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As life expectancy continues to rise, age-related diseases are becoming more prevalent. For example, proteinuric glomerular diseases typified by podocyte injury have worse outcomes in the elderly compared with young patients. However, the reasons are not well understood. We hypothesized that injury to nonaged podocytes induces senescence, which in turn augments their aging processes. In primary cultured human podocytes, injury induced by a cytopathic antipodocyte antibody, adriamycin, or puromycin aminonucleoside increased the senescence-related genes CDKN2A (p16INK4a/p14ARF), CDKN2D (p19INK4d), and CDKN1A (p21). Podocyte injury in human kidney organoids was accompanied by increased expression of CDKN2A, CDKN2D, and CDKN1A. In young mice, experimental focal segmental glomerulosclerosis (FSGS) induced by adriamycin and antipodocyte antibody increased the glomerular expression of p16, p21, and senescence-associated β-galactosidase (SA-β-gal). To assess the long-term effects of early podocyte injury-induced senescence, we temporally followed young mice with experimental FSGS through adulthood (12 m of age) and middle age (18 m of age). p16 and Sudan blackstaining were higher at middle age in mice with earlier FSGS compared with age-matched mice that did not get FSGS when young. This was accompanied by lower podocyte density, reduced canonical podocyte protein expression, and increased glomerular scarring. These results are consistent with injury-induced senescence in young podocytes, leading to increased senescence of podocytes by middle age accompanied by lower podocyte lifespan and health span. [ABSTRACT FROM AUTHOR]

Histone protein modifications control fate determination during normal development and dedifferentiation during disease. Here, we set out to determine the extent to which dynamic changes to histones affect the differentiated phenotype of ordinarily quiescent adult glomerular podocytes. To do this, we examined the consequences of shifting the balance of the repressive histone H3 lysine 27 trimethylation (H3K27me3) mark in podocytes. Adriamycin nephrotoxicity and subtotal nephrectomy (SNx) studies indicated that deletion of the histone methylating enzyme EZH2 from podocytes decreased H3K27me3 levels and sensitized mice to glomerular disease. H3K27me3 was enriched at the promoter region of the Notch ligand Jag1 in podocytes, and derepression of Jag1 by EZH2 inhibition or knockdown facilitated podocyte dedifferentiation. Conversely, inhibition of the Jumonji C domain-containing demethylases Jmjd3 and UTX increased the H3K27me3 content of podocytes and attenuated glomerular disease in adriamycin nephrotoxicity, SNx, and diabetes. Podocytes in glomeruli from humans with focal segmental glomerulosclerosis or diabetic nephropathy exhibited diminished H3K27me3 and heightened UTX content. Analogous to human disease, inhibition of Jmjd3 and UTX abated nephropathy progression in mice with established glomerular injury and reduced H3K27me3 levels. Together, these findings indicate that ostensibly stable chromatin modifications can be dynamically regulated in quiescent cells and that epigenetic reprogramming can improve outcomes in glomerular disease by repressing the reactivation of developmental pathways., Introduction The glomerulus is the basic filtering unit of the kidney. Its sieving action is made possible by highly specialized cells, termed podocytes, that reside within it. Arising from the [...]

The effect of ANG II on intracellular pH (pHi) recovery rate and [AT.sub.1] receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin H receptor isoform 1 ([hAT.sub.1]) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 [+ or-] 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 [+ or-] 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II ([10.sup.-9] M) hut not with ANG II ([10.sup.-6] M). Losartan ([10.sup-7] M) and dimethyl-BAPTA-AM ([10.sup.-7] M) decreased significantly the stimulatory effect of ANG II ([10.sup.-9] M) and induced an increase in [Na.sup.+]/[H.sup.+] exchanger 1 (NHE-1) activity with ANG II ([10.sup.-6] M). Immunofluorescence studies indicated that in control situation, the [hAT.sub.1] receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II ([10.sup.-9] M) and internalized with ANG II ([10.sup.-6] M). Losartan ([10.sup.-7] M) induced [hAT.sub.l] translocation to plasma membrane in all studied groups. DimethylBAPTA-AM ([10.sup.-7] M) did not change the effect of ANG II ([10.sup.-9] M) on the hAT1 receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II ([10.sup.-6] M). With ionomycin ([10.sup.-6] M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to [AT.sub.1] receptor and intracellular signaling events related to [AT.sub.1] translocation. cytosolic [Ca.sup.2+] levels; Madin-Darby canine kidney cells; [Na.sup.+]/[H.sup.+] exchanger

Blood glucose-lowering therapies can positively or negatively affect heart function in type 2 diabetes, or they can have neutral effects. Dipeptidyl peptidase 4 (DPP-4) inhibitors lower blood glucose by preventing the proteolytic inactivation of glucagon-like peptide 1 (GLP-1). However, GLP-1 is not the only peptide substrate of DPP-4. Here, we investigated the GLP-1-independent cardiac effects of DPP-4 substrates. Pointing to GLP-1 receptor (GLP-1R)-independent actions, DPP-4 inhibition prevented systolic dysfunction equally in pressure-overloaded wild-type and GLP-1R knockout mice. Likewise, DPP-4 inhibition or the DPP-4 substrates substance P or C-X-C motif chemokine ligand 12 (CXCL12) improved contractile recovery after no-flow ischemia in the hearts of otherwise healthy young adult mice. Either DPP-4 inhibition or CXCL12 increased phosphorylation of the Ca2+ regulatory protein phospholamban (PLN), and CXCL12 directly enhanced cardiomyocyte Ca2+ flux. In contrast, hearts of aged obese diabetic mice (which may better mimic the comorbid patient population) had diminished levels of PLN phosphorylation. In this setting, CXCL12 paradoxically impaired cardiac contractility in a phosphoinositide 3-kinase γ-dependent manner. These findings indicate that the cardiac effects of DPP-4 inhibition primarily occur through GLP-1R-independent processes and that ostensibly beneficial DPP-4 substrates can paradoxically worsen heart function in the presence of comorbid diabetes. [ABSTRACT FROM AUTHOR]

Background/Aims: To assess the possible contribution of the β-adrenergic overstimulation in early stages of renal injury, the present study evaluated, in rats, the effects of the β-adrenoceptor agonist isoproterenol (ISO) on renal function and morphology, as well as the renal mRNA and protein expression of the NADPH oxidase isoform 4 (Nox 4) and subunit p22phox, endoplasmic reticulum (ER) stress, pro-inflammatory, pro-apoptotic and renin-angiotensin system (RAS) components. Methods: Wistar rats received ISO (0.3 mg.kg-1.day-1 s.c.) or vehicle (control) for eight days. At the end of the treatment, food and water intake, urine output and body weight gain were evaluated and renal function studies were performed. Renal tissue was used for the morphological, quantitative PCR and immunohistochemical studies. Results: ISO did not change metabolic parameters or urine output. However it induced a decrease in renal blood flow and an increase in the filtration fraction. These changes were accompanied by increased cortical mRNA and protein expression for the renal oxidative stress components including Nox 4 and p22phox; ER stress, pro-inflamatory, pro-apoptotic as well as RAS components. ISO also induced a significant increase in medullar renin protein expression. Conclusion: These findings support relevant information regarding the contribution of specific β-adrenergic hyperactivity in early stage of renal injury, indicating the reactive oxygen species, ER stress and intrarenal RAS as important factors in this process. [ABSTRACT FROM AUTHOR]

Chronic angiotensin II (ANG II) infusion for 1 or 2 wk leads to progressive hypertension and induces inward hypertrophic remodeling in preglomerular vessels, which is associated with increased renal vascular resistance (RVR) and decreased glomerular perfusion. Considering the ability of preglomerular vessels to exhibit adaptive responses, the present study was performed to evaluate glomerular perfusion and renal function after 6 wk of ANG II infusion. To address this study, male Wistar rats were submitted to sham surgery (control) or osmotic minipump insertion (ANG II 200 ng·kg-1·min-1, 42 days). A group of animals was treated or cotreated with losartan (10 mg·kg-1·day-1), an AT1 receptor antagonist, between days 28 and 42. Chronic ANG II infusion increased systolic blood pressure to 185 ± 4 compared with 108 ± 2 mmHg in control rats. Concomitantly, ANG II-induced hypertension increased intrarenal ANG II level and consequently, preglomerular and glomerular injury. Under this condition, ANG II enhanced the total renal plasma flow (RPF), glomerular filtration rate (GFR), urine flow and induced pressure natriuresis. These changes were accompanied by lower RVR and enlargement of the lumen of interlobular arteries and afferent arterioles, consistent with impairment of renal autoregulatory capability and outward preglomerular remodeling. The glomerular injury culminated with podocyte effacement, albuminuria, tubulointerstitial macrophage infiltration and intrarenal extracellular matrix accumulation. Losartan attenuated most of the effects of ANG II. Our findings provide new information regarding the contribution of ANG II infusion over 2 wk to renal hemodynamics and function via the AT1 receptor. [ABSTRACT FROM AUTHOR]

Aims: Thioredoxin interacting protein (TXNIP), an inhibitor of antioxidant thioredoxin (Trx), is upregulated by hyperglycemia and implicated in pathogenesis of diabetes complications. We evaluated mRNA expressions of genes encoding TXNIP and Trx (TXN) in urinary sediment and peripheral blood mononuclear cells (PBMC) of type 1 diabetes (T1D) patients with different degrees of chronic complications.Methods: qPCR was employed to quantify target genes in urinary sediment (n = 55) and PBMC (n = 161) from patients sorted by presence or absence of diabetic nephropathy (DN), retinopathy, peripheral and cardiovascular neuropathy; 26 healthy controls and 13 patients presenting non-diabetic nephropathy (focal and segmental glomerulosclerosis, FSGS) were also included.Results: Regarding the urinary sediment,TXNIP(but notTXN) expression was higher in T1D (p = 0.0023) and FSGS (p = 0.0027) patientsversuscontrols. Expressions ofTXNIPandTXNwere higher, respectively, in T1D patients withversuswithout DN (p = 0.032) and in those with estimated glomerular filtration rate (eGFR) < 60versus ≥60 mL/min/1.73 m2(p = 0.008). eGFR negatively correlated withTXNIP(p = 0.04,r = −0.28) andTXN(p = 0.04,r = −0.30) expressions. T1D patients who lost ≥5 mL/min/1.73 m2yearly of eGFR presented higher basalTXNIPexpression than those who lost <5 mL/min/1.73 m2yearly after median follow-up of 24 months.TXNIP(p < 0.0001) andTXN(p = 0.002) expressions in PBMC of T1D patients were significantly higher than in controls but no differences were observed between patients with or without chronic complications.Conclusions:TXNIPandTXNare upregulated in urinary sediment of T1D patients with diabetic kidney disease (DKD), but onlyTXNIPexpression is associated with magnitude of eGFR decline. [ABSTRACT FROM AUTHOR]

The role of hyperleptinemia in cardiovascular diseases is well known; however, in the renal tissue, the exact site of leptin’s action has not been established. This study was conducted to assess the effect of leptin treatment for 7 and 28 days on renal function and morphology and the participation of angiotensin II (Ang II), through its AT1 receptor. Rats were divided into four groups: sham, losartan (10 mg/kg/day, s.c.), leptin (0.5 mg/kg/day for the 7 days group and 0.25 mg/kg/day for the 28 days group) and leptin plus losartan. Plasma leptin, Ang II and endothelin 1 (ET-1) levels were measured using an enzymatic immuno assay. The systolic blood pressure (SBP) was evaluated using the tail-cuff method. The renal plasma flow (RPF) and the glomerular filtration rate (GFR) were determined by p-aminohippuric acid and inulin clearance, respectively. Urinary Na+ and K+ levels were also analyzed. Renal morphological analyses, desmin and ED-1 immunostaining were performed. Proteinuria was analyzed by silver staining. mRNA expression of renin-angiotensin system (RAS) components, TNF-α and collagen type III was analyzed by quantitative PCR. Our results showed that leptin treatment increased Ang II plasma levels and progressively increased the SBP, achieving a pre-hypertension state. Rats treated with leptin 7 days showed a normal RPF and GFR, but increased filtration fraction (FF) and natriuresis. However, rats treated with leptin for 28 showed a decrease in the RPF, an increase in the FF and no changes in the GFR or tubular function. Leptin treatment-induced renal injury was demonstrated by: glomerular hypertrophy, increased desmin staining, macrophage infiltration in the renal tissue, TNF-α and collagen type III mRNA expression and proteinuria. In conclusion, our study demonstrated the progressive renal morphological changes in experimental hyperleptinemia and the interaction between leptin and the RAS on these effects. [ABSTRACT FROM AUTHOR]

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca2+/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA1K3R/4E and SB1K3R/4E. Wild type and mutants were transfected into PS120 cells and their activity was examined by H+ flux (JH+). The basal JH+ of wild type was 4.71 ± 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10-12 and 10-9 M) increased the JH+ in wild type and SB. Ang II (10-6 M) increased this parameter only in SA. Ang II (10-9 M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10-6 M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10-7 M), a calcium chelator, suppressed the effect of Ang II (10-9 M) in wild type. With Ang II (10-6 M), Bapta failed to affect wild type or SA, but it increased the JH+ in SB. W13 or calmidazolium chloride (10-5 M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10-9 M) in wild type or SB. With Ang II (10-6 M), W13 or calmidazolium chloride decreased the JH+ in wild type or SA and increased it in SB. Thus, with Ang II (10-12 and 10-9 M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10-6 M), site B is important to maintain its basal activity. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The effect of ANG II on intracellular pH (pHi) recovery rate and AT1 receptor translocation was investigated in transfected MDCK cells. The pHi recovery rate was evaluated by fluorescence microscopy using the fluorescent probe BCECF-AM. The human angiotensin II receptor isoform 1 (hAT1) translocation was analyzed by immunofluorescence and confocal microscope. Our data show that transfected cells in control situation have a pHi recovery rate of 0.219 ± 0.017 pH U/min (n = 11). This value was similar to nontransfected cells [0.211 ± 0.009 pH U/min (n = 12)]. Both values were significantly increased with ANG II (10-9 M) but not with ANG II (10-6 M). Losartan (10-7 M) and dimethyl-BAPTA-AM (10-7 M) decreased significantly the stimulatory effect of ANG II (10-9 M) and induced an increase in Na+/H+ exchanger 1 (NHE-1) activity with ANG II (10-6 M). Immunofluorescence studies indicated that in control situation, the hAT1 receptor was predominantly expressed in cytosol. However, it was translocated to plasma membrane with ANG II (10-9 M) and internalized with ANG II (10-6 M). Losartan (10-7 M) induced hAT1 translocation to plasma membrane in all studied groups. Dimethyl- BAPTA-AM (10-7 M) did not change the effect of ANG II (10-9 M) on the hAT1 receptor distribution but induced its accumulation at plasma membrane in cells treated with ANG II (10-6 M). With ionomycin (10-6 M), the receptor was accumulated in cytosol. The results indicate that, in MDCK cells, the effect of ANG II on NHE-1 activity is associated with ligand binding to AT1 receptor and intracellular signaling events related to AT1 translocation. [ABSTRACT FROM AUTHOR]