular profile was similar in both groups, suggesting that the increase in tubular volume is due to hypertrophy and not to hyperplasia. Conclusions Obesity-related glomerular hyperfiltration is associated with proximal tubular epithelial hypertrophy and increased glomerular and tubular urinary space volume in subjects with proteinuria. The expanded glomerular and urinary space is probably a direct consequence of glomerular hyperfiltration. These effects may be involved in the pathogenesis of obesity-related renal disease.

Regression of left ventricular hypertrophy in patients with primary aldosteronism/low-renin hypertension on low-dose spironolactone

BACKGROUND The incidence of left ventricular hypertrophy (LVH) in primary aldosteronism (PA) is higher than in essential hypertension. LVH is an independent cardiovascular risk factor. Treatment of PA with mineralocorticoid receptor blockers (MRBs) improves LVH. Previous studies included relatively small groups, low incidence of LVH and used high MRB dose. We tested the hypothesis that long-term regression of LVH in PA/low-renin hypertension may be achieved with low-dose MRB. METHODS Forty-eight patients (male/female 28/20, age 61.4 years, range 47-84) had PA (low renin, high aldosterone and high aldosterone/renin ratio, n=24) or low-renin hypertension (low renin, normal aldosterone and high aldosterone/renin ratio, n=24). All had either LVH or concentric remodelling. All had an echocardiogram both at baseline and at 1 year after the initiation of spironolactone. A subgroup of 29 patients had an echocardiogram at baseline, 1 year (range 0.5-1.5) and 3 years (range 1.8-7). RESULTS At baseline, spironolactone was commenced in all patients. The dose was 33.3±13.7 and 29.0±11.7 mg/day at 1 year and 3 years, respectively. A total of 73% of the patients received ≤37.5 mg/day. Introduction of spironolactone enabled the reduction of other antihypertensive medications (from 2.6±1.2 to 1.5±1.0 at 1 year). At 1 year, systolic and diastolic blood pressure decreased (149.3±14.1 to 126.2±12.0 mmHg, P<0.001, and 88.2±9.8 to 78.3±7.1 mmHg, P<0.001, respectively). At baseline, LVH was present in 39 of the 48 (81%) patients, and concentric remodelling, i.e. increased relative wall thickness (RWT) with a normal left ventricular mass index (LVMI), in 36 (75%). At 1 year, LVMI decreased in 44 of the 48 (92%) patients (142.9±25.4 versus 117.7±20.4 g/m2, P<0.001). LVH normalized in 16 of the 39 (41%) patients. RWT normalized in 36% of the patients. The changes in blood pressure and LVMI did not correlate. At 3 years, LVH decreased further and normalized in 57% of the patients. CONCLUSIONS In patients with PA/low-renin hypertension, long-term regression of LVH may be achieved with low-dose MRB.

TGFβ1-dependent podocyte dysfunction.

Purpose of reviewThe glomerular filtration barrier is a unique structure characterized by a specialized framework of podocytes. Transforming growth factor-&bgr;1 (TGF&bgr;1) upregulation occurs in virtually all chronic kidney diseases and is associated with podocyte injury and proteinuria. This review is aimed at describing the latest advances made in the understanding of TGF&bgr;-induced podocyte injury. Recent findingsDuring the past decade, progress has been made in understanding the biology and mechanisms of TGF&bgr;-induced podocyte injury. Most forms of glomerular diseases, including diabetic nephropathy, are associated with increased TGF&bgr;1 signaling and thus TGF&bgr;1 plays a central role in the pathogenesis of podocytopathy. The mechanism of podocyte injury is complex, involving a number of independent and overlapping cellular and molecular pathways. This review will examine these direct and indirect effects of TGF&bgr;1 on podocyte dysregulation as reflected in their growth, differentiation, and motility. SummaryThese new developments in understanding the podocyte response to injury are critical for establishing better therapeutic interventions that target specific pathways, which otherwise could lead to irreversible injury.

Effect of Acetazolamide on Obesity-Induced Glomerular Hyperfiltration: A Randomized Controlled Trial

Aims Obesity is an important risk factor for the development of chronic kidney disease. One of the major factors involved in the pathogenesis of obesity-associated kidney disease is glomerular hyperfiltration. Increasing salt-delivery to the macula densa is expected to decrease glomerular filtration rate (GFR) by activating tubuloglomerular feedback. Acetazolamide, a carbonic anhydrase inhibitor which inhibits salt reabsorption in the proximal tubule, increases distal salt delivery. Its effects on obesity-related glomerular hyperfiltration have not previously been studied. The aim of this investigation was to evaluate whether administration of acetazolamide to obese non diabetic subjects reduces glomerular hyperfiltration. Materials and Methods The study was performed using a randomized double-blind crossover design. Obese non-diabetic men with glomerular hyperfiltration were randomized to receive intravenously either acetazolamide or furosemide at equipotent doses. Twelve subjects received the allocated medications. Two weeks later, the same subjects received the drug which they had not received during the first study. Inulin clearance, p-aminohippuric acid clearance and fractional lithium excretion were measured before and after medications administration. The primary end point was a decrease in GFR, measured as inulin clearance. Results GFR decreased by 21% following acetazolamide and did not decrease following furosemide. Renal vascular resistance increased by 12% following acetazolamide, while it remained unchanged following furosemide administration. Natriuresis increased similarly following acetazolamide and furosemide administration. Sodium balance was similar in both groups. Conclusions Intravenous acetazolamide decreased GFR in obese non-diabetic men with glomerular hyperfiltration. Furosemide, administered at equipotent dose, did not affect GFR, suggesting that acetazolamide reduced glomerular hyperfiltration by activating tubuloglomerular feedback. Trial Registration ClinicalTrials.gov NCT01146288