Dimitrios S. Goumenos

Transforming Growth Factor-β1 and Myofibroblasts: A Potential Pathway towards Renal Scarring in Human Glomerular Disease

Background/Aims: The cellular and humoral factors involved in the development and progression of renal scarring have not been fully investigated. Transforming growth factor-β (TGF-β1) is considered to be the main fibrogenic growth factor and it is implicated in the pathogenesis of renal fibrosis in experimental and clinical nephropathies. On the other hand, collagen III is an important component of the extracellular matrix. In this study we attempted to identify any possible links between TGF-β1 and collagen III synthesis and expression with the expression of myofibroblasts in the evolution of renal scarring in human glomerular diseases. Methods: We studied retrospectively 40 patients with various types of primary and secondary glomerulonephritis (GN), with either proliferative or nonproliferative pattern, with emphasis on the renal synthesis of TGF-β1 and collagen III (detected by in situ hybridization) and their expression (detected by immunohistochemistry) as well as myofibroblast expression. The possible links of TGF-β1 expression with myofibroblast distribution (α-smooth muscle actin, α-SMA(+) cells) and with conventional histopathology and renal function was also examined. Results: TGF-β1 protein and mRNA were detected in the renal tubular epithelial cells and interstitium and to a lesser extent within glomeruli of patients with GN. Collagen III was mainly detected in the interstitium (peritubular and periglomerular areas) and to a lesser extent in the glomeruli. Messenger RNA for collagen III followed a similar peritubular and periglomerular distribution to that of TGF-β1 and α-SMA(+) interstitial cells. The intensity of interstitial TGF-β1 protein expression was significantly related to the degree of interstitial fibrosis (r = 0.628, p < 0.01), tubular atrophy (r = 0.612, p < 0.01), interstitial collagen III expression (r = 0.478, p < 0.05), and serum creatinine values (r = 0.722, p < 0.001). Also there was a close positive correlation between the severity of interstitial myofibroblast expression and interstitial TGF-β1 (r = 0.412, p < 0.05), as well as collagen III (r = 0.409, p < 0.05). In addition, a significant correlation was found between glomerular TGF-β1 expression and severity of glomerulosclerosis (r = 0.620, p < 0.01). Conclusion: The results of this study suggest that TGF-β1 plays an important role in the pathogenesis of fibrosis developing in human kidney, during the evolution of glomerular disease. Interstitial myofibroblasts may contribute to interstitial fibrosis through the synthesis and release of both TGF-β1 and collagen III.

Early histological changes in the kidney of people with morbid obesity

BACKGROUND Morbid obesity represents a major health problem with increasing incidence worldwide. The clinical manifestation of renal involvement in obesity is proteinuria, and the histological feature is glomerulomegaly with or without focal and segmental glomerulosclerosis (FSGS). In this study, we have investigated the very early histological changes in kidneys of people with morbid obesity and no proteinuria. Patients and methods. Eighteen patients with body mass index (BMI) >50 kg/m(2) who underwent a variant of biliopancreatic diversion with Roux-en-Y reconstruction (BPD-RYGBP) and consented to undergo a renal biopsy during the surgical procedure were included in the study. The estimation of early histological changes was performed on light (n = 18) and electron microscopy (n = 13). RESULTS The mean glomerular cross-sectional area was 30 943 +/- 10,984 microm(2) that is higher than that observed in non-obese individuals. In 21% of the examined glomeruli, the glomerular planar surface area (GPSA) was >40,000 microm(2). Thickening of the glomerular basement membrane (GBM) and scattered paramesangial deposits were identified in 9 of 13 patients (70%) whose renal tissue was examined by electron microscopy. A reduction in the slit pore frequency was observed in obese patients due to extensive foot process effacement. Significant positive correlations between mean GPSA and body weight (r = 0.462, P = 0.05), and between GBM thickness and HbA1c, serum total cholesterol and triglyceride levels (r = 0.60, P = 0.05; r = 0.789, P = 0.004; r = 0.70, P = 0.016, respectively), were observed. CONCLUSIONS Glomerulomegaly as well as histological lesions resembling those of early diabetic nephropathy are observed in kidney biopsies of patients with morbid obesity even before the appearance of microalbuminuria. The potential regression of these changes after weight loss needs to be clarified.

Accurate Measurement and Clinical Significance of Urinary Transforming Growth Factor-Beta1

Transforming growth factorβ1 (TGF-β1) is the main modulator of the healing process after tissue injury. In the kidney, if TGF-β1 release is not switched off, extracellular matrix components (ECM) are accumulated and tissue fibrosis occurs. Urinary TGF-β1 levels reflect its renal production and it has been determined in various types of glomerular disease. In this review, a critical analysis of the different immunoassays that have been used for the measurement of TGF-β1 in the urine is presented and the importance of the serial determination of urinary TGF-β1 levels in patients with various types of renal disease is discussed.

Corticosteroids and ciclosporin A in idiopathic membranous nephropathy: higher remission rates of nephrotic syndrome and less adverse reactions than after traditional treatment with cytotoxic drugs.

Background/Aim: Idiopathic membranous nephropathy, the most common cause of nephrotic syndrome in adults, has been traditionally treated with corticosteroids and cytotoxic drugs. Ciclosporin A (CsA) is used in resistant cases, but also as a first-line treatment, due to the serious side effects of cytotoxic drugs. In this study, the remission rates of nephrotic syndrome and the incidence of side effects of corticosteroids and low CsA doses are compared with those after treatment with cytotoxic drugs. Methods: Seventy-seven nephrotic patients with well-preserved renal function who were treated with methylprednisolone and CsA (n = 46) or cytotoxic drugs (n = 31) were studied. The effects of treatments were estimated on the basis of remission rates of nephrotic syndrome and preservation of the renal function. Results: Remission (complete or partial) of nephrotic syndrome was observed in 85% of the patients treated with CsA and in 55% of the patients treated with cytotoxic drugs (p < 0.01). Deterioration of the renal function, more common in patients with multiple relapses and interstitial fibrosis, was observed in 26 and 23% of the patients, respectively (p = NS). Serious side effects and discontinuation of treatment were more frequent in patients treated with cytotoxic drugs (10 vs. 4%). Conclusion: The combination of corticosteroids with CsA represents a better regimen for patients having idiopathic membranous nephropathy, since it is associated with higher remission rates of nephrotic syndrome and less severe side effects than corticosteroids and cytotoxic drugs.

Endothelin-1 in the kidney and urine of patients with glomerular disease and proteinuria.

BACKGROUND Endothelin-1 (ET-1) is a strong vasoconstrictive peptide that is involved in the pathogenesis of arterial hypertension. There is increasing evidence, based on studies in experimental animals, that endothelin-1 is produced by tubular epithelial cells in response to activation by filtered protein and is involved in the development of renal scarring. The aim of this study is to examine the distribution of ET-1 in the renal tissue of patients with heavy proteinuria and to estimate the changes in its urinary excretion after immunosuppressive therapy. PATIENTS AND METHODS Twenty-four patients with severe proteinuria (7.5 +/- 6.5 g/24 h) due to different types of glomerular disease and normal renal function (creatinine clearance 91 +/- 14 ml/ min) were investigated. All patients underwent a renal biopsy and commenced on immunosuppressive therapy with corticosteroids and cyclosporin A. The localization of ET-1 in the renal tissue was examined by immunohistochemistry and compared to control renal tissue from 9 patients who underwent nephrectomies because of hypernephroma. In patients with proteinuria, endothelin-1 excretion in the urine at diagnosis was determined by radioimmunoassay and compared to that of 14 healthy subjects. A second measurement of urinary ET-1 excretion was performed after remission of proteinuria or 6 months after the initiation of treatment in patients with persistent nephrotic syndrome. RESULTS ET-1 in renal tissue of patients and controls was localized within the cytoplasm of endothelial cells. In nephrotic patients, it was also localized within the cytoplasm of tubular epithelial cells. Urinary ET-1 levels were higher in nephrotic patients compared to healthy subjects (746 +/- 180 ng/24 h vs 410 +/- 112 ng/ml, p < 0.001). In 17 of 24 patients who showed remission of proteinuria with immunosuppressive therapy, the urinary ET-1 levels decreased (from 803 +/- 168 ng/24 h to 511 +/- 80 ng/24 h, p < 0.001) whereas in 7 patients with persistent proteinuria, urinary ET-1 excretion remained elevated. CONCLUSIONS The increased urinary excretion of ET-1 in patients with severe proteinuria followed by a significant decrease after remission ofproteinuria with immunosuppressive treatment, along with its expression within tubular epithelial cells, suggests a possible relationship between proteinuria and renal ET-1 production.

Immunosuppressive treatment of idiopathic focal segmental glomerulosclerosis : A five-year follow-up study

Background/Aims: Focal segmental glomerulosclerosis (FSGS) is a common type of glomerular disease that can lead to chronic renal failure. Various therapeutic regimens have been used in nephrotic FSGS patients. The effect of treatment with prednisolone alone or its combination with azathioprine and cyclosporin and parameters related to a poor outcome are studied. Methods: Fifty-one patients with idiopathic FSGS and a follow-up period of 5 years were included. Twenty-five were treated with prednisolone alone (1 mg/kg BW/day) or combination of prednisolone (0.5 mg/kg BW/day) with azathioprine (2 mg/kg BW/day) or cyclosporine (3 mg/kg BW/day) in gradually reduced doses whereas 26 patients received no immunosuppressive drugs. Lower prednisolone dose regimens were used as initial treatment in obese, borderline diabetics or patients with bone disease. The clinical course was estimated using the end-points of 50% or doubling of baseline serum creatinine and/or end-stage renal failure. The contribution of clinical and histological parameters in the clinical outcome was estimated by univariate and multivariate analyses. Results: Increase of baseline serum creatinine by 50% during the follow-up period was observed in 2 treated and 9 untreated patients (8% vs. 35%, p = 0.03) whereas doubling of serum creatinine in 2 and 5 patients respectively (8% vs. 19%, p = NS). End-stage renal failure developed in 4 of 51 patients (8%), 2 treated and 2 untreated (p = NS). Parameters related to a poor outcome were baseline serum creatinine and severity of glomerulosclerosis (multivariate analysis OR = 1.08, p = 0.01). Most of patients (68%) who reached end-points had persistent nephrotic syndrome during the follow-up. Remission of nephrotic syndrome was observed more frequently among treated (75 vs. 30.7%, p = 0.05). Prednisolone alone was followed by remission of nephrotic syndrome in 62.5% whereas combination of lower prednisolone dose with azathioprine and cyclosporin in 80 and 85.7% of patients. No serious side-effects were observed. Conclusion: This and previous studies suggest that steroid and/or immunosuppressive therapy have a role in amelioration of the clinical course and remission of nephrotic syndrome in patients with FSGS A combination of low predisolone dose with cyclosporine could be used as initial treatment in patients with higher risk for side-effects from the usual prednisolone dose.

Prednisolone and Azathioprine in IgA Nephropathy

Background: Immunoglobulin A nephropathy (IgAN) is one of the most common primary glomerular diseases. Although its clinical course is usually benign, some patients develop end-stage renal failure (ESRF). The role of immunosuppressive drugs in the treatment of IgAN remains controversial. The effect of treatment with prednisolone and azathioprine and the clinical and histological parameters related to a poor outcome are examined retrospectively in this analysis. Methods: Seventy-four patients with IgAN and a follow-up period of 10 years were included in this study. Forty-one were treated with prednisolone (initially 60 mg/day) and azathioprine (initially 2 mg/kg BW/day) in gradually reduced doses for 24 ± 9 months, whereas 33 patients received no immunosuppressive drugs. The clinical course was estimated using the end-points of doubling of baseline serum creatinine and/or ESRF. The contribution of clinical and histological parameters in the clinical outcome was estimated by univariate and multivariate analyses. Results: The overall clinical courses of both groups of patients showed a rather similar pattern. Doubling of serum baseline creatinine was observed in 9 of 41 treated (22%) and in 10 of 33 untreated (30%), whereas ESRF developed in 6 treated (15%) and 6 untreated patients (18%) (p = NS). However, treated patients with heavy proteinuria (>3 g/24 h) had a significantly better outcome compared to untreated (doubling of serum creatinine in 29 vs. 78% and ESRF in 17 vs. 55%, p < 0.05). Proteinuria (p < 0.01), mean blood pressure (p < 0.02), baseline serum creatinine (p = 0.02) and severity of interstitial myofibroblast expression (p = 0.02) were identified as independent risk factors related to a poor outcome by multivariate analysis. Side effects of treatment were not uncommomn and observed in 10 (24%) patients. Conclusion: Treatment with prednisolone and azathioprine is beneficial in ameliorating the clinical course of a subset of IgAN patients with heavy proteinuria or impaired renal function. Patients with advanced renal failure and severe chronic histological lesions should not be treated by this regimen as no benefit is expected and there is a risk of side effects.

The remission of nephrotic syndrome with cyclosporin treatment does not attenuate the progression of idiopathic membranous nephropathy

BACKGROUND Idiopathic membranous nephropathy (IMN), a common cause of nephrotic syndrome in adults, is usually treated by combination of corticosteroids with cytotoxic drugs. In cases resistant to this regimen, the use of cyclosporin A (CsA) is followed by frequent remissions of the nephrotic syndrome. AIM The purpose of this study was to estimate the effectiveness of prednisolone and small doses of CsA as first-line treatment of nephrotic patients with IMN, in relation to the progression of the disease, based on functional and histological changes. PATIENTS AND METHODS Sixteen patients, with nephrotic syndrome due to IMN and well-preserved renal function, were treated with prednisolone (starting dose: 0.5 mg/kg bw/day) and CsA (starting dose: 3 mg/kg bw/day) for 24 months. A repeat renal biopsy was performed after 18 months of treatment in 10 patients with remission of nephrotic syndrome, to estimate the activity of the disease and to identify any features of CsA toxicity. RESULTS Remission of the nephrotic syndrome was observed in 14 out of 16 patients after 5 +/- 2 months of treatment. Complete remission was observed in 8 and partial remission in 6 patients (urinary protein was reduced from 6.9 +/- 3.4-0.2 +/- 0.06 g/24 h and 1.2 +/- 1.0 g/24 h, respectively, p < 0.01). The renal function was well preserved in 13 out of 16 patients over a 24-month period of treatment. Deterioration of renal function was observed in 3 patients (creatinine clearance reduced from 86 +/- 21-37 +/- 17 ml/min, p < 0.05) who had either persistent nephrotic syndrome or frequent relapses. Relapses of the nephrotic syndrome were observed in 5 of 14 patients. Repeat renal biopsies showed that glomerular sclerosis, tubulointerstitial injury, vascular hyalinosis and stage of the disease were deteriorated in most patients. Isometric vacuolization of tubular epithelial cells was observed in 2 of 10 patients. CONCLUSION IMN nephrotic patients treated with prednisolone and low doses of cyclosporin A showed a high remission rate of nephrotic syndrome. However, progression of chronic histological lesions was found in repeat renal biopsies. This suggests that cyclosporin can frequently induce remission of nephrotic syndrome in IMN patients, but even low doses of the drug are not free of potential renal toxicity.

Keratins are novel markers of renal epithelial cell injury

Keratins, the intermediate filaments of the epithelial cell cytoskeleton, are up-regulated and post-translationally modified in stress situations. Renal tubular epithelial cell stress is a common finding in progressive kidney diseases, but little is known about keratin expression and phosphorylation. Here, we comprehensively describe keratin expression in healthy and diseased kidneys. In healthy mice, the major renal keratins, K7, K8, K18, and K19, were expressed in the collecting ducts and K8, K18 in the glomerular parietal epithelial cells. Tubular expression of all 4 keratins increased by 20- to 40-fold in 5 different models of renal tubular injury as assessed by immunohistochemistry, Western blot, and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The up-regulation became significant early after disease induction, increased with disease progression, was found de novo in distal tubules and was accompanied by altered subcellular localization. Phosphorylation of K8 and K18 increased under stress. In humans, injured tubules also exhibited increased keratin expression. Urinary K18 was only detected in mice and patients with tubular cell injury. Keratins labeled glomerular parietal epithelial cells forming crescents in patients and animals. Thus, all 4 major renal keratins are significantly, early, and progressively up-regulated upon tubular injury regardless of the underlying disease and may be novel sensitive markers of renal tubular cell stress.

Apoptosis and Myofibroblast Expression in Human Glomerular Disease: A Possible Link with Transforming Growth Factor-Beta-1

Background/Aims: The pathophysiological pathways involved in the pathogenesis and evolution of renal fibrosis, have not been fully elucidated. Transforming growth factor-beta1 (TGF-β1) is involved in the development of renal scarring. Apoptosis is responsible for intrinsic cell deletion observed in end-stage kidney disease. Myofibroblasts are involved in the development of renal fibrosis. This study investigates whether there is a potential relationship between apoptosis, myofibroblast infiltration and TGF-β1 expression in the kidney of patients with glomerulonephritis (GN). Methods: Forty patients with various types of GN were included in the study. Myofibroblasts and TGF-β1 positive cells were detected in kidney biopsies by immunohistochemistry, while apoptotic cells were detected by the in situ end labelling of fragmented DNA. Results: Myofibroblasts were identified in the glomeruli of some patients with severe mesangioproliferative GN and glomerulosclerosis but a more intensive myofibroblast expression was found in the renal interstitium. TGF-β1 was expressed in the cytoplasm of tubular epithelial cells, in the renal interstitium and in the glomeruli of patients with GN. Apoptotic cells were mainly detected in the tubules and interstitium and were present in areas with intense myofibroblast infiltration. Positive correlations were observed between the intensity of myofibroblast expression in the interstitium and apoptosis in the tubulointerstitial area (r = 0.521, p < 0.01) as well as TGF-β1 expression (r = 0.462, p < 0.05) and degree of renal impairment (r = 0.430, p < 0.05). Conclusions: These observations suggest that myofibroblast infiltration and apoptosis along with TGF-β1 expression are associated with the development of interstitial fibrosis in patients with glomerular disease.