Urinary GSTP1-1 excretion is markedly increased in normotensive pregnancy as well as in preeclampsia

BACKGROUND: Glutathione S-transferases (GSTs) are present in large amounts in the human kidney, where they demonstrate a specific distribution. The assessment of urinary excretion of GST alpha (proximal tubules) and pi (distal and collecting tubules) could be helpful in determining if, and to what degree renal tubular damage is present in preeclampsia and whether this damage is in the proximal or distal region. METHODS: Urine samples were collected from 22 women with severe preeclampsia and/or HELLP syndrome (PE), from 30 non-pregnant women with a history of severe preeclampsia (HPE), from 18 women with uncomplicated pregnancies (PC) and from 30 non-pregnant women with a history of uncomplicated pregnancies (HPC). GSTA1-1 and GSTP1-1 were assayed by ELISA and were expressed as nanograms per 10 mmol creatinine (Cr). RESULTS: Median urinary GSTP1-1 concentrations were significantly (p<0.001) higher in women with preeclampsia [62.2 (4.3-291.2) ng/10 mmol Cr] compared to non-pregnant women with a history of preeclampsia [22.3 (0-142.6) ng/10 mmol Cr]). In addition, in normotensive pregnant women, urinary GSTP1-1 concentrations were significantly (p<0.01) higher [82.6 (8.3-206.7) ng/10 mmol Cr]) compared to non-pregnant controls [5.1 (0-66.7) ng/10 mmol Cr]. No difference in GSTP1-1 concentrations was found between women with preeclampsia and normotensive pregnant women. GSTA1-1 concentrations were not significantly different between the four groups of women investigated. There were no correlations between the degree of proteinuria and urinary GSTP1-1 or GSTA1-1 concentrations. CONCLUSION: GSTP1-1 metabolism in the distal tubule changes during normotensive as well as preeclamptic pregnancy. Whether this is due to tubular cell damage, disturbed resorption or an increase in cellular levels cannot be determined as yet.     

Assessment of Low-flow Sevoflurane and Isoflurane Effects on Renal Function Using Sensitive Markers of Tubular Toxicity

Background: Carbon dioxide absorbents degrade sevoflurane, particularly at low gas flow rates, to fluoromethyl-2,2-difluoro-1-(trifluoromethyl)vinyl ether (compound A). Compound A causes renal proximal tubular injury in rats but has had no effect on blood urea nitrogen (BUN) or creatinine concentrations in patients. This investigation compared the effects of low-flow sevoflurane and isoflurane on renal tubular function in surgical patients using conventional (BUN and creatinine) and finer indices of renal injury, specifically those biomarkers sensitive for compound A toxicity in rats (glucosuria, proteinuria, and enzymuria [N-acetyl-beta-D-glucosaminidase (NAG) and alpha-glutathione-S-transferase (alpha GST)]).

Methods: Consenting patients with normal preoperative renal function at two institutions were randomized to receive sevoflurane (n = 36) or isoflurane (n = 37) in oxygen and air. Total gas flow was 1 l/min, opioid doses were minimized, and barium hydroxide lime was used to maximize anesthetic degradation. Inspiratory and expiratory compound A concentrations were quantified every 30-60 min. Blood and urine were obtained before and 24-72 h after anesthesia for laboratory evaluation.

Results: Sevoflurane and isoflurane groups were similar with respect to age, weight, sex, American Society of Anesthesiologists status, anesthetic duration (3.7 or 3.9 h), and anesthetic exposure (3.6 or 3 minimum alveolar concentration [MAC]-hour). Maximum inspired compound A concentration (mean +/- standard deviation) was 27 +/- 13 ppm (range, 10-67 ppm). Areas under the inspired and expired compound A concentration versus time curves (AUC) were 79 +/- 54-ppm-h (range, 10-223 ppm-h) and 53 +/- 40 ppm-h (range, 6-159 ppm-h), respectively. There was no significant difference between anesthetic groups in postoperative serum creatinine or BUN, or urinary excretion of protein, glucose, NAG, proximal tubular alpha GST, or distal tubular pi GST. There was no significant correlation between compound A exposure (AUC) and protein, glucose, NAG, alpha GST, or pi GST excretion. Postoperative alanine and aspartate aminotransferase concentrations were not different between the anesthetic groups, and there were no significant correlations between compound A exposure and alanine or aspartate aminotransferase concentrations.     

Heterogeneity of glutathione S-transferase isoenzyme expression in renal disease.

Recent work has suggested that glutathione S-transferase (GST) enzymuria may be used to assess renal injury in transplant kidneys. There is little work investigating the possibility of using glutathione S-transferase enzymuria to assess other renal diseases and this study was undertaken to evaluate the localisation of GST isoenzymes in various glomerular and tubular pathologies so that the specificity of these enzymes as markers of tubular injury could be defined. Immunostaining was carried out to establish the location of alpha and pi class GST in renal biopsies from patients with a wide variety of renal diseases including membranous glomerulonephritis, minimal-lesion glomerulonephritis, mesangial proliferative glomerulonephritis, loin pain haematuria syndrome, and renal allograft rejection. In the cases of glomerulonephritis studied, alpha class GST was detected in proximal tubules and pi class GST in distal convoluted tubules, podocytes, and Bowman's capsule. The majority of cases of glomerulonephritis showed a heterogeneous pattern of expression of both isoenzymes: that is, there was variation in intensity of staining both in single tubules and also between tubules as opposed to the uniform staining pattern observed in normal kidneys. The location of enzymes in the cases of glomerulonephritis was the same as that in normal kidneys and we were unable to demonstrate any de novo expression of GST isoenzymes. However, seven out of ten renal allograft biopsies showed expression of pi class GST in proximal tubules which were atrophic. Provided there is no significant tubular atrophy, urinary release of these enzymes may be used to localise renal tubular injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Renal Cysteine Conjugate beta-Lyase in the Mechanism of Compound A Nephrotoxicity in Rats

Background: The sevoflurane degradation product compound A is nephrotoxic in rats, in which it undergoes extensive metabolism to glutathione and cysteine S-conjugates. The mechanism of compound A nephrotoxicity in rats is unknown. Compound A nephrotoxicity has not been observed in humans. The authors tested the hypothesis that renal uptake of compound A S-conjugates and metabolism by renal cysteine conjugate beta-lyase mediate compound A nephrotoxicity in rats.

Methods: Compound A (0-0.3 mmol/kg in initial dose-response experiments and 0.2 mmol/kg in subsequent inhibitor experiments) was administered to Fischer 344 rats by intraperitoneal injection. Inhibitor experiments consisted of three groups: inhibitor (control), compound A, or inhibitor plus compound A. The inhibitors were probenecid (0.5 mmol/kg, repeated 10 h later), an inhibitor of renal organic anion transport and S-conjugate uptake; acivicin (10 mg/kg and 5 mg/kg 10 h later), an inhibitor of gamma-glutamyl transferase, an enzyme that cleaves glutathione conjugates to cysteine conjugates; and aminooxyacetic acid (0.5 mmol/kg and 0.25 mmol/kg 10 h later), an inhibitor of renal cysteine conjugate beta-lyase. Urine was collected for 24 h and then the animals were killed. Nephrotoxicity was assessed by light microscopic examination and biochemical markers (serum urea nitrogen and creatinine concentration, urine volume and urine excretion of protein, glucose, and alpha-glutathione-S-transferase [alpha GST], a marker of tubular necrosis).

Results: Compound A caused dose-related nephrotoxicity, as shown by selective proximal tubular cell necrosis at the corticomedullary junction, diuresis, proteinuria, glucosuria, and increased alpha GST excretion. Probenecid pretreatment significantly (P < 0.05) diminished compound A-induced increases (mean +/- SE) in urine excretion of protein (45.5 +/- 3.8 mg/24 h vs. 25.9 +/- 1.7 mg/24 h), glucose (28.8 +/- 6.2 mg/24 h vs. 10.9 +/- 3.2 mg/24 h), and alpha GST (6.3 +/- 0.8 micro gram/24 h vs. 1.0 +/- 0.2 micro gram/24 h) and completely prevented proximal tubular cell necrosis. Aminooxyacetic acid pretreatment significantly diminished compound A-induced increases in urine volume (19.7 +/- 3.5 ml/24 h vs. 9.8 +/- 0.8 ml/24 h), protein excretion (37.2 +/- 2.7 mg/24 h vs. 22.2 +/- 1.8 mg/24 h), and alpha GST excretion (5.8 +/- 1.5 vs. 2.3 micro gram/24 h +/- 0.8 micro gram/24 h) but did not significantly alter the histologic pattern of injury. In contrast, acivicin pretreatment increased the compound A-induced histologic and biochemical markers of injury. Compound A-related increases in urine fluoride excretion, reflecting compound A metabolism, were not substantially altered by any of the inhibitor treatments.     

Increased urinary excretion of alpha1-microglobulin at 6 months after transplantation is associated with urinary excretion of transforming growth factor-beta1 and indicates poor long-term renal outcome

BACKGROUND: Albumin and alpha1-microglobulin (alpha1M) are absorbed by two specific receptors in tubular epithelial cells. Any cell injury will disturb the reabsorption of these proteins, The increased urinary excretions of albumin or alpha1M could thus serve as a marker of subclinical graft lesions and as an early indicator of chronic allograft dysfunction. METHODS: We measured 24-hour urinary excretions of albumin, alpha1M, and transforming growth factor (TGF)-beta1 at 6 months after transplantation in 79 renal-graft recipients and recorded the changes in 24-hour creatinine clearance an average 51 (range 14-72) posttransplant follow-up months. RESULTS: At 6 months from transplantation, 46 of 79 (58%) patients were normoalbuminuric, 25 (32%) microalbuminuric, and 8 (10%) macroalbuminuric. In normoalbuminuric patients, urinary alpha1M/creatinine ratio was 10 times, and TGF-beta1/creatinine ratio approximately 5 times, higher than in the healthy subjects but lower than in albuminuric patients. In all patients, urinary alpha1M correlated with urinary TGF-beta1 (r=0.508, P<0.001), with albumin (r=0.220, P<0.05), and with the annual changes in 24-hour creatinine clearance (r=-0.273, P<0.05). During follow-up, renal function deteriorated in 20 of 33 (60%) patients with alpha1M/creatinine ratio greater than 5 mg/mmol, but only in 1 of 46 (2%) patients whose ratio was less than 5 mg/mmol (P<0.01), giving the ratio 5 mg/mmol or greater a 95% sensitivity to detect patients with poor long-term outcome. CONCLUSIONS: We show proximal tubular injury, measured by increased urinary alpha1M, to be present even in normoalbuminuric patients and to be associated with increased excretion of TGF-beta1 and with the annual deterioration of glomerular filtration rate. These findings show increased alpha1M/creatinine ratio to be an early and sensitive indicator of poor long-term outcome in renal-transplant patients.     

Are urinary enzymes useful markers of kidney damage in obstructive jaundice? An experimental study on Sprague-Dawley rats

To evaluate the reliability of urinary enzymes as markers of renal tubular damage in obstructive jaundice, research was carried out on 26 Sprague-Dawley rats submitted to bile duct ligation and on 16 sham-operated rats. The fractional clearances of lysozyme (CfrLYS) and of malto-dehydrogenase (CfrMDH)-indices of tubular function-and the fractional excretions of gamma-glutamyltransferase (UfrGGT) and of alpha-glucosidase (UfrAGL)-indices of tubular anatomic damage - were measured 5, 10, 20 and 30 days after operation. Creatinine clearance, urinary sodium excretion, urinary potassium excretion, proteinuria, plasma bilirubin and bile acids were also measured. Kidneys were taken for histology. All rats submitted to common bile duct ligation had high levels of bilirubin and bile acids; proximal tubules were damaged and the extent of the lesions increased with time. However, creatinine clearance, urinary sodium excretion, proteinuria, CfrMDH and UfrAGL gave no indication of renal lesions, whereas CfrLYS and UfrGGT were significantly higher 20 and 30 days after bile duct ligation, respectively. These findings show that CfrLYS and UfrGGT could be useful tests for renal tubular lesions in jaundice.     

Urinary thymidine glycol as a biomarker for oxidative stress after kidney transplantation

Reactive oxygen species are generated during ischemia-reperfusion tissue injury. Oxidation of thymidine by hydroxyl radicals (HO*) causes formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol excreted in urine can be used as a biomarker of oxidative DNA damage. The aim of this study was to investigate the oxidative DNA damage in patients showing immediate allograft function after kidney transplantation, and to find out whether this damage correlates with glomerular and tubular lesions. Time dependent changes in urinary excretion rates of thymidine glycol, but also of total protein, albumin, low molecular weight (alpha1-microglobulin, beta2-microglobulin) and high molecular weight proteins (transferrin, IgG, alpha2-macroglobulin) were analyzed quantitatively and by polyacrylamide-gel electrophoresis in six patients. Urinary thymidine glycol was determined by a fluorimetric assay in combination with affinity chromatography and HPLC. After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum within the first 48 hours (16.56+/-11.3 nmol/m mol creatinine, ref. 4.3+/-0.97). Severe proteinuria with an excretion rate of up to 7.2 g/mmol creatinine was observed and declined within the first 24 hours of allograft function (0.35+/-0.26 g/mmol creatinine). The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The initial nonselective glomerular proteinuria disappeared within 48 hours, changing to a mild selective glomerular proteinuria. In this period (12-48 hours) higher levels of thymidine glycol excretion were observed, when compared to the initial posttransplant phase (13.66+/-9.76 vs. 4.31+/-3.61 nmol/mmol creatinine; p<0.05). An increased excretion of thymidine glycol is seen after kidney transplantation and is explained by the ischemia-reperfusion induced oxidative DNA damage in the kidney. In the second phase higher levels of excretion were observed parallel to the change from a nonselective to a selective glomerular and tubular proteinuria. An explanation may be sought in the repair process of DNA in the glomerular and tubular epithelial cells, appearing simultaneously with functional recovery.     

Tubular proteinuria defined by a study of Dent's (CLCN5 mutation) and other tubular diseases

Tubular proteinuria defined by a study of Dent's ( CLCN5 mutation) and other tubular diseases. BACKGROUND: The term "tubular proteinuria" is often used interchangeably with "low molecular weight proteinuria" (LMWP), although the former implies a definite etiology. A specific quantitative definition of tubular proteinuria is needed, and we address this by studying five different renal disorders. METHODS: Tubular proteinuria was assessed by measuring urinary retinol-binding protein (RBP), beta2-microglobulin (beta2M), alpha1-microglobulin (alpha1M), and albumin in 138 patients: 26 affected males and 24 female carriers of the X-linked syndrome "Dent's disease," 6 patients with other Fanconi syndromes, 17 with distal renal tubular acidosis (dRTA), 39 with glomerulonephritis (GN), and 26 with Chinese herbs nephropathy (CHN). RESULTS: RBP was better than beta2M or alpha1M in identifying the tubular proteinuria of Dent's disease. Median urinary RBP levels in mg/mmol creatinine were: affected male Dent's, 18.2, N = 26; carrier female Dent's, 0. 30, N = 24; dRTA, 0.027, N = 17; GN, 0.077, N = 39; and normal adults, 0.0079, N = 61. Elevated urinary RBP (>0.017) and albumin < (10 x RBP) + 2 identified all patients with the LMWP of Dent's disease and clearly distinguished their LMWP from that of dRTA and GN. This is a quantitative definition of tubular proteinuria. Consistent with this definition, 80% of those patients with CHN who had an elevated RBP had tubular proteinuria. Urinary RBP and albumin in carriers of Dent's disease were strikingly correlated over a 100-fold range (R = 0.933). CONCLUSION: The combination of elevated urinary RBP (>0.017) and albumin < (10 x RBP) + 2 (mg protein/mmol creatinine) is a quantitative definition of tubular proteinuria. Furthermore, our findings suggest that a shared defect in tubular RBP and albumin reuptake causes this form of proteinuria.     

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

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

Urinary N-acetyl-beta-glucosaminidase excretion is a marker of tubular cell dysfunction and a predictor of outcome in primary glomerulonephritis.

BACKGROUND: The urinary excretion of N-acetyl-beta-glucosamynidase (NAG) is increased in subjects exposed to substances toxic for renal tubular cells. In experimental and human glomerular diseases, its increased excretion is probably due to the dysfunction of tubular epithelial cells induced by increased traffic of proteins in the tubular lumen. The first aim of this study was to evaluate whether NAG excretion is correlated not only with the amount of proteinuria but also with some proteinuric components which reflect both glomerular capillary wall damage (IgG) and an impairment of tubular reabsorption of microproteins (alpha(1) microglobulin). The second aim was to assess whether NAG excretion has a predictive value on functional outcome and response to therapy. METHODS: In 136 patients with primary glomerulonephritis [74 with idiopathic membranous nephropathy (IMN), 44 with primary focal segmental glomerulosclerosis (FSGS) and 18 with minimal change disease (MCD)] urinary NAG excretion was measured by a colorimetric method and expressed in units per gram of urinary creatinine. RESULTS: Using univariate linear regression analysis NAG excretion in all 136 patients was significantly dependent on IgG excretion, 24-h proteinuria, fractional excretion of alpha(1) microglobulin (FE alpha(1)m) and diagnosis. Using multiple linear regression analysis, NAG excretion was significantly dependent only on IgG excretion and 24-h proteinuria. Limiting the analysis to 67 patients with nephrotic syndrome (NS) and baseline normal renal function, by multiple linear regression, NAG excretion was significantly dependent on IgG excretion (P=0.0004), 24-h proteinuria (P=0.0067) and FE alpha(1)-m (P=0.0032) (R(2)=0.63). In 66 patients with NS and normal baseline renal function (MCD 10 patients; FSGS 20 patients; IMN 36 patients), according to values below or above defined cut-offs (IMN, 18 U/g urinary Cr; FSGS and MCD, 24 U/g urinary Cr), NAG excretion predicted remission in 86 vs 27% of IMN patients (P=0.0002) and 77 vs 14% of FSGS patients (P=0.005). Progression to chronic renal failure (CRF) was 0 vs 47% in IMN patients (P=0.001) and 8 vs 57% in FSGS patients (P=0.03). Using Cox model, in IMN patients only NAG excretion (P=0.01, RR 5.8), but not 24-h proteinuria, predicted progression to CRF. All MCD patients had NAG excretion values below the chosen cut-off, and 90% of them developed remission. Response to immunosuppressive therapy was significantly different in patients with NAG excretion values below or above the cut-offs. CONCLUSION: Urinary NAG excretion can be considered as a reliable marker of the tubulo-toxicity of proteinuria in the early stage of IMN, FSGS and MCD; the excretion values show a significant relationship with 24-h proteinuria, IgG excretion and FE alpha(1)m. Its determination may be a non-invasive, useful test for the early identification of patients who will subsequently develop CRF or clinical remission and responsiveness to therapy.     

Increased urinary excretion of monocyte chemoattractant protein-1 in proteinuric renal diseases

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is produced mainly by tubular epithelial cells in kidney and contributes to renal interstitial inflammation and fibrosis. More recently, we have demonstrated that urinary MCP-1 excretion is increased in proportion to the degree of albuminuria (proteinuria) and positively correlated with urinary N-acetylglucosaminidase (NAG) levels in type 2 diabetic patients. Based on these findings, we have suggested that heavy proteinuria, itself, probably aggravates renal tubular damage and accelerates the disease progression in diabetic nephropathy by increasing the MCP-1 expression in renal tubuli. In the present study, to evaluate whether urinary MCP-1 excretion is increased in the proteinuric states not only in diabetic nephropathy but also in other renal diseases, we examined urinary MCP-1 levels in IgA nephropathy patients with macroalbuminuria (IgAN group; n = 6), and compared the results with the data obtained from type 2 diabetic patients with overt diabetic nephropathy (DN group; n = 23) and those without diabetic nephropathy (non-DN group; n = 27). Urinary MCP-1 excretion levels in non-DN, DN, IgAN groups were 157.2 (52.8-378.5), 346.1 (147.0-1276.7), and 274.4 (162.2-994.5) ng/g creatinine, median (range), respectively. Expectedly, urinary MCP-1 and NAG excretion levels in DN and IgAN groups were significantly elevated as compared with non-DN group. Therefore, we suggest that MCP-1 expression in renal tubuli is enhanced in proteinuric states,irrespective of the types of renal disease, and that increased MCP-1 expression probably contributes to renal tubular damage in proteinuric states.     

Urinary excretion of alpha1-microglobulin and albumin in acute myocardial infarction. Correlation with plasma concentrations of troponin I and C-reactive protein

OBJECTIVES: Microalbuminuria associated with myocardial infarction (MI) is normally ascribed to glomerular factors, but could just as well involve pathophysiological processes located in other parts of the nephron, e.g. proximal tubules where plasma albumin normally filtered through the glomerulus is almost completely reabsorbed. The aims of this study were to establish whether impaired tubular reabsorption of filtered albumin contributes to microalbuminuria during MI, and whether the urinary excretion of protein relates to plasma levels of C-reactive protein (CRP) and troponin I. MATERIAL AND METHODS: We monitored plasma CRP and troponin I as well as the urinary excretion of albumin and alpha1-microglobulin, a low-molecular-weight plasma protein and marker of tubular proteinuria, in 18 patients with MI. RESULTS AND CONCLUSIONS: The urinary excretion of alpha1-microglobulin and albumin was significantly elevated in nine patients, and the urinary excretion of the two proteins correlated significantly. A decrease in tubular reabsorption of albumin may thus be at least one of the causative factors underlying microalbuminuria during MI. The plasma levels of CRP and troponin I were also significantly higher in this group of nine patients, and correlated with urinary protein excretion, suggesting an interrelationship between inflammatory response and tubular dysfunction. Extrarenal inflammatory processes seem to affect renal tubular function, thereby contributing to microalbuminuria during MI.     

Influence of albumin infusion on the urinary excretion of beta2-microglobulin in patients with proteinuria

Most filtered proteins are reabsorbed by the renal proximal tubule by a mechanism that involves binding to the brush border membrane and endocytosis. Under normal conditions the low-molecular-weight protein beta2-microglobulin (beta2M), which is used to detect tubular injury, is reabsorbed almost completely. However, in proteinuric patients an increased urinary excretion of beta2M may not simply reflect tubular damage but might also result from a decreased tubular reabsorption due to competitive mechanisms. To examine the magnitude of such an effect we have studied the renal effects of albumin infusion (40 g in 2 h of a 20% solution) in 10 patients with a glomerular disease and proteinuria >3.5 g/24 h. Before, during and after albumin infusion the GFR (inulin clearance), RPF (PAH clearance), blood pressure and the urinary excretion of albumin, IgG, transferrin and beta2M were measured. Albumin infusion resulted in a slight decrease of the GFR (72 +/- 11 ml/min before and 67 +/- 10 ml/min after infusion), an increase of the RPF (379 +/- 66 ml/min before and 445 +/- 83 ml/min after), a decrease of the filtration fraction (0.20 before and 0.17 after), and hemodilution. After infusion the urinary excretion of albumin increased from 4.5 +/- 0.7 to 8.4 +/- 1.6 mg/min (p < 0.05). The urinary excretion of IgG and transferrin increased, probably reflecting a change in glomerular size-selectivity. In contrast, the urinary excretion of beta2M did not change significantly (baseline 12 +/- 5 microg/min, end 13 +/- 6 microg/min, percentage change 16.8 +/- 11%). To correct for changes in tubular load we calculated the fractional reabsorption of beta2M. The initial rise in albuminuria during infusion did not affect fractional tubular reabsorption (Delta%: 0. 72 +/- 0.52%, median 0.005%). In the period after infusion a slight decrease was noted (median -0.33%, p < 0.01). A decrease in the fractional reabsorption was particularly observed in patients with pre-existing tubular damage. In conclusion: infusion of albumin in proteinuric patients has no clinically relevant effect on the tubular reabsorption of beta2M. Therefore, beta2M is useful as a parameter to detect tubular injury and alterations in tubular handling of proteins in patients with proteinuria and glomerular diseases.     

Decreased urinary excretion of vascular endothelial growth factor in idiopathic membranous glomerulonephritis

BACKGROUND: Membranous glomerulonephritis (MGN) has, for unknown reasons, an unpredictable and highly variable clinical course. Vascular endothelial growth factor (VEGF) enhances endothelial cell proliferation, angiogenesis, microvascular permeability, and monocyte chemotaxis, and it activates proteinases. In normal kidneys, it is predominantly expressed by glomerular podocytes, where its physiological function and role in development of renal diseases are obscure. This study was designed to evaluate the urinary excretion of VEGF in MGN compared with several other glomerular disease and to asses its relationships to the clinical activity of MGN. METHODS: Urinary VEGF was studied during renal biopsy using a sandwich enzyme immunoassay from 30 patients with idiopathic MGN, 8 with minimal change glomerulonephritis, 10 with focal segmental glomerulosclerosis (FSGS), 8 with necrotizing glomerulonephritis associated with systemic vasculitis, and 12 with diabetic nephropathy. In addition, 33 healthy controls were examined. Fifteen patients with MGN were re-evaluated 12 months later, and the evolution of proteinuria was compared with changes in urinary VEGF excretion. RESULTS: In healthy control subjects, urinary VEGF excretion was 68 +/- 10 (95% CI, 49 to 88) ng/mmol creatinine (UCr). In MGN, the excretion was decreased to 16 +/- 3 (CI, 10 to 23) ng/mmol crea (P < 0.0001, ANOVA), whereas in minimal change glomerulonephritis and diabetic nephropathy, it was unchanged [55 +/- 14 (CI, 24 to 86) and 101 +/- 25 (CI, 45 to 156) ng/mmol UCr, respectively, P = NS]. In vasculitis and FSGS patients, the excretion was higher than normal [184 +/- 68 (CI, 24 to 344), P = 0.01, and 160 +/- 29 (CI 95 to 226), P = 0.002 ng/mmol UCr, respectively]. The excretion did not correlate with serum VEGF, renal function, or proteinuria. In the follow-up of 15 patients, improving MGN (decreasing proteinuria) was associated with increasing VEGF excretion, while persistent disease (no change or increase of proteinuria) was associated with constantly low urinary VEGF excretion. The change in urinary protein excretion over one year correlated inversely with the change in urinary VEGF (r = -0.57, P = 0.026). CONCLUSIONS: MGN is associated with decreased urinary VEGF compared with normal subjects, which is in contrast with other proteinuric diseases. Moreover, decreasing clinical activity (proteinuria) is accompanied by increasing VEGF excretion. Urinary VEGF may serve as an indicator of activity of MGN.     

Study on urinary splitting enzymes and proteins. III. Effect of non-ionic contrast medium on renal function

Renal toxicity of non-ionic contrast medium (iohexol) for drip infused pyelography (DIP) was studied in a randomized trial of nine patients with normal renal function. Urine samples were collected before and immediately after DIP, and analyzed for albumin, an index of glomerular permeability; gamma-glutamyl transpeptidase (gamma-GTP), a brush-border enzyme; N-acetyl-beta-glucosaminidase (NAG), a lysosomal enzyme; alpha 1 microglobulin (alpha 1MG) and beta 2 microglobulin (beta 2MG), an index to tubular proteinuria; and creatinine. The urinary excretion of enzymes and proteins was compared with urinary creatinine. Urinary excretion of gamma-GTP and NAG increased significantly (P less than 0.001, 0.02) after DIP. Urinary alpha 1 MG and beta 2-MG did not change significantly. The change of urinary albumin was mild. Our data suggest that non-ionic, low osmolal radiocontrast medium ioheol shows a lower renal tubular toxicity, and the brush-border enzyme gamma-GTP and lysosomal enzyme NAG are considered as a good index for renal tubular damage.     

Increased Urinary Excretion of Monocyte Chemoattractant Protein-1 in Proteinuric Renal Diseases

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is produced mainly by tubular epithelial cells in kidney and contributes to renal interstitial inflammation and fibrosis. More recently, we have demonstrated that urinary MCP-1 excretion is increased in proportion to the degree of albuminuria (proteinuria) and positively correlated with urinary N-acetylglucosaminidase (NAG) levels in type 2 diabetic patients. Based on these findings, we have suggested that heavy proteinuria, itself, probably aggravates renal tubular damage and accelerates the disease progression in diabetic nephropathy by increasing the MCP-1 expression in renal tubuli. In the present study, to evaluate whether urinary MCP-1 excretion is increased in the proteinuric states not only in diabetic nephropathy but also in other renal diseases, we examined urinary MCP-1 levels in IgA nephropathy patients with macroalbuminuria (IgAN group; n = 6), and compared the results with the data obtained from type 2 diabetic patients with overt diabetic nephropathy (DN group; n = 23) and those without diabetic nephropathy (non-DN group; n = 27). Urinary MCP-1 excretion levels in non-DN, DN, IgAN groups were 157.2 (52.8–378.5), 346.1 (147.0–1276.7), and 274.4 (162.2–994.5) ng/g creatinine, median (range), respectively. Expectedly, urinary MCP-1 and NAG excretion levels in DN and IgAN groups were significantly elevated as compared with non-DN group. Therefore, we suggest that MCP-1 expression in renal tubuli is enhanced in proteinuric states, irrespective of the types of renal disease, and that increased MCP-1 expression probably contributes to renal tubular damage in proteinuric states.     

Reversible low-molecular-weight proteinuria in patients with distal renal tubular acidosis

Four patients with untreated renal tubular acidosis had a urinary excretion of low-molecular-weight (LMW) proteins which was restored to normal by alkali therapy. Hypokalaemic proximal tubular damage in untreated patients with distal renal tubular acidosis is believed to be the cause of LMW proteinuria. An examination of urinary excretion of LMW proteins is useful for determining hypokalaemic proximal tubular dysfunction, as well as the efficiency of alkali therapy.     

In vivo localization of C3 on the brush border of proximal tubules of kidneys from nephrotic patients

Deposits of C3 but not of C1q and C4 were detected on the proximal tubules of kidneys from nephrotic patients with non-selective proteinuria. The incidence of tubular C3 deposits was significantly higher in patients with membranous glomerulonephritis, focal glomerulosclerosis, membrano-proliferative glomerulonephritis and non-selective proteinuria than in patients with minimal change disease, nephrotic syndrome and selective proteinuria or in patients with glomerular disease, but without nephrotic syndrome. The occurrence of tubular C3 deposits was positively correlated with the amount of urinary C3 excretion. In vitro studies showed that the human normal kidney as well as pathologic specimens negative for in vivo tubular C3 deposits were able to bind C3 on the brush border of proximal tubules when incubated with fresh heterologous serum. In contrast, in patients with non-selective proteinuria and in vivo tubular C3 deposits, the binding of heterologous C3 to the brush border of proximal tubules was markedly reduced. The positive correlation between the occurrence of tubular C3 deposits and the urinary complement excretion, together with the detection of the C3 breakdown products in the urines further supported the hypothesis that complement components, once filtrated through the glomerular barrier, might be activated by the brush border of the proximal tubule.     

High urinary excretion of kidney injury molecule-1 is an independent predictor of graft loss in renal transplant recipients

BACKGROUND: Chronic transplant dysfunction is characterized by renal function decline and proteinuria. Kidney injury molecule (KIM)-1, a transmembrane tubular protein with unknown function, is undetectable in normal kidneys, but markedly induced after injury. Urinary KIM-1 excretion has been quantified as biomarker of renal damage. We prospectively studied whether urinary KIM-1 predicts graft loss, independent of renal function and proteinuria. METHODS: Renal transplant recipients (n=145) visiting our outpatient clinic between August 2001 and July 2003 collected 24-hour urine samples for assessment of baseline urinary KIM-1 excretion (microsphere-based Luminex technology), and were followed for graft loss. RESULTS: Recipients participated at a median (interquartile range) of 6.0 (2.5-12.0) years posttransplant in baseline measurements. Follow-up beyond baseline was 4.0 (3.2-4.5) years. Urinary KIM-1 excretion was 0.72 (0.42-1.37) ng per 24 hours. Occurrence of graft loss increased over tertiles of KIM-1 excretion: 3 (6.3%), 11 (22.4%), and 17 cases (35.4%; P=0.001), respectively. High KIM-1 excretion was associated with proteinuria, low creatinine clearance, and high donor age (all P<0.01). In multivariate Cox regression analyses, prediction of graft loss by KIM-1 appeared independent of creatinine clearance, proteinuria, and donor age. Hazard ratios (95% CI) for the second and third tertile of KIM-1 excretion were 3.6 (0.9-13.5) and 5.1 (1.5-17.8) in the final model. CONCLUSIONS: Urinary excretion of KIM-1 is an independent predictor of long-term graft loss and therefore a promising new biomarker in early prediction of graft loss.     

URINARY THYMIDINE GLYCOL AS A BIOMARKER FOR OXIDATIVE STRESS AFTER KIDNEY TRANSPLANTATION

Reactive oxygen species are generated during ischemia-reperfusion tissue injury. Oxidation of thymidine by hydroxyl radicals (HO^˙) causes formation of 5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol). Thymidine glycol excreted in urine can be used as a biomarker of oxidative DNA damage. The aim of this study was to investigate the oxidative DNA damage in patients showing immediate allograft function after kidney transplantation, and to find out whether this damage correlates with glomerular and tubular lesions.

Time dependent changes in urinary excretion rates of thymidine glycol, but also of total protein, albumin, low molecular weight (α₁-microglobulin, β₂-microglobulin) and high molecular weight proteins (transferrin, IgG, α₂-macroglobulin) were analyzed quantitatively and by polyacrylamide-gel electrophoresis in six patients. Urinary thymidine glycol was determined by a fluorimetric assay in combination with affinity chromatography and HPLC.

After kidney transplantation the urinary excretion rate of thymidine glycol increased gradually reaching a maximum within the first 48 hours (16.56 ± 11.3 nmol/mmol creatinine, ref. 4.3 ± 0.97). Severe proteinuria with an excretion rate of up to 7.2 g/mmol creatinine was observed and declined within the first 24 hours of allograft function (0.35 ± 0.26 g/mmol creatinine). The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The initial nonselective glomerular proteinuria disappeared within 48 hours, changing to a mild selective glomerular proteinuria. In this period (12–48 hours) higher levels of thymidine glycol excretion were observed, when compared to the initial posttransplant phase (13.66 ± 9.76 vs. 4.31 ± 3.61 nmol/mmol creatinine; p < 0.05).

An increased excretion of thymidine glycol is seen after kidney transplantation and is explained by the ischemia-reperfusion induced oxidative DNA damage in the kidney. In the second phase higher levels of excretion were observed parallel to the change from a nonselective to a selective glomerular and tubular proteinuria. An explanation may be sought in the repair process of DNA in the glomerular and tubular epithelial cells, appearing simultaneously with functional recovery.