Antiproteinuric therapy while preventing the abnormal protein traffic in proximal tubule abrogates protein- and complement-dependent interstitial inflammation in experimental renal disease

In proteinuric glomerulopathies, the excess traffic of proteins into the renal tubule is a candidate trigger of interstitial inflammatory and immune events leading to progressive injury, and a key target for the renoprotective action of antiproteinuric drugs. Among proteins trafficked to the proximal tubule, the third component of complement (C3) can be activated locally and contribute to inflammation at sites of protein reabsorption. Experiments were performed in rats with renal mass reduction (RMR, 5/6 nephrectomy) with the following aims: (1) to study Ig (IgG) and complement deposition in proximal tubules, and interstitial macrophage infiltration and MHC class II expression at intervals after surgery by double immunofluorescence analysis; (2) to assess whether lisinopril (angiotensin-converting enzyme inhibitor [ACEi], 25 mg/L in the drinking water, from either day 1 or day 7) limited IgG and C3 accumulation and interstitial inflammation at day 30. In 7-d remnant kidneys, intracellular staining for both IgG and C3 was detectable in proximal tubules in focal areas; C3 was restricted to IgG-positive tubular cells, and there were no interstitial ED-1 macrophage and MHC II-positive cellular infiltrates. In 14-d and 30-d remnant kidneys, proximal tubular IgG and C3 staining was associated with the appearance of interstitial infiltrates that preferentially localized to areas of tubules positive for both proteins. RMR rats given ACEi had no or limited increases in levels of urinary protein excretion, tubular IgG, and C3 reactivity, and interstitial cellular infiltrates in kidneys at 30 d, even when ACEi was started from day 7 after surgery. These findings document that (1) in RMR, IgG and C3 accumulation in proximal tubular cells is followed by leukocyte infiltration and MHC II overexpression in the adjacent interstitium; (2) ACEi while preventing proteinuria limits both tubular accumulation of IgG and C3 and interstitial inflammation. The data suggest that ACE inhibition can be renoprotective by limiting the early abnormal protein traffic in proximal tubule and consequent deleterious effects of excess protein reabsorption, including the accumulation and local activation of complement as well as the induction of chemokines and endothelin genes known to promote interstitial inflammation and fibrosis.     

Protein overload activates proximal tubular cells to release vasoactive and inflammatory mediators

Chronic renal diseases with highly enhanced glomerular permeability to proteins are accompanied by tubulointerstitial inflammation and scarring and progression to renal failure. As a consequence of increased glomerular permeability, proteins filtered through the glomerular capillary in excessive amount have intrinsic renal toxicity at least partially linked to their accumulation in the proximal tubular cell cytoplasm during the process of reabsorption along the nephron. Experimental evidence is available showing that protein overload per se activates proximal tubular epithelial cells in culture to upregulate genes encoding for endothelin, chemokines and cytokines. These vasoactive and inflammatory substances, formed in excessive quantities by the tubular cells, are released mainly into the basolateral compartment, a pattern of secretion that in the kidney would favor recruitment and activation of inflammatory cells into the renal interstitium and fibrogenic reaction leading to renal scarring.     

Role of atrophic changes in proximal tubular cells in the peritubular deposition of type IV collagen in a rat renal ablation model.

BACKGROUND: Tubular atrophy, dilation and interstitial fibrosis are common in tubulointerstitial lesions, but the precise roles and inter-relationships of these components in the development of interstitial lesions have not been determined. This study focused on the origin and roles of atrophic tubules in the peritubular deposition of type IV collagen in a rat renal ablation model. METHODS: Male Wistar rats underwent 5/6 nephrectomy or sham operation, and then were sacrificed at 4, 8 or 12 weeks, their remaining kidneys removed for histological and immuno-histochemical studies as well as in situ hybridization for type IV collagen mRNA. RESULTS: Immuno-histochemistry demonstrated the positive staining of atrophic tubules to vimentin, platelet-derived growth factor-B chain (PDGF) and heat shock protein 47 (HSP47). Cells positive to one or more of PDGF receptor beta, alpha-smooth muscle actin (alpha-SMA), and HSP47 accumulated around atrophic tubules. Type IV collagen was also increased in the proximity of the atrophic tubules. These intimate relationships were more clearly demonstrated in 'mosaic tubules', which are composed of both intact and atrophic proximal tubular epithelial cells, and which had a mixed pattern of staining with vimentin, PDGF and HSP47. The interstitial cells positive to alpha-SMA or HSP47, or both, were in close contact with atrophic but not with intact epithelial cells. Type IV collagen was exclusively deposited between atrophic tubules and HSP47-positive interstitial cells. In situ hybridization of type IV collagen mRNA demonstrated predominant expression in atrophic tubular epithelial cells, but not in surrounding interstitial cells. CONCLUSIONS: These findings suggest that atrophic proximal tubular cells are active in the development of collagen deposition in the peritubular space, i.e. in this model type IV collagen in the interstitial fibrotic area may be produced mainly by atrophic proximal tubules.     

Wegener's granulomatosis: inflammatory cells and markers of repair and fibrosis in renal biopsies--a clinicopathological study.

OBJECTIVE: This study aimed to quantitate inflammatory cells in renal biopsies from patients with Wegener's granulomatosis (WG) and to identify cells participating in early fibrogenesis. The goal was to determine whether these cells correlated with the severity of renal disease and whether their presence had a bearing on renal prognosis. MATERIAL AND METHODS: Sixty-one patients with WG who had a renal biopsy taken at the time of diagnosis were included in the study. Immunostaining with monoclonal antibodies towards macrophages (CD68), T- and B-lymphocytes, alpha-smooth muscle actin (alpha-SMA) and vimentin was done. RESULTS: The dominating intraglomerular leucocytes were macrophages (29.9 +/- 15 cells/glomerular cross-section) and to a lesser extent T-cells (2.57 +/- 1.8 cells/glomerular cross-section). No B-lymphocytes were detected in the glomeruli. More than two-thirds of the T-cells were CD8+ (cytotoxic) cells. Macrophages and T-lymphocytes were distributed equally in the renal interstitium and were numerous around crescentic glomeruli. Glomerular and interstitial macrophages and interstitial T-cells correlated significantly with serum (S-) creatinine at the time of biopsy but not after 1 year. S-creatinine at the time of biopsy and after 1 year differed significantly among the three levels of interstitial alpha-SMA staining. S-creatinine at biopsy was highest when tubular vimentin staining was strongest, and tubular vimentin staining was strongest in patients with acute tubular damage. CONCLUSIONS: Evidence was found for a cellular type IV immune response in WG, with CD8+ T-lymphocytes and macrophages dominating the cellular infiltrate. The detection of interstitial alpha-SMA, probably staining myofibroblasts implicated in renal fibrogenesis, indicated a low glomerular filtration rate 1 year after renal biopsy.     

Transforming growth factor-beta(1) 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-beta (TGF-beta(1)) 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-beta(1) 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-beta(1) and collagen III (detected by in situ hybridization) and their expression (detected by immunohistochemistry) as well as myofibroblast expression. The possible links of TGF-beta(1) expression with myofibroblast distribution (alpha-smooth muscle actin, alpha-SMA(+) cells) and with conventional histopathology and renal function was also examined. RESULTS: TGF-beta(1) 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-beta(1) and alpha-SMA(+) interstitial cells. The intensity of interstitial TGF-beta(1) 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-beta(1) (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-beta(1) expression and severity of glomerulosclerosis (r = 0.620, p < 0.01). CONCLUSION: The results of this study suggest that TGF-beta(1) 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-beta1 and collagen III.     

Predominant tubulointerstitial nephritis in a patient with systemic lupus erythematosus: phenotype of infiltrating cells

A 63-year-old man with systemic lupus erythematosus developed tubular proteinuria. All subclasses of serum IgG increased, and the largest IgG subclass increase was IgG4. A renal biopsy showed lupus nephritis (Class II) with severe tubulointerstitial nephritis (so-called predominant tubulointerstitial lupus nephritis, an unusual form of lupus nephritis). Immunofluorescence microscopy revealed positive granular staining for IgG, C3 and C1q in the mesangium and peritubular interstitium, and along the tubular basement membranes (TBM). Electron microscopy also showed electron-dense deposits in the mesangium and TBM. Immunophenotyping of interstitial infiltrating cells disclosed a predominance of T cells. CD8-positive cytotoxic T cells infiltrated the peritubular interstitium, and some of these cells infiltrated the tubules. B cell-rich lymphoid follicles were also observed. IgG subclass analyses showed glomerular IgG1, IgG2 and IgG4 deposition, positive staining of IgG4 in the peritubular interstitium and along the TBM, and abundant IgG1-, IgG3- and IgG4-positive plasma cells in the interstitium. The patient responded well to moderate-dose steroid therapy. This is the first report of immunophenotyping of interstitial infiltrates in predominant tubulointerstitial lupus nephritis. The results suggest CD8-positive cytotoxic T cell-mediated tubular injury. Furthermore, immune complexes containing IgG4 might be one of etiologic factors.     

Complement-mediated dysfunction of glomerular filtration barrier accelerates progressive renal injury

Intrarenal complement activation leads to chronic tubulointerstitial injury in animal models of proteinuric nephropathies, making this process a potential target for therapy. This study investigated whether a C3-mediated pathway promotes renal injury in the protein overload model and whether the abnormal exposure of proximal tubular cells to filtered complement could trigger the resulting inflammatory response. Mice with C3 deficiency were protected to a significant degree against the protein overload-induced interstitial inflammatory response and tissue damage, and they had less severe podocyte injury and less proteinuria. When the same injury was induced in wild-type (WT) mice, antiproteinuric treatment with the angiotensin-converting enzyme inhibitor lisinopril reduced the amount of plasma protein filtered, decreased the accumulation of C3 by proximal tubular cells, and protected against interstitial inflammation and damage. For determination of the injurious role of plasma-derived C3, as opposed to tubular cell-derived C3, C3-deficient kidneys were transplanted into WT mice. Protein overload led to the development of glomerular injury, accumulation of C3 in podocytes and proximal tubules, and tubulointerstitial changes. Conversely, when WT kidneys were transplanted into C3-deficient mice, protein overload led to a more mild disease and abnormal C3 deposition was not observed. These data suggest that the presence of C3 increases the glomerular filtration barrier's susceptibility to injury, ultrafiltered C3 contributes more to tubulointerstitial damage induced by protein overload than locally synthesized C3, and local C3 synthesis is irrelevant to the development of proteinuria. It is speculated that therapies targeting complement combined with interventions to minimize proteinuria would more effectively prevent the progression of renal disease.     

Role of apoptosis and Bcl-2/Bax in the development of tubulointerstitial fibrosis during experimental obstructive nephropathy

BACKGROUND/AIMS: To examine the role of apoptosis in experimental unilateral ureteral obstruction (UUO). METHODS: Rat kidneys were examined 3, 7 and 11 days following UUO or sham operation (SO). Tissue was immunohistochemically stained for alpha-smooth muscle actin (alpha-SMA), proliferating cell nuclear antigen (PCNA), Bcl-2 and Bax proteins. Apoptotic analysis was carried out in kidney sections using in situ end labelling of endonuclease cleaved DNA. RESULTS: The relative volume (Vv) of cortical interstitium and interstitial alpha-SMA increased progressively following UUO. ED1-positive monocytes/macrophages peaked at day 7 and significantly decreased at day 11. PCNA-positive cells in tubulointerstitium were significantly increased on day 3. Staining returned to the level of the SO group by day 11, meanwhile those in the interstitium remained much higher than baseline. TUNEL-positive cells were persistently raised following UUO. Transient tubular cell proliferation seemed unable to counteract the apoptosis since tubular atrophy was apparently present by day 11 of UUO. However, interstitial cell proliferation was high enough to overwhelm apoptosis, particularly with respect ot myofibroblasts, since alpha-SMA immunostaining and Vv remained elevated. The ratio of the number of PCNA-positive cells to apoptotic cells formed a predictive pattern for the staining score of interstitial alpha-SMA (R(2) = 47.23%, p < 0.05) and Vv (R(2) = 49.93%, p < 0.05). Tubular Bcl-2 immunostaining peaked on day 3, and then gradually decreased to baseline by day 11. The expression of Bax protein was inhibited on day 3 when compared with that of the SO group, but increased with time following UUO. CONCLUSION: These findings suggest an important role for apoptosis and its regulatory proteins in the processes of tubular atrophy and fibrogenesis following UUO.     

Intraperitoneal protein injection in the axolotl: the amphibian kidney as a novel model to study tubulointerstitial activation

BACKGROUND: A substantial body of experimental evidence suggests that protein loading causes activation of proximal tubular epithelial cells with consecutive interstitial fibrosis. These studies have mostly been performed using mammalian in vivo models of glomerular damage or tissue cultures of mammalian tubulointerstitial cells. The kidney of the axolotl contains not only closed nephrons, but also nephrons with ciliated peritoneal funnels called nephrostomes that have access to the peritoneal fluid. Injection of protein into the peritoneal cavity fails to expose closed nephrons to a protein load, but causes selective uptake and transient storage of proteins in tubular epithelial cells of nephrons with nephrostomes. The purpose of the present study was to determine whether (a) the axolotl kidney can be used as a model to assess protein uptake by tubular cells in vivo in the absence of glomerular damage, and (b) this is accompanied by any evidence of tubular epithelial cell activation and interstitial fibrosis. METHODS: Male and female axolotl (80 to 120 g of weight) were given a daily intraperitoneal injection of 1.5 mL endotoxin-free calf serum or saline as control. Kidneys were harvested after 4 or 10 days using perfusion fixation for light microscopy (fibrous tissue stain) and saline perfusion for immunohistochemistry (fibronectin, TGF-beta and collagen I). RESULTS: The findings document selective storage of protein and lipids, progressive with time, in proximal tubular epithelial cells of nephrons draining the coelomic cavity. In addition, progressive focal accumulation of fibrous tissue was noted around protein-storing tubules. Immunohistochemical staining demonstrated the presence of fibronectin and TGF-beta in the tubular epithelial cells and interstitial cells. CONCLUSION: The axolotl kidney provides a novel in vivo model to study tubulointerstitial activation and induction of interstitial fibrosis by protein loading. The findings are independent of alterations of glomerular function that may have potential confounding effects on peritubular hemodynamics, pO2, cell traffic, etc.     

Molecular basis of renal fibrosis

All progressive renal diseases are the consequence of a process of destructive fibrosis. This review will focus on tubulointerstitial fibrosis, the pathophysiology of which will be divided into four arbitrary phases. First is the cellular activation and injury phase. The tubules are activated, the peritubular capillary endothelium facilitates migration of mononuclear cells into the interstitium where they mature into macrophages, and myo-fibroblasts/activated fibroblasts begin to populate the interstitium. Each of these cells releases soluble products that contribute to ongoing inflammation and ultimately fibrosis. The second phase, the fibrogenic signaling phase, is characterized by the release of soluble factors that have fibrosis-promoting effects. Several growth factors and cytokines have been implicated, with primary roles suggested for transforming growth factor-β, connective tissue growth factor, angiotensin II and endothelin-1. Additional factors may participate including platelet-derived growth factor, basic fibroblast growth factor, tumor necrosis factor-α and interleukin-1, while interferon-γ and hepatocyte growth factor may elicit antifibrotic responses. Third is the fibrogenic phase when matrix proteins, both normal and novel to the renal interstitium, begin to accumulate. During this time both increased matrix protein synthesis and impaired matrix turnover are evident. The latter is due to the renal production of protease inhibitors such as the tissue inhibitors of metalloproteinases and plasminogen activator inhibitors which inactivate the renal proteases that normally regulate matrix turnover. Fourth is the phase of renal destruction, the ultimate sequel to excessive matrix accumulation. During this time the tubules and peritubular capillaries are obliterated. The number of intact nephrons progressively declines resulting in a continuous reduction in glomerular filtration. Received: 9 March 2000 / Revised: 24 July 2000 / Accepted: 26 July 2000     

Progression in chronic kidney disease

The pathogenic mechanisms that lead to chronic kidney disease (CKD) converge on a common pathway that results in progressive interstitial fibrosis, peritubular capillary loss with hypoxia, and destruction of functioning nephrons because of tubular atrophy. Interstitial recruitment of inflammatory leukocytes and myofibroblasts occurs early in kidneys destined to develop fibrosis. Circulating monocytes are recruited by locally secreted chemoattractant molecules, facilitated by leukocyte adhesion molecules. Functionally heterogeneous macrophages secrete many fibrosis-promoting molecules, but under some circumstances they may also serve a protective scavenging role. Excessive extracellular matrix production occurs primarily within interstitial myofibroblasts, a population of cells that appears to have more than 1 origin, including the resident interstitial fibroblasts, trans-differentiated tubular epithelial cells, and bone marrow-derived cells. Impaired activity of the endogenous renal matrix-degrading proteases may enhance interstitial matrix accumulation, but the specific pathways that are involved remain unclear. Tubules, inflammatory cells, and myofibroblasts synthesize the molecules that activate the fibrogenic cascades, the most important of which is transforming growth factor beta (TGF-beta). TGF-beta may direct cells to assume a pro-fibrotic phenotype or it may do so indirectly after stimulating synthesis of other fibrogenic molecules such as connective tissue growth factor and plasminogen activator inhibitor-1. Reduced levels of antifibrotic factors that are normally produced in the kidney such as hepatocyte growth factor and bone morphogenic protein-7 may accelerate fibrosis and its destructive consequences. Development of new therapeutic agents for CKD looks promising, but several agents that target different components of the fibrogenic cascade will almost certainly be necessary.     

The role of alpha-smooth muscle actin and platelet-derived growth factor-beta receptor in the progression of renal damage in human IgA nephropathy

BACKGROUND: The degree of tubulointerstitial damage can be considered a better indicator of renal function outcome in IgA nephropathy (N) than the extent of glomerular sclerosis. MATERIALS AND METHODS: To investigate the pathogenetic mechanisms of interstitial injury in IgAN, we used immunohistochemistry and in situ hybridization to evaluate the glomerular and tubolointerstitial expression of PDGF-beta receptor (R) and alpha-smooth muscle actin (SMA), two markers of mesenchymal cell activation, and correlated these findings with the histopathologic and clinical features of the disease. We studied 155 IgAN patients, divided into three groups based on the histological findings (mild, moderate and severe histological lesions). RESULTS: In normal kidneys and in patients with mild histological lesions, the interstitial areas showed scattered peritubular cells positive for PDGF-betaR and alpha-SMA, with a distribution resembling the capillary network. In the glomeruli several cells (mainly in the mesangial area) stained for PDGF-betaR, but only very few cells were positive for alpha-SMA. Alpha-SMA and PDGF-betaR staining, as expected, was also observed in vascular smooth muscle cells. Compared to patients with mild histological lesions, alpha-SMA expression was strikingly increased in patients with moderate to severe lesions, particularly in areas of tubulointerstitial fibrosis. In these patients, PDGF-betaR gene and protein expression, at the tubulointerstitial level, paralleled that in alpha-SMA. Both signals were significantly correlated with the interstitial damage (interstitial infiltrate and fibrosis). Interestingly, these patients showed a different pattern of distribution of alpha-SMA and PDGF-betaR in the glomeruli: PDGF-betaR expression was upregulated, whereas no changes were seen in alpha-SMA staining. In addition, glomerular PDGF-betaR staining was significantly correlated with mesangial cell proliferation, while alpha-SMA was not. Image analysis showed that 40.2+/-10.3/1,000 microm2 of interstitial cells were positive to both PDGF-betaR and alpha-SMA, but only 2.8+/-1.8/1,000 microm of glomerular cells expressed both signals. CONCLUSIONS: Our study supports the hypothesis that interstitial PDGF-betaR and alpha-SMA positive cells may play a key role in the pathogenesis of tubulointerstitial damage.     

Fibroblast-specific protein 1 is a specific prognostic marker for renal survival in patients with IgAN

BACKGROUND: There is little direct evidence that fibroblasts are involved in the progression of the renal interstitial fibrosis in human glomerulonephritis. With the availability of a new specific marker for fibroblasts, we determined the presence of fibroblasts in kidneys with IgA nephropathy (IgAN) and correlated their numbers with various clinical parameters. In particular, we also prospectively asked if the number of fibroblasts in the renal interstitium correlates with prognosis. METHODS: Cells positive for fibroblast-specific protein 1 (FSP1) were localized in renal biopsy specimens using immunohistochemistry with anti-FSP1 antibody. Clinical features were analyzed by one-way analysis of variance (ANOVA) with the Bonferroni correction. To assess the prognostic impact of the number of FSP1+ fibroblasts on renal survival in 142 patients with normal serum creatinine, the relationship between covariates to renal survival were evaluated univariately using the log-rank test and multivariately using Cox proportional hazards. RESULTS: Fibroblasts identified by their expression of FSP1 accumulate in areas showing severe interstitial fibrosis. Some tubular epithelial cells undergoing epithelial-mesenchymal transition (EMT) in fibrotic areas also express FSP1. Numbers of FSP1+ fibroblasts directly correlate with serum creatinine (r = 0.74, P < 0.0001) and inversely correlate with estimated creatinine clearance (r = -0.54, P < 0.0001), and by multivariate analysis, the clinical factors influencing renal survival are urinary protein excretion [> or = 1.0 g/day, relative risk (RR) = 4.20, P= 0.032], hypertension (RR 5.85, P = 0.0027), and > or = 20 FSP1+ fibroblasts per high power field (HPF) (RR 7.39, P = 0.0015). Staining for FSP1+ fibroblasts is largely nonoverlapping with alpha-smooth muscle actin+ (alpha-SMA) cells in the interstitium. CONCLUSION: The target protein FSP1 identifies human fibroblasts and tubular epithelium undergoing EMT, and distinguishes them from the diaspora of alpha-SMA+ vascular smooth muscle cells. FSP1+ fibroblasts are critically related to the progression of IgAN; consequently, staining FSP1 in renal biopsy specimens provides a valuable histologic index of progression.     

Expression of adhesion molecules in kidney with experimental chronic obstructive uropathy: the pathogenic role of ICAM-1 and VCAM-1

BACKGROUND: Chronic obstructive uropathy induced by maintained unilateral ureter ligation in the rat is characterized morphologically by interstitial inflammation, interstitial fibrosis, and tubular atrophy. Infiltrating mononuclear inflammatory cells, particularly T lymphocytes and macrophages, may contribute to the progression of this lesion by mediating tubular injury and by the activation of interstitial fibroblasts, with resultant tubular atrophy and interstitial fibrosis, respectively. Altered expression and activation of adhesion molecules by leukocytes, vascular endothelial cells, and parenchymal cells likely contributes both to the infiltration of inflammatory cells into the tubulointerstitial compartment and to the interaction of activated inflammatory cells with parenchymal cells. METHODS: In the current study, we examined changes in the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in a 90-day model of maintained unilateral ureter ligation in male Sprague-Dawley rats. RESULTS: Rat kidneys showed constitutive expression of ICAM-1 mRNA and constitutive immunostaining for ICAM-1 in peritubular capillaries, glomeruli, and a small percentage of cortical tubules. Ureter ligation resulted in a rapid increase in ICAM-1 mRNA, which was almost 2-fold greater than those of the contralateral and control kidneys as early as 3 h and which was maintained at a 4- to 6-fold higher level in the ligated vs. contralateral kidneys throughout the entire 90-day time course. There was a marked increase in ICAM-1 immunostaining within the tubular epithelium, with up to 80% of both cortical and medullary tubular cross-sections showing strong apical immunostaining from day 6 to 25, with a subsequent decrease throughout the remainder of the experiment. ICAM-1 immunostaining in the expanding interstitium in the ligated kidneys showed a gradual increase throughout the duration of the experiment. In contrast, glomerular immunostaining for ICAM-1 was decreased in the ligated compared to the contralateral kidneys throughout the entire experiment. There was a later but prominent increase in VCAM-1 mRNA in ligated kidneys, which was first evident at 2 days and which was maintained 2- to 10-fold greater than the contralateral kidneys throughout the entire time course. VCAM-1 immunostaining increased in the expanding interstitium, but decreased in glomeruli in obstructed vs. contralateral kidneys. Tubular staining for VCAM-1 did not change after ureter ligation. CONCLUSION: Increased ICAM-1 and VCAM-1 may contribute to the prominent inflammatory cell infiltration in the chronic tubulointerstitial nephritis accompanying maintained unilateral ligation. Tubule expression of ICAM-1, which occurs during a similar time course as previously documented for tubular cell proliferation and especially tubular cell apoptosis in this model, may contribute to injurious interactions of activated inflammatory cells with tubular epithelium.     

Overexpression of chemokines, fibrogenic cytokines, and myofibroblasts in human membranous nephropathy

Overexpression of chemokines, fibrogenic cytokines, and myofibroblasts in human membranous nephropathy. BACKGROUND: Proteinuria plays a central role in the progression of glomerular disease, and there is growing evidence suggesting that it may determine tubular cell activation with release of chemokines and fibrogenic factors, leading to interstitial inflammatory reaction. However, most studies on this subject have been performed in experimental models, and the experience in human kidney biopsies has been scarce. We analyzed the tissue sections of patients with idiopathic membranous nephropathy (IMN), a noninflammatory glomerular disease that may follow a progressive disease with heavy persistent proteinuria, interstitial cell infiltration, and decline of renal function. METHODS: Paraffin-embedded biopsy specimens from 25 patients with IMN (13 progressive and 12 nonprogressive) were retrospectively studied by immunohistochemistry [monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted chemokine (RANTES), osteopontin (OPN), platelet-derived growth factor-BB (PD-GF-BB)] and in situ hybridization [MCP-1, RANTES, PDGF-BB, transforming growth factor-beta1 (TGF-beta1)]. Moreover, we studied the presence of myofibroblasts, which were identified by the expression of alpha-smooth muscle actin (alpha-SMA), the monocytes/macrophages (CD68-positive cells), and T-cell infiltration (CD4+ and CD8+ cells). All of the patients were nephrotic and without treatment at time of the biopsy. RESULTS: A strong up-regulation of MCP-1, RANTES, and OPN expression was observed, mainly in tubular epithelial cells, with a significant major intensity in the progressive IMN patients. A strong correlation between the mRNA expression and the corresponding protein was noted. The presence of these chemokines and OPN was associated with interstitial cell infiltration. TGF-beta and PDGF were also up-regulated, mainly in tubular epithelial cells, with a stronger expression in the progressive IMN, and an association with the presence of myofibroblasts was found. CONCLUSIONS: Patients with severe proteinuria and progressive IMN have an overexpression in tubular epithelial cells of the chemokines MCP-1, RANTES, and OPN and the profibrogenic cytokines PDGF-BB and TGF-beta. Because this up-regulation was associated with an interstitial accumulation of mononuclear cells and an increase in myofibroblastic activity, it is suggested that those mediators are potential predictors of progression in IMN. Finally, based on experimental data and the findings of this article, we speculate that severe proteinuria is the main factor responsible for the up-regulation of these factors in tubular epithelial cells.     

Mast cells and fibrosis on testicular biopsies in male infertility

Testicular dysfunction correlates with increased testicular mast cells. Mast cells can activate fibroblasts and promote collagen synthesis. The aim of the study was to examine testicular mast cells containing tryptase, and the relationship between mast cells and different fibrosis stages of interstitium and peritubular region of testes. Testicular biopsies obtained from 33 infertile men were assigned to 2 groups: normal spermatogenesis (n = 10) and defective spermatogenesis (n = 23). Total, interstitial, and peritubular mast cells were examined immunohistochemically using antihuman tryptase. The fibrosis stage was evaluated using vimentin and alpha-smooth muscle actin. The ratio of tubules with sclerosis to total tubules was also calculated. In all cases, mast cells were mainly localized in the interstitium. The number of total mast cells was significantly higher in defective spermatogenesis than in normal spermatogenesis (p = .048). In both groups, interstitial mast cells were higher than peritubular mast cells. However, the increase in peritubular region was much higher than the increase in interstitium. Total, peritubular, and interstitial mast cell counts were not different from each other, according to the changing fibrosis stages. Total and interstitial mast cells were significantly higher in the cases with sclerosing seminiferous tubules than in the cases with no sclerosis (p = .04 and p = .024, respectively). The mast cells and the mast cell product tryptase could be involved in the etiology of defective spermatogenesis, especially whenever the last stage (tubular hyalinization and sclerosis) takes place.