Glomerular Protein Research Articles

C3G overexpression in glomerular epithelial cells during anti-GBM-induced glomerulonephritis

The guanine nucleotide exchange factor C3G, along with the CrkII adaptor protein, mediates GTP activation of the small GTPase proteins Rap1 and R-Ras, facilitating their activation of downstream signaling pathways, which had been found to be important in the pathogenesis of glomerulonephritis. We found that expression of C3G protein was upregulated in glomerular epithelial cells in an experimental model of accelerated anti-GBM antibody-induced glomerulonephritis expression. To determine the consequence of its increased expression, we transfected C3G (using adenoviral constructs) into cultured glomerular epithelial cells and measured the activated forms (i.e., GTP-bound) forms of Rap1 and R-Ras. Activation of Rap1 was not affected by C3G; however, the basal level of GTP-bound R-Ras was decreased. Further, C3G over-expression enhanced the activation of R-Ras in response to endothelin. Overexpression of C3G also led to a significant reduction in glomerular epithelial cell spreading and decreased the cells' E-cadherin expression and augmented their migration. We found that C3G was overexpressed in accelerated anti-GBM antibody-induced glomerulonephritis and suggest that this modulates glomerular epithelial cell morphology and behavior.

Contributions of blood pressure to proteinuria and renal function in the puerperium

Background. Increasing evidence indicates that hypertension in pregnancy plays a key role in cardiovascular disease. However, the correlation between blood pressure (BP) and proteinuria as well as renal function in the puerperium has not been established. Patients. We evaluated the estimated glomerular filtration rate (eGFR) and 24-h urine total protein excretion (TPE) during the second postpartum week in 852 pregnant women with normal BPs and 114 pre-eclamptic women with BPs ≥140/90 mmHg. The 852 pregnant women with normal BPs were divided into two groups based on the mean arterial pressure (MAP): 684 subjects with an MAP<90 mmHg and 168 subjects with an MAP≥90 mmHg. Results. The eGFR was significantly decreased in pre-eclamptic women (112±41 ml/min/1.73 m2) compared with healthy women with an MAP<90 mmHg (131±35 ml/min/1.73 m2, p<0.01) and an MAP≥90 mmHg (128±34 ml/min/1.73 m2, p<0.01), while the TPE was significantly increased compared with healthy women with an MAP<90 mmHg and an MAP≥90 mmHg (1790±1422 vs 124±148 and 255±427 mg/24 h, respectively; p<0.001). Although the eGFR did not reveal a difference between the two groups of healthy women (131±35 vs 128±34 ml/min/1.73 m2, p>0.05), the TPE was significantly higher in subjects with an MAP≥90 mmHg than in subjects with an MAP<90 mmHg (255±427 vs 124±148 mg, p=0.004). Conclusions. Pre-eclampsia induces significant renal injury characterized by an elevation of TPE and a reduction in GFR. BP is closely related to urinary protein excretion, even in healthy women (BP <140/90 mmHg) in the puerperium.

Discrepancy between Intrarenal Messenger RNA and Protein Expression of ACE and ACE2 in Human Diabetic Nephropathy

Background: The intrarenal angiotensin-converting enzyme (ACE) and type 2 ACE (ACE2) play important roles in the pathogenesis of diabetic nephropathy, but human data are limited. We studied glomerular and tubulointerstitial mRNA and the protein expression of ACE and ACE2 in patients with diabetic nephropathy. Methods: We studied renal biopsy specimens of 22 patients with diabetic nephropathy and 11 transplant donors as normal controls. Intrarenal mRNA expression of ACE and ACE2 was measured by laser microdissection and real-time quantitative polymerase chain reaction; expression at the protein level was determined by immunostaining. Results: Glomerular and tubulointerstitial mRNA expression levels of ACE and ACE2 were significantly higher in patients with diabetic nephropathy than in normal controls (p < 0.001 for all comparisons). Glomerular ACE and ACE2 protein levels of patients with diabetic nephropathy were significantly higher than those of kidney donors (4.90 ± 2.55% vs. 2.64 ± 0.98%, p = 0.022, and 7.40 ± 3.36% vs. 4.37 ± 2.36%, p = 0.017, respectively). The tubulointerstitial ACE at the protein level, however, was similar between diabetic patients and controls (8.76 ± 4.18% vs. 10.44 ± 6.61%, p = 0.453), and the tubulointerstitial ACE2 at the protein level was significantly lower in diabetic nephropathy (16.48 ± 7.68% vs. 23.23 ± 7.65%, p = 0.025). Conclusion: The mRNA expression of ACE and ACE2 increased in both the glomerular and tubulointerstitial area of diabetic nephropathy. However, the tubulointerstitial ACE expression at the protein level remained unchanged, while that of ACE2 actually decreased. Our results suggest a posttranscriptional modulation of tubulointerstitial ACE and ACE2 expression. Experimental data of intrarenal mRNA expression of ACE and ACE2 should be interpreted with caution.

Excretion of Proteins by Rat Kidney during Various Types of Diuresis

Experiments on healthy rats showed that increased diuresis induced by administration of water, polyethylene glycol 400, furosemide, or 1-desamino-arginine-vasotocin is associated with increased protein excretion by the kidneys. The results can be explained by enhanced filtration of plasma proteins in glomeruli during polyuria of various geneses.

Changes in protein profiles during course of experimental glomerulonephritis

Better characterization of the molecular mechanisms underlying glomerular cell proliferation may improve our understanding of the pathogenesis of glomerulonephritis and yield disease-specific markers. We used two-dimensional gel electrophoresis (2DE) and mass spectrometry (MS) to generate expression profiles of glomerular proteins in the course of anti-Thy-1 nephritis. Glomeruli were isolated from Wistar rats by sieving, and proteins were separated by 2DE. In preliminary studies using normal rats, we identified known glomerular proteins from microfilaments [tropomyosin (Tm)] and intermediate filaments (vimentin and lamin A), proteins involved in assembly (alpha-actinin-4, F-actin capping protein) and membrane cytoskeletal linking (ezrin), as well as several enzymes (protein disulfide isomerase, ATP synthase, and aldehyde dehydrogenase). Comparison of glomerular protein abundance between normal rats and rats in the early phase of anti-Thy-1 nephritis yielded 28 differentially expressed protein spots. MS analysis identified 16 differentially expressed proteins including Tm. Altered Tm abundance in the course of anti-Thy-1 nephritis was confirmed, and specific isoforms were characterized by Western blotting. We demonstrated a complex change in Tm isoform abundance in the course of anti-Thy-1 nephritis. The early mesangiolytic phase of the disease was characterized by decreased abundance of low-molecular-weight isoforms Tm5a/5b and increased abundance of high-molecular-weight isoforms Tm6, Tm1, Tm2, and Tm3. The late proliferative phase of the disease was associated with increased abundance of isoforms Tm5a/5b, Tm6, and Tm1 and decreased abundance of Tm3. Isoforms Tm4 and Tm5 remained unchanged in the course of this model of experimental glomerulonephritis. Characterization of Tm isoform abundance in the course of clinical glomerulonephritis may identify disease-specific markers.

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Involvement of hyperglycemia in deposition of aggregated protein in glomeruli of diabetic mice

The aim of this study was to clarify the influence of hyperglycemia on the deposition of aggregated protein in the glomeruli of diabetic mice. KK-A y mice injected with aggregated bovine serum albumin accumulated more of it in the glomeruli than did ICR mice. There were no histological alterations in the glomeruli of KK-A y mice. KK-A y mice given voglibose in mouse-chow for 2 weeks had significantly reduced blood glucose, glycated albumin, and hemoglobinA 1C levels compared with control mice. The voglibose-treated KK-A y mice were injected with aggregated bovine serum albumin and accumulated significantly less albumin in the glomeruli than did the control mice. Pioglitazone decreased blood glucose levels compared with the control, and reduced the glomerular deposition of aggregated albumin. Glomerular aggregated bovine serum albumin levels and blood glucose levels were reduced significantly by the injection of insulin. Six times more advanced glycation endproducts were produced from aggregated bovine albumin than from non-aggregated bovine albumin on incubation with glucose and L-lysine in vitro. Glucose-loaded ICR mice generated more advanced glycation endproducts from aggregated albumin, and had more aggregated bovine albumin in the glomeruli. It was suggested that hyperglycemia contributes to an increase in the deposition of aggregated protein in glomeruli even early on in diabetes.

Therapeutic benefit of treatment with anti-thymocyte globulin and latent TGF-β1 in the MRL/lpr lupus mouse model

The pathogenesis of systemic lupus erythematosus is believed to involve defects in regulatory T cell (Treg) activity and abnormal activation of B and T lymphocytes. The purpose of this study was to test the therapeutic potential of rabbit anti-mouse thymocyte globulin (ATG), a lymphocyte-depleting agent, in conjunction with transforming growth factor (TGF)-beta1, a factor involved in the induction and expansion of Tregs. MRL/lpr mice with active disease were treated with ATG followed by a 12-day course of latent TGF-beta1 during the period of lymphocyte repopulation. Treatment with ATG + latent TGF-beta1 synergistically inhibited the progression of proteinuria and albuminuria and provided a significant improvement in long-term survival. This therapeutic benefit correlated histologically with reduced glomerular pathology and protein cast formation. The mechanism of action did not involve suppression of autoantibody formation but may involve the activity of CD4+CD25+FoxP3+ Tregs, which were found to be induced by ATG + TGF-beta1 treatment in vitro.

Glomerular angiotensinogen protein is enhanced in pediatric IgA nephropathy

Enhanced intrarenal renin-angiotensin system (RAS) is implicated in the development and progression of renal injury. To investigate whether angiotensinogen (AGT) expression is involved in glomerular RAS activity and glomerular injury, we examined glomerular AGT expression and its correlation with expression of other RAS components, and levels of glomerular injury in samples from patients with immunoglobulin A nephropathy (IgAN) (23) and minor glomerular abnormalities (MGA) (8). Immunohistochemistry showed that AGT protein was highly expressed by glomerular endothelial cells (GEC) and mesangial cells in nephritic glomeruli of IgAN compared with glomeruli of MGA. Levels of glomerular AGT protein were well correlated with levels of glomerular angiotensin II (ang II), transforming growth factor-β (TGF-β), α-smooth-muscle actin, glomerular cell number, and glomerulosclerosis score but not with those of glomerular angiotensin-converting enzyme and ang II type 1 receptor. Real-time polymerase chain reaction (RT-PCR) and Western blot analyses using cultured human GEC indicated that ang II upregulated AGT messenger ribonucleic acid (mRNA) and protein expression in a dose- and time-dependent manner. These data suggest that activated glomerular AGT expression is likely involved in elevated local ang II production and, thereby, may contribute to increased TGF-β production and development of glomerular injury in IgAN. Augmentation of GEC-AGT production with ang II stimulation might drive further glomerular injury in a positive-feedback loop.

The glomerular proteome in a model of chronic kidney disease

Adequate kidney function is crucial in sustaining vertebrate homeostasis. Certain diseases can diminish renal function and lead to end-stage renal disease. Diabetes mellitus and hypertension are the main causes of glomerulosclerosis and albuminuria in adults. The molecular mechanisms that trigger these maladaptive changes are still unsatisfyingly described. We previously introduced 2-D DIGE in combination with focused tissue isolation methods to analyze protein expression in glomeruli. Glomeruli, the crucial compartments in albuminuric renal diseases, were extracted using magnetic particles from subtotally nephrectomized FVB mice (n = 6); this 5/6 nephrectomy in FVB mice is a model of chronic kidney disease. Analysis of protein expression levels from glomerular protein lysates was performed using 2-D DIGE and compared with glomerular protein lysates from mice that underwent sham surgery. The comparison of about 2100 detectable spots between both groups revealed 48 protein spots that showed significant differential expression. Of those, 33 proteins could be identified using nanoLC-ESI MS. The metalloproteinase meprin 1 alpha, the beta galactoside-binding-lectin galectin-1 and dimethylarginine dimethylaminohydrolase 1, a key enzyme in NO metabolism, were found to be differentially regulated, thus implying a role in the pathogenesis and pathophysiology of progressive kidney disease. In conclusion, 2-D DIGE protein analysis of smallest sample sizes from specific organ compartments provides focused protein expression results, which help in gaining an understanding of the molecular mechanisms of chronic kidney disease.

Membrane protein identifications by mass spectrometry using electrocapture-based separation as part of a two-dimensional fractionation system

A two-dimensional (2D) separation method was used to decrease sample complexity in analysis of tryptic peptides from glomerular membrane proteins by tandem mass spectrometry (MS/MS). The first dimension was carried out by electrocapture (EC), which fractionates peptides according to electrophoretic mobility. The second dimension was reverse-phase liquid chromatography (RP–LC), in which EC fractions were further separated and analyzed online by MS/MS. Using this methodology, we now identify 102 glomerular proteins (57 membrane proteins). Many peptides were possible to observe and select for MS/MS only using the 2D approach. Others were detectable in both one-dimensional (1D, without the EC step) and 2D experiments but were selectable for sequence analysis only from the 2D separations because the decrease in complexity then gives time for the mass analyzer to select the peptide and switch to the MS/MS mode. A minority of the peptides were detectable only in the 1D mode (presumably because of handling losses), but at the end this did not decrease the number of proteins identified by the 2D separation. After a database search, the combination of EC and RP–LC MS/MS versus a 1D RP–LC MS/MS separation resulted in a threefold increase in the number of proteins identified and improved the sequence coverage in the identifications, bringing our proteome-identified glomerular proteins to 282.

The protective role of uteroglobin through the modulation of tissue transglutaminase in the experimental crescentic glomerulonephritis

Tissue transglutaminase (tTG) may induce pro-inflammatory cytokines and produce irreversible end-products, thus promoting renal scarring. It has recently been confirmed that the crescent formation in murine experimental crescentic glomerulonephritis (ecGN) has been inhibited by the administration of recombinant uteroglobin (rUG). However, the ability of UG on tTG modulation has not been thoroughly assessed. In this study, we investigated the feasible protective role of UG in murine ecGN through the modulation of tTG and TGF-beta1 expressions. ecGN was induced by the administration of anti-GBM Ab into C57BL/6 mice. Both proteinuria and BUN levels were distinctively lower in rUG-treated mice compared to those of disease control mice. Glomerular injuries such as mesangial proliferation, matrix production and crescent formation were lessened with the rUG treatment, and these findings were parallel with the attenuated expression of tTG and TGF-beta1. tTG and TGF-beta1 were expressed mainly on mesangial areas by the induction of ecGN and rUG treatment markedly attenuated the expressions of these proteins in glomeruli without spatial changes. With the addition of LPS to mesangial cells, the expressions of tTG and TGF-beta1 were up-regulated, whilst the addition of cysteamine, tTG inhibitor, attenuated the expression of tTG and TGF-beta1 as well as the cellular proliferation which was further induced by LPS. We demonstrate for the first time that rUG is able to attenuate the renal injury through the modulation of expressions of tTG and TGF-beta1 in ecGN and further suggest a wide range of feasible molecular targets to reduce the severity of human glomerulonephritis.

The pattern of proteinuria following karate (Kumite) competitions.

Sir, Despite the large amount of proteins in the plasma, the urine is virtually protein free due to the selectivity of the glomerular barrier [1]. The major component of urinary protein is a tubular protein (Tamm-Horsfall) while albumin constitutes 30–40% of the total urinary protein. Various physiologic settings, including exercise, can induce a transient increase in the urinary protein excretion that is usually benign and reversible. The type of post-exercise proteinuria depends on the intensity of exercise rather than its duration, so that moderate exercise induces glomerular and heavy exercise glomerular-tubular mixed-type protein loss [2]. Post-exercise proteinuria may be due to the loss of the charge selectivity from the glomerular capillary wall, a relative preservation of the glomerular filtration rate, proteinuria out of proportion of maximal tubular reabsorption capacity following heavy exercise [2,3] and oxidative stress produced by free radicals due to enhanced oxygen consumption in muscles [4]. The purpose of our study was to compare the amount and pattern of proteinuria before and after karate (Kumite) competition in 18 male practitioners, aged 18–21 years, with similar physical characteristics. All practitioners competed in three rounds, each lasted 3 min, with a 10 min resting interval between them. Urine samples were collected just before the competition and during 24 h thereafter. Total urinary protein, urinary beta2-microglobulin B2M as tubular and albumin as glomerular protein were assayed. Before competition, the mean value of total urinary protein, albumin and B2M as basal levels were 70.68 ± 12.5, 4.84 ± 3.17 and 0.0217 ± 0.0133 mg/day, respectively. After competition, the mean values of 24-h total urine protein (196.05 ± 70.88 mg/day), albumin (34.07 ± 32.88 mg/day) and B2M (0.0933 ± 0.0372 mg/day) levels were significantly increased (P = 0.023, P = 0.001 and P < 0.001). This study revealed significantly increased proteinuria of a mixed type (albumin and B2M) in all practitioners following exhaustive short-term competition. Despite the mixed type of proteinuria, we observed a six-fold increase compared to the basal level in urine albumin (versus four-fold increase for urinary B2M) suggesting a more prominent role of glomerular proteinuria probably due to glomerular membrane permeability changing factors, such as sympathetic overactivity and competitive stress. Conflict of interest statement. None.

RhoA/Rho-Kinase Contribute to the Pathogenesis of Diabetic Renal Disease

Accumulation of glomerular matrix proteins is central to the pathogenesis of diabetic nephropathy, with resident mesangial cells (MCs) known to upregulate matrix protein synthesis in response to high glucose. Because activation of the GTPase RhoA has been implicated in matrix upregulation, we studied its role in induction of the matrix protein fibronectin in diabetic MCs and in vivo in diabetic nephropathy. Glucose (30 mmol/l)-induced RhoA/Rho-kinase, AP-1 activation, and fibronectin upregulation were assessed by immunoblotting, luciferase, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, real-time PCR, Northern blots, and immunofluorescence. Streptozotocin-induced diabetic rats were treated with the rho-kinase inhibitor fasudil, which was compared with enalapril, and functional and pathologic parameters were assessed. Glucose led to RhoA and downstream Rho-kinase activation. Mannitol was without effect. Activity of the transcription factor AP-1, increased in diabetic MCs and kidneys, is important in the profibrotic effects of glucose, and this was dependent on Rho-kinase signaling. Upregulation of fibronectin by glucose, shown to be mediated by activator protein-1 (AP-1), was prevented by Rho-kinase inhibition. RhoA siRNA and dominant-negative RhoA also markedly attenuated fibronectin upregulation by high glucose. Applicability of these findings were tested in vivo. Fasudil prevented glomerular fibronectin upregulation, glomerular sclerosis, and proteinuria in diabetic rats, with effectiveness similar to enalapril. High glucose activates RhoA/Rho-kinase in MCs, leading to downstream AP-1 activation and fibronectin induction. Inhibition of this pathway in vivo prevents the pathologic changes of diabetic nephropathy, supporting a potential role for inhibitors of RhoA/Rho in the treatment of diabetic renal disease.

The mTOR Inhibitor Everolimus Attenuates the Time Course of Chronic Anti-Thy1 Nephritis in the Rat

Background: The antiproliferative immunosuppressant everolimus adversely affects the acute reversible anti-Thy1 nephritis model. We hypothesized that everolimus treatment started after the acute proliferative phase could even be beneficial in the chronic anti-Thy1 nephritis model in the rat. Methods: Chronic anti-Thy1 nephritis was induced by injection of the monoclonal antibody 1-22-3 in 20 male Sprague-Dawley rats 7 days after uninephrectomy. Two weeks after disease induction, rats were randomly treated with either everolimus or vehicle for 14 weeks. Changes in progression of renal disease were investigated by immunohistochemistry and real-time PCR 16 weeks after disease induction. Results: During chronic anti-Thy1 nephritis, the formation of focal segmental glomerulosclerosis lesions, the degree of interstitial fibrosis as well as the increase in proteinuria over 14 weeks was ameliorated by everolimus treatment. Increased glomerular hypertrophy observed in the vehicle-treated rats was completely prevented in the everolimus-treated nephritic rats. Increased glomerular fibronectin mRNA and protein as well as the renal influx of monocytes/macrophages was significantly reduced in the everolimus group. Everolimus reduced the pro-angiogenic factor vascular endothelial growth factor (VEGF) and VEGF mRNA in glomeruli, while the transforming growth factor-β signaling pathway was not affected. Conclusion: ‘Late’ start of everolimus treatment demonstrates beneficial effects on the time course of chronic anti-Thy1 nephritis.

Glomerular Hypertrophy in Offspring of Subtotally Nephrectomized Ewes

We have shown that fetuses whose mothers underwent subtotal nephrectomy (STNx) before pregnancy had high urine flow rates and sodium excretions, but lower hematocrits, plasma chloride, and plasma renin levels compared with controls. To see if these functional differences in utero persist after birth and are the result of altered renal development, we studied 8 lambs born to STNx mothers (STNxL) and 10 controls (ConL) in the second week of life. These lambs were of similar body weights, nose-rump lengths and abdominal girths. Their kidney weights were not different (ConL 36.1 +/- 1.9 vs. STNxL 39.8 +/- 3.3 g), nor were kidney dimensions or glomerular number (ConL 423,520 +/- 22,194 vs. STNxL 429,530 +/- 27,471 glomeruli). However, STNxL had 30% larger glomerular volumes (both mean and total, P < 0.01) and there was a positive relationship between total glomerular volume and urinary protein excretion (P < 0.05) in STNxL. Despite this change in glomerular morphology, glomerular filtration rate, tubular function, urine flow, and sodium excretion rates were not different between STNxL and ConL, nor were plasma electrolytes, osmolality, and plasma renin levels. Thus while many of the functional differences seen in late gestation were not present at 1-2 weeks after birth, the alteration in glomerular size and its relationship to protein excretion suggests that exposure to this altered intrauterine environment may predispose offspring of mothers with renal dysfunction to renal disease in adult life.

The mTOR Inhibitor Everolimus Induces Proteinuria and Renal Deterioration in the Remnant Kidney Model in the Rat

Under certain circumstances the nonnephrotoxic, antiproliferative, immunosuppressive mammalian target of rapamycin (mTOR) inhibitors can cause renal deterioration and proteinuria after conversion from calcineurin inhibitors, especially in long-term renal transplant patients with low glomerular filtration rates. The mTOR inhibitors also show an impaired glomerular healing reaction during acute renal injury in experimental mesangial proliferative glomerulonephritis. In this study, everolimus treatment was investigated in a low nephron number model, the remnant kidney model in rats. The remnant kidney model was induced by uninephrectomy and infarction of 2/3 of the remaining kidney in 31 male Sprague-Dawley rats. Three days after disease induction, rats were randomly treated either with everolimus or vehicle. Changes in progression of renal disease were investigated by immunohistochemistry on days 22 and 38 after disease induction. In the remnant kidney model, everolimus treatment worsened chronic disease progression as assessed by increased proteinuria, glomerulosclerosis, interstitial fibrosis, glomerular inflammation as well as decreased creatinine-clearance. This result was due to a markedly increased fraction of glomeruli with a defective glomerular architecture in the everolimus group. Everolimus apparently inhibited the chronic glomerular repair reaction via inhibition of the proliferative but not apoptotic activity of the glomerular endothelial and mesangial cells, which was associated with reduced glomerular vascular endothelial growth factor mRNA and protein. In contrast, the fraction of glomeruli with an intact glomerular architecture within the everolimus group showed clearly less glomerular enlargement compared to vehicle-treated nephrectomy rats. This study demonstrates potential mechanisms of mTOR inhibitor induced renal deterioration and proteinuria in the low nephron number remnant kidney model.

Interactions between maternal subtotal nephrectomy and salt: effects on renal function and the composition of plasma in the late gestation sheep fetus

Effects of altered maternal salt intake between 122 and 127 days gestation (term is 150 days) were studied in eight fetuses carried by ewes which had renal insufficiency caused by subtotal nephrectomy (STNxF) and seven fetuses carried by intact ewes (IntF). Plasma sodium and osmolality were increased in ewes with subtotal nephrectomy on a high-salt intake (0.17 m NaCl in place of drinking water for 5 days; P < 0.05). The STNxF had normal body weights. A high maternal salt intake did not affect fetal blood pressure or heart rate. Plasma osmolality was higher in STNxF (P < 0.001), and plasma sodium and osmolality were increased by high salt (P < 0.001 and P < 0.04, respectively). The STNxF had higher urinary osmolalities (P = 0.002), which were also increased by a high maternal salt intake (P = 0.03). Renal blood flow fell in STNxF in response to a high maternal salt intake, but increased in IntF (P = 0.003). In STNxF but not IntF, glomerular filtration rate and urinary protein excretion were positively related to fetal plasma renin levels (P < or = 0.01). It is concluded that the salt intake of pregnant ewes with renal insufficiency affects maternal and fetal osmolar balance, fetal plasma sodium and fetal renal function. Since STNxF also had altered renal haemodynamic responses to high maternal salt and evidence of renin-dependent glomerular filtration and protein excretion, we suggest that interactions between dietary salt and pre-existing maternal renal disease impair glomerular integrity and function in the fetus.

Basic science meets clinical medicine: identification of a CD2AP-deficient patient

Recent years have witnessed an explosion of research into the molecular basis of glomerular disease resulting in nephrotic- range urinary protein leak using both human genetics and animal models. Löwik et al. describe the first case report of an early-onset nephrotic syndrome presenting in conjunction with a homozygous CD2AP mutation. These data demonstrate the convergence between basic and clinical approaches and their potential to transform our understanding of the pathogenetic mechanisms underlying human glomerular disease.

Monocyte chemoattractant protein-1 has prosclerotic effects both in a mouse model of experimental diabetes and in vitro in human mesangial cells

Diabetic nephropathy is characterised by mesangial extracellular matrix accumulation. Monocyte chemoattractant protein-1 (MCP-1), a chemokine promoting monocyte infiltration, is upregulated in the diabetic glomerulus. We performed in vitro and in vivo studies to examine whether MCP-1 may have prosclerotic actions in the setting of diabetes, presumably via its receptor, chemokine (C-C motif) receptor 2 (CCR2), which has been described in mesangial cells. Human mesangial cells were exposed to recombinant human (rh)-MCP-1 (100 ng/ml) for 12, 24 and 48 h and to rh-MCP-1 (10, 100 and 200 ng/ml) for 24 h. Fibronectin, collagen IV and transforming growth factor, beta 1 (TGF-beta1) protein levels were measured by ELISA and pericellular polymeric fibronectin levels by western blotting. The intracellular mechanisms were investigated using specific inhibitors for CCR2, nuclear factor kappa B (NF-kappaB), p38 mitogen-activated protein kinase and protein kinase C, and an anti-TGF-beta1 blocking antibody. In both non-diabetic and streptozotocin-induced diabetic mice that were deficient or not in MCP-1, glomerular fibronectin accumulation was examined by immunohistochemistry, while cortical Tgf-beta1 (also known as Tgfb1) and fibronectin mRNA and protein levels were examined by real-time PCR and western blotting. In mesangial cells, MCP-1 binding to CCR2 induced a 2.5-fold increase in fibronectin protein levels at 24 h followed by a rise in pericellular fibronectin, whereas no changes were seen in collagen IV production. MCP-1-induced fibronectin production was TGF-beta1- and NF-kappaB-dependent. In diabetic mice, loss of MCP-1 diminished glomerular fibronectin protein production and both renal cortical Tgf-beta1 and fibronectin mRNA and protein levels. Our in vitro and in vivo findings indicate a role for the MCP-1/CCR2 system in fibronectin deposition in the diabetic glomerulus, providing a new therapeutic target for diabetic nephropathy.