Valentina Kon

Glomerular actions of endothelin in vivo.

In Munich-Wistar rats, a micropipette was inserted into a first-order branch of the left main renal artery and continuously infused with human/porcine endothelin (0.4 ng/min). Micropuncture measurements revealed substantial differences within the cortical microcirculation of the same left kidney: SNGFR was some 35% lower in glomeruli exposed to endothelin compared with non-endothelin-perfused glomeruli (P less than 0.005). Similarly, glomerular plasma flow rate was some 38% lower in the endothelin-exposed glomeruli (P less than 0.001). The hypoperfusion and hypofiltration in the endothelin-exposed glomeruli reflected an increase in resistances in the afferent and efferent arterioles. There was no difference in the value of the glomerular capillary ultrafiltration coefficient between the two populations of glomeruli. We also studied kidneys that underwent 25 min of renal artery clamping 48 h before study. Antiendothelin antibody infused into one of the branches of the main renal artery ameliorated the vasoconstriction characteristic of postischemic nephrons: within the cortical microcirculation, the SNGFR in glomeruli exposed to antiendothelin antibody was 27.0 +/- 3.1 nl/min as compared with 17.4 +/- 1.7 measured in glomeruli not perfused with the antibody (P less than 0.001). Similarly, glomerular plasma flow rate was higher in the glomeruli exposed to antiendothelin antibody (128.7 +/- 14.4 nl/min vs. 66.6 +/- 5.6, P less than 0.005). Resistances in both the afferent and efferent arterioles were substantially lower in the antibody-exposed glomeruli. It is, therefore, suggested that endothelin, presumably released from damaged endothelium, may play an important intermediary role in the hypoperfusion and hypofiltration observed in postischemic kidneys.

Endotoxin stimulates endothelin-release in vivo and in vitro as determined by radioimmunoassay

A marked increase in immunoreactive endothelin was observed in rat serum collected within 10-15 min after infusion of endotoxin. Endothelin level was 117 +/- 11.5 pg/ml (mean +/- S.E., N = 4) in rats exposed to endotoxin as compared with undetectable levels (less than 2 pg/ml, N = 4) in controls. We have also observed a significant stimulation of endothelin-release by endotoxin from cultured bovine transformed thoractic aortic endothelial cells at concentrations of endotoxin ranging between 0.1 and 10.0 micrograms/ml. Serum was indispensable for the stimulating effect of endotoxin, although serum itself did not show any effect at the concentration used (1%). These results suggest that endothelin plays an important role in mediation of pathophysiological responses caused by endotoxin. The levels of endothelin were measured by radioimmunoassay with high sensitivity.

Conditional Knockout of Macrophage PPARγIncreases Atherosclerosis in C57BL/6 and Low-Density Lipoprotein Receptor–Deficient Mice

Objective—Peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in macrophage-derived foam cells of atherosclerotic lesions, and its expression may have a dramatic impact on atherosclerosis. Methods and Results—To investigate the contribution of macrophage PPARγ expression on atherogenesis in vivo, we generated macrophage-specific PPARγ knockout (MacPPARγKO) mice. C57BL/6 and low-density lipoprotein (LDL) receptor–deficient (LDLR−/−) mice were reconstituted with MacPPARγKO or wild-type marrow and challenged with an atherogenic diet. No differences were found in serum lipids between recipients reconstituted with MacPPARγKO and wild-type marrow. In contrast, both C57BL/6 and LDLR−/− mice transplanted with MacPPARγKO marrow had significantly larger atherosclerotic lesions than control recipients. In addition, MacPPARγKO→LDLR−/− mice had higher numbers of macrophages in atherosclerotic lesions compared with controls. Peritoneal macrophages isolated from the MacPPARγKO mice had decreased uptake of oxidized but not acetylated LDL and showed no changes in either cholesterol efflux or inflammatory cytokine expression. Macrophages from MacPPARγKO mice had increased levels of migration and CC chemokine receptor 2 (CCR2) expression compared with wild-type macrophages. Conclusion—Thus, macrophage PPARγ deficiency increases atherosclerosis under conditions of mild and severe hypercholesterolemia, indicating an antiatherogenic role for PPARγ, which may be caused, at least in part, by modulation of CCR2 expression and monocyte recruitment.

Absence of angiotensin II type 1 receptor in bone marrow–derived cells is detrimental in the evolution of renal fibrosis

We examined the in vivo function of the angiotensin II type 1 receptor (Agtr1) on macrophages in renal fibrosis. Fourteen days after the induction of unilateral ureteral obstruction (UUO), wild-type mice reconstituted with marrow lacking the Agtr1 gene (Agtr1(-/-)) developed more severe interstitial fibrosis with fewer interstitial macrophages than those in mice reconstituted with Agtr1(+/+) marrow. These differences were not observed at day 5 of UUO. The expression of profibrotic genes - including TGF-beta1, alpha1(I) collagen, and alpha1(III) collagen - was substantially higher in the obstructed kidneys of mice with Agtr1(-/-) marrow than in those with Agtr1(+/+) marrow at day 14 but not at day 5 of UUO. Mice with Agtr1(-/-) marrow were characterized by reduced numbers of peripheral-blood monocytes and macrophage progenitors in bone marrow. In vivo assays revealed a significantly impaired phagocytic capability in Agtr1(-/-) macrophages. In vivo treatment of Agtr1(+/+) mice with losartan reduced phagocytic capability of Agtr1(+/+) macrophages to a level comparable to that of Agtr1(-/-) macrophages. Thus, during urinary tract obstruction, the Agtr1 on bone marrow-derived macrophages functions to preserve the renal parenchymal architecture, and this function depends in part on its modulatory effect on phagocytosis.

Dysfunctional High-Density Lipoprotein in Patients on Chronic Hemodialysis

OBJECTIVES This study examined the functionality of high-density lipoprotein (HDL) in individuals with end-stage renal disease on dialysis (ESRD-HD). BACKGROUND The high rate of cardiovascular disease (CVD) in chronic kidney disease is not explained by standard risk factors, especially in patients with ESRD-HD who appear resistant to benefits of statin therapy. HDL is antiatherogenic because it extracts tissue cholesterol and reduces inflammation. METHODS Cellular cholesterol efflux and inflammatory response were assessed in macrophages exposed to HDL of patients with ESRD-HD or controls. RESULTS HDL from patients with ESRD-HD was dramatically less effective than normal HDL in accepting cholesterol from macrophages (median 6.9%; interquartile range [IQR]: 1.4% to 10.2%) versus control (median 14.9%; IQR: 9.8% to 17.8%; p < 0.001). The profound efflux impairment was also seen in patients with ESRD-HD and diabetes compared with patients with diabetes without renal disease (median 8.1%; IQR: 3.3% to 12.9%) versus control (median 13.6%; IQR: 11.0% to 15.9%; p = 0.009). In vitro activation of cellular cholesterol transporters increased cholesterol efflux to both normal and uremic HDL. HDL of patients with ESRD-HD had reduced antichemotactic ability and increased macrophage cytokine response (tumor necrosis factor-alpha, interleukin-6, and interleukin-1-beta). HDL of patients with ESRD-HD on statin therapy had reduced inflammatory response while maintaining impaired cholesterol acceptor function. Interestingly, impaired HDL-mediated efflux did not correlate with circulating C-reactive protein levels or cellular inflammatory response. CONCLUSIONS These findings suggest that abnormal HDL capacity to mediate cholesterol efflux is a key driver of excess CVD in patients on chronic hemodialysis and may explain why statins have limited effect to decrease CV events. The findings also suggest cellular cholesterol transporters as potential therapeutic targets to decrease CV risk in this population.

Proteomic Patterns and Prediction of Glomerulosclerosis and Its Mechanisms

Protein expression profiles linked to sclerosis in the 5/6 nephrectomy (Nx) rat model of focal segmental glomerulosclerosis were investigated. Sections of control glomeruli from normal baseline Nx tissue and nonsclerotic and sclerotic glomeruli from 12 wk after 5/6 Nx were isolated by laser capture microdissection. Protein profiles were acquired directly by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Classification accuracy was 99.2% for distinguishing normal versus sclerotic glomeruli and 96.7 and 97.8% for nonsclerotic versus normal and sclerotic glomeruli, respectively. The proteomic pattern of the nonsclerotic glomeruli was more similar to sclerotic than normal glomeruli (P < 0.0001). Thymosin beta4, a protein with relevant interactions with plasminogen activator inhibitor-1, angiogenesis, and wound healing, was identified as a key differentially expressed protein. Thymosin beta4 immunostaining was increased in sclerotic glomeruli, predominantly in endothelial cells. Downregulation of thymosin beta4 by RNAi in cultured glomerular endothelial cells decreased angiotensin II-induced plasminogen activator inhibitor-1 expression. In conclusion, proteomic patterns can accurately distinguish normal versus nonsclerotic versus sclerotic glomeruli. The closely related proteomic patterns of nonsclerotic and sclerotic glomeruli suggest early activation of prosclerotic mechanisms even in seemingly intact glomeruli. Thymosin beta4 is a marker of such early events and may even contribute to sclerosis.

Combined antagonism of endothelin A/B receptors links endothelin to vasoconstriction whereas angiotensin II effects fibrosis. Studies in chronic cyclosporine nephrotoxicity in rats.

Both functional and structural damage characterize nephrotoxicity due to cyclosporine (CsA) with accumulating evidence for dissociation of mechanisms that lead to each of these processes. We studied the role of endothelin (Et) and angiotensin II (AII), since each of these peptides can modulate vasoconstriction as well as parenchymal destruction. Salt-depleted rats were treated with daily CsA (15 mg/kg s.c.) for 5 weeks (group 1, CsA, n = 13). Separate groups of CsA-treated rats received either a combined antagonist of both EtA/EtB receptors (group 2, CsA+aEtA/B, 100 mg/kg/day p.o., n = 6) or angiotensin I-converting enzyme inhibitor (group 3, CsA+ACEI, enalapril 200 mg/L drinking water, n = 8). Glomerular filtration rate (GFR) was assessed by creatinine clearance (Ccr) in conscious rats at 3 and 5 weeks. At 3 weeks, serum creatinine was 1.5 +/- 0.1 mg/dl in group 1 rats, 1.2 +/- 0.2 mg/dl in group 2 rats (P < 0.05 vs. CsA), and 2.3 +/- 0.8 mg/dl in group 3 rats. Ccr was 0.87 +/- 0.08 ml/min in group 1. In group 2, GFR was remarkably preserved (1.14 +/- 0.11 ml/min, P < 0.05 vs. group 1). By contrast, GFR in group 3 rats was lower (0.31 +/- 0.08 ml/min) than either aEtA/B-treated or even CsA-treated rats (P < 0.0005 vs. group 1, P < 0.0005 vs. group 2). At 5 weeks, the same pattern emerged; serum creatinine was 2.5 +/- 0.2 mg/dl in group 1, 1.2 +/- 0.1 in group 2 (P < 0.0005 vs. CsA), and 3.4 +/- 0.9 in group 3 (P < 0.025 vs. CsA+aEtA/B). Ccr had decreased dramatically in CsA-treated rats to 0.18 +/- 0.03 ml/min. GFR was preserved in CsA+aEtA/B rats (0.51 +/- 0.03 ml/min, P < 0.0005 vs. group 1), while profound hypofiltration was apparent in CsA+ACEI rats (0.12 +/- 0.03 ml/min, P < 0.0005 vs. group 2). In salt-depleted control animals, GFR was 0.62 +/- 0.02 ml/min. Despite striking functional preservation in response to antagonism of Et receptors, tubular vacuolization/dilatation, as well as arteriolopathy, was not different among the CsA-treated groups. Tubulointerstitial fibrosis was also not different between CsA and CsA+aEtA/B rats (on a 0-4 scale, 1.05 +/- 0.14 vs. 0.87 +/- 0.14, P = NS). In contrast, both tubular vacuolization/dilatation and interstitial fibrosis were significantly greater in all CsA-treated groups compared with salt-depleted controls. However, in the CsA+ACEI group that had the most severe hypofiltration at each time point, tubulointerstitial fibrosis was 0.69 +/- 0.06 (P < 0.05 vs. CsA).(ABSTRACT TRUNCATED AT 400 WORDS)

Scope and Mechanisms of Obesity-Related Renal Disease

Purpose of reviewObesity is established as an important contributor of increased diabetes mellitus, hypertension, and cardiovascular disease, all of which can promote chronic kidney disease (CKD). Recently, there is a growing appreciation that, even in the absence of these risks, obesity itself significantly increases CKD and accelerates its progression. Recent findingsExperimental and clinical studies reveal that adipose tissue, especially visceral fat, elaborates bioactive substances that contribute to the pathophysiologic renal hemodynamic and structural changes leading to obesity-related nephropathy. Adipocytes contain all the components of the renin–angiotensin–aldosterone system, plasminogen activator inhibitor, as well as adipocyte-specific metabolites such as free fatty acids, leptin, and adiponectin, which affect renal function and structure. In addition, fat is infiltrated by macrophages that can alter their phenotype and foster a proinflammatory milieu, which advances pathophysiologic changes in the kidney associated with obesity. SummaryObesity is an independent risk factor for development and progression of renal damage. Although the current therapies aimed at slowing progressive renal damage include reduction in weight and rely on inhibition of the renin–angiotensin system, the approach will likely be supplemented by interventions aimed at obesity-specific targets including adipocyte-driven cytokines and inflammatory factors.

Inhibition of 11β–hydroxysteroid dehydrogenase type II selectively blocks the tumor COX-2 pathway and suppresses colon carcinogenesis in mice and humans

Colorectal cancer (CRC) is a leading cause of cancer death, yet primary prevention remains the best approach to reducing overall morbidity and mortality. Studies have shown that COX-2-derived PGE2 promotes CRC progression, and both nonselective COX inhibitors (NSAIDs) and selective COX-2 inhibitors (such as glucocorticoids) reduce the number and size of colonic adenomas. However, increased gastrointestinal side effects of NSAIDs and increased cardiovascular risks of selective COX-2 inhibitors limit their use in chemoprevention of CRC. We found that expression of 11beta-hydroxysteroid dehydrogenase type II (11betaHSD2), which converts active glucocorticoids to inactive keto-forms, increased in human colonic and Apc+/min mouse intestinal adenomas and correlated with increased COX-2 expression and activity. Furthermore, pharmacologic inhibition or gene silencing of 11betaHSD2 inhibited COX-2-mediated PGE2 production in tumors and prevented adenoma formation, tumor growth, and metastasis in mice. Inhibition of 11betaHSD2 did not reduce systemic prostacyclin production or accelerate atherosclerosis in mice, thereby avoiding the major cardiovascular side effects seen with systemic COX-2 inhibitors. Therefore, 11betaHSD2 inhibition represents what we believe to be a novel approach for CRC chemoprevention and therapy by increasing tumor glucocorticoid activity, which in turn selectively blocks local COX-2 activity.

Angiotensin type 1 receptor modulates macrophage polarization and renal injury in obesity

The mechanisms for increased risk of chronic kidney disease (CKD) in obesity remain unclear. The renin-angiotensin system is implicated in the pathogenesis of both adiposity and CKD. We investigated whether the angiotensin type 1 (AT(1)) receptor, composed of dominant AT(1a) and less expressed AT(1b) in wild-type (WT) mice, modulates development and progression of kidney injury in a high-fat diet (HFD)-induced obesity model. WT mice had increased body weight, body fat, and insulin levels and decreased adiponectin levels after 24 wk of a high-fat diet. Identically fed AT(1a) knockout (AT1aKO) mice gained weight similarly to WT mice, but had lower body fat and higher plasma cholesterol. Both obese AT1aKO and obese WT mice had increased visceral fat and kidney macrophage infiltration, with more proinflammatory M1 macrophage markers as well as increased mesangial expansion and tubular vacuolization, compared with lean mice. These abnormalities were heightened in the obese AT1aKO mice, with downregulated M2 macrophage markers and increased macrophage AT(1b) receptor. Treatment with an AT(1) receptor blocker, which affects both AT(1a) and AT(1b), abolished renal macrophage infiltration with inhibition of renal M1 and upregulation of M2 macrophage markers in obese WT mice. Our data suggest obesity accelerates kidney injury, linked to augmented inflammation in adipose and kidney tissues and a proinflammatory shift in macrophage and M1/M2 balance.