[Show abstract][Hide abstract] ABSTRACT: Diabetic nephropathy is characterised by structural changes known to be associated in non-diabetic nephropathies with the expression of the cytoskeletal proteins a-smooth muscle actin and vimentin. We aimed to investigate the expression of cytoskeletal proteins in experimental diabetic nephropathy.
Rats were made diabetic by an injection of streptozotocin (45 mg/kg). Groups of rats (n = 6) and their respective controls (n = 4) were killed at different time intervals. (days 7, 15, 30, 60, 90 and 120). We also studied two groups of diabetic rats treated with a long-acting insulin; the first (n = 8) was treated from the induction of diabetes and the second (n = 8) received insulin from day 15 onward. At each time-point, kidney function, proteinuria and histology were evaluated. Cytoskeletal proteins and collagens III and IV deposition was determined by immunohistochemistry. Changes in the transcription of the cytoskeletal proteins was determined by northern blot analysis.
Although normal glomeruli did not express alpha-smooth muscle actin until late in the time course, it was detected in diabetic mesangium from day 7 onward. In the interstitium, it appeared in a perivascular and peritubular distribution. Vimentin was detectable within normal glomerular epithelial cells and increased rapidly (days 7 and 15) in diabetic rats. Vimentin also appeared early within the lining of the peritubular capillaries and damaged diabetic tubules. These changes were associated with a delayed increased transcription of alpha-smooth muscle actin and vimentin. Treatment with insulin (early or late) attenuated and reversed respectively the expression of cytoskeletal proteins and collagens within diabetic kidneys. Close correlations were noted between the number of alpha-smooth muscle actin positive cells within diabetic glomeruli and mesangial expansion (r = 0.46, p < 0.02) as well as interstitial alpha-smooth muscle actin positive cells and interstitial fibrosis (r = 0.51, p < 0.002).
Changes in the expression of cytoskeletal proteins within the kidneys of diabetic rats suggest a role for alpha-smooth muscle actin and vimentin in the pathogenesis of diabetic kidney disease.
[Show abstract][Hide abstract] ABSTRACT: Myofibroblasts play an important role in wound healing in a variety of tissue injuries. They have also been implicated in tissue fibrosis including renal scarring. This study was aimed at defining their role in one of the commonest forms of nephrotic syndrome in adults, namely membranous nephropathy. We have studied 21 patients with biopsy proven idiopathic membranous nephropathy who were treated with glucocorticoids, attempting to define the role of myofibroblasts (alpha-smooth muscle actin-positive as well as vimentin-positive cells) in the progression of this form of nephropathy. There were 13 non-progressors (NP) and 8 progressors (P). The clinical, histological, and immunohistochemical characteristics of both groups were compared. Immunohistochemical staining for myofibroblasts cytoplasmic markers a-smooth muscle actin (alpha-SMA) and vimentin relied on an avidin-biotin-peroxidase method. The level of blood pressure, degree of proteinuria, severity of interstitial infiltrate and interstitial fibrosis did not differentiate P from NP. However, vascular sclerosis was more severe in P compared to NP (p < 0.016) and its severity predicted the subsequent functional outcome (slope of the 1/serum creatinine against time; r2 = 0.618, p < 0.01). Mesangial alpha-SMA was significantly higher in P (31 +/- 18.6%) than in NP (14.5 +/- 9.8%), p < 0.015. Interstitial alpha-SMA immunostain was also higher in P but did not reach statistical significance. However, the number of interstitial myofibroblasts (alpha-SMA positive cells) closely predicted the subsequent rate of the progression of chronic renal failure (r2 = 0.919, p < 0.0001). Mesangial vimentin expression was not different between both groups. By contrast, interstitial vimentin immunostain was higher in P (19.1 +/- 8.8%) compared to NP (7.9+/-5.6 %), p < 0.002. These data suggest that the expression of mesangial and interstitial cytoskeletal proteins (alpha-SMA and vimentin) may have useful prognostic implications as they appear to differentiate between patients with membranous nephropathy who respond to immunosuppression and those who continue to progress.
[Show abstract][Hide abstract] ABSTRACT: Myofibroblasts have been implicated in the pathogenesis of wound healing and tissue fibrosis. A role has also been put forward for these cells in the development of experimental and clinical renal scarring.
We examined the expression of myofibroblast phenotypes by immunohistochemistry, relying on an avidin-biotin-peroxidase method, during the course of renal scarring in rats submitted to subtotal (5/6) nephrectomy (SNx). We also attempted to identify changes in immunoreactive transforming growth factor-beta (TGF-beta) and collagen (III and IV) within remnant kidneys in order to determine their association with the expression of the myofibroblasts.
In normal sham-operated rats, alpha-smooth muscle actin (alpha-SMA) was confined to the media of renal arteries and arterioles. In contrast, in rats with renal ablation we observed the early (day 7) appearance of myofibroblasts expressing alpha-SMA (A) in the interstitium of remnant kidneys particularly around vessels. Interstitial cells expressing alpha-SMA increased with time as tubulointerstitial fibrosis progressed. By day 30 some interstitial cells also expressed vimentin (V). Various interstitial myofibroblast phenotypes (A, V, VA) were expressed during the course of experimental renal scarring. Interstitial myofibroblasts appeared to be associated with TGF-beta as these cells' cytoplasm stained for both this growth factor and alpha-SMA. Interstitial fibrosis was also associated with increased interstitial expression of both collagen III and IV. Some atrophic tubular cells showed positive immunostaining for vimentin during the late stages of renal scarring (days 90-150). In the glomeruli, a segmental expression of alpha-SMA was noted from day 21 after SNx onward. Normal glomerular endothelial cells appeared to express vimentin while epithelial cells expressed both vimentin and desmin (D). The glomerular immunostain for vimentin increased with time but decreased as glomerulosclerosis progressed. In contrast, glomerular desmin and alpha-SMA immunostain continued to rise with progressive glomerulosclerosis. This was associated with the appearance of type III collagen within scarred glomeruli. Both vimentin and desmin appeared within the walls of the renal arterioles and increased with time from day 7 and 15, respectively. Vimentin was also expressed in the peritubular capillaries of remnant kidneys. By contrast, alpha-SMA, normally present in the media of arterioles, decreased as arteriolar sclerosis progressed. These changes cannot be exclusively attributed to systemic hypertension as they were absent in a group of age-matched, sham-operated, spontaneously hypertensive rats.
Myofibroblasts may play a role in the pathogenesis of glomerulosclerosis, tubulointerstitial fibrosis and vascular sclerosis. Further, the acquisition of new myofibroblastic phenotypes by glomerular and tubular cells may contribute to renal fibrosis.
[Show abstract][Hide abstract] ABSTRACT: The progression of chronic renal failure is characterised histologically by glomerulosclerosis, tubulointerstitial fibrosis and vascular sclerosis. Recent research has identified common mechanisms underlying these fibrotic processes. In particular, the scarring process within the glomeruli and the tubulointerstitium involves the infiltration by inflammatory cells including monocytes, the activation of intrinsic renal cells as well as interactions between infiltrating and resident cells. These interactions depend, to a large extent, on the release by these cells of chemokines, cytokines and growth factors. These factors are in turn involved in the induction of cellular proliferation within the kidney and the stimulation of the synthesis and deposition of extracellular collagenous matrix. Fibrosis is believed to result from excessive synthesis of extracellular matrix and a concommitant decrease in its breakdown. This fibrotic process resulting in end stage renal insufficiency bears strong similarities to that taking place within cirrhotic livers or fibrotic lungs. The new insights in our understanding of renal fibrosis have opened the way to new interventions aimed at its prevention. This may ultimately slow the progression of chronic renal insufficiency and decrease the number of patients requiring dialysis replacement therapy.
The International Journal of Biochemistry & Cell Biology 02/1997; 29(1):55-62. DOI:10.1016/S1357-2725(96)00119-7 · 4.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cellular mediators of progressive renal fibrosis in diabetic nephropathy remain unknown. Myofibroblasts have been implicated in the pathogenesis of experimental and clinical renal fibrosis. Their role in the progression of diabetic nephropathy is the subject of this study.
We have studied by immuno-histochemistry the expression of cytoskeletal proteins associated with the activation of myofibroblasts; alpha-smooth-muscle actin (alpha-SMA), vimentin (Vi) and desmin (D), in the kidneys of 25 patients with diabetic nephropathy (5 patients had a superimposed glomerulonephritis). Comparisons were made with normal tissue from three kidneys removed for renal-cell carcinoma. Correlations were studied between clinical and biochemical parameters with the expression of renal cytoskeletal proteins.
In normal kidneys, cells expressing alpha-SMA were confined to the vascular media and adventitia while immunoreactive Vi was detected in glomerular epithelial cells. In diabetic kidneys, cells expressing alpha-SMA were detected primarily in the renal interstitium and to a lesser extent in some glomeruli in association with mesangial proliferation. Vimentin immunostain decreased in glomeruli displaying diabetic hyalinosis and sclerosis. By contrast, strong Vi immunoreactivity was noted in atrophic diabetic tubules and to a lesser extent in the interstitium. Desmin was not detected in either normal or diabetic kidneys. Close correlations were observed between the expression of renal cytoskeletal proteins and the progression of renal insufficiency. Interstitial alpha-SMA proved to be a predictor of progressive diabetic nephropathy (R2 for 1/serum Cr slope = 0.608, P = 0.00001). This predictive value was superior to, and independent from, that of the best conventional histological predictive parameters; tubular atrophy (R2 = 0.477, P = 0.00004) and interstitial fibrosis (R2 = 0.28, P = 0.001).
We have demonstrated in this study the neoexpression of cytoskeletal proteins within diabetic kidneys. This has allowed the identification of new predicting histological markers for the progression of diabetic nephropathy.
[Show abstract][Hide abstract] ABSTRACT: We have studied the acute changes (up to 30 days) in the expression of epidermal growth factor (EGF) and its receptor (EGFr) in the kidneys of adult male Wistar rats made diabetic by a single intravenous injection of streptozotocin (55 mg/kg) using a combination of immunocytochemical staining and in situ hybridization histochemistry. In the absence of insulin treatment, diabetic rats displayed renal growth (hypertrophy and hyperplasia). It was accompanied by an increase in immunostainable EGF within the thick ascending limb (TAL) of the loops of Henle which was apparent within 24 h of the onset of diabetes, reached a peak by day 7 and persisted to the end of the experimental period (day 30). In situ hybridization histochemistry revealed that these changes were preceded by a rapid rise in EGF mRNA in the cells of the TAL, which was highest after 1 day but declined to control levels by day 7. Increased immunostainable EGFr was evident in both the proximal and TAL and in the cortical collecting ducts from day 1. Staining of the proximal tubules declined rapidly after day 1 but that of the TAL and collecting ducts persisted until day 7 and declined thereafter. These results are discussed in light of the role of EGF in the hypertrophy and repair of the diabetic kidney.
[Show abstract][Hide abstract] ABSTRACT: We have studied the distribution of immunoreactive growth factors, by an avidin-biotin-peroxidase technique, throughout the course of progressive renal scarring in rats submitted to extensive renal ablation. Groups of rats (n = 6) were sacrificed at Days 7, 15, 21, 30, 90 and 150 following subtotal nephrectomy (SNx) by ligation and resection of the renal poles. During the early stages, when compensatory renal growth took place, increased renal immunostaining for insulin-like growth factor-I (IGF-I) and epidermal growth factor (EGF) was detected within the collecting ducts and distal tubules, respectively. As renal scarring became established by Days 90 and 150, these two growth factors were detected within the cells of damaged and vacuolated distal tubules. By contrast, a progressive increase immunostain for platelet-derived growth factor (PDGF)-AB was apparent within the glomeruli from Day 15 onward preceding the onset of glomerulosclerosis. A third staining pattern was apparent by Day 15 for transforming growth factor-beta (TGF-beta) and by Day 30 for IGF-I consisting of a perivascular and interstitial distribution coinciding with adventitial expansion and tubulo-interstitial fibrosis, respectively. A mosaic of growth factors is expressed within the kidneys of rats submitted to extensive renal ablation.
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of experimental renal growth. This study was designed to investigate the quantitative and qualitative changes in renal IGF-I which occur during the course of progressive renal scarring in rats submitted to extensive renal ablation. Analyses were carried out at 7, 15, 21, 30, 90 and 150 days after subtotal nephrectomy. Compensatory renal growth occurred over the first 30 days, amounting to a 161% increase in the protein content per remnant kidney (272 +/- 22 vs. 104 +/- 3 mg at the outset, p < 0.001, means +/- SD, n = 6) but DNA per remnant kidney continued to rise, reaching a 285% increase by day 90 (5.12 +/- 0.06 vs. 1.33 +/- 0.06 mg at the outset, p < 0.001), probably reflecting the continued recruitment of inflammatory cells. Changes in extractable renal IGF-I (eIGF-I) were measured by radioimmunoassay. Seven days after subtotal nephrectomy eIGF-I had increased by 70% compared with controls (2.37 +/- 0.22 ng/mg protein vs. 1.39 +/- 0.2 ng/mg at the outset, p < 0.001) but had returned to normal by 30 days. This coincided with a transient increase in the immunostainable IGF-I (iIGF-I) within the cortical collecting ducts which, again, had subsided by 30 days. However, at 90 and 150 days, as severe scarring was detected, a different pattern of immunostaining (iIGF-I) was noted. Strong staining was evident in the injured cells of the distal tubules and within the fibrosed interstitium.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: We have compared the kidneys of two inbred strains of rats (Lewis and Lewis-Dwarf) 7 days after the induction of diabetes mellitus with streptozotocin, in order to examine the influence of a selective growth hormone (GH) deficiency on diabetic renal growth and insulin-like growth factor-I (IGF-I) content of the kidneys. Insulin-like growth factor-I (IGF-I) content of the kidneys. Insulin-like growth factor-I was measured by radioimmunoassay and its distribution within the kidney by immunohistochemical staining. We detected a significant increase in both the wet weight (32.9 +/- 5.3%, P = 0.0085) and dry weight (16.3 +/- 6.3%, P = 0.046) of the kidneys of diabetic Lewis rats but dwarf rats, selectively deficient in GH, did not show a significant increase in either parameter. Extractable IGF-I increased within the kidneys of diabetic rats of both strains but to a lesser extent in the dwarf rats (+105 +/- 28% and +65 +/- 21% respectively, P < 0.01). In diabetic Lewis rats a positive correlation was noted between the severity of glycaemia and kidney IGF-I content (r = 0.604, P < 0.05) but no such correlation was noted in dwarf rats. Inulin-like growth factor-I immunostaining increased in diabetic rats of both strains, mainly within cells of the thick ascending limb of the loop of Henle including damaged and vacuolated cells. However, morphometric analysis of the staining showed that it was significantly less widespread in the diabetic dwarf rats (P = 0.026).(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: Insulin-like growth factor-I (IGF-I) has been implicated in the pathogenesis of experimental diabetic renal growth. In this study, we have examined the serial changes of renal IGF-I, by radioimmunoassay (RIA) and immunohistochemistry, in rats made diabetic by a single intravenous injection of streptozotocin (STZ; 55 mg/kg). The kidney IGF-I content, as determined by RIA, increased within 48 h of the induction of diabetes mellitus, peaked on day 4 and returned to normal levels by day 30. Renal IGF-I correlated positively with the severity of hyperglycaemia on day 7 (r = 0.685, p < 0.001). Immunostaining showed IGF-I to be located within the cortical collecting ducts of normal rats. In diabetic rats, IGF-I also appeared within the cells of the thick ascending limbs of the loops of Henle, in particular those undergoing cytoplasmic vacuolation. The changes in immunoreactive IGF-I assessed stereologically were consistent with the amounts of extractable IGF-I. They were not observed in normoglycaemic, STZ-injected, rats treated with high-dose insulin. This study suggests that IGF-I is involved in diabetic tubular injury and growth.
[Show abstract][Hide abstract] ABSTRACT: The role of cGMP in compensatory renal growth (CRG) is uncertain, since inconsistent changes in renal cGMP have been reported following uninephrectomy (UNx) in the rat. The aim of this study was to reassess the change in cGMP following UNx in the conscious rat by sequential measurements of cGMP excretion and to determine the contribution of nitric oxide, an activator of guanylate cyclase, using L-NAME an inhibitor of nitric oxide synthase. In the conscious rat a sustained increase in the urinary excretion of cGMP was produced in the 7-day period following UNx. L-NAME (20 mg/kg per day) prevented the increase in cGMP excretion following UNx, but not compensatory renal growth. Total kidney DNA, however, was reduced by L-NAME. These observations dissociate the increase in cGMP after UNx from the process of renal hypertrophy.