Characterization of the urinary albumin degradation pathway in the isolated perfused rat kidney.

This study examines the existence of the urinary albumin degradation pathway and the proposed role of receptor-mediated endocytosis in this process using the isolated perfused rat kidney (IPK) model. Albumin-derived peptides in IPK urine are analyzed in terms of their relative size distribution using radioactivity and absorbance at 214 nm, and their susceptibility to trypsin digestion. The effects of perfusing kidneys with concanamycin A and myristoyl trimethyl ammonium bromide (MTMAB), inhibitors of the receptor-mediated endocytosis regulators vacuolar-type H(+) ATPase (v-ATPase) and dynamin GTPase, respectively, are examined. Normal IPK urine contains mildly degraded (defined as approximately 10-40 kDa; 43.0 +/- 8.3%) and heavily degraded (defined as <10 kDa; 22.6 +/- 7.7%) albumin peptides as well as intact albumin (34.5 +/- 4.1%). The relative size distribution of the peptides is similar by radioactivity and absorbance at 214 nm, and both profiles are reduced to very small peptides following trypsin digestion. Administration of concanamycin A or MTMAB causes a significant increase in the proportion of intact albumin (concanamycin A: 55.8 +/- 11.6%; MTMAB: 50.0 +/- 11.9%) excreted compared with normal IPK urine. This coincides with a reduction in the proportion of mildly (concanamycin A: 27.6 +/- 9.8%; MTMAB: 39.9 +/- 11.5%) and heavily degraded (concanamycin A: 16.6 +/- 7.4%; MTMAB: 10.0 +/- 2.5%) albumin present and is not associated with changes in glomerular permeability to albumin because no significant change is observed in the fractional clearance of Ficoll (radius range 20-60 A) in the presence of concanamycin A. This study demonstrates the existence of albumin peptides in IPK urine and suggests that receptor-mediated endocytosis plays a role in urinary albumin degradation.

The analysis and characterisation of immuno-unreactive urinary albumin in healthy volunteers.

OBJECTIVES: To compare the analysis of different forms of intact albumin in urine from healthy volunteers. To determine contamination by common non-albumin proteins on HPLC analysis of urinary albumin and of purified immuno-unreactive albumin. DESIGN AND METHODS: Overnight urine samples collected from healthy volunteers were analysed for total albumin (immunoreactive plus immuno-unreactive) by HPLC and densitometry following native PAGE separation and for immunoreactive albumin by RIA. The contamination by non-albumin proteins of the HPLC analysis of urinary albumin and of immuno-unreactive albumin preparations was determined by ELISA. Immuno-unreactive albumin was tested for Co2+-binding capacity. RESULTS AND CONCLUSIONS: HPLC analysis of healthy urine generates higher ACR values than immunological methods due to the presence of immuno-unreactive albumin. Immuno-unreactive albumin cannot be accounted for by the non-albumin urinary proteins tested. Isolated immuno-unreactive albumin is not recognised by antibodies to common urinary proteins or by an array of anti-albumin antibodies and behaves like serum albumin in terms of HPLC elution, native PAGE migration, and cobalt ion binding.

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.

Urinary-peptide excretion by patients with and volunteers without diabetes.

Large quantities of peptides (1-4 g) are excreted in human urine each day. In this study we sought to analyze how peptide excretion varies with increasing albuminuria associated with diabetes, as well as to characterize the size distribution of albumin-derived peptides in urine from volunteers without diabetes and from patients with macroalbuminuria and diabetes. We detected albumin-derived peptides by injecting tritiated albumin intravenously into human volunteers and patients with diabetes. Urine was collected after 24 hours and fractionated on a size-exclusion column. This fractionation revealed peptides with molecular weights ranging from 300 to 500 Da in volunteers without diabetes. The albumin-derived peptides were of higher molecular weight in the urine of a patient with macroalbuminuria and diabetes. The molecular-weight distribution of the peptides derived from tritiated albumin peptides was paralleled by the distribution of all protein peptides (including albumin) as determined with the use of the Biuret protein assay or absorbance at 214 nm. We determined peptide-excretion rates by filtering urine from patients with diabetes through a 10,000 Da molecular-weight-cutoff membrane and then measuring the filtrate with the use of the Biuret assay. This analysis revealed that the peptide-excretion rate increased with increasing total protein excretion, regardless of whether the patient demonstrated normoalbuminuria or microalbuminuria. Among patients with macroalbuminuria, the peptide-excretion rate leveled off and even decreased in the face of an increasing albumin concentration or protein-excretion rate. This study confirms that albumin-derived and protein-derived peptides exist at high concentrations in urine. Although peptide-excretion rates are maintained at similar levels up to macroalbuminuric states, the relative proportion of peptide excretion is significantly reduced compared with total protein.

Detection of urinary albumin.

Microalbuminuria is an important clinical marker in patients with diabetes and cardiovascular disease. The concentration of albumin in urine has traditionally been measured by semiquantitative dipsticks or by various quantitative immunochemical methods such as immunonephelometry, immunoturbidimetry, and radioimmunoassay. However, until recently, urinary albumin not reabsorbed by proximal tubular cells was assumed to be excreted intact. Studies have now revealed that the nature of urinary albumin is complex and is excreted as a mixture of intact albumin, albumin-derived peptides that are not detected by routine dipstick and antibody-based tests, and a species of intact albumin (immunounreactive albumin), also not detected by dipstick and antibody-based tests. A new test, Accumin, based on high-performance liquid chromatography analysis, is able to detect all the immunoreactive intact albumin and immunounreactive intact albumin (total intact albumin) in urine. The advantage in the use of Accumin over a conventional dipstick test or antibody-based laboratory method for detecting microalbuminuria is that false negatives are reduced and a relatively earlier diagnosis of incipient kidney disease can be achieved. The introduction of Accumin has, therefore, highlighted the need for a global standard in the detection and measurement of microalbuminuria. By detecting all of the immunoreactive and immunounreactive intact albumin in urine, Accumin has virtually invalidated the use of dye and immunologically-based dipstick tests and immunologically-based laboratory methods in screening for microalbuminuria in diabetic patients and in identifying microalbuminuria as a risk factor for cardiovascular disease.

Albumin-like material in urine.

Recent studies have demonstrated the presence, in both rat and human urine, of a modified form of albumin not detected by conventional antibodies. This modified albumin behaves physicochemically as intact albumin under nondenaturing conditions. We have demonstrated this form of albumin to be disproportionately excreted in microalbuminuric states in diabetes. Quantitation of this modified form of albumin leads to the prediction of the onset of microalbuminuria in diabetic patients on average 3 to 4 years earlier than when measured by conventional immunoassays.

Mechanism of albuminuria associated with cardiovascular disease and kidney disease.

The major underlying factors associated with tissue damage and fibrosis in cardiovascular and kidney disease are the up-regulation and action of growth factors such as transforming growth factor-beta (TGF-beta) and cytokines produced in response to changes in systemic factors, particularly blood pressure or hyperglycemia. This study identifies the relationship of elevated levels of TGF-beta to increased levels of intact albumin in the urine (micro- and macroalbuminuria). This mechanism may be directly linked to the effect of TGF-beta on albumin uptake and the lysosomal breakdown of filtered albumin by proximal tubular cells prior to excretion.

Characterization of immunochemically nonreactive urinary albumin.

BACKGROUND: Conventional immunoassays underestimate the urinary albumin concentration because intact albumin in urine exists in two forms, immunoreactive and immunochemically nonreactive. METHODS: Urinary albumin concentration measured by HPLC (which measures total albumin, i.e., the sum of immunoreactive albumin + immunochemically nonreactive albumin) or RIA was compared with densitometric analysis of albumin bands in diabetic urine samples separated by either native polyacrylamide gel electrophoresis (PAGE) or reducing sodium dodecyl sulfate (SDS)-PAGE. Immunochemically nonreactive albumin was also isolated from diabetic urine (relative amount detected, 70-80% of the expected) and was tested for contamination by common urinary proteins by native PAGE, ELISA, and capillary electrophoresis. RESULTS: Urinary albumin concentrations measured by native PAGE and HPLC were better correlated (r(2) = 0.83) than concentrations measured by native PAGE and RIA (r(2) = 0.62) because under native conditions both native PAGE and HPLC detect total albumin and not only the immunoreactive albumin alone that is measured by RIA. Urinary albumin concentrations measured by reducing SDS-PAGE and RIA were better correlated (r(2) = 0.84) than concentrations measured by reducing SDS-PAGE and HPLC (r(2) = 0.65) because under reducing conditions immunochemically nonreactive albumin is unstable and fragments into many smaller peptides. The partially purified preparation was found to contain <1% contamination by common urinary proteins and is stable to freezing and frequent freeze/thaw cycles. CONCLUSIONS: The results are consistent with the interpretation that immunochemically nonreactive albumin has a limited number of polypeptide chain scissions and is held together by noncovalent intrachain bonding and disulfide bonds. Detection of this molecule is likely to be of clinical importance in diagnosing kidney disease as well as cardiovascular disease.

Earlier detection of microalbuminuria in diabetic patients using a new urinary albumin assay.

BACKGROUND: Microalbuminuria is regarded as the most important predictor of high risk for the development of diabetic nephropathy. Early detection may allow treatment to prevent progression to persistent albuminuria and renal failure. Recent studies have shown that conventional immunoassays underestimate urinary albumin concentration, as albumin in urine may exist in two forms, immuno-reactive and immuno-unreactive. The present study examines the differential lead-time for the development of microalbuminuria as measured by both conventional radioimmunoassay (RIA; measures immuno-reactive) and high-performance liquid chromatography (HPLC; measures total albumin = immuno-reactive plus immuno-unreactive) analysis in both type 1 and type 2 diabetic patients. METHODS: Analysis was performed on 511 stored urine samples collected over the last 13 years from type 1 diabetic patients who either progressed from normo- to microalbuminuria (progressors, N= 17), or who remained normoalbuminuric (nonprogressors, N= 25) as defined by RIA, and on 634 urine samples collected from patients with type 2 diabetes defined as either progressors (N= 24) or nonprogressors (N= 25). RESULTS: For type 1 progressors, the mean lead-time for the HPLC assay versus the RIA was 3.9 years, with a 95% CI of 2.1 to 5.6 years. For type 2 progressors, the mean lead-time was 2.4 years with a 95% CI of 1.2 to 3.5 years. There was no significant difference between the lead-time analysis between type 1 and type 2 diabetic patients. CONCLUSION: These results demonstrate that measurement of total albumin may allow earlier detection of microalbuminuria associated with diabetic nephropathy.

Renal processing of serum proteins in an albumin-deficient environment: an in vivo study of glomerulonephritis in the Nagase analbuminaemic rat.

BACKGROUND: Plasma albumin has been considered as important for governing glomerular permselectivity as well as being tubulotoxic in proteinuric states. The purpose of this study was to examine glomerular permselectivity and protein clearance of plasma albumin-deficient Nagase analbuminaemic rats (NAR) in normal and proteinuric states associated with puromycin aminonucleoside nephrosis (PAN) and anti-glomerular basement membrane glomerulonephritis (anti-GBM GN) and to compare the results with those of previous studies using Sprague-Dawley rats. METHODS: Glomerular permselectivity was measured using tritium-labelled polydisperse Ficoll. In vivo fractional clearance (FC) of albumin, transferrin and immunoglobulin G was measured to include both intact and degraded forms of filtered material. Endogenous protein clearance was analysed using two-dimensional electrophoresis in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. RESULTS: FCs of proteins and Ficoll in control NAR were similar to those found in Sprague-Dawley rats. Despite the lack of serum albumin in NAR, proteinuria and morphological changes observed were also similar to those found in Sprague-Dawley rats, with total protein excretion increasing 6-fold in PAN rats and 4-fold in anti-GBM GN rats with respect to controls. Two-dimensional electrophoresis in combination with MALDI mass spectrometry identified the major proteins being excreted as transferrin and a group of mildly acidic proteins in the MW range 40-50 kDa, namely antithrombin III, kininogen, alpha-1-antiproteinase, haemopexin and vitamin D-binding protein. CONCLUSIONS: Both diseases exhibited similar effects to those observed in Sprague-Dawley rats despite the lack of serum albumin, including inhibition of renal protein degradation. The net changes in protein FC, particularly in the range of radii of 36-55 A, could not be accounted for by changes in size selectivity as Ficoll FC was little affected by the disease states. This emphasizes the need to reassess the relative importance of changes in renal tubular handling vs changes in glomerular capillary barrier in proteinuric states. These studies also demonstrate that albumin is not a critical factor in governing glomerular permselectivity or proteinuria.

Differences in urinary albumin detected by four immunoassays and high-performance liquid chromatography.

OBJECTIVES: To compare the analysis of urinary albumin from diabetic patients by four conventional immunoassays including radioimmunoassay (RIA), immunonephelometry (IN), and two different methods of immunoturbidimetry (IT), as well as by high-performance liquid chromatography (HPLC). DESIGN AND METHODS: Urines were collected over a 24-h period and stored at -20 degrees C until assay. Urinary albumin concentration was determined by an in-house RIA, by IN using a Beckman Array Analyser with reagents from Beckman Diagnostics (Sydney, Australia), by IT using a Dade-Behring Turbitimer with reagents from Dade-Behring (Marburg, Germany), by IT using a Dade-Behring Dimension R x L Chemistry Analyser with reagents from DiaSorin (Stillwater, OK, USA), and by HPLC using a Zorbax Bio series preparative GF-250 column. Regression lines were calculated using a least squares method to determine the correlation between the assays studied. Bland-Altman bias plots including limits of agreement were also calculated. RESULTS: The correlation coefficients calculated were high (>0.85) indicating a strong linear relationship between all assays studied. The slopes calculated for the comparisons demonstrate that each assay can vary from one another (up to threefold) and have a slope significantly different from an ideal slope of 1 (P < 0.001). These results were confirmed by Bland-Altman bias plots and calculation of the limits of agreement that were all large. CONCLUSIONS: At this time, there is no global standard by which urinary albumin assays may be standardized. This study suggests the need for such standards.

Additive effects of hypertension and diabetes on renal cortical expression of PKC-alpha and -epsilon and alpha-tubulin but not PKC-beta 1 and -beta 2.

OBJECTIVE: This study examined the separate and combined effects of hypertension and diabetes on renal cortical expression of protein kinase C (PKC) isoforms -beta 1, -beta 2, -alpha and -epsilon, to determine whether albuminuria is the result of an increase in the expression of one or a combination of PKC isoforms. Corresponding changes in renal microtubules were also assessed. METHODS: Diabetes (D) was induced in Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) by streptozotocin. After 24 weeks, PKC expression was determined by Western blot and microtubules were assessed by immunohistochemistry for alpha-tubulin protein. RESULTS: Diabetes was characterized by significant increases in glycated haemoglobin (HbA1c) as compared to controls (C). There was a significant increase of three- to four-fold in PKC protein content for all four isoforms in renal cortex from SHR-C and WKY-D, and similar and significant levels of albuminuria (approximately 10 mg/24 h) observed in these groups in comparison to WKY-C (approximately 1 mg/24 h). Interestingly, PKC-alpha and -epsilon but not PKC-beta 1 and -beta 2 protein content was doubled in SHR-D, and albuminuria increased tenfold (approximately 100 mg/24 h) in comparison to SHR-C and WKY-D. These changes were paralleled by a significant decrease in alpha-tubulin protein content of approximately 50% in SHR-C and approximately 33% in WKY-D compared to WKY-C, with a further decrease of approximately 67% in SHR-D compared to WKY-C. CONCLUSION: These findings indicate that PKC expression can be increased by either diabetes or hypertension, and that there are further specific increases in the expression of PKC isoforms -alpha and -epsilon in the model of combined diabetes and hypertension. In addition, the degree of disruption in microtubular cytoskeleton appears to be correlated with PKC activation and levels of albuminuria.

Reduced tubular cation transport in diabetes: prevented by ACE inhibition.

BACKGROUND: The renal clearance of organic cations is important for the homeostasis of a number of exogenous and endogenous compounds. The organic cation transporters (OCTs) situated on the basolateral surface of proximal tubular cells mediate active cation excretion. Alterations of cation transport may occur in diabetes, although the role of the OCTs has not been previously assessed. METHODS: Experimental diabetes was induced in rats with streptozotocin (55 mg/kg) and animals were randomly assigned to receive ramipril (3 mg/mL) in drinking water for 24 weeks. In a second protocol, rats were infused with angiotensin II (Ang II) at a dose of 58.3 ng/kg/min for 2 weeks via an implanted osmotic pump. Expression of the OCTs and renal clearance of the endogenous cation N-methyl-nicotinamide (NMN) was assessed. RESULTS: Diabetes was associated with a reduction in gene and protein expression of both OCT-1 and OCT-2 and a reduction in NMN clearance. These effects were prevented by ramipril, associated with the prevention of albuminuria and tubular injury as demonstrated by the expression of osteopontin and glutathione peroxidase 3 (GPX-3). An infusion of Ang II also reduced NMN clearance but without altering the renal expression of OCTs. CONCLUSION: We hypothesize that reduced expression of OCTs in diabetes may be a marker of tubular injury. However, Ang II may also directly augment renal cation clearance independent of changes in transporter expression. Together these effects may provide additional mechanism to explain treatment-related improvements in creatinine clearance and renoprotection in diabetes following blockade of the renin-angiotensin system (RAS).

Reversible angiotensin II-mediated albuminuria in rat kidneys is dynamically associated with cytoskeletal organization.

Angiotensin II (ANG II) is intimately involved in normal renal function, and is estimated to exist at a normal physiological range of 6-10 nM within the renal tubules. The potential role that intrarenal ANG II may play in renal disease was assessed by perfusing isolated rat kidneys with or without excess intratubular levels of ANG II, which may mimic changes in the intrarenal RAS under pathological conditions. The effects of increased systemic ANG II were also determined by infusing rats with ANG II by osmotic pump. In isolated perfused kidneys, ANG II significantly and specifically increased the fractional clearance of albumin to clinical levels, as determined by using radiolabelled albumin. This effect was reversible, as removing ANG II from the perfusate caused the albumin fractional clearance to decrease to pre-ANG II exposure levels. The increase in fractional clearance of albumin was not correlated with renal hemodynamic changes, nor glomerular permeability alterations as measured by the fractional clearance of 36 A Ficoll and immunoglobulin G. Immunochemical analysis using anti-alpha-tubulin antibody of perfused kidney sections revealed that ANG II caused a marked disruption of tubular epithelial cytoskeletal components, through disassembly and reorganization of alpha-tubulin. This disruption was reversible. In vivo, osmotic pump delivery of ANG II at less potent dosage caused a proteinuria (Biuret) and an albuminuria (radioimmunoassay) in rats, from as early as 2 days after pump implantation. These results demonstrate that ANG II may reversibly induce clinical levels of albuminuria. These data point to an important role for renal tubules and the intratubular lumen concentrations of ANG II in the renal processing of albumin.

The effect of ramipril on albumin excretion in diabetes and hypertension: the role of increased lysosomal activity and decreased transforming growth factor-beta expression.

OBJECTIVES: Albumin excretion is modulated post-filtration by lysosomal processing that produces a spectrum of albumin-derived material in urine, much of which is not detected by conventional immunoassays. This study aimed to determine the efficacy of ramipril treatment (+ RAM) after 24 weeks on total albumin excretion (intact plus albumin-derived peptides) in spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats with (d) and without (c) diabetes. METHODS: Intact albumin excretion was analysed by radioimmunoassay and total albumin excretion was analysed by measuring radioactivity derived from circulating [ C]albumin. Renal lysosomal activity was determined by urinary [ H]dextran sulphate desulphation. Renal transforming growth factor-beta 1 (TGF-beta 1), TGF-beta inducible gene-h3 (beta ig-h3) and angiotensinogen mRNA production were analysed by real time reverse transcriptase-polymerase chain reaction. RESULTS: Hypertension (SHR-c and SHR-d) resulted in a significant increase in intact albumin excretion, which was significantly reduced by ramipril treatment (P < 0.05 for SHR-c + RAM and 0.001 for SHR-d + RAM compared to non-treated). This was accompanied by a significant decrease in blood pressure (P < 0.001 for SHR-c + RAM and SHR-d + RAM), renal beta ig-h3 mRNA production (P < 0.05 for SHR-c + RAM and SHR-d + RAM), and an increase in lysosomal activity. Diabetes (WKY-d and SHR-d) primarily caused a significant increase in total albumin excretion, predominantly in the form of albumin-derived fragments in the WKY-d group and intact albumin in the SHR-d group. Ramipril treatment reduced total albumin excretion in the WKY-d + RAM group (P < 0.001). CONCLUSIONS: Ramipril prevents increases in both intact albumin and total albumin excretion in hypertensive and diabetic states, respectively.

High prevalence of immuno-unreactive intact albumin in urine of diabetic patients.

BACKGROUND: Intact albumin in urine may exist in two forms, immunoreactive and immuno-unreactive. Previous estimates of albuminuria in diabetic urine have only detected immunoreactive forms. METHODS: High performance liquid chromatography (HPLC) was used in this study to measure both forms of intact albumin (termed total intact albumin) to provide a more accurate measurement of albuminuria compared with radioimmunoassay (RIA) on 97 fresh urine samples from patients with diabetes. Eighty-six control urine samples from volunteers without diabetes were also tested. RESULTS: There was no significant difference between the two methods for nondiabetic controls. For diabetic urine samples, 91.6% of samples showed a greater concentration of albumin measured by HPLC than RIA. For normoalbuminuric diabetic samples, HPLC gave a mean albumin excretion rate of 12.5 +/- 4.4 microgram/min (SD), whereas RIA gave a rate of 8.0 +/- 6.7 microgram/min (P = 0.004; N = 28). For microalbuminuric samples, there also was a statistically significant difference: HPLC albumin excretion rate, 82.0 +/- 49.9 microgram/min, and RIA, 49.0 +/- 34.6 microgram/min (P = 0.004; N = 30). Thirty-two urine samples were normoalbuminuric by RIA (albumin, 11.4 +/- 3.9 microgram/min), but in the microalbuminuric range as determined by HPLC (albumin, 38.5 +/- 14.4 microgram/min). For urine samples in the macroalbuminuric range, there was no statistically significant difference between HPLC and RIA. Immuno-unreactive albumin was confirmed as albumin, analyzed by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry. CONCLUSION: These studies show that to determine microalbuminuria accurately, there is a need to assess urinary total intact albumin, rather than simply immunoreactive albumin. Am J Kidney Dis 41:336-342.

Ramipril prevents microtubular changes in proximal tubules from streptozotocin diabetic rats.

This study has investigated the microtubular cytoskeleton in rat glomerular and proximal tubule cells in experimental diabetes. The effect of treatment with ramipril on the relationship between microtubule organization and albuminuria in diabetes has also been examined. Diabetes was induced in male Sprague-Dawley rats by administration of streptozotocin (50 mg/kg, i.v.). Rats were treated with or without ramipril in their drinking water for 12 weeks. Diabetes was characterized by an increase in blood glucose level, glomerular filtration rate, and albumin excretion rate. Treatment of diabetic rats with ramipril did not affect glycaemic control, but reduced systolic blood pressure and prevented the rise in albuminuria and glomerular filtration rate. Immunohistochemistry was performed by using the ARK Peroxidase method with alpha-tubulin antibody. The regular, grainy staining pattern of the microtubules present in the renal proximal tubules from control kidneys was altered in diabetic animals, and appeared fragmented and striated. This was prevented by treatment with ramipril. Quantitative morphometric analysis revealed an increase in the percent proportional staining for alpha-tubulin in the proximal tubules of untreated diabetic rats (33.3 +/- 3.3%, n = 8, P < 0.05 vs control) compared with control rats (11.7 +/- 1.7%, n = 6), which was reduced by ramipril treatment (26.7 +/- 2.1%, n = 6, P < 0.05 vs untreated diabetic). Staining for alpha-tubulin in glomerular cells was unchanged in all groups. There was no significant difference in renal alpha-tubulin expression among all groups, as determined by real-time reverse transcription-polymerase chain reaction. These results raise the possibility that diabetes-induced changes in microtubules in the renal proximal tubules may contribute, in part, to the increase in albuminuria observed in diabetes.