Tanya M. Osicka

Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)

Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-beta and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of (125)I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Abs to RAGE or to TGF-beta. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-beta expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.

Modulation of Soluble Receptor for Advanced Glycation End Products by Angiotensin-Converting Enzyme-1 Inhibition in Diabetic Nephropathy

Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocin-induced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.

Angiotensin converting enzyme inhibition reduces retinal overexpression of vascular endothelial growth factor and hyperpermeability in experimental diabetes.

Aims/hypothesis. Angiotensin converting enzyme (ACE) inhibition has been recently suggested to have retinoprotective actions in diabetic patients but the mechanism of this effect is not known. In vitro, angiotensin II stimulates expression of vascular endothelial growth factor (VEGF), a permeability-inducing and endothelial cell specific angiogenic factor which has been implicated in the pathogenesis of diabetic retinopathy in humans and in experimental animals. We sought to determine the effects of ACE inhibition on retinal VEGF expression and permeability in experimental diabetic retinopathy.¶Methods. Streptozotocin-induced diabetic rats and control animals were assigned at random to receive ACE inhibitor treatment or vehicle. At 24 weeks the retinal VEGF protein gene expression was assessed by northern blot analysis and in situ hybridisation. Retinal permeability to albumin was measured using a double isotope technique.¶Results. Experimental diabetes was associated with cell specific two to fourfold increase in retinal VEGF protein gene expression (p < 0.01) and a 2-fold increase in retinal vascular permeability to albumin (p < 0.01). The localization of VEGF expression in the retina was not altered in animals with experimental diabetes. Angiotensin converting enzyme inhibitor treatment of diabetic rats reduced diabetes-associated changes in VEGF gene expression and vascular permeability.¶Conclusion/interpretation. These findings implicate the renin-angiotensin system in the VEGF overexpression and hyperpermeability which accompany diabetic retinopathy and provide a potential mechanism for the beneficial effects of ACE inhibition in diabetic retinal disease. [Diabetologia (2000) 43: 1360–1367]

Glomerular capillary wall permeability to albumin and horseradish peroxidase

Summary: Tritium labelled albumin present in urine fractions from in vivo rat filtration studies and isolated perfused rat kidneys (IPK) has been shown by gel chromatographic analysis to be severely degraded (approximately 90%) to small peptides. the in vivo degradation of rat serum albumin and bovine serum albumin has been shown to be similar. the degradation has a marked influence on the fractional clearance analysis. the fractional clearance for albumin in the isolated perfused kidney as determined by Lowry assay was 0.00180 ± 0.0007, 0.0060 ± 0.0033 by Biuret assay and 0.0075 ± 0.0039 as measured by total radioactivity in the urine. Specific analysis of intact albumin by gel chromatography gave a fractional clearance of 0.00039 ± 0.00016, which is comparable to in vivo values. Degradation of albumin in the urine from IPK experiments was inhibited by 42% by including 150 mmol/L lysine, an inhibitor of tubular cell uptake, in the perfusate. Inhibition of degradation of anionic horseradish peroxidase was also observed. These studies demonstrate that albumin is degraded during renal passage probably by tubular cells. In a series of isolated perfused kidney systems using different inhibitors of protein tubular uptake, namely lysine, ammonium chloride, chloroquine and cytochalasin B, we estimate the glomerular sieving coefficient of albumin to be approximately 0.074 as determined using [3H]‐albumin. This glomerular sieving coefficient is considerably higher than originally thought, yet it is in accord with the size selectivity characteristics of the glomerular capillary wall as determined by transport probes calibrated for hydrodynamic size by gel exclusion chromatography. the tubular inhibitors also destroyed charge selectivity of the anionic and neutral forms of albumin and horseradish peroxidase. the tubular inhibitors were characterized in terms of their effect on tubular lysozyme uptake and glomerular size selectivity as determined using polydisperse dextran fractions. By accounting for the destination of all the tritium labelled albumin in the urine, perfusate, whole kidney and isolated glomeruli we conclude that a major rapid transtubular cell pathway must exist that directs albumin from the tubular lumen to either the renal lymphatics or peritubular capillaries or both. This pathway appears to be an overload pathway because since the albumin concentration in the perfusate was lowered from 50 mg/mL to 2.0 mg/mL it was not active. the pathway is also inactive for other proteins, such as horseradish peroxidase, that are at relatively low concentration.

Modulation of nephrin in the diabetic kidney: association with systemic hypertension and increasing albuminuria.

Objective Nephrin, a cytoskeletal protein which localizes to the slit pore of podocytes, may play a role in proteinuria. This study examines the possible relationship between nephrin expression and albuminuria in normotensive and hypertensive diabetic rats. Methods Streptozotocin diabetes was induced in both Wistar–Kyoto (WKY) and spontaneously hypertensive (SHR) rats. Diabetic and control animals were sacrificed and the kidneys obtained after 8, 16 and 24 weeks. The glomerular filtration rate (GFR) and albuminuria were also measured. Glycaemic control was assessed by measurement of plasma glucose and glycated haemoglobin (HbA1c). Nephrin gene expression was quantitated by real-time polymerase chain reaction (PCR) and localized by in situ hybridization. Nephrin protein expression was localized by immunohistochemistry and quantitated. Results Following a transient rise at 8 weeks in the diabetic SHR (P < 0.05 versus control SHRs), nephrin gene expression, as determined by real-time PCR, was significantly decreased at 16 and 24 weeks (P < 0.05 versus control SHRs). In situ hybridization confirmed similar changes in nephrin gene expression, which were confined to the glomeruli. This reduction in glomerular nephrin gene expression was associated with increasing albuminuria at 16 and 24 weeks in diabetic SHRs. There were no significant changes in nephrin gene expression, either by real-time reverse transcription polymerase chain reaction or in situ hybridization, observed in normotensive diabetic WKY rats, in the context of much less albuminuria in this group. Immunohistochemistry for nephrin protein revealed a greater depletion in renal nephrin content in SHR than in WKY rats after 24 weeks of diabetes. Conclusion Reduction in renal nephrin gene and protein expression is closely associated with the development of albuminuria, as observed in an experimental model of diabetes and hypertension.

Aminoguanidine and ramipril prevent diabetes-induced increases in protein kinase C activity in glomeruli, retina and mesenteric artery

This study investigated the effects of insulin therapy, inhibition of advanced glycation end-product formation with aminoguanidine and angiotensin-converting enzyme inhibition with ramipril on diabetes-related increases in protein kinase C (PKC) activity in the streptozotocin-diabetic rat. PKC activity in the glomeruli, retina and mesenteric artery was increased by 1.5-2-fold after induction of diabetes, and this increase was maintained over 24 weeks. Treatment with insulin at 2 units or 6 units per day attenuated glomerular PKC in proportion to the level of glycohaemoglobin after 4 weeks of diabetes (r=0.68, P<0.0001). The higher dose of insulin prevented the diabetes-related increase in glomerular PKC activity, although blood glucose levels were not normalized. After 8 weeks of diabetes, ramipril completely prevented the diabetes-related increases in PKC activity in the glomeruli, retina and mesenteric artery. By contrast, aminoguanidine treatment resulted in no inhibition of glomerular PKC activity, partial inhibition of retinal PKC activity and complete inhibition of mesenteric artery PKC activity. After 24 weeks of diabetes, both aminoguanidine and ramipril prevented the diabetes-related increases in PKC activity in all three tissues, in parallel with suppression of albuminuria by both agents. Aminoguanidine also prevented diabetes-related increases in retinal permeability at 16 weeks. These results suggest that the organ-protective effects of insulin, aminoguanidine and ramipril in diabetes may be mediated, at least in part, through the differential inhibition of PKC activity in various tissues.

Renal expression of angiotensin receptors in long-term diabetes and the effects of angiotensin type 1 receptor blockade.

Objective The aims of this study were to assess the renal expression of angiotensin type 1 (AT1) and type 2 (AT2) receptors in diabetic spontaneously hypertensive rats (SHR) and the effect of AT1 receptor blockade on the expression of these receptors. Design Diabetes was induced by injection of streptozotocin in SHRs. Irbesartan, an AT1 receptor antagonist, was given to diabetic SHRs for 32 weeks (15 mg/kg per day, n = 10). Diabetic (n = 10) and non-diabetic SHRs (n = 10) were studied concurrently. A separate group of control and diabetic Wistar–Kyoto (WKY) rats were also evaluated. Methods Gene and protein expressions of the AT1 and AT2 receptor were assessed by reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry with specific antibodies and in vitro autoradiography with [125I]Sar(1), Ile(8) angiotensin II or [125I]CGP42112B. Results Both AT1 and AT2 receptor mRNA levels in the kidney were reduced in diabetic SHRs compared to non-diabetic SHRs. Immunohistochemistry staining with specific antibodies showed a similar reduction in glomerular and tubulo-interstitial staining for both AT1 and AT2 receptors. Reduced binding for the AT1 and AT2 receptor was found in the kidney of diabetic SHRs. Diabetic SHRs developed albuminuria and had glomerular and tubulo-interstitial injury, which were prevented by treatment with irbesartan. Reduced expression of the AT1 receptor, but not the AT2 receptor, in diabetic SHRs was prevented by treatment with irbesartan. In diabetic WKY rats no such reduction in AT1 expression was observed, although there was a trend for reduced AT2 receptor expression. Conclusions These findings demonstrated that renal expression of both AT1 and AT2 receptor was reduced in long-term diabetic SHRs and that blockade of the AT1 receptor had disparate effects on expression of angiotensin II receptor subtypes.

Ramipril and aminoguanidine restore renal lysosomal processing in streptozotocin diabetic rats

Aims/hypothesis. We aimed to examine the time course for the diabetes-related changes in renal lysosomal processing and to determine whether these changes can be prevented by aminoguanidine or ramipril treatment. Methods. The percentage desulphation of intravenously injected tritium labelled dextran sulphate ([3H]DSO4) in the urine, as determined by ion-exchange chromatography, was used as a marker of lysosomal sulphatase activity. Sulphatase activity was determined 1, 2, 3 and 4 weeks after the onset of diabetes in rats as well as in rats treated with either aminoguanidine or ramipril for twelve weeks. Results. The amount of totally desulphated [3H]DSO4 in urine collected from control rats was 65.6 ± 0.8 %. This was significantly reduced in diabetic rats two (57.4 ± 1.4 % desulphated), three (56.8 ± 1.3 % desulphated) and four (52.9 ± 2.2 % desulphated) weeks after the onset of diabetes. The significant decrease in the amount of totally desulphated [3H]DSO4 in the urine also found at 12 weeks after the onset of diabetes was not affected by drug treatment. There was no significant difference in the amount of partially desulphated [3H]DSO4 in the urine between all the study groups. However, the increase in totally sulphated [3H]DSO4 in the urine collected from diabetic rats (8.7 ± 1.7 % sulphated) compared with that of control rats (2.2 ± 0.5 % sulphated) was normalised by treatment with both aminoguanidine (4.8 ± 1.6 % sulphated) or ramipril (4.5 ± 0.8 % sulphated). Conclusions/interpretation.These results raise the possibility that the diabetes-induced changes in renal lysosomal processing may be one of the initial events in the development of diabetic nephropathy. Aminoguanidine and ramipril, known for their different mechanism of action, seem to prevent diabetes-induced changes in lysosomal processing either through their effects on enzyme activity within the lysosome or through their effects on the trafficking of molecules to and from the lysosome. [Diabetologia (2001) 44: 230–236]

Renal Processing of Albumin in Diabetes and Hypertension in Rats

Background/Aims: Recent studies show that albuminuria may be the result of changes in post-glomerular cellular uptake and processing of albumin. This study aims to determine whether this processing is disrupted in diabetes and/or hypertension. Methods: Diabetes (d) was induced using streptozotocin in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) and studied after 8, 16 and 24 weeks of disease. Intact albumin excretion was determined by radioimmunoassay. Total albumin was determined by [14C]albumin. Lysosomal activity was determined by dextran sulfate desulfation. Renal TGF-β1 and transforming growth factor-β1 inducible gene-h3 mRNA (βig-h3) expression was determined by real time RT-PCR. Results: SHR-c rats exhibited an increase in intact albuminuria without significant change in total albumin excretion (intact plus albumin-derived peptides). For WKY-d rats, intact albuminuria developed initially, followed by an increase in total albumin excretion primarily in the form of albumin peptides (peptiduria). SHR-d rats exhibited both increases in peptiduria and intact albuminuria. There was no increase in glomerular permeability at 24 weeks for polydisperse [3H]Ficoll in all groups. Increased renal TGF-β1 and βig-h3 expression was correlated with a decrease in dextran sulfate desulfation and increased intact albuminuria independent of peptiduria. Conclusion: Increased albumin excretion in hypertension and/or diabetes is manifested in different forms independent of glomerular permeability.

Tubular inhibition destroys charge selectivity for anionic and neutral horseradish peroxidase

The fractional clearance of [3H]anionic HRP and [3H]neutral HRP in the isolated perfused kidney as determined by radioactivity analysis was 0.0160+/-0.0028 (n=6) and 0.0388+/-0.0076 (n=8) respectively. The apparent charge selectivity for both the anionic and neutral forms of HRP observed was destroyed with the inclusion of the tubular uptake inhibitors, 150 mM lysine and 10 mM NH4Cl, in the perfusate. In the presence of 150 mM lysine, the clearances of [3H]anionic HRP and [3H]neutral HRP were 0.0645+/-0.0110 (n=4) and 0. 0784+/-0.0120 (n=4) respectively, and 0.0564+/-0.0035 (n=4) and 0. 0694+/-0.0054 (n=4) respectively in the presence of 10 mM NH4Cl. The clearance for both the anionic and neutral HRP probes in these tubular uptake inhibited systems fits precisely the size selective characteristics of the glomerular capillary wall as determined by transport probes calibrated for hydrodynamic size by size exclusion chromatography. The tubular uptake inhibitors were observed not to alter glomerular permselectivity as determined using polydisperse dextran fractions and the behaviour of neutral HRP. This study demonstrates that charge selectivity for differently charged proteins is not as great as originally thought and suggests that the clearance differences between anionic and neutral forms may be due to differential handling by the tubules.