HK-2 cell response to TGF-ß highly depends on cell culture medium formulations.

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-ß) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-ß-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-ß1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-ß1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

Pioglitazone Protects Tubular Epithelial Cells during Kidney Fibrosis by Attenuating miRNA Dysregulation and Autophagy Dysfunction Induced by TGF-ß.

Excessive renal TGF-ß production and pro-fibrotic miRNAs are important drivers of kidney fibrosis that lack any efficient treatment. Dysfunctional autophagy might play an important role in the pathogenesis. We aimed to study the yet unknown effects of peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone (Pio) on renal autophagy and miRNA dysregulation during fibrosis. Mouse primary tubular epithelial cells (PTEC) were isolated, pre-treated with 5 µM pioglitazone, and then stimulated with 10 ng/mL TGF-ß1 for 24 h. Male 10-week-old C57Bl6 control (CTL) and TGF-ß overexpressing mice were fed with regular chow (TGF) or Pio-containing chow (20 mg/kg/day) for 5 weeks (TGF + Pio). PTEC and kidneys were evaluated for mRNA and protein expression. In PTEC, pioglitazone attenuated (p < 0.05) the TGF-ß-induced up-regulation of Col1a1 (1.4-fold), Tgfb1 (2.2-fold), Ctgf (1.5-fold), Egr2 (2.5-fold) mRNAs, miR-130a (1.6-fold), and miR-199a (1.5-fold), inhibited epithelial-to-mesenchymal transition, and rescued autophagy function. In TGF mice, pioglitazone greatly improved kidney fibrosis and related dysfunctional autophagy (increased LC3-II/I ratio and reduced SQSTM1 protein content (p < 0.05)). These were accompanied by 5-fold, 3-fold, 12-fold, and 2-fold suppression (p < 0.05) of renal Ccl2, Il6, C3, and Lgals3 mRNA expression, respectively. Our results implicate that pioglitazone counteracts multiple pro-fibrotic processes in the kidney, including autophagy dysfunction and miRNA dysregulation.

Zymosan Particle-Induced Hemodynamic, Cytokine and Blood Cell Changes in Pigs: An Innate Immune Stimulation Model with Relevance to Cytokine Storm Syndrome and Severe COVID-19.

Hemodynamic disturbance, a rise in neutrophil-to-lymphocyte ratio (NLR) and release of inflammatory cytokines into blood, is a bad prognostic indicator in severe COVID-19 and other diseases involving cytokine storm syndrome (CSS). The purpose of this study was to explore if zymosan, a known stimulator of the innate immune system, could reproduce these changes in pigs. Pigs were instrumented for hemodynamic analysis and, after i.v. administration of zymosan, serial blood samples were taken to measure blood cell changes, cytokine gene transcription in PBMC and blood levels of inflammatory cytokines, using qPCR and ELISA. Zymosan bolus (0.1 mg/kg) elicited transient hemodynamic disturbance within minutes without detectable cytokine or blood cell changes. In contrast, infusion of 1 mg/kg zymosan triggered maximal pulmonary hypertension with tachycardia, lasting for 30 min. This was followed by a transient granulopenia and then, up to 6 h, major granulocytosis, resulting in a 3-4-fold increase in NLR. These changes were paralleled by massive transcription and/or rise in IL-6, TNF-alpha, CCL-2, CXCL-10, and IL-1RA in blood. There was significant correlation between lymphopenia and IL-6 gene expression. We conclude that the presented model may enable mechanistic studies on late-stage COVID-19 and CSS, as well as streamlined drug testing against these conditions.

Susceptibility to kidney fibrosis in mice is associated with early growth response-2 protein and tissue inhibitor of metalloproteinase-1 expression.

Patients with chronic kidney disease and experimental animal models of kidney fibrosis manifest diverse progression rates. Genetic susceptibility may contribute to this diversity, but the causes remain largely unknown. We have previously described kidney fibrosis with a mild or severe phenotype in mice expressing transforming growth factor-beta1 (TGF-ß1) under the control of a mouse albumin promoter (Alb/TGF-ß1), on a mixed genetic background with CBAxC57Bl6 mice. Here, we aimed to examine how genetic background may influence kidney fibrosis in TGF-ß1 transgenic mice, and in the unilateral ureteral obstruction (UUO) and subtotal nephrectomy (SNX) mouse models. Congenic C57Bl6(B6)-TGFß and CBAxB6-TGFß (F1) transgenic mice were generated and survival, proteinuria, kidney histology, transcriptome and protein expressions were analyzed. We investigated the kidneys of B6 and CBA mice subjected to UUO and SNX, and the effects of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) neutralization on the fibrotic process. CBAxB6-TGFß mice developed severe kidney fibrosis and premature death, while B6-TGF-ß mice had mild fibrosis and prolonged survival. Kidney early growth response factor-2 (EGR2) and TIMP-1 expression were induced only in CBAxB6-TGFß mice. Similar strain-dependent early changes in EGR2 and TIMP-1 of mice subjected to UUO or SNX were observed. TIMP-1 neutralization in vivo hindered fibrosis both in transgenic mice and the SNX model. EGR2 over-expression in cultured HEK293 cells induced TIMP-1 while EGR2 silencing hindered TGF-ß induced TIMP-1 production in HK-2 cells and ureteral obstructed kidneys. Finally, EGR2 and TIMP1 was increased in human kidneys manifesting focal segmental glomerulosclerosis suggesting a correlation between animal studies and patient clinical settings. Thus, our observations demonstrate a strong relationship between genetic background and the progression of kidney fibrosis, which might involve early altered EGR2 and TIMP-1 response, but the relationship to patient genetics remains to be explored.

PPARγ and TGFß-Major Regulators of Metabolism, Inflammation, and Fibrosis in the Lungs and Kidneys.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a type II nuclear receptor, initially recognized in adipose tissue for its role in fatty acid storage and glucose metabolism. It promotes lipid uptake and adipogenesis by increasing insulin sensitivity and adiponectin release. Later, PPARγ was implicated in cardiac development and in critical conditions such as pulmonary arterial hypertension (PAH) and kidney failure. Recently, a cluster of different papers linked PPARγ signaling with another superfamily, the transforming growth factor beta (TGFß), and its receptors, all of which play a major role in PAH and kidney failure. TGFß is a multifunctional cytokine that drives inflammation, fibrosis, and cell differentiation while PPARγ activation reverses these adverse events in many models. Such opposite biological effects emphasize the delicate balance and complex crosstalk between PPARγ and TGFß. Based on solid experimental and clinical evidence, the present review summarizes connections and their implications for PAH and kidney failure, highlighting the similarities and differences between lung and kidney mechanisms as well as discussing the therapeutic potential of PPARγ agonist pioglitazone.

PPARγ is a gatekeeper for extracellular matrix and vascular cell homeostasis: beneficial role in pulmonary hypertension and renal/cardiac/pulmonary fibrosis.

PURPOSE OF REVIEW: Pulmonary arterial hypertension (PAH) is characterized by pulmonary arterial endothelial cell (PAEC) dysfunction and apoptosis, pulmonary arterial smooth muscle cell (PASMC) proliferation, inflammation, vasoconstriction, and metabolic disturbances that include disrupted bone morphogenetic protein receptor (BMPR2)-peroxisome proliferator-activated receptor gamma (PPARγ) axis and DNA damage. Activation of PPARγ improves many of these mechanisms, although erroneous reports on potential adverse effects of thiazolidinedione (TZD)-class PPARγ agonists reduced their clinical use in the past decade. Here, we review recent findings in heart, lung, and kidney research related to the pathobiology of vascular remodeling and tissue fibrosis, and also potential therapeutic effects of the PPARγ agonist pioglitazone. RECENT FINDINGS: Independent of its metabolic effects (improved insulin sensitivity and fatty acid handling), PPARγ activation rescues BMPR2 dysfunction, inhibits TGFß/Smad3/CTGF and TGFß/pSTAT3/pFoxO1 pathways, and induces the PPARγ/apoE axis, inhibiting vascular remodeling. PPARγ activation dampens mtDNA damage via PPARγ/UBR5/ATM pathway, improves function of endothelial progenitor cells (EPCs), and decrease renal fibrosis by repressing TGFß/pSTAT3 and TGFß/EGR1. SUMMARY: Pharmacological PPARγ activation improves many hallmarks of PAH, including dysfunction of BMPR2-PPARγ axis, PAEC, PASMC, EPC, mitochondria/metabolism, and inflammation. Recent randomized controlled trials, including IRIS (Insulin Resistance Intervention After Stroke Trial), emphasize the beneficial effects of PPARγ agonists in PAH patients, leading to recent revival for clinical use.

The PPARγ agonist pioglitazone prevents TGF-ß induced renal fibrosis by repressing EGR-1 and STAT3.

BACKGROUND: It has been proposed that peroxisome proliferator-activated receptor-γ (PPARγ) agonists might reduce renal fibrosis, however, several studies had contradictory results. Moreover, the possible interaction of TGF-ß1, PPARγ, and transcription factors in renal fibrosis have not been investigated. We hypothesized that oral pioglitazone treatment would inhibit TGF-ß-driven renal fibrosis and its progression, by modulating profibrotic transcription factors in TGF-ß1 transgenic mice. METHODS: Male C57Bl/6 J mice (control, CTL, n = 14) and TGF-ß overexpressing transgenic mice (TGFß, n = 14, having elevated plasma TGF-ß1 level) were divided in two sets at 10 weeks of age. Mice in the first set were fed with regular rodent chow (CTL and TGFß, n = 7/group). Mice in the second set were fed with chow containing pioglitazone (at a dose of 20 mg/kg/day, CTL + Pio and TGFß+Pio, n = 7/group). After 5 weeks of treatment, blood pressure was assessed and urine samples were collected, and the kidneys were analyzed for histology, mRNA and protein expression. RESULTS: TGF-ß1 induced glomerulosclerosis and tubulointerstitial damage were significantly reduced by pioglitazone. Pioglitazone inhibited renal mRNA expression of all the profibrotic effectors: type-III collagen, TGF-ß1, CTGF and TIMP-1, and alike transcription factors cFos/cJun and protein expression of EGR-1, and STAT3 protein phosphorylation. CONCLUSIONS: Oral administration of PPARγ agonist pioglitazone significantly reduces TGF-ß1-driven renal fibrosis, via the attenuation of EGR-1, STAT3 and AP-1. This implies that PPARγ agonists might be effective in the treatment of chronic kidney disease patients.

The Effect of Combined Treatment with the (Pro)Renin Receptor Blocker HRP and Quinapril in Type 1 Diabetic Rats.

BACKGROUND/AIMS: Diabetic nephropathy remains a major clinical problem. The effects of prorenin might be adverse, but the literature data are controversial. We compared the renal effects of the (pro)renin receptor ((P)RR) blockade and angiotensin converting enzyme (ACE) inhibition on the progression of diabetic nephropathy in rats. METHODS: Diabetes (DM) was induced by ip. streptozotocin administration in adult male Sprague-Dawley rats, followed by eight weeks of treatment with the (P)RR blocker "handle region" decoy peptide (HRP, 0,1 mg/kg/day) or with the ACE inhibitor Quinapril (Q, 50 mg/kg/day) and grouped as follows: 1. Control (n=10); 2. DM (n=8); 3. DM+HRP (n=6); 4. DM+Q (n=10); 5. DM+Q+HRP (n=10). Renal functional parameters, histology and gene expressions were evaluated. RESULTS: HRP reduced glomerulosclerosis and podocyte desmin expression, but did not affect proteinuria and tubular ERK(1/2) phosphorylation. Both Q and Q+HRP treatment reduced proteinuria, glomerular and tubular damage, tubular TGF-ß1 expression and ERK(1/2) phosphorylation to the same extent. CONCLUSION: The effects of HRP were partially beneficial on diabetic kidney lesions as HRP reduced damage but did not improve tubular damage and failed to reduce ERK(1/2) phosphorylation in rats. The combination of HRP with Quinapril had no additive effects over Quinapril monotherapy on the progression of diabetic nephropathy.

Sustained hyperosmolarity increses TGF-ß1 and Egr-1 expression in the rat renal medulla.

BACKGROUND: Although TGF-ß and the transcription factor Egr-1 play an important role in both kidney fibrosis and in response to acute changes of renal medullary osmolarity, their role under sustained hypo- or hyperosmolar conditions has not been elucidated. We investigated the effects of chronic hypertonicity and hypotonicity on the renal medullary TGF-ß and Egr-1 expression. METHODS: Male adult Sprague Dawley rats (n = 6/group) were treated with 15 mg/day furosemide, or the rats were water restricted to 15 ml/200 g body weight per day. Control rats had free access to water and rodent chow. Kidneys were harvested after 5 days of treament. In cultured inner medullary collecting duct (IMCD) cells, osmolarity was increased from 330 mOsm to 900 mOsm over 6 days. Analyses were performed at 330, 600 and 900 mOsm. RESULTS: Urine osmolarity has not changed due to furosemide treatment but increased 2-fold after water restriction (p < 0.05). Gene expression of TGF-ß and Egr-1 increased by 1.9-fold and 7-fold in the hypertonic medulla, respectively (p < 0.05), accompanied by 6-fold and 2-fold increased c-Fos and TIMP-1 expression, respectively (p < 0.05) and positive immunostaining for TGF-ß and Egr-1 (p < 0.05). Similarly, hyperosmolarity led to overexpression of TGF-ß and Egr-1 mRNA in IMCD cells (2.5-fold and 3.5-fold increase from 330 to 900 mOsm, respectively (p < 0.05)) accompanied by significant c-Fos and c-Jun overexpressions (p < 0.01), and increased Col3a1 and Col4a1 mRNA expression. CONCLUSION: We conclude that both TGF-ß and Egr-1 are upregulated by sustained hyperosmolarity in the rat renal medulla, and it favors the expression of extracellular matrix components.

Simple, readily available clinical indices predict early and late mortality among patients with ANCA-associated vasculitis.

BACKGROUND: The early identification of patients with ANCA-associated vasculitis (AAV) who are at increased risk for inferior clinical outcome at the time of diagnosis might help to optimize the immunosuppressive therapy. In this study we wanted to determine the predictive value of simple clinical characteristics, which may be applicable for early risk-stratification of patients with AAV. METHODS: We retrospectively analyzed the outcome of 101 consecutive patients with AAV receiving a protocolized immunosuppressive therapy. Baseline Birmingham Vasculitis Activity Score (BVAS) and non-vasculitic comorbidities were computed, then predictors of early (<90 days) and late (>90 days) mortality, infectious death, relapse and end stage kidney disease (ESKD) were evaluated. RESULTS: The baseline comorbidity score independently predicted early mortality (HR 1.622, CI 1.006-2.614), and showed association with infectious mortality (HR 2.056, CI 1.247-3.392). Patients with BVAS at or above median (=21) had worse early mortality in univariable analysis (HR 3.57, CI 1.039-12.243) (p = 0.031), and had more frequent relapses (p = 0.01) compared to patients with BVAS below median. CONCLUSIONS: Assessing baseline comorbidities, beside clinical indices characterizing the severity and extension of AAV, might help clinicians in risk-stratification of patients. Future prospective studies are needed to investigate whether therapies based on risk-stratification could improve both short term and long term survival.

The soluble guanylate cyclase activator cinaciguat prevents cardiac dysfunction in a rat model of type-1 diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) leads to the development of diabetic cardiomyopathy, which is associated with altered nitric oxide (NO)--soluble guanylate cyclase (sGC)--cyclic guanosine monophosphate (cGMP) signalling. Cardioprotective effects of elevated intracellular cGMP-levels have been described in different heart diseases. In the current study we aimed at investigating the effects of pharmacological activation of sGC in diabetic cardiomyopathy. METHODS: Type-1 DM was induced in rats by streptozotocin. Animals were treated either with the sGC activator cinaciguat (10 mg/kg/day) or with placebo orally for 8 weeks. Left ventricular (LV) pressure-volume (P-V) analysis was used to assess cardiac performance. Additionally, gene expression (qRT-PCR) and protein expression analysis (western blot) were performed. Cardiac structure, markers of fibrotic remodelling and DNA damage were examined by histology, immunohistochemistry and TUNEL assay, respectively. RESULTS: DM was associated with deteriorated cGMP signalling in the myocardium (elevated phosphodiesterase-5 expression, lower cGMP-level and impaired PKG activity). Cardiomyocyte hypertrophy, fibrotic remodelling and DNA fragmentation were present in DM that was associated with impaired LV contractility (preload recruitable stroke work (PRSW): 49.5 ± 3.3 vs. 83.0 ± 5.5 mmHg, P < 0.05) and diastolic function (time constant of LV pressure decay (Tau): 17.3 ± 0.8 vs. 10.3 ± 0.3 ms, P < 0.05). Cinaciguat treatment effectively prevented DM related molecular, histological alterations and significantly improved systolic (PRSW: 66.8 ± 3.6 mmHg) and diastolic (Tau: 14.9 ± 0.6 ms) function. CONCLUSIONS: Cinaciguat prevented structural, molecular alterations and improved cardiac performance of the diabetic heart. Pharmacological activation of sGC might represent a new therapy approach for diabetic cardiomyopathy.

Characterization and role of SCAI during renal fibrosis and epithelial-to-mesenchymal transition.

During progressive tubulointerstitial fibrosis, renal tubular epithelial cells transform into α-smooth muscle actin (SMA)-expressing myofibroblasts via epithelial-to-mesenchymal transition (EMT). SMA expression is regulated by transforming growth factor (TGF)-ß1 and cell contact disruption, through signaling events targeting the serum response factor-myocardin-related transcription factor (MRTF) complex. MRTFs are important regulators of fibrosis, tumor cell invasion, and metastasis. Consistent with the role of MRTFs in tumor progression, suppressor of cancer cell invasion (SCAI) was recently identified as a negative regulator of MRTF. Herein, we studied the role of SCAI in a fibrotic EMT model established on LLC-PK1 cells. SCAI overexpression prevented SMA promoter activation induced by TGF-ß1. When co-expressed, it inhibited the stimulatory effects of MRTF-A, MRTF-B or the constitutive active forms of RhoA, Rac1, or Cdc42 on the SMA promoter. SCAI interfered with TGF-ß1-induced SMA, connective tissue growth factor, and calponin protein expression; it rescued TGF-ß1-induced E-cadherin down-regulation. IHC studies on human kidneys showed that SCAI expression is reduced during fibrosis. Kidneys of diabetic rats and mice with unilateral ureteral obstruction depicted significant loss of SCAI expression. In parallel with the decrease of SCAI protein expression, diabetic rat and mouse kidneys with unilateral ureteral obstruction showed SMA expression, as evidenced by using Western blot analysis. Finally, TGF-ß1 treatment of LLC-PK1 cells attenuated SCAI protein expression. These data suggest that SCAI is a novel transcriptional cofactor that regulates EMT and renal fibrosis.

A Systematic Review and Meta-Analysis of microRNA Profiling Studies in Chronic Kidney Diseases.

Chronic kidney disease (CKD) represents an increasing health burden. Evidence suggests the importance of miRNA in diagnosing CKD, yet the reports are inconsistent. This study aimed to determine novel miRNA biomarkers and potential therapeutic targets from hypothesis-free miRNA profiling studies in human and murine CKDs. Comprehensive literature searches were conducted on five databases. Subgroup analyses of kidney diseases, sample types, disease stages, and species were conducted. A total of 38 human and 12 murine eligible studies were analyzed using Robust Rank Aggregation (RRA) and vote-counting analyses. Gene set enrichment analyses of miRNA signatures in each kidney disease were conducted using DIANA-miRPath v4.0 and MIENTURNET. As a result, top target genes, Gene Ontology terms, the interaction network between miRNA and target genes, and molecular pathways in each kidney disease were identified. According to vote-counting analysis, 145 miRNAs were dysregulated in human kidney diseases, and 32 were dysregulated in murine CKD models. By RRA, miR-26a-5p was significantly reduced in the kidney tissue of Lupus nephritis (LN), while miR-107 was decreased in LN patients' blood samples. In both species, epithelial-mesenchymal transition, Notch, mTOR signaling, apoptosis, G2/M checkpoint, and hypoxia were the most enriched pathways. These miRNA signatures and their target genes must be validated in large patient cohort studies.

Selective phosphodiesterase-5 (PDE-5) inhibitor vardenafil ameliorates renal damage in type 1 diabetic rats by restoring cyclic 3',5' guanosine monophosphate (cGMP) level in podocytes.

BACKGROUND: Diabetic nephropathy (DN) is characterized by podocyte damage and increased phosphodiesterase-5 (PDE-5) activity-exacerbating nitric oxide (NO)-cyclic 3',5' guanosine monophosphate (cGMP) pathway dysfunction. It has been shown that PDE-5 inhibition ameliorates DN. The role of podocytes in this mechanism remains unclear. We investigated how selective PDE-5 inhibition influences podocyte damage in streptozotocin (STZ) diabetic rats. METHODS: Male Sprague-Dawley rats (250-300 g) were injected with STZ and divided into two groups: (i) STZ control (non-treated, STZ, n=6) and (ii) STZ+vardenafil treatment (10 mg/kg/day, STZ-Vard, n=8). Non-diabetic rats served as negative controls (Control, n=7). Following 8 weeks of treatment, immunohistochemical and molecular analysis of the kidneys were performed. RESULTS: Diabetic rats had proteinuria, increased renal transforming growth factor (TGF)-ß1 expression and podocyte damage when compared with controls. Vardenafil treatment resulted in preserved podocyte cGMP levels, less proteinuria, reduced renal TGF-ß1 expression, desmin immunostaining in podocytes and restored both nephrin and podocin mRNA expression. Diabetes led to increased glomerular nitrotyrosine formation and renal neuronal nitric oxide synthase and endothelial nitric oxide synthase mRNA expression, but vardenafil did not influence these parameters. CONCLUSIONS: Our data suggest that a dysfunctional NO-cGMP pathway exacerbates podocyte damage in diabetes. In conclusion, vardenafil treatment preserves podocyte function and reduces glomerular damage, which indicates therapeutic potential in patients with DN.

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice.

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 µg/kg) or paricalcitol (0.1 µg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 µm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-ß1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-ß expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).

Elevated systemic TGF-beta impairs aortic vasomotor function through activation of NADPH oxidase-driven superoxide production and leads to hypertension, myocardial remodeling, and increased plaque formation in apoE(-/-) mice.

The role of circulating, systemic TGF-beta levels in endothelial function is not clear. TGF-beta(1) may cause endothelial dysfunction in apolipoprotein E-deficient (apoE(-/-)) mice via stimulation of reactive oxygen species (ROS) production by the NADPH oxidase (NOX) system and aggravate aortic and heart remodeling and hypertension. Thoracic aorta (TA) were isolated from 4-mo-old control (C57Bl/6), apoE(-/-), TGF-beta(1)-overexpressing (TGFbeta(1)), and crossbred apoE(-/-) x TGFbeta(1) mice. Endothelium-dependent relaxation was measured before and after incubation with apocynin (NOX inhibitor) or superoxide dismutase (SOD; ROS scavenger). Superoxide production within the vessel wall was determined by dihydroethidine staining under confocal microscope. In 8-mo-old mice, aortic and myocardial morphometric changes, plaque formation by en face fat staining, and blood pressure were determined. Serum TGF-beta(1) levels (ELISA) were elevated in TGFbeta(1) mice without downregulation of TGF-beta-I receptor (immunohistochemistry). In the aortic wall, superoxide production was enhanced and NO-dependent relaxation diminished in apoE(-/-) x TGFbeta(1) mice but improved significantly after apocynin or SOD. Myocardial capillary density was reduced, fibrocyte density increased, aortic wall was thicker, combined lesion area was greater, and blood pressure was higher in the apoE(-/-) x TGFbeta vs. C57Bl/6 mice. Our results demonstrate that elevated circulating TGF-beta(1) causes endothelial dysfunction through NOX activation-induced oxidative stress, accelerating atherosclerosis and hypertension in apoE(-/-) mice. These findings may provide a mechanism explaining accelerated atherosclerosis in patients with elevated plasma TGFbeta(1).

The Rowett rat strain is resistant to renal fibrosis.

BACKGROUND: Genetic susceptibility to renal fibrosis may determine the individual rate of progression to renal failure. We aimed to study the progression in Rowett (RO) rats, a strain we found resistant to subtotal nephrectomy (SNX), comparing to Sprague-Dawley (SD) rats, a strain with established sensitivity in a radical ablation/infarction and diet-induced SNX model. METHODS: Eight-week-old male RO (RO-SNX) and SD (SD-SNX, n = 5/group) rats underwent SNX and were kept on high protein and salt diet. Kidney function was monitored and the kidneys were evaluated by histology and immunohistochemistry 5 weeks after SNX. RESULTS: RO-SNX rats had only mild proteinuria and less glomerulosclerosis, accompanied by less fibronectin and TGF-beta staining as compared to SD-SNX rats. Glomerular nitrotyrosine staining was less intense in RO-SNX vs SD-SNX, accompanied by less podocyte damage as demonstrated by desmin staining. CONCLUSION: Our results demonstrate the importance of podocyte damage in glomerulosclerosis and that Rowett rats are protected from renal fibrosis. To our knowledge, this is the first strain of rats with unknown genetic resistance, which makes the strain attractive for studying the genetic background of renal fibrosis.

Preliminary findings on the efficacy of selamectin in the treatment of dogs naturally infected with Dirofilaria repens.

Subcutaneous dirofilariosis caused by Dirofilaria repens is common in dogs and it is an emerging helminthozoonosis in Europe, Asia, Africa and also in Hungary. Macrocyclic lactones are used for preventing the infection; however, their activity against the microfilariae and mature stages of this species is questionable. Selamectin is widely used for the prophylaxis of heartworm (D. immitis) infection. The objective of the present study was to test the microfilaricidal efficacy of the topical formulation of selamectin in dogs naturally infected with D. repens . A total of 78 Beagle dogs were examined for the presence of circulating microfilariae by Knott's test. Twenty-three of the microfilaraemic dogs were divided into four groups and included in the trial. The dogs received monthly or biweekly selamectin treatment and were subjected to monthly blood testing for a period of 252 or 336 days. At the end of the study, 65% of the dogs were not microfilaraemic and the rest had low number of microfilariae in their blood. These results indicate that chronic spot-on selamectin treatment may be a useful tool also in the control of canine subcutaneous dirofilariosis.

Increased renoprotection with ACE inhibitor plus aldosterone antagonist as compared to monotherapies--the effect on podocytes.

BACKGROUND: Blockade of the renin-angiotensin-aldosterone system (RAAS) does not completely prevent progression of renal disease. Mineralocorticoid receptor blockade provides additional renoprotection over ACE-inhibition monotherapy. We examined the mechanisms underlying superior renoprotection in the subtotal nephrectomy (SNX) model. METHODS: Sprague-Dawley rats were randomized into six groups: (1) sham-op, (2) SNX without treatment, (3) SNX + quinapril (Q), (4) SNX + spironolactone (S), (5) SNX + combination therapy (Q+S), (6) SNX + combination hydrochlorothiazide + reserpin + hydralazine (HRH). Albuminuria and blood pressure were monitored, and kidneys were examined by morphometric and molecular methods. RESULTS: In SNX rats, albumin excretion was significantly higher than in sham-op rats. Blood pressure reduction was not significantly different between the treatment groups. All therapies (S, Q, Q+S and HRH) reduced albuminuria; the values were lowest in animals treated with Q+S. The volume density of glomerular matrix and the number of mesangial cells were significantly increased in SNX and were lowest in SNX treated with Q+S. The number of podocytes was reduced in SNX, but was normalized in SNX treated with Q+S. Glomerular volumes and podocyte volumes were significantly higher in SNX than in sham-op. Both volumes were reduced by all interventions, but almost normalized by treatment with Q+S. Expression of collagen IV, TGF-beta(1) and desmin was increased after SNX and significantly reduced by treatment with Q and Q+S. CONCLUSIONS: In subtotally nephrectomized rats, mineralocorticoid blockade provided additional renoprotection over and above ACE inhibition. Such benefit was paralleled by major changes in podocyte number and morphology and was not blood pressure dependent.