Estrogen receptor alpha expression in podocytes mediates protection against apoptosis in-vitro and in-vivo.

CONTEXT/OBJECTIVE: Epidemiological studies have demonstrated that women have a significantly better prognosis in chronic renal diseases compared to men. This suggests critical influences of gender hormones on glomerular structure and function. We examined potential direct protective effects of estradiol on podocytes. METHODS: Expression of estrogen receptor alpha (ERα) was examined in podocytes in vitro and in vivo. Receptor localization was shown using Western blot of separated nuclear and cytoplasmatic protein fractions. Podocytes were treated with Puromycin aminonucleoside (PAN, apoptosis induction), estradiol, or both in combination. Apoptotic cells were detected with Hoechst nuclear staining and Annexin-FITC flow cytometry. To visualize mitochondrial membrane potential depolarization as an indicator for apoptosis, cells were stained with tetramethyl rhodamine methylester (TMRM). Estradiol-induced phosphorylation of ERK1/2 and p38 MAPK was examined by Western blot. Glomeruli of ERα knock-out mice and wild-type controls were analysed by histomorphometry and immunohistochemistry. RESULTS: ERα was consistently expressed in human and murine podocytes. Estradiol stimulated ERα protein expression, reduced PAN-induced apoptosis in vitro by 26.5±24.6% or 56.6±5.9% (flow cytometry or Hoechst-staining, respectively; both p<0.05), and restored PAN-induced mitochondrial membrane potential depolarization. Estradiol enhanced ERK1/2 phosphorylation. In ERα knockout mice, podocyte number was reduced compared to controls (female/male: 80/86 vs. 132/135 podocytes per glomerulus, p<0.05). Podocyte volume was enhanced in ERα knockout mice (female/male: 429/371 µm(3) vs. 264/223 µm(3) in controls, p<0.05). Tgfß1 and collagen type IV expression were increased in knockout mice, indicating glomerular damage. CONCLUSIONS: Podocytes express ERα, whose activation leads to a significant protection against experimentally induced apoptosis. Possible underlying mechanisms include stabilization of mitochondrial membrane potential and activation of MAPK signalling. Characteristic morphological changes indicating glomerulopathy in ERα knock-out mice support the in vivo relevance of the ERα for podocyte viability and function. Thus, our findings provide a novel model for the protective influence of female gender on chronic glomerular diseases.

Human polyomaviruses and other human viruses in neuroendocrine tumors.

BACKGROUND: While the association of the Merkel cell polyomavirus (MCV) with the neuroendocrine Merkel cell carcinomas (MCC) has been shown recently, it is unknown whether other human polyomaviruses (HPyV) may be associated with neuroendocrine tumours (NETs) of distinct entities. METHODS: Using novel, highly sensitive polyomavirus genotyping assays, we evaluated the prevalence of eight distinct HPyVs in a selection of 51 NETs from different entities. In addition, we analyzed these NETs for the presence of DNA from 12 adeno-associated virus (AAV) genotypes, adeno virus-5, 27 mucosal human papillomavirus (HPV) genotypes, hepatitis B (HBV), 8 human herpes viruses (HHV), and xenotropic murine leukemia virus-related virus (XMRV). RESULTS: 43 of the 50 (86%) NETs were positive for the DNA integrity control. Of these, 2 of 3 MCCs (67%) were positive for MCV. NETs from other entities, however, were negative for all HPyVs. Only a small subset of lung and appendix NETs were positive for EBV, HHV-6, and -7. CONCLUSION: While the association of MCV with MCC was confirmed, other human viruses could not be identified as potentially causative agents of other NETs. IMPACT: Our findings suggest that the human viruses tested for in this study do not play a comparable role in NETs like the polyomavirus MCV in MCC.

Both high and low maternal salt intake in pregnancy alter kidney development in the offspring.

In humans, low glomerular numbers are related to hypertension, cardiovascular, and renal disease in adult life. The present study was designed 1) to explore whether above- or below-normal dietary salt intake during pregnancy influences nephron number and blood pressure in the offspring and 2) to identify potential mechanisms in kidney development modified by maternal sodium intake. Sprague-Dawley rats were fed low (0.07%)-, intermediate (0.51%)-, or high (3.0%)-sodium diets during pregnancy and lactation. The offspring were weaned at 4 wk and subsequently kept on a 0.51% sodium diet. The kidney structure was assessed at postnatal weeks 1 and 12 and the expression of proteins of interest at term and at week 1. Blood pressure was measured in male offspring by telemetry from postnatal month 2 to postnatal month 9. The numbers of glomeruli at weeks 1 and 12 were significantly lower and, in males, telemetrically measured mean arterial blood pressure after month 5 was higher in offspring of dams on a high- or low- compared with intermediate-sodium diet. A high-salt diet was paralleled by higher concentrations of marinobufagenin in the amniotic fluid and an increase in the expression of both sprouty-1 and glial cell-derived neutrophic factor in the offspring's kidney. The expression of FGF-10 was lower in offspring of dams on a low-sodium diet, and the expression of Pax-2 and FGF-2 was lower in offspring of dams on a high-sodium diet. Both excessively high and excessively low sodium intakes during pregnancy modify protein expression in offspring kidneys and reduce the final number of glomeruli, predisposing the risk of hypertension later in life.

Is there an obesity-metabolic syndrome related glomerulopathy?

PURPOSE OF REVIEW: There is an increasing evidence for a specific form of focal segmental glomerulosclerosis (FSGS) related to obesity. Its prevalence has progressively increased in past decades. This form of FSGS represents the tip of an iceberg: a much broader spectrum of renal malfunction is linked to visceral obesity, which is closely connected to, but not completely identical with, the concept of 'metabolic syndrome'. RECENT FINDINGS: The obesity-associated FSGS (obFSGS) is characterized by massive proteinuria and glomerular lesions which are similar to but less pronounced than in idiopathic FSGS, but the long-term prognosis is still dubious. The patholophysiology underlying obesity-associated renal pathology includes insulin resistance and salt sensitivity of blood pressure (BP); more recently adiponectin deficiency, hyperaldosteronism and many other pathogenetic factors have been identified. The abnormalities of renal structure in obese and morbidly obese individuals include increased kidney weight, glomerulomegaly, disorder of podocytes, mesangial expansion and more recently also abnormalities of the renal interstitium. This is accompanied by functional abnormalities, that is renal hyperperfusion, increased filtration fraction and albuminuria. Both obesity and metabolic syndrome have been identified as powerful predictors of chronic kidney disease (CKD) and end-stage renal disease (ESRD). This correlation is not fully explained by associated hypertension and prediabetes/diabetes. SUMMARY: The link between progressive kidney disease and visceral obesity is of enormous public health importance. Apart from causing obFSGS, obesity aggravates most primary kidney diseases. Beyond standard therapy and weight loss, bariatric surgery has recently emerged as a successful intervention for obFSGS.

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).

Vasculotropic effects of calcimimetics.

PURPOSE OF REVIEW: At all stages of chronic kidney disease (CKD) cardiovascular death is the most prominent cause of mortality. Current treatment options are still not completely satisfactory in this group of high cardiovascular risk patients. Experimental data and clinical observations suggest a role of secondary hyperparathyroidism, hyperphosphatemia, and hypercalcemia in the genesis of cardiovascular complications of CKD. The ubiquitous expression of the calcium-sensing receptor, which is targeted by calcimimetics and the pleiotropic effects of calcimimetics, make this class of drugs potential candidates for cardiovascular intervention. RECENT FINDINGS: Recent experimental studies suggest that calcimimetics interfere with the development of vascular abnormalities in CKD and to some extent even reverse them. The effects of calcimimetics on the vasculature are, at least partially, independent of their effects on calcemia, phosphatemia, and parathyroid hormone concentration. The beneficial effects of calcimimetics on vascular calcification, arteriolar thickening, atherogenesis, and myocardial capillarization are well documented. In addition they have hypotensive and renoprotective actions. SUMMARY: Experimental models suggest beneficial effects of calcimimetics on cardiovascular disease. Although prospective clinical data are still lacking, retrospective data suggest cardiovascular benefit of calcimimetics even in humans. Clinical trials with calcimimetics evaluating hard cardiovascular end-points would be desirable.

Arterial calcification in patients with chronic kidney disease.

OBJECTIVE: In patients with chronic kidney disease (CKD), aortic calcification is more frequent and severe and it is also predictive of adverse cardiovascular outcome. The aim of the present study was to characterize aortic calcification in renal compared with non-renal patients. METHODS: Aortas of 31 patients with advanced CKD and of 31 age-and gender-matched controls were obtained at autopsy. Calcium and phosphorus content in the aorta was quantitated using x-ray analysis. The expression of calcification-promoting and calcification-inhibiting proteins was assessed using immunohistochemistry. RESULTS: The calcium and phosphorus content of the aorta was higher in CKD patients than in controls. Even in non-calcified aortic specimens of CKD, staining for Msx-2, BMP-2, bone sialo-protein, TNF-alpha and nitrotyrosine was significantly more marked compared to controls. The same proteins were immunodetected in calcified aortic specimens of both CKD and controls. In contrast, staining for transglutaminase-2 and Fetuin A was significantly reduced in CKD. Higher expression of cbfa-1 and Pit-1 was observed in all calcified aortas with no difference between CKD and controls. The expression of TNF-alpha, phospho-p38 and Msx-2 was correlated to the intensity of upregulation of BMP-2 and osteoblastic transdifferentiation by VSMC even in non-calcified areas of the aortas of CKD. CONCLUSION: The expression of markers characteristic for calcification is not different in calcified aorta of CKD patients compared to controls, but in CKD patients, evidence of inflammation, transformation to an osteoblastic phenotype and reduced expression of transglutaminase are also found even in non-calcified aorta.

A calcimimetic (R-568), but not calcitriol, prevents vascular remodeling in uremia.

Renal insufficiency increases cardiovascular risk, accelerates atherogenesis, and causes vascular wall remodeling. Here we evaluated the effect of the calcimimetic R-568 and non-hypercalcemic doses of calcitriol on vascular structure. Subtotal nephrectomy was produced in Sprague-Dawley rats followed by treatment with R-568, calcitriol, or vehicle for 12 weeks. The aortic wall was significantly thicker in vehicle-treated uremic rats than in those with a sham-operation but R-568-treated uremic rats had a lower value. In contrast, calcitriol increased wall thickness in both the sham-operated and uremic groups. The calcification score, measured by von Kossa staining, and the number of proliferating cells in the intima and media were significantly higher in the calcitriol-treated uremic group. The expression of the calcium sensing receptor was higher in the intima of sham-operated and uremic rats treated with R-568 compared to animals treated with vehicle or calcitriol, while the expression of the vitamin D receptor was upregulated by both calcitriol and R-568. Our study shows that in uremic rats, calcitriol increased while R-568 attenuated media calcification and proliferation of vascular smooth muscle and endothelial cells.

Comparative and morphological analysis of commonly used autografts for anterior cruciate ligament reconstruction with the native ACL: an electron, microscopic and morphologic study.

Ligaments and tendons are similar in composition but differ in proportion and arrangement. Tendons are being used as grafts for the ACL reconstruction. Their microscopic structure has not been sufficiently studied and compared to the native ACL. A null hypothesis was declared stating that the anterior cruciate ligament should be histological, morphologically and functionally different from the tendon grafts used for ACL reconstruction. We investigated similarities and differences of the structure of ACL and tendons used as a graft tissue for ACL reconstruction. In this study, standardized samples of quadriceps, hamstrings (semitendinosus and gracilis) and patellar tendons, and the ACL were harvested from 26 autopsies (average age 36.4) and were investigated using light and electron microscopy, immunohistochemistry and morphometry. The thickness of the collagen fibrils, collagen organization and diameter, the fibril/interstitium ratio, density of fibroblasts and blood vessels, and distribution of the collagen type I, III and V fibrils were analyzed. The semitendinosus showed the highest density of fibroblasts and blood vessels, while the gracilis the highest fibril/interstitium ratio. No differences regarding the thickness of collagen fibrils and distribution of fibrils were found. The ACL had the highest concentration of type III and V collagen fibrils as well as elastic fibers. The histological and ultrastructural appearance of the ACL differs from those of the tendons used as graft, for ACL reconstruction. Its ultrastructure is varied and complex, with its collagen fibers bundles lying in many directions.

Degenerative changes of the interface membrane as a possible reason for prosthesis loosening.

OBJECTIVE: The aim of the present study was to perform a comparative evaluation of septic and aseptic interface membranes, assessing histological features, inflammatory infiltrate, and expression of inflammatory cytokines. METHODS: Septic and aseptic interface membranes from 102 patients were examined by histology, histochemistry, and immunohistochemistry (tissue arrays). The cell subpopulations were characterized by quantification of CD3, CD4, CD8, CD20, and CD163 positive cells. Additionally, a semiquantitative evaluation of inflammatory cytokines (TNFalpha, TGF-beta1, IL-1, IL-6, CRP, MMP-1, MMP-6) was performed to complete the analysis of inflammatory infiltrates. RESULTS: The histological analysis revealed three different types of aseptic interface membranes: wear particle, degenerative, and mixed type. The expression of inflammatory molecules did not differ between septic and wear particle interface membranes. Significantly lower expression of cytokines, MMPs and CRP was observed, however, in degenerative interface membranes compared to other types. No expression of TNFalpha was observed in the degenerative interface membranes. Over 88% of patients with degenerative interface membranes had had a clinical record of osteoarthritis. CONCLUSION: Aseptic interface membranes were represented by wear particle, degenerative and mixed type. The expression of inflammatory factors in wear particle type is similar to this in septic membranes and can contribute to the bone destruction and prosthesis loosening. These factors seem not to play a major role in the degenerative membranes.

Regression of glomerulosclerosis in subtotally nephrectomized rats: effects of monotherapy with losartan, spironolactone, and their combination.

Angiotensin II accelerates and renin-angiotensin system blockade halts progression; blockade with high doses even reverses established glomerulosclerosis. Aldosterone also accelerates progression of glomerulosclerosis, partially independently of angiotensin II. The purpose of this study was to assess the relative ability of an angiotensin receptor type 1 (AT1) blocker, a mineralocorticoid receptor blocker, and their combination to reverse glomerulosclerosis. Sprague-Dawley rats were subjected to subtotal renal ablation (SNX) or sham operation. Eight weeks after surgery, they were either euthanized or allocated to treatment with vehicle, losartan, spironolactone, their combination, or unspecific antihypertensive treatment (dihydralazine) for 4 wk. Renal morphology was evaluated by stereology in tissues obtained using pressure-controlled perfusion fixation. Systolic blood pressure was significantly higher in SNX compared with sham-operated animals and decreased in all treatment groups. Compared with wk 8 after SNX, the glomerulosclerosis index (GSI) had increased further by week 12 in the vehicle- and dihydralazine-treated groups but was significantly lowered in the SNX+losartan as well as in the SNX+losartan+spironolactone groups and had not progressed further in the SNX+spironolactone group. The study confirms the partial regression of established glomerulosclerosis in subtotally nephrectomized rats after high-dose AT1 receptor blockade. Nonhyperkalemic doses of spironolactone prevented the increase but failed to decrease the GSI below the 8-wk level and preserved podocyte numbers. Combining the AT1 blocker with mineralocorticoid receptor blockade failed to further increase the regression of glomerulosclerosis.

High expression level of bone degrading proteins as a possible inducer of osteolytic features in pigmented villonodular synovitis.

Protein expression of osteopontin (OPN), osteoprotegerin (OPG), bone sialoprotein (BSP), osteocalcin (OC), RANKL and PTHrP was determined by use of immunohistochemical analysis on tissue arrays (48 cases of PVNS, 20 cases of active (a-RA), non-active rheumatoid arthritis (na-RA), and osteoarthritis (OA)). Additionally, gene expression was analysed using complimentary DNA (cDNA) microarrays. All PVNS cases showed a higher level of both protein and gene expression of RANKL, OPN and BSP in comparison with OA cases. Expression of OPG was not significantly different in PVNS compared to OA. The RANKL/OPG expression ratio was significantly higher in PVNS than in OA. High expressions level of proteins involved in bone degradation in PVNS may promote an intra-osseous propagation of the lesion. This evidence suggests that PVNS might respond to treatment using specific inhibitors of RANKL, OPN and BSP.

Comparative analysis of the morphologic structure of quadriceps and patellar tendon: a descriptive laboratory study.

PURPOSE: The difference in ultimate strength between the quadriceps and patellar tendon could be reflected by different morphologic features. METHODS: Standardized samples of quadriceps and patellar tendons were harvested from 20 cadavers and were investigated via light and electron microscopy, immunohistochemistry, and morphometry. The thickness of collagen fibrils, fibril-interstitium ratio, density of blood vessels, density of fibroblasts, and distribution of collagen fibrils were analyzed. RESULTS: In comparison with the patellar tendon, the quadriceps tendon showed a significantly higher fibril-interstitium ratio (P = .0004) and a higher fibroblast density (P = .0011). No differences regarding the thickness of collagen fibrils, density of blood vessels, and distribution of fibrils were found. CONCLUSIONS: The quadriceps tendon graft can provide approximately 20% more collagen than the patellar tendon graft with the same thickness. This fact can play an important role in the better ultimate strength of the quadriceps tendon. CLINICAL RELEVANCE: The detailed anatomic description of the quadriceps tendon and patellar tendon explains the difference in their ultimate strength.

Morphologic and histologic comparison between the patella and hamstring tendons grafts: a descriptive and anatomic study.

PURPOSE: Morphologic and histologic comparison of patella and hamstring tendon grafts. METHODS: Hamstring tendons (semitendinosus and gracilis) and patellar tendons were taken from 20 cadaveric knees and were investigated by using light and electron microscopy, immunohistochemistry, and morphometry. The thickness of collagen fibrils, fibril/interstitum ratio, density of blood vessels, density of fibroblasts, and distribution of the collagen fibrils were analyzed. RESULTS: The semitendinosus and gracilis tendons provide 20% and 30% more fibril/interstitum ratio compared with the patella tendon (P = .0056 and .0028). Also, the density of fibroblasts was 50% and 35% more (P = .0061 and .0050). No differences regarding the thickness of the collagen fibrils, density of blood vessels, and distribution of the fibrils were found. CONCLUSIONS: Both semitendinous and gracilis tendons provide significantly more density of collagen fibrils as well as density of fibroblasts in comparison with patellar tendons. These findings provide a potential advantage of the hamstrings group on better remodelling and regeneration of the tissue. CLINICAL RELEVANCE: These grafts have been used as autografts for anterior cruciate ligament reconstruction. Despite the interest on these tendons, their microscopic structure has not been sufficiently investigated yet.