Glomerular Area Research Articles

Morphometric Analysis of Nonsclerosed Glomeruli Size and Connective Tissue Content during the Aging Process

Number of sclerotic glomeruli increases during the aging process. Consequently, majority of remained nonsclerosed glomeruli become hypertrophic and some of them sclerotic, too. The aim of this study was to quantify the size and connective tissue content of nonsclerosed glomeruli and to evaluate the percentage of hypertrophic ones in examined human cases during the aging. Material was right kidney's tissue of 30 cadavers obtained during routine autopsies. Cadavers were without previously diagnosed kidney disease, diabetes, hypertension, or any other systemic disease. Tissue specimens were routinely prepared for histological and morphometric analysis. Images of the histological slices were analyzed and captured under 400x magnification with digital camera. Further they were morphometrically and statistically analyzed with ImageJ and NCSS-PASS software. Multiple and linear regression of obtained morphometric parameters showed significant increase of glomerular connective tissue area and percentage. Cluster analysis showed the presence of two types of glomeruli. Second type was characterized with significantly larger size, connective tissue content, and significantly lower cellularity, in relation to the first type. Such glomeruli might be considered as hypertrophic. First type of glomeruli was predominant in younger cases, while second type of glomeruli was predominant in cases older than 55 years.

Effect of Cordyceps sinensis on the expression of HIF-1α and NGAL in rats with renal ischemia-reperfusion injury

To observe the level of urinary neutrophil gelatinase-associated lipocalin (NGAL), the expression of hypoxia inducible factor-1α (HIF-1α) and NGAL in rat kidney after renal ischemia and reperfusion (I/R), before and after the treatment with Cordyceps Sinensis (C. sinensis), and to explore the mechanism of C. sinensis against I/R injury. A total of 45 healthy male Sprague-Dawley rats were randomly divided into a sham group, a renal I/R model group, and a C. sinensis group (15 in each group).The rats in the sham group and the renal I/R model group were intragastrically administered saline (2 mL/d), and rats in the treatment group were intragastricabby administered of C. sinensis [5.0 g/(kg.d)]. The rats were sacrificed at 24, 48, and 72 h, respectively after the reperfusion and urinary N-acetyl-β-D-glucosaminidase (NAG) level was measured, renal function in rats was detected, and the pathological changes were observed with HE staining. We determined the urinary NGAL levels in the rats by ELISA, the expression of HIF-1α mRNA by RT-PCR, and the expressions of HIF-1α and NGAL proteins by confocal immunofluorescence. Compared with the sham group, the levels of BUN, SCr, levels of NAG and NGAL in urine were increased in the I/R group and the C. sinensis group, reached a peak at 24 h after the reperfusion and slowly declined at 48 and 72 h. Glomerular and tubulointerstitial areas in the sham group did not show any pathological change. Induced pathological changes included tubular cell necrosis, focal areas of proximal tubular dilation, distal tubular casts, effacement and loss of proximal tubule brush border, etc. Compared with the sham group, the expression of HIF-1α and NGAL in the kidney tissues of the I/R group and the C. sinensis group increased. C. sinensis can lower the level of NAG and NGAL in the urine and the expression of NGAL protein in the kidney tissues. It up-regulated the expression of HIF-1α mRNA and protein in the kidney tissues whilst attenuated the pathological changes. Renal I/R injury in rats can lead to pathological changes in renal tubular epithelial cells and renal interstitial damage, which are consistent with the pathological features of acute kidney injury (AKI).The level of urinary NAGL increases after the I/R, and positively correlates with the level of urinary NAG and pathological changes, suggesting that urinary NGAL may serve as a urinary biomarker for specific detection of tubular injury in AKI. C. sinensis can attenuate the renal I/ R-induced AKI. Its mechanism may be associated with up-regulating the expression of HIF-1α and down-regulating the expression of NGAL in the kidney tissues.

A Novel Diabetes Mellitus Mouse Model, MAFA-Deficient and Beta Cell-Specific MAFK-Overexpressing Hybrid Transgenic Mice, Developed Severe Diabetic Nephropathy and Improved with TCV-116 (Candesartan Cilexetil) Treatment

Many models of diabetic nephropathy have been reported. However, it is rare that the characteristic findings of severe human diabetic nephropathy, such as diffuse, nodular, and exudative lesions, are all detected in one model mouse. Previously, we reported that MAFA-deficient and beta cell-specific MAFK-overexpressing hybrid transgenic (Mafa(-/-)Mafk (+)) mice develop diabetes mellitus and, after uninephrectomy, demonstrate these characteristic lesions. In this study, we administered TCV-116 (candesartan cilexetil) to Mafa(-/-)Mafk (+) mice after uninephrectomy and examined whether TCV-116 ameliorated the diabetic nephropathy. We also evaluated the utility of these mice as a model for developing treatments for diabetic nephropathy. We performed uninephrectomy of the Mafa(-/-)Mafk (+) mice at 8 weeks old. We then divided these mice into two groups as follows: 1) an untreated group and 2) a group treated with TCV-116 at 5 µg/g/day from 10 to 20 weeks. TCV-116 treatment did not affect serum glucose levels. However, in the treated group, urinary protein excretion, mesangial matrix expansion, enlargement of the kidney, and glomerular surface area were all improved relative to untreated mice. Oxidative stress is known to be increased in diabetic nephropathy and to be suppressed by TCV-116. The urinary level of 8-OHdG, an oxidative stress marker, at 20 weeks was lower in the TCV-116-treated group than in the untreated group. From these results, we concluded that the Mafa(-/-)Mafk (+) mouse is a useful model to analyze diabetic nephropathy and a useful tool for the development of new drugs to treat diabetic nephropathy.

Cytological organization of the alpha component of the anterior olfactory nucleus and olfactory limbus

This study describes the microscopic organization of a wedge-shaped area at the intersection of the main (MOB) and accessory olfactory bulbs (AOBs), or olfactory limbus (OL), and an additional component of the anterior olfactory nucleus or alpha AON that lies underneath of the AOB. The OL consists of a modified bulbar cortex bounded anteriorly by the MOB and posteriorly by the AOB. In Nissl-stained specimens the OL differs from the MOB by a progressive, antero-posterior decrease in thickness or absence of the external plexiform, mitral/tufted cell, and granule cell layers. On cytoarchitectual grounds the OL is divided from rostral to caudal into three distinct components: a stripe of glomerular-free cortex or preolfactory area (PA), a second or necklace glomerular area, and a wedge-shaped or interstitial area (INA) crowned by the so-called modified glomeruli that appear to belong to the anterior AOB. The strategic location and interactions with the main and AOBs, together with the previously noted functional and connectional evidence, suggest that the OL may be related to both sensory modalities. The alpha component of the anterior olfactory nucleus, a slender cellular cluster (i.e., 650 × 150 μm) paralleling the base of the AOB, contains two neuron types: a pyramidal-like neuron and an interneuron. Dendrites of pyramidal-like cells (P-L) organize into a single bundle that ascends avoiding the AOB to resolve in a trigone bounded by the edge of the OL, the AOB and the dorsal part of the anterior olfactory nucleus. Utrastructurally, the neuropil of the alpha component contains three types of synaptic terminals; one of them immunoreactive to the enzyme glutamate decarboxylase, isoform 67.

Histomorphological classification of focal segmental glomerulosclerosis: A critical evaluation of clinical, histologic and morphometric features

Primary focal segmental glomerulosclerosis (FSGS) has recently been divided into five subtypes by the Columbia classification. However, little is known about the incidence of these subtypes in the Indian population. In addition, there are very few studies evaluating the clinico-pathologic features with morphometric parameters in these subtypes. This study was aimed at evaluating the clinical, histopathological and morphometric parameters in various subtypes of FSGS at our referral center. Sixty-five (65) cases of idiopathic FSGS, diagnosed over two years (2006-2007), were included in the study. Detailed clinical and biochemical investigations were noted. Histological sections were reviewed and cases classified according to the Columbia classification and various glomerular and tubulo-interstitial features were noted. Glomerular morphometry on digitized images was performed using image analysis software. Renal biopsies with minimal change disease were used as controls for morphometric evaluation. In this study, FSGS not otherwise specified (NOS) was the most common subtype (44.6%), followed by perihilar FSGS (24.6%), collapsing (13.8%), tip (12.3%) and cellular FSGS (4.6%). Collapsing subtype showed significantly shorter duration of symptoms and higher degree of proteinuria, mean serum urea and creatinine compared with the other subtypes. On histologic analysis, features like glomerular hyalinosis, capsular adhesion, mesangial proliferation and visceral epithelial cell prominence (VEP) were frequently seen. The cases with VEP had a shorter duration of symptoms and a higher mean serum creatinine and 24-h urine protein excretion compared with those without VEP. The morphometric study revealed a significant higher mean glomerular area in the NOS, perihilar and collapsing variants as compared with the control biopsies. The present study highlights the differences in the prevalence in the FSGS subtypes in our population compared with the western data. Also, the significant differences in the clinical, biochemical and histological parameters reaffirm the utility of the Columbia classification of FSGS in routine reporting of renal biopsies. We found VEP (without causing collapse of the tuft) to be associated with higher serum creatinine at presentation. This feature needs to be evaluated in further studies for its potential significance.

Quantitative Histological Analysis of SM22α (Transgelin) in an Adriamycin-Induced Focal Segmental Glomerulosclerosis Model

Background/Aims: SM22α, transgelin, has been revealed to be specifically expressed in glomerular epithelial cells and interstitial cells, according to the nature of the renal injury. In this study, quantitative analyses of SM22α positivity were performed to investigate the pathological significance of its expression. Methods: Kidney samples of adriamycin nephropathy underwent immunohistochemistry with a newly established anti-SM22α monoclonal antibody. The SM22α positivity was quantified by an image analyzer. The correlation of the histological values with biochemical data was investigated statistically. Microstructural localization of SM22α was studied by immunoelectron microscopy. Results: SM22α was expressed along the dense basal microfilaments of degenerating podocytes, and diffusely in interstitial cells. Both the extent and intensity of SM22α expression in glomerular and tubulointerstitial area were correlated with the deterioration of renal function and the severity of proteinuria. Stepwise multiple linear regression analysis revealed that the extent of its positivity in glomerular or tubulointerstitial area was the determinant of the amount of proteinuria or the deterioration of creatinine clearance (Ccr), respectively. Inversely, the deterioration of Ccr was the most important predictor of SM22α expression. Conclusion: SM22α expression in podocytes and interstitial cells represented the severity of proteinuria and the deterioration of renal function. SM22α expression in renal tissues might be a hallmark of kidney diseases.

De novo expression of podocyte proteins in parietal epithelial cells in experimental aging nephropathy

A progressive decrease in podocyte number underlies the development of glomerulosclerosis and reduced kidney function in aging nephropathy. Recent data suggest that under certain disease states, parietal epithelial cells (PECs) begin to express proteins considered specific to podocytes. To determine whether this phenomenon increases in aging kidneys, 4-, 12-, and 20-mo ad libitum-fed and 20-mo calorie-restricted (CR) rats were studied. Single and double immunostaining were performed with antibodies to the PEC protein paired box gene 2 (PAX2) and tight junction protein claudin-1, the podocyte-specific protein Wilms' tumor 1 (WT-1), and the proliferating cell protein (Ki-67). ImageJ software measured Bowman's basement membrane (BBM) length and glomerular tuft area in individual glomeruli from each animal to assess glomerular size. The results showed that in aged ad libitum rats, the decrease in number of podocytes/glomerular tuft area was accompanied by an increase in the number of PECs/BBM length at 12 and 20 mo (P < 0.01 vs. 4 mo). The increase in PEC number was due to proliferation (increase in PAX2/Ki-67 double-positive cells). Aging was accompanied by a progressive increase in the number of glomerular cells double staining for PAX2 and WT-1. In contrast, the control 20-mo-old CR rats had no increase in glomerular size, and podocyte and PEC number were not altered. These results suggest that although the number of PECs and PECs expressing podocyte proteins increase in aging nephropathy, they are likely not sufficient to compensate for the decrease in podocyte number.

Postnatal Renal Abnormalities in Rats Exposed to Losartan during Lactation

Background/Aims: Rats exposed to losartan during lactation exhibit progressive changes in renal function and structure. This study analyzed the early events in pups from dams that received losartan during lactation. Methods: Male Wistar rats from dams that received 2% sucrose (control, n = 25) or losartan (100 mg/kg/day) diluted in 2% sucrose (n = 33) during lactation were anesthetized 21 days after birth. Blood and urine samples were collected to assess renal function, and kidneys were removed for histological, immunohistochemical, Western blot, lipid peroxidation and glutathione analyses. Results: The group exposed to losartan exhibited increased albuminuria and fractional sodium and potassium excretion, decreased glomerular area and interstitial expansion. Immunohistochemical analyses demonstrated increased tubulointerstitial macrophage infiltration, apoptosis and increased vimentin and α-smooth-muscle-actin expression in animals exposed to losartan. In addition, the glomeruli of animals exposed to losartan exhibited increased peripheral desmin expression and reduced glomerular epithelial protein 1 and podocin expression compared to controls. Lastly, renal lipid peroxidation and glutathione levels were higher in the losartan-treated pups. Conclusion: Pups exposed to losartan during lactation exhibited adverse changes in renal function and structure, and tubulointerstitial inflammation at 21 days of age that were associated with apoptosis and oxidative stress.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M r of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes.

Intrauterine growth restriction is associated with persistent aortic wall thickening and glomerular proteinuria during infancy

Low birth weight, caused either by preterm birth or by intrauterine growth restriction, has recently been associated with increased rates of adult renal and cardiovascular disease. Since aortic intima–media thickening is a noninvasive marker of preclinical vascular disease, we compared abdominal aortic intima–media thickness among intrauterine growth restricted and equivalent gestational age fetuses in utero and at 18 months of age. The relationship between intrauterine growth restriction, fetal aortic thickening, and glomerular function during infancy was measured by enrolling 44 mothers with single-fetus pregnancies at 32 weeks gestation: 23 growth restricted and 21 of appropriate gestational age as controls. Abdominal aortic intima–media thickness was measured by ultrasound at enrollment and again at 18 months of age. Fetuses with intrauterine growth restriction had significantly higher abdominal aortic intima–media thickness compared with age controls when measured both in utero and at 18 months. At 18 months, the median urinary microalbumin and median albumin–creatinine ratio were significantly higher in those infants who experienced intrauterine growth restriction compared to the controls. Our results show that intrauterine growth restriction is associated with persistent aortic wall thickening and significantly higher microalbuminuria during infancy.

Bradykinin receptor 1 activation exacerbates experimental focal and segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is one of the most important causes of end-stage renal failure. The bradykinin B1 receptor has been associated with tissue inflammation and renal fibrosis. To test for a role of the bradykinin B1 receptor in podocyte injury, we pharmacologically modulated its activity at different time points in an adriamycin-induced mouse model of FSGS. Estimated albuminuria and urinary protein to creatinine ratios correlated with podocytopathy. Adriamycin injection led to loss of body weight, proteinuria, and upregulation of B1 receptor mRNA. Early treatment with a B1 antagonist reduced albuminuria and glomerulosclerosis, and inhibited the adriamycin-induced downregulation of podocin, nephrin, and α-actinin-4 expression. Moreover, delayed treatment with antagonist also induced podocyte protection. Conversely, a B1 agonist aggravated renal dysfunction and even further suppressed the levels of podocyte-related molecules. Thus, we propose that kinin has a crucial role in the pathogenesis of FSGS operating through bradykinin B1 receptor signaling.

Association of Kidney Function and Metabolic Risk Factors With Density of Glomeruli on Renal Biopsy Samples From Living Donors

To test the hypothesis that kidney function and metabolic risk factors are associated with glomerular density on renal biopsy samples from healthy adults. This study compared glomerular density with predonation kidney function, blood pressure, and metabolic risk factors in living kidney donors at Mayo Clinic in Rochester, MN, from May 10, 1999, to February 4, 2009. During implantation of the kidney allograft, an 18-gauge core needle biopsy sample of the renal cortex was obtained, sectioned, and examined by pathologists. Glomerular density was determined by the number of glomeruli (normal and sclerotic) divided by area of cortex. The study sample of 1046 kidney donors had a mean of 21 glomeruli (0.8 sclerotic glomeruli) and a glomerular density of 2.3 glomeruli per square millimeter. In a subset of 54 donors, glomerular density inversely correlated with the mean glomerular area (r(s) = -0.28). Independent predictors of decreased glomerular density were older age, increased glomerular filtration rate, family history of end-stage renal disease, increased serum uric acid, and increased body mass index. Increased urine albumin excretion, hypertension, decreased high-density lipoprotein cholesterol, and metabolic syndrome were also associated with decreased glomerular density after age-sex adjustment. These associations were not explained by the presence of glomerulosclerosis, tubular atrophy, interstitial fibrosis, or arteriosclerosis on the renal biopsy sample. In older donors, decreased glomerular density was attenuated by an increased prevalence of glomerulosclerosis and tubular atrophy. Decreased glomerular density is associated with many different kidney function and metabolic risk factors among relatively healthy adults and may represent an early state of increased risk of parenchymal injury.

Loss of olfactory cell adhesion molecule reduces the synchrony of mitral cell activity in olfactory glomeruli

Odours generate activity in olfactory receptor neurons, whose axons contact the dendritic tufts of mitral cells within olfactory bulb glomeruli. These axodendritic synapses are anatomically separated from dendrodendritic synapses within each glomerulus. Mitral cells within a glomerulus show highly synchronized activity as assessed with whole-cell recording from pairs of mitral cells. We examined glomerular activity in mice lacking the olfactory cell adhesion molecule (OCAM). Glomeruli in mice lacking OCAM show a redistribution of synaptic subcompartments, but the total area occupied by axonal inputs was similar to wild-type mice. Stimulation of olfactory nerve bundles showed that excitatory synaptic input to mitral cells as well as dendrodendritic inhibition was unaffected in the knockout. However, correlated spiking in mitral cells was significantly reduced, as was electrical coupling between apical dendrites. To analyse slow network dynamics we induced slow oscillations with a glutamate uptake blocker. Evoked and spontaneous slow oscillations in mitral cells and external tufted cells were broader and had multiple peaks in OCAM knockout mice, indicating that synchrony of slow glomerular activity was also reduced. To assess the degree of shared activity between mitral cells under physiological conditions, we analysed spontaneous sub-threshold voltage oscillations using coherence analysis. Coherent activity was markedly reduced in cells from OCAM knockout mice across a broad range of frequencies consistent with a decrease in tightly time-locked activity. We suggest that synchronous activity within each glomerulus is dependent on segregation of synaptic subcompartments.

Digital pathology evaluation of complement C4d component deposition in the kidney allograft biopsies is a useful tool to improve reproducibility of the scoring

Complement C4d component deposition in kidney allograft biopsies is an established marker of antibody-mediated rejection. In the Banff 07 classification of renal allograft pathology, semi-quantitative evaluation of the proportion of C4d-positive peritubular capilaries (PTC) is used. We aimed to explore the potential of digital pathology tools to obtain quantitative and reproducible measure of C4d deposition in the renal allograft tissue.34 routine kidney allograft biopsies immunohistochemically stained for C4d were included in the study and were evaluated by a qualified pathologist twice, recording an approximate percentage of positive PTC and glomerular area. The same slides were scanned by Aperio ScanScope scanner. Two layers of annotations were created: layer of glomeruli and the remaining non-glomerular area. Image analysis was performed with Aperio Positive Pixel Count algorithm to quantify the proportion of C4d-positive pixels in the area analysed. The percentage of positive (defined as 2+ and 3+) pixels in glomeruli and non-glomerular area was obtained and compared to the percentage of C4d-positive PTC and C4d-positive area of glomeruli recorded by the pathologist.The correlation of digital and manual C4d-positive area scoring in glomeruli was very high (r= 0.89, p<0.0001), while the correlation for non-glomerular (digital) and PTC (manual) area was moderate (r=0.60, p<0.001). The correlation between digital and manual evaulation of C4d in non-glomerular area after exclusion of C4d-positive arterioles from analysis did not improve substantially (r = 0.59, p < 0.001). Reproducibility of digital and manual results was evaluated. For C4d deposition in PTC, agreement between the first and the second digital C4d evaluation (after re-drawing annotations) was perfect (κ=0.96, CI 0.91÷1.00) while agreement between two subsequent manual C4d scorings was substantial (κ = 0.67, CI 0.47 ÷ 0.88). Similarly, for C4d deposition in the glomeruli, agreement of digital evaluation was perfect (κ=1) while for manual scorings it was substantial (κ = 0.76, CI 0.64 ÷ 0.88).Digital evaluation of C4d deposition in allograft kidney correlates with pathologist‘s scoring and exceeds the latter in reproducibilty. Therefore, it provides a useful tool to control for intraobserver and interobserver variability and may serve as quality assurance measure for allograft pathology diagnosis and research.

The significance of caveolae in the glomeruli in glomerular disease

AimsThe aim of this study was to demonstrate expression of cell membrane invagination ‘caveolae’ in glomeruli and to correlate this with functional and structural characteristics of the human glomerular diseases.MethodsThe...

Fibronectin Glomerulopathy 4家系5症例の臨床病理学的特徴と形態学的検討

Fibronectin glomerulopathy (FNG) is an inherited disease, characterized by massive fibronectin (FN) deposits in the glomeruli. We semiquantitatively analyzed glomerular lesions and their progression in 5 cases with FNG from 4 different families: a 4 year-old male, a 19 year-old female, a 27 year-old male, a 58 year-old male (the father of the former case) and a 75 year-old male. All subjects showed a 201 times higher value of mean glomerular-tuft area (GA) and a 2.0 times higher mean number of mesangial cells (No. of MC) relative to control (p < 0.001). Strong positive correlations were observed between GA and the No. of MC (r = 0.86, p < 0.001). The younger cases showed markedly higher value of GA and No. of MC than the older cases. Mean individual capillary luminal area was decreased in all but one case and the mean total capillary luminal area, which is roughly estimated as the glomerular filtration area, was less changed compared with the control. The number of capillary loops tended to increase, indicating elongation of the capillary loops. The fractional area of FN (%FN), collagen IV (%Coll. IV) and laminin (%Lam) were high in all cases except for the first Bx in the 27 year-old case. The %FN strongly correlated with %Coll. IV and %Lam (r = 0.86, r = 0.69, p < 0.001, respectively). Serial biopsy (Bx) with a 10 year-interval was examined in the 27 year-old case and his father: GA and No. of MC were increased 1.4 and 1.9 times in the son, compared with his first Bx (p < 0.001, respectively), while no change was observed in the father. The %FN, %Coll. IV and %Lam were significantly increased in their second Bx (p < 0.001). These results suggest that 1) enlargement of the glomeruli in FNG is caused by intraglomerular accumulation of FN, Coll. IV and Lam and proliferated mesangial cells, 2) there is a strong influence from the aging factor, and 3) compensatory elongation of the loops (increase in the capillary luminal area) may maintain the glomerular filtration.

The involvement of Neuropilin-1 and immune Semaphorin's in lupus nephritis

Background and objectivesNeuropilin-1(NP-1), a known vascular endothelial growth factor (VEGF) receptor, is important to the interaction between activated DCs and resting T cells. It is also expressed on Treg cells,...

Renal protective effects of N-acetyl-Ser-Asp-Lys-Pro in deoxycorticosterone acetate–salt hypertensive mice

Hypertension-induced renal injury is characterized by inflammation, fibrosis and proteinuria. Previous studies have demonstrated that N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP) inhibits renal damage following diabetes mellitus and antiglomerular basement membrane nephritis. However, its effects on low-renin hypertensive nephropathy are not known. Thus, we hypothesized that Ac-SDKP has renal protective effects on deoxycorticosterone acetate (DOCA)-salt hypertensive mice, decreasing inflammatory cell infiltration, matrix deposition and albuminuria. We uninephrectomized 16-week-old C57BL/6J mice and treated them with either placebo, DCOA (10 mg/10 g body weight subcutaneous) and 1% sodium chloride with 0.2% potassium chloride in drinking water (DOCA-salt) or DOCA-salt with Ac-SDKP (800 μg/kg per day) for 12 weeks. We measured blood pressure, urine albumin, glomerular matrix, renal collagen content, monocyte/macrophage infiltration and glomerular nephrin expression. Treatment with DOCA-salt significantly increased blood pressure (P < 0.01), which remained unaltered by Ac-SDKP. Ac-SDKP decreased DOCA-salt-induced renal collagen deposition, glomerular matrix expansion and monocyte/macrophage infiltration. Moreover, DOCA-salt-induced increase in albuminuria was normalized by Ac-SDKP (controls, 10.8 ± 1.7; DOCA-salt, 41 ± 5; DOCA-salt + Ac-SDKP, 13 ± 3 μg/10 g body weight per 24 h; P < 0.001, DOCA-salt vs. DOCA-salt + Ac-SDKP). Loss of nephrin reportedly causes excess urinary protein excretion; therefore, we determined whether Ac-SDKP inhibits proteinuria by restoring nephrin expression in the glomerulus of hypertensive mice. DOCA-salt significantly downregulated glomerular nephrin expression (controls, 37 ± 8; DOCA-salt, 10 ± 1.5% of glomerular area; P < 0.01), which was partially reversed by Ac-SDKP (23 ± 4.0% of glomerular area; P = 0.065, DOCA-salt vs. DOCA-salt + Ac-SDKP). We concluded that Ac-SDKP prevents hypertension-induced inflammatory cell infiltration, collagen deposition, nephrin downregulation and albuminuria, which could lead to renoprotection in hypertensive mice.

Mean Glomerular Volume After Renal Transplantation in Patients Receiving Sirolimus and Cyclosporine A Compared With Elimination of Cyclosporine A at 3 Months

Glomerular enlargement after renal transplantation is associated with kidney allograft preservation. Increased glomerular size in donor biopsies is associated with donor age and with increased serum creatinine during follow-up, suggesting that older donors with larger glomeruli have exhausted their capacity for further glomerular adaptation (1). Allografts from younger donors with smaller glomeruli experience further increase of glomerular size after transplantation (2), which is associated with an improvement of renal function. The use of anticalcineurinic agents is associated with vasoconstriction and decreased glomerular filtration rate (GFR) (3). Sirolimus (SRL) has allowed withdrawing cyclosporine A (CsA) early after transplantation as it has been observed in the Rapamune Maintenance Regimen (RMR) study in which a steady improvement of GFR was observed in patients in the CsA withdrawal arm in comparison with the CsA maintenance group (4). We hypothesized that amelioration of GFR in patients after CsA withdrawal is related to glomerular size increase after transplantation. Sixty-four patients from a substudy (5) of the RMR trial conducted in Spain and Portugal with a donor and 1-year paired biopsies were reviewed. Details of the RMR study design and clinical results were published elsewhere (4–6). Of the 64 patients reviewed for this study, only 15 contained at least 10 glomerular sections in both biopsies (6 randomized to CsA elimination at 3 months and 9 maintained with CsA and SRL during the first year). Banff acute and chronic scores were blindly evaluated by a central pathologist. Vg was estimated according to the Weibel and Gomez formula using the mean glomerular area evaluated with an optical pen. Absolute differences between chronic Banff scores and mean glomerular volume (Vg) in both biopsies were calculated. Unpaired comparisons between means were done by Mann-Whitney U test and paired comparisons by Wilcoxon t test. The Vg in the donor and 1-year protocol biopsies in both groups is shown in Figure 1. In the CsA withdrawal group, a significant glomerular enlargement was observed (2.15±0.73× 106 μ3 in the donor and 2.66±0.78×106 μ3 in the 1-year biopsy, P=0.009) whereas in patients maintaining CsA, Vg decreased (2.87±0.81×106 μ3 in the donor and 2.27±0.70×106 μ3 in the 1-year biopsy, P=0.026). The Vg difference between the 1-year and the donor biopsy was significantly different between treatment arms (+0.51±0.31 vs. −0.60± 0.66×106 μ3, P=0.002).FIGURE 1.: Mean glomerular volume in donor and 1-year biopsies from patients randomized to cyclosporine A (CsA) maintenance or elimination at 3 months. Thin lines represent evolution of individual kidneys, and thick lines represent the mean for each group. SRL, sirolimus; P, prednisone.There was a positive correlation between Vg in the donor and 1-year biopsies (R=0.53; P=0.042), and this relationship was closer in patients eliminating CsA at 3 months (R=0.92, P=0.009) than in those maintaining CsA during the first year (R=0.65, P=0.059). The capsular tuft volume fraction was not different between both groups in the donor biopsy (0.58±0.13 vs. 0.61±0.07), whereas in the 1-year biopsy, it was lower in the CsA maintaining group (0.55±0.11 vs. 0.66± 0.34; P=0.045). Glomerular enlargement was associated with a lower progression of chronic lesions (Banff chronic score −0.7±1.0 vs. 2.5±1.7, P<0.01), whereas acute lesions at 1 year were not different between groups. Our data suggest that CsA prevents glomerular adaptation after transplantation and may contribute to explain why estimated GFR was superior in the CsA withdrawal group at 1 and 3 years of follow-up (4). In our study, it is important to remark that in patients receiving CsA and SRL during the first year, glomerular size significantly decreased, which may be the result of a synergistic effect of CsA and SRL impairing glomerular adaptation after renal transplantation by two different mechanisms: renal ischemia and inhibition of glomerular hypertrophy. This hypothesis is further sustained by the observation that 1-year capsule tuft volume fraction was lower in CsA- and SRL-treated patients. The higher degree of chronic tubulointerstitial damage observed in patients treated with CsA and SRL may constitute a third mechanism contributing to the impeded glomerular adaptation in patients receiving CsA and SRL. We have previously shown that interstitial inflammation and interstitial fibrosis are two major predictors of glomerular adaptation after transplantation (7). Thus, worse preservation of interstitial structure in CsA maintenance group may also account for the lack of glomerular adaptation because of peritubular capillary damage associated with interstitial fibrosis and inflammation. In summary, despite the number of cases included is low and consequently, these results should be interpreted with caution, we show that in stable grafts, the combination of CsA and SRL prevents glomerular enlargement after transplantation when compared with CsA withdrawal and SRL continuation. Joana Sellarés1 Francesc Moreso2 Juan Carlos Ruiz3 Daniel Seron2 1 Nephrology Department Hospital Universitari Bellvitge L'Hospitalet, Barcelona, Spain 2 Nephrology Department Hospital Vall d'Hebron Universitat Autonoma Barcelona Barcelona, Spain 3 Nephrology Department Hospital Universitario Marqués de Valdecilla Santander, Spain

Amelioration of renal alterations in obese type 2 diabetic mice by vasohibin-1, a negative feedback regulator of angiogenesis

The involvement of VEGF-A as well as the therapeutic efficacy of angiogenesis inhibitors in diabetic nephropathy have been reported. We recently reported the therapeutic effects of vasohibin-1 (VASH-1), an endogenous angiogenesis inhibitor, in a type 1 diabetic nephropathy model (Nasu T, Maeshima Y, Kinomura M, Hirokoshi-Kawahara K, Tanabe K, Sugiyama H, Sonoda H, Sato Y, Makino H. Diabetes 58: 2365-2375, 2009). In this study, we investigated the therapeutic efficacy of VASH-1 on renal alterations in obese mice with type 2 diabetes. Diabetic db/db mice received intravenous injections of adenoviral vectors encoding human VASH-1 (AdhVASH-1) and were euthanized 8 wk later. AdhVASH-1 treatment resulted in significant suppression of glomerular hypertrophy, glomerular hyperfiltration, albuminuria, increase in the CD31(+) glomerular endothelial area, F4/80(+) monocyte/macrophage infiltration, the accumulation of type IV collagen, and mesangial matrix. An increase in the renal levels of VEGF-A, VEGFR-2, transforming growth factor (TGF)-β1, and monocyte chemoattractant protein-1 in diabetic animals was significantly suppressed by AdhVASH-1 (immunoblotting). AdhVASH-1 treatment significantly recovered the loss and altered the distribution patterns of nephrin and zonula occludens (ZO)-1 and suppressed the increase in the number of fibroblast-specific protein-1 (FSP-1(+)) and desmin(+) podocytes in diabetic mice. In vitro, recombinant human VASH-1 (rhVASH-1) dose dependently suppressed the upregulation of VEGF induced by high ambient glucose (25 mM) in cultured mouse podocytes. In addition, rhVASH-1 significantly recovered the mRNA levels of nephrin and the protein levels of ZO-1 and P-cadherin and suppressed the increase in protein levels of desmin, FSP-1, Snail, and Slug in podocytes under high-glucose condition. Taken together, these results suggest the potential use of VASH-1 as a novel therapeutic agent in type 2 diabetic nephropathy mediated via antiangiogenic effects and maintenance of podocyte phenotype in association with antiproteinuric effects.