Glomerular Barrier Research Articles

Why do we not all have proteinuria? An update of our current understanding of the glomerular barrier.

The key question is not why some patients have proteinuria but rather why not all people have it. In the present review, we will present an update on the glomerular barrier after the recent breakthroughs in podocyte biology. In particular, we will discuss the role of the endothelium, which seems to be a neglected part of the glomerular membrane.

The focal segmental glomerulosclerosis permeability factor: biochemical characteristics and biological effects.

Focal segmental glomerulosclerosis (FSGS) is characterized by steroid resistant nephrotic syndrome and progression to end-stage renal disease. Proteinuria in certain patients with FSGS may be caused by a circulating factor (FSGS permeability factor [FSPF]). The current report documents the biochemical characteristics and the biological and molecular effects of 70% ammonium sulfate supernatant of plasma from patients with recurrence of FSGS after transplantation (FSGS 70% supernatant). FS permeability activity, defined as the capacity of plasma from patients with FSGS to increase albumin permeability (P(alb)) of isolated glomeruli, was assessed in vitro. Permeability activity was not affected by lyophilization. FSPF bound strongly to matrices containing Mono-Q anion exchanger or protein A. It eluted from matrix-bound Cibacron blue F3GA over a wide range of salt concentrations, indicating a potential binding with other proteins, such as albumin. FSPF caused a maximal increase in P(alb) within 2 mins of incubation in vitro. Cellular proteins isolated from glomeruli with increased P(alb) showed decreased tyrosine phosphorylation of focal adhesion kinase, paxillin, and other proteins. Tyrosine phosphatase ]inhibition prevented the increase in P(alb). Intravenous administration of as little as 3 mg protein in FSGS 70% supernatant increased P(alb), while 9 mg or more were required to produce proteinuria. We conclude that FSPF is a low-molecular-weight protein, carries an anionic charge, and binds to protein A. Effects of FSPF on the glomerular permeability barrier are rapid and dose dependent and involve signaling through altered phosphorylation of cellular proteins. Identification of these biochemical and biological characteristics may be used to design strategies for removing FSPF from circulation and for purification and identification of this factor.

Dynamic (re)organization of the podocyte actin cytoskeleton in the nephrotic syndrome.

The visceral glomerular epithelial cell, also known as the podocyte, plays an important role in the maintenance of renal glomerular function. This cell type is highly specialized and its foot processes together with the interposed slit diaphragm (SD) form the final barrier to urinary protein loss. Effacement of foot processes is associated with the development of proteinuria and-if not reversed in a certain time-with permanent deterioration of the glomerular filter. To maintain an intact glomerular filter barrier, podocyte-podocyte interactions and podocyte interactions with the glomerular basement membrane (GBM) are essential. Recent years have highlighted podocyte functions by unraveling the molecular composition of the SD, but have also clarified the important role of the podocyte actin cytoskeleton, and the podocyte-GBM interaction in the development of foot process (FP) effacement. This review provides an update of podocyte functions with respect to novel podocyte-specific proteins and also focuses on the dynamic interaction between the actin cytoskeleton of podocytes, their cell surface receptors and the GBM.

CD2-associated protein and glomerular disease.

Proteinuria is a major cause of progression in renal disease. The glomerular ultrafiltration barrier, containing highly differentiated podocytes, normally restricts protein access to the urine. Patients with urinary protein in the nephrotic range (>3.5 g daily) often have effaced podocyte foot-processes. Slit diaphragms span the gaps between foot processes as a barrier to macromolecules. Nephrin and podocin are slit-diaphragm proteins identified in families with congenital nephrotic syndromes. CD2-associated protein (CD2AP) is an adapter protein originally identified as a novel ligand interacting with the T-cell-adhesion protein CD2. CD2AP knockout (-/-) mice develop a congenital nephrotic syndrome with podocyte foot-process effacements and die at 6 weeks of age from renal failure. CD2AP localises to the slit diaphragm and links nephrin and podocin to phosphoinositide 3-OH kinase; this complex has cell-signalling properties. The CD2AP +/- heterozygous mice developed by Jeong Kim and colleagues (Science 2003; 300: 1298-300) are haploinsufficient and develop glomerular changes at 9 months of age with a histological pattern similar to that in human focal segmental glomerulosclerosis. These researchers found that 2 of 30 African-American patients with idiopathic focal segmental glomerulosclerosis had a CD2AP mutation that ablated expression of one allele. WHERE NEXT? Further studies should address the normal distribution of the CD2AP heterozygous mutation in different ethnic populations, because the association with human idiopathic focal segmental glomerulosclerosis could be accidental. Decreased expression of CD2AP in podocytes of individuals with the CD2AP heterozygous mutation would help to understand how the haploinsufficiency translates into increased susceptibility to renal disease. Transfection of podocytes with mutated CD2AP or study of cultured podocytes from CD2AP +/- mice would provide further insight into whether the nephrin-podocin-CD2AP signal-transduction pathway is altered and leads to increased apoptosis of podocytes. Assuming that a decrease in CD2AP attenuates clearance of glomerular immune complexes, patients with other types of idiopathic glomerulonephritis should also have a CD2AP mutation. However, first studies looking at the most common form of glomerulonephritis, IgA nephropathy, have failed to show decreased renal CD2AP expression.

Clustering-induced tyrosine phosphorylation of nephrin by Src family kinases

Nephrin is a recently discovered protein of the immunoglobulin (Ig) superfamily. In the kidney, it is located at the slit diaphragm, which forms the decisive size-selective filter of glomerular ultrafiltration barrier and locates between the interdigitating foot processes of podocytes. Nephrin is mutated in congenital nephrosis of the Finnish type (NPHS1) and has been demonstrated to be an essential component of the slit diaphragm. Based on its domain structure, nephrin is likely to be a cell-cell or cell-matrix adhesion protein that may have a signaling function. In this study, we hypothesized that the clustering of nephrin with antibodies on cell surface mimics the situation where the interaction between nephrin and its extracellular ligand(s) is altered. Nephrin was clustered on the surface of stably transfected HEK293 cells by a monoclonal antinephrin antibody and polyclonal secondary antibody. Clusters were visualized by immunofluorescence microscopy. Changes in protein phosphorylation were studied employing immunoprecipitations and Western blot analysis. A specific inhibitor and cotransfection experiments were used to investigate role of Src family kinases in nephrin phosphorylation. Clustering of nephrin induced its own tyrosine phosphorylation. This phosphorylation was inhibited by PP2, an inhibitor of Src family kinases. Several members of Src family kinases were able to induce nephrin phosphorylation when cotransfected to HEK293 cells with nephrin. Moreover, the Src family kinase Fyn was consistently found to be coimmunoprecipitated with nephrin. Interestingly, clustering of nephrin induced also tyrosine phosphorylation of a 46 kD protein that was as well found to be coimmunoprecipitated with nephrin. Nephrin is a signaling protein phosphorylated by Src family kinases.

Targeted Downregulation of Extracellular Nephrin in Human IgA Nephropathy

Background: The identification of nephrin and CD2AP, podocyte proteins which modulate the properties of glomerular barrier selectivity, has made it possible to unravel the mechanisms undergoing foot process effacement and proteinuria. Here we explored the role of nephrin and CD2AP together with the integrity of the slit diaphragm in the pathogenesis of proteinuria in patients with acquired glomerular diseases. Methods: Nephrin mRNA and protein expression were systematically evaluated in 28 renal biopsy samples from adult patients with primary glomerular disease and proteinuria by in situ hybridization and immunohistochemistry using antibodies directed against extra- and intracellular nephrin and compared with biopsy samples from normal controls. CD2AP protein expression by immunohistochemistry was also assessed. Morphometrical analysis of the filtration slit was performed by transmission electron microscopy. Results: Nephrin mRNA and expression of the extracellular nephrin were markedly reduced in IgA nephropathy. No changes were found in patients with minimal change nephrosis and focal segmental glomerulosclerosis. The staining of intracellular nephrin and CD2AP did not change among patients. A comparable frequency of the filtration slits was observed in all patient groups, except in minimal change disease patients, due to extensive foot process effacement. The percentage of slit diaphragms with a filamentous image was markedly reduced in IgA nephropathy, but was comparable to controls in minimal change disease. Slit pore width showed a tendency to decrease both in patients with minimal change disease and IgA nephropathy. Conclusions: These results indicate that the ultrastructure of the filtration slit diaphragm is altered in patients with IgA nephropathy as a consequence of targeted downregulation of extracellular nephrin. Further studies are needed to evaluate the pathophysiological meaning of nephrin abnormality in IgA nephropathy and how these changes can be modulated by antiproteinuric therapy.

Dynamic alterations of glomerular charge density in fixed rat kidneys suggest involvement of endothelial cell coat.

In a previous paper, we found that low ionic strength (I) reversibly reduced the glomerular charge density, suggesting increased volume of the charge-selective barrier. Because glutaraldehyde makes most structures rigid, we considered the isolated, perfusion-fixed rat kidney to be an ideal model for further analysis. The fixed kidneys were perfused with albumin solutions containing FITC-Ficoll at two different Is (I = 151 and 34 mM). At normal I, the fractional clearance () for albumin was 0.0049 (SE -0.0017, +0.0027, n = 6), whereas for neutral Ficoll35.5A of similar size was significantly higher 0.104 (SE 0.010, n = 5, P < 0.001). At low I, for albumin was 0.0030 (SE -0.0011, +0.0018, n = 6, not significant from albumin at normal I) and for Ficoll35.5A was identical to that at normal I, 0.104 (SE 0.015, n = 6, P < 0.01 compared with albumin at low I). According to a heterogeneous charged fiber model, low I reduced the fiber density from 0.056 to 0.0315, suggesting a 78% gel volume expansion. We conclude that 1) there is a significant glomerular charge barrier. 2) Solutions with low I increase the volume of the charge barrier even in kidneys fixed with glutaraldehyde. Our findings suggest that polysaccharide-rich structures, such as the endothelial cell coat, are key components in the glomerular barrier.

Proteinuria and phenotypic change of proximal tubular cells.

Terminal renal failure is the final common fate of chronic nephropathies independent of the type of initial insult. The outstanding review by Brenner et al. (1) in 1982 introduced the idea that glomerular hemodynamic changes that develop as compensatory adaptation to irreversible nephron damage cause progressive deterioration of function and structure of the remaining nephrons. After reduction of renal mass in rats, remnant intact nephrons undergo sudden hypertrophy with concomitant lowering of arteriolar resistance and increased glomerular plasma flow that results from a differential decrease in vessel tone that is less in afferent than efferent arterioles. This fosters adaptive increase in glomerular capillary hydraulic pressure and more filtrate per nephron, changes, however, that are detrimental in the long term. The increase in intraglomerular capillary pressure alters the size-selective function of the glomerular barrier and causes protein ultrafiltration. Abnormally filtered proteins have an intrinsic renal toxicity linked to their over-reabsorption by proximal tubular cells and activation of tubular-dependent pathways of interstitial inflammation and fibrosis (2,3). The functional importance of tubulointerstitial events in progressive renal disease is supported by evidence that the severity of tubular interstitial damage strongly correlates with the risk of renal failure, even better than do the glomerular lesions. The relationship between proteinuria and tubulointerstitial damage was suggested by early studies in renal biopsies of rats with age-related proteinuria (4) and adriamycin nephrosis (5), showing that protein reabsorption droplets accumulate in the cytoplasm of proximal tubular cells with focal breaks of the tubular basement membrane and extravasation of the tubular content into the renal interstitium, followed by an inflammatory reaction and tubulointerstitial lesions. Furthermore, in models of overload proteinuria, repeated injections of albumin in the rat increased glomerular barrier permeability and caused massive proteinuria (6,7) and tubular changes with heavy macrophage and T-lymphocyte infiltration into the renal interstitium (8). That excess protein reabsorption by proximal tubular cells may play a role in the development of interstitial inflammation has been documented by a recent study of our group in two different models of proteinuric nephropathies (9). In rats with 5/6 nephrectomy, albumin and IgG accumulation in proximal tubular cells was visualized in the early stage, preceding the interstitial infiltration of major histocompatability complex II–positive cells and macrophages. The infiltrating cells concentrated almost exclusively in regions containing IgG-positive proximal tubules and tubules with luminal casts. Similar patterns were found in the immune model of passive Heymann nephritis indicating that the interstitial inflammatory reaction develops at the sites of protein overload, regardless of the type of glomerular injury. Protein Traffic as a Fosterer of Tubular Cell Dysfunction Filtered albumin and other proteins that accumulate within intracellular compartments of proximal tubular cells perturb cell function by several mechanisms. Studies have contributed to define the biochemical pathways specifically activated by excessive tubular reabsorption, and evidence is now available that protein overloading of proximal tubular cells in culture activates the transcription of a number of genes encoding for vasoactive, inflammatory, and fibrogenic molecules with potential toxic effects on the kidney.

Renal Epithelial Cells

Epithelia, which make up the nephron, exert critical functions in the kidney. Within the glomerulus, podocytes maintain normal glomerular architecture and barrier function. Along the tubule, epithelial cells participate in the conversion of glomerular filtrate into a concentrated urine whose

Renal function in proteinuric glomerular diseases correlates to the changes in urine IgM excretion but not to the changes in the degree of albuminuria.

Renal function in proteinuric glomerular diseases correlates to the changes in urine IgM but not to the changes in the degree of albuminuria. Albuminuria is believed to correlate to the progression of renal failure in glomerular diseases. Nevertheless, many patients with glomerular disorders maintain their renal function despite persistent albuminuria. In previous studies, we found that the baseline urine excretion of IgM, rather than the degree of albuminuria, predicts the renal outcome in glomerulopathies. In the present study, we examine correlations between changes in the content and in the amount of urine proteins and renal survival during a follow-up time of 3.5 years. An observational study of a mean of 44 (+/- 3.6) months was conducted in 37 proteinuric patients (21 males and 16 females) with biopsy-verified primary glomerular disease. The patients were subdivided, according to the findings at the end of the study, into 3 groups, 1 group with decreasing albuminuria (by more than 50%), 1 group with persisting albuminuria and low (< 0.04 mg/mmol creatinine) urinary IgM excretion and 1 group with persisting albuminuria and with high (> or = 0.04 mg/mmol) urinary IgM excretion. All patients that showed remission of albuminuria had also low IgM excretion at the end of the study. All these patients, except 1, maintained their renal function. Patients with persistent albuminuria and high urinary IgM excretion showed a decrease in the glomerular filtration rate (GFR) of a mean of 9.6 ml/min/year compared to a mean GFR increase by 1.5 ml/min/year in patients with low IgM excretion and the same degree of albuminuria (p < 0.01). Seven out of the 9 patients in the former group fall in GFR by more than 5 ml/min/year compared to only 1 of the 10 patients in the latter group. Furthermore, the GFR alterations that occurred during follow-up time correlated in a higher degree to the changes in urinary IgM excretion (r = 0.6, p < 0.01) than to the changes in the degree of albuminuria, (r = 0.4, p < 0.05). A stepwise regression analysis indicated that increased urine IgM excretion is a strong predictor of the GFR decline (r = 0.73, p < 0.001). High urinary IgM excretion correlates to decreased GFR in primary glomerular diseases regardless of the degree of albuminuria. In parallel, low urinary IgM excretion indicates beneficial prognosis in these diseases. Since IgM passes the glomerular barrier entirely through large shunts or defects in the glomerular capillary wall, decreased urine content of IgM might be considered as a sign of recovery in the glomerular damage.

Glomerular filtration rate dependence of sieving of albumin and some neutral proteins in rat kidneys

The size and charge-selective properties of the glomerular barrier are partly controversial. Glomerular sieving coefficients (theta) for proteins have rarely been determined noninvasively before in vivo. Therefore, theta was assessed vs. glomerular filtration rate (GFR; (51)Cr-EDTA clearance) in intact rats for radiolabeled myoglobin, kappa-dimer, neutral horseradish peroxidase (nHRP), neutral human serum albumin (nHSA), and native albumin (HSA). To obtain theta, glomerular tracer clearance, assessed from the 7- to 8-min kidney uptake of protein, was divided by the GFR. The data were fitted with a two-pore model of glomerular permeability, where the small-pore radius was 37.35 +/- 1.11 (SE) A, and the "unrestricted pore area over diffusion path length" (A(0)/DeltaX) 1.84 +/- 0.43 x 10(6) cm. Although seemingly horizontal for nHRP and nHSA, the log theta vs. GFR curves showed slightly negative slopes for the proteins investigated in the GFR interval of 2-4.5 ml/min. Strong negative (linear) correlations between (log) theta and GFR were obtained for myoglobin (P = 0.002) and HSA (P = 0.006), whereas they were relatively weak for nHRP and nHSA and nonsignificant for kappa-dimer. Theta for nHSA was markedly higher than that for HSA. In conclusion, there were no indications of increases in theta vs. GFR, as indicative of concentration polarization, for the proteins investigated at high GFRs. Furthermore, the glomerular small-pore radius assessed from endogenous (neutral) protein sieving data was found to be smaller than previously determined using dextran or Ficoll as test molecules.

Pathophysiology of proteinuria

Proteinuria is consequence of two mechanisms: the abnormal transglomerular passage of proteins due to increased permeability of glomerular capillary wall and their subsequent impaired reabsorption by the epithelial cells of the proximal tubuli. In the various glomerular diseases, the severity of disruption of the structural integrity of the glomerular capillary wall correlates with the area of the glomerular barrier being permeated by "large" pores, permitting the passage in the tubular lumen of high-molecular-weight (HMW) proteins, to which the barrier is normally impermeable. The increased load of such proteins in the tubular lumen leads to the saturation of the reabsorptive mechanism by the tubular cells, and, in the most severe or chronic conditions, to their toxic damage, that favors the increased urinary excretion of all proteins, including low-molecular-weight (LMW) proteins, which are completely reabsorbed in physiologic conditions. Recent clinical studies showed that in patients with glomerular diseases the urinary excretion of some HMW proteins [immunoglobulins G and M (IgG and IgM)] and of some LMW proteins, alpha1-microglobulin, beta2-microglobulin, correlates with the severity of the histologic lesions, and may predict, better than the quantity of proteinuria, the natural course, the outcome, and the response to treatment. It is suggested that some patients have already, at the time of clinical presentation, a structural damage of the glomerular capillary wall (injury of podocytes) and of the tubulointerstitium, the severity and scarce reversibility of which are reliably indicated by an elevated urinary excretion of HMW and LMW proteins.

Glomerular ultrafiltration in normal and preeclamptic pregnancy.

GFR and renal plasma flow (RPF) decrease in preeclampsia, a serious hypertensive complication of pregnancy. Serial data derived in late pregnancy (LP) and >5 mo postpartum (PP) in 13 healthy controls and 10 preeclamptic women (13 and 5, respectively) returning PP for theoretical analysis of neutral dextran sieving curves (theta(D)), are presented and are used to calculate the key determinants of glomerular ultrafiltration. Normal LP hyperfiltration was associated with increases in RPF and the ultrafiltration coefficient (K(f)), as well as in the nondiscriminatory shunt pathway (omega(0)) and the SD of pore size (S). Preeclamptic LP showed the largest omega(0) and S values, indicating a loss of size-selectivity, accompanying reduced K(f) and RPF, both of which are implicated in the relative hypofiltration. Despite a 100-fold increase in urinary albumin excretion (UAE), LP preeclamptic theta(D) values were reduced for the equivalent neutral dextran (36A), providing indirect evidence for a loss of glomerular barrier charge-selectivity. All the determinants of GFR and all modeled parameters were comparable across both groups PP, strong evidence that preeclamptic glomerular dysfunction resolves.

Final common pathways of progression of renal diseases

Complement activation plays a critical role in the pathogenesis of many forms of glomerulonephritis. Studies utilizing cultured glomerular cells and animal models have clarified the important role of imbalance between complement activation and complement regulatory proteins in the pathogenesis of immune-mediated glomerular injury. Glomerular injury results not only from glomerulonephritis but occurs through a variety of mechanisms, including those associated with hypertension and diabetes mellitus. However, once glomerular damage reaches a certain threshold, progression of renal disease is consistent and irreversible. Pathological studies have revealed a close correlation between tubulointerstitial damage and poor prognosis, emphasizing the crucial role of tubulointerstitial injury in eventual kidney failure. One common mechanism that leads to renal failure via tubulointerstitial injury is massive proteinuria. Specific components of the filtered protein may be toxic to tubular cells. Among various harmful proteins filtered through the glomerular barrier are complement components, which are activated by ammonium resulting from excess delivery of protein to tubular cells. Another common mechanism is chronic ischemia in the tubulointerstitium. Tubulointerstitial damage results in the loss of peritubular capillaries and induces chronic hypoxia in this compartment, producing a vicious cycle. Therapeutic approaches that target these final common pathways may prove valuable in many kinds of renal disease.

Transforming Growth Factor-β1 Is Up-Regulated by Podocytes in Response to Excess Intraglomerular Passage of Proteins: A Central Pathway in Progressive Glomerulosclerosis

Chronic diseases of the kidney have a progressive course toward organ failure. Common pathway mechanisms of progressive injury, irrespectively of the etiology of the underlying diseases, include glomerular capillary hypertension and enhanced passage of plasma proteins across the glomerular capillary barrier because of impaired permselective function. These changes are associated with podocyte injury and glomerular sclerosis. Direct evidence for causal roles is lacking, particularly for the link between intraglomerular protein deposition and sclerosing reaction. Because transforming growth factor-beta1 (TGF-beta1) is the putative central mediator of scarring, we hypothesized that TGF-beta1 can be up-regulated by protein overload of podocytes thereby contributing to sclerosis. In rats with renal mass reduction, protein accumulation in podocytes as a consequence of enhanced transcapillary passage preceded podocyte dedifferentiation and injury, increase in TGF-beta1 expression in podocytes, and TGF-beta1-dependent activation of mesangial cells. Angiotensin-converting enzyme inhibitor prevented both accumulation of plasma proteins and TGF-beta1 overexpression in podocytes and sclerosis. Albumin load on podocytes in vitro caused loss of the synaptopodin differentiation marker and enhanced TGF-beta1 mRNA and protein. Conditioned medium of albumin-stimulated podocytes induced a sclerosing phenotype in mesangial cells, an effect mimicked by TGF-beta1 and blocked by anti-TGF-beta1 antibodies. Thus, the passage of excess plasma proteins across the glomerular capillary wall is the trigger of podocyte dysfunction and of a TGF-beta1-mediated mechanism underlying sclerosis. Agents to reduce TGF-beta1, possibly combined with angiotensin blockade, should have priority in novel approaches to treatment of progressive nephropathies.

Role of nephrin in renal disease including diabetic nephropathy.

Nephrin, a newly described protein, has been localized to the slit membrane between adjacent podocytes of the glomerulus. Its discovery followed the demonstration of the gene NPHS1 and its mutation, resulting in the absence of the protein product, nephrin, in the congenital nephrotic syndrome of the Finnish type. The link between permutations in nephrin expression and proteinuria has been shown in animal models by using neutralizing antibodies or studying mice with inactivation of the nephrin gene. Moreover, the expression of nephrin has been shown to be reduced in various animal models of proteinuric renal disease. The relationship between changes in nephrin expression and proteinuric renal disease in humans is not fully elucidated, with a reduction in expression of this protein reported in a range of renal diseases. Diabetic nephropathy, one of the major causes of end-stage renal disease, is associated with substantial proteinuria and in experimental models with a reduction in slit pore density. In experimental models of diabetes, nephrin expression has been described as being transiently increased in the first 8 weeks of diabetes, followed in longer-term studies with reduced nephrin expression in association with increasing proteinuria. An angiotensin II-receptor blocker has been shown to prevent depletion in glomerular nephrin expression in the diabetic kidney. Human studies in both type 1 and type 2 diabetes suggest down-regulation of nephrin expression in the diabetic kidney and it has been postulated that these changes may play a role in the pathogenesis of diabetic nephropathy, specifically the development of proteinuria in this condition. Although there are other proteins involved in the structure of the epithelial podocyte and specifically the slit pore, nephrin seems to play a pivotal role in preventing passage of protein through the glomerular barrier. Furthermore, it is suggested that the antiproteinuric effects of inhibition of the renin-angiotensin system may partly relate to the effects of these agents on nephrin expression.

Glucocorticoid regulation of proteoglycan synthesis in mesangial cells

Proteoglycans are integral components of the mesangial matrix and glomerular permeability barrier. Recent studies have shown that changes in glomerular proteoglycan expression may play a major role in the pathogenesis of renal disease. Steroid hormones are used as first-choice therapy for the treatment of glomerular diseases, however, the effects of glucocorticoids on expression of glomerular proteoglycans are unknown. This study examined the effects of in vitro and in vivo administration of dexamethasone on proteoglycan synthesis and gene expression of proteoglycan core proteins using rat (RMC) and human (HMC) mesangial cells. Treatment of cultured RMC with dexamethasone resulted in a dose- and time-dependent decrease (P < 0.05) in both cell-associated and secreted proteoglycan synthesis to approximately 50% of control levels. This effect was inhibited by the glucocorticoid antagonist mifepristone, and mimicked by prednisolone or corticosterone treatment. Separation of proteoglycans by ion-exchange and gel permeation chromatography suggested that chondroitin sulfate/dermatan sulfate proteoglycans were down-regulated after steroid treatment. Northern blot analysis, RT-PCR, Western blot, and promoter activity assays revealed that dexamethasone caused a significant decrease in decorin mRNA (to 61 +/- 8% of controls), whereas biglycan expression and promoter activity were increased after steroid treatment. A similar trend was found in glomeruli isolated from rats treated in vivo with dexamethasone. These results demonstrate that treatment of mesangial cells with steroids results in a decrease in total proteoglycan synthesis, as well as subtype-specific changes in proteoglycan core protein gene expression by transcriptional control, furthering our understanding of the effects of steroid treatment on the renal glomeruli.

Effect of high dose ramipril with or without indomethacin on glomerular selectivity

Despite the accumulating evidence of their efficacy, angiotensin-converting enzyme inhibitors (ACEi) still provide imperfect renoprotection. Up-titration above conventional doses and combined therapy with other antiproteinuric agents may serve to achieve renoprotection in patients at risk of rapid disease progression. The effect of maximum tolerated ACEi doses (ramipril 15 mg/day, range 5 to 20) alone or combined with indomethacin (75 mg x 2/day) on urinary protein excretion (UPE) and glomerular barrier size-selective function was evaluated in 19 patients with chronic non-diabetic nephropathies and persistent proteinuria. Maximum ramipril doses decreased UPE more effectively than non-ACEi therapy. Proteinuria reduction was associated with significant reduction (>50%) of the non-selective glomerular membrane shunt, but did not correlate with concomitant changes in arterial pressure and renal hemodynamics, nor was it influenced by treatment duration. The reduction in UPE and sieving coefficient of the largest neutral dextrans exceeded by twofold the reduction achieved by conventional ACEi doses in historical controls with similar renal dysfunction and proteinuria, previously studied under identical experimental conditions. Indomethacin did not influence renal effects of maximum ramipril doses and was prematurely withdrawn in six patients because of reversible side effects. Serum potassium significantly increased only in combination with indomethacin and never required treatment withdrawal. Up-titration to maximally tolerated doses safely increases ACEi antiproteinuric effect and may serve to achieve maximum renoprotection in the long-term. Combination with indomethacin is poorly tolerated and ineffective. Innovative approaches are needed to use ACEi more effectively.

Proximal tubular cells promote fibrogenesis by TGF-β1–mediated induction of peritubular myofibroblasts

In proteinuric nephropathies with increasingly severe defects of the glomerular filtering barrier, interstitial fibrogenesis is a major effector of scarring. An early event in this process is the peritubular accumulation of myofibroblasts that express alpha-smooth muscle actin (alpha-SMA) and contribute to abnormal matrix production. Common trigger factors are poorly understood. Enhanced protein trafficking may play a role by up-regulating inflammatory and fibrogenic genes in proximal tubular cells. The remnant kidney model in rats was used to (1) analyze interactions between activated proximal tubular cells, peritubular cells expressing the myofibroblast marker, and inflammatory cells at time intervals (days 7, 14, and 30) after surgery, and (2) evaluate the effects of angiotensin-converting enzyme inhibitor (ACEi) on protein trafficking, fibrogenic signaling, and alpha-SMA expression. Abnormal uptake of ultrafiltered proteins by proximal tubular cells (IgG staining) occurred at an early stage (day 7) and was subsequently associated with macrophage and alpha-SMA+ cell accumulation into the peritubular interstitium. alpha-SMA+ cells clustered with macrophages into the interstitium. These changes were associated with appearance of transforming growth factor-beta1 (TGF-beta1) mRNA in proximal tubular cells and in the infiltrating cells with time. At day 30, focal alpha-SMA staining also was found in the tubular cells and in peritubular endothelial cells on semithin ultracryosections. ACEi prevented both proteinuria and abnormal protein accumulation in tubular cells, as well as the inflammatory and fibrogenic reaction with peritubular alpha-SMA expression. Profibrogenic signaling from both proximal tubular cells on challenge with filtered protein and inflammatory cells is implicated as a key candidate trigger of progressive tubulointerstitial injury.

Chronic renal diseases: renoprotective benefits of renin-angiotensin system inhibition.

Progression to renal parenchymal damage and end-stage renal disease, which seems to be largely independent of the initial insult, is the final common pathway for chronic, proteinuric nephropathies in animals and humans. The key event is enhanced glomerular capillary pressure; this impairs glomerular permeability to proteins and permits excessive amounts of proteins to reach the lumen of the proximal tubule. The secondary process of reabsorption of filtered proteins can contribute to renal interstitial injury by activating intracellular events, including upregulation of the genes encoding vasoactive and inflammatory mediators. Both interstitial inflammation and progression of disease can be controlled by such drugs as angiotensin-converting enzyme inhibitors, which strengthen the glomerular permeability barrier to proteins and thereby limit proteinuria and filtered protein-dependent inflammatory signals. Clinical data strongly suggest that remission can now be achieved in some patients with chronic renal disease. Because of the current lag time between starting treatment and remission, however, a substantial proportion of patients still progress to end-stage renal disease before renal function begins to stabilize. A multimodal approach that centers on reducing or removing all risk factors associated with the progression of renal disease may decrease the time to remission of the disease for most patients with proteinuric nephropathies.