Gianfranco Tramonti

REAPPRAISAL OF SERUM β2-MICROGLOBULIN AS MARKER OF GFR

Introduction. Beta 2 microglobulin(β2M) is filtered by the glomeruli and reabsorbed by the proximal tubularcells where it is metabolized. Its plasma concentration increases with decreasingrenal function. Aim. To compare serum creatinine(Cr) and serum β2M as markers of GFR. Patientsand Methods. In 160 adult patients, with various kidney diseasesand different GFR, serum Cr (autoanalyzer), serum β2M (RIA) and GFR (bladdercumulative method using 99mTc-DTPA as glomerulartracer) were measured in the same day. Results.A linear relationship was observed between ln GFR and both ln serum Cr (lnCr= 3.112–0.716lnGFR; r = 0.92) and ln serum β2M (lnβ2M = 4.274–0.814lnGFR; r = 0.90). With decreasing GFR the increase in serum β2M was higherthan that of serum Cr (see regression coefficients that are significantlydifferent). The normal upper limit of serum Cr corresponds to a GFR 48.1 mL/minwhile that of serum β2M to a GFR 65.0. With decreasing GFR the increaseof serum β2M occurs before than that of serum Cr. Conclusions.With declining renal function, serum β2M increases more and before thanserum Cr. Serum β2M is a good endogenous marker of GFR, better than serumCr.

Measurement of Glomerular Filtration Rate in Man Using DTP A-99mTc

The aim of this study was the evaluation of the mechanism of renal excretion of DTPA-99mTc in man. For this purpose the renal clearances of DTPA-99mTc and diatrizoate-131

RELATIONSHIP BETWEEN RENAL FUNCTION AND BLOOD LEVEL OF CHROMOGRANIN A

Chromogranin A (CGA) is a low MW (49,000) acidic hydrophilic protein. It is synthesized in the chromaffm granules of the neuroendocrine cells, and has been found circulating in the blood of healthy subjects. The aim of this study was to assess the relationship between serum levels of CGA and renal function. One hundred two renal patients (45 M and 57 F; age 14–76 years, mean 52) participated in the study. Glomerular filtration rate (GFR) was measured by the bladder cumulative method, using 99mTc-DTPA as a tracer. Blood CGA was determined by RIA. Plasma creatinine, β2microglobulin (β2m) and tumor associated trypsin inhibitor (TATI) were also determined. The reduction in renal function was associated with an increase in all of the above studied parameters. In patients with advanced renal failure (GFR < 20 mL/min) CGA levels increased by 22-fold as compared to the patients with normal renal function (GFR > 100 mL/min). The other studied parameters were also increased but to a lesser degree, e.g., TATI 14-, β2m 8- and creatinine 5-fold. The results of this study demonstrate that renal handling of the CGA is similar to other low MW proteins, and it accumulates in the blood in renal failure.

STATUS OF GLUCOSE TRANSPORTERS IN THE MAMMALIAN KIDNEY AND RENAL DEVELOPMENT

Glucose is the main source of energy for the mammalian cells and its entry is mediated via various transporters. About 7 facilitative (GULT-1 to -7) and 2 concentrative glucose transporters (SGLT-1 and -2) have been identified. The facilitative glucose transporters allow the glucose entry into the cell interior due to the concentration gradient and the latter via the Na+-dependent electrochemical gradient. They have similar structural motifs with 12–14 putative transmembrane domains with a predicted protein size varying from 50 to 76 kDa. Some of the facilitative glucose transporters (GLUT-1, -2, -4 and -5) and both the sodium glucose co-transporters (SGLT-1 and -2) are expressed in the kidney. The transporters that are involved in the major transport of glucose in the kidney include GLUT-2 and SGLT-2. They are of high capacity and low affinity type and are expressed in the S1 segment of the proximal tubule. All the transporters expressed in the kidney are developmentally regulated. The mRNA expression of renal GLUTs is variable during the fetal and postnatal periods. On the other hand the mRNA of SGLTs increases steadily from the fetal period to maturity along with the increase in their functional activity, i.e., glucose uptake. Recent studies indicate that the SGLTs are believed to selectively regulate tubulogenesis since they are expressed in the metanephric tubules very early in the embryonic life in mammals.

Review and discussion of tubular biomarkers in the diagnosis and management of diabetic nephropathy

The prevalence of diabetic nephropathy has tremendously increased with the relentless rise in the incidence of diabetes over the last couple decades. Diabetic nephropathy is a leading cause of morbidity and mortality, and it invariably leads to an end-stage renal disease (ESRD). In an effort to delay the onset of ESRD systematic screening and appropriate management are needed to evaluate the progression of renal damage in diabetic nephropathy. The reliability of current tests in predicting the onset, progression and response to various regimens for diabetic nephropathy is still under debate; and it has engendered a search for more sensitive and specific urinary biomarkers, especially those reflective of tubular dysfunctions. It is well-known that there is a good correlation between the degree of damage to the tubulo-interstitial compartment and the deterioration of renal functions. In view of this, the utility of urinary biomarkers, reflective of tubular injury, reported in the literature is discussed in this brief review.

P-GLYCOPROTEIN IN HK-2 PROXIMAL TUBULE CELL LINE

P-glycoprotein (PGP) is an efflux pump physiologically expressed in the apical membrane of the proximal tubular cells. PGP may play a role in the elimination of exogenous substances such as chemotherapeutic drugs, calcium channel blockers and immunosuppressors. The involvement of renal PGP in the transport of endogenous substrates is under investigation. HK-2 is an immortalized proximal tubule cell line from normal adult human kidney, reported to retain a phenotype indicative of a well-differentiated state. No data regarding expression and/or activity of PGP in this cell line are available. The aim of this study was to ascertain the usefulness of HK-2 cell line to investigate the properties and roles of PGP in proximal tubular cells. PGP expression in HK-2 cells was determined by immunoblotting analysis using the monoclonal antibody C219. The activity of PGP was assessed by measuring the transport of the fluorescent probe Rhodamine 123 (R-123) in intact cell monostrates. The interactions of putative PGP modulators, including verapamil and cyclosporin A were also evaluated. Western blot revealed a C219 immunoreactive band of about 150kDa consistent with the presence of PGP. HK-2 cells preloaded with R-123 rapidly effluxed the dye, the efflux being inhibited by verapamil. Verapamil and, to a major extent cyclosporin A, significantly increased R-123 intracellular accumulation. PGP immunoblottable amount was increased when cells were cultured in the presence of either cyclosporin A or dexamethasone. The results suggest that the HK-2 cells, among the various differentiation features of proximal tubules, retain also the expression of a functional PGP in their membranes and that both PGP activity and expression may be modulated by drugs. Therefore, HK-2 line appears a suitable and promising tool for the study in vitro of renal transport processes dependent on PGP.

99mTc-aprotinin: A new tracer for kidney morphology and function

AbstractAprotinin (Ap), a low-molecular-weight polypeptide (6500 dalton), is a protease inhibitor which is electively and stably accumulated in the kidney. In 112 adult patients, with either uni- or bilateral renal disease with different degrees of renal impairment (from normal GFR to advanced renal failure), renal scans were performed by means of Ap labelled with99mTc. Highly satisfactory renal scans were obtained in all patients. In 20 patients with renal failure (serum creatinine 1.8–8.5 mg/dl, mean 4.7) a comparison was made of the renal scans obtained with99mTc-Ap and with99mTc-DMSA.99mTc-Ap was slightly better than99mTc-DMSA, especially in patients with far advanced renal failure.Some aspects of the pharmacokinetics of99mTc-Ap were studied in 72 cases. In 22 of these patients plasma clearance of99mTc-Ap was determined by the single injection method using a two-compartment model. In patients with GFR>90 ml/min plasma clearance of99mTc-Ap was 67.6±8.4 SD ml/min. A good correlation was observed between plasma clearance of99mTc-Ap and GFR (r=0.74). After IV injection99mTc-Ap was stably fixed by the kidney. Renal radioactivity remained stable between the second and eighth hour after the injection. Urinary excretion of radioactivity measured in 35 patients in the first and in the second 2-h interval after IV injection of99mTc-Ap was negligible in all patients (2.7±1.5 SD percent of the dose in the first 2 h; 2.8±1.4 SD between the second and fourth hour). 99mTc-Ap is an excellent agent for renal imaging. It also seems promising for renal function studies.

Gamma-glutamyltransferase is a reliable marker for tubular effects of contrast media.

The aim of this study was to evaluate the usefulness of the measurement of urinary excretion of the brush-border enzyme gamma glutamyl-transferase (GGT), in comparison with that of alanine aminopeptidase (AAP), as a marker for tubular toxicity due to contrast media (CM). Urinary activities of AAP and GGT were measured prior to the administration of CM and 1, 3 and 5 days after in forty-nine adult renal patients undergoing a radiological examination with intravascular administration of CM. The behavior of GGT was similar to that of AAP. In fact, urinary activities of both AAP and GGT increased greatly after CM. This effect was maximal on the 1st day and statistically significant for both enzymes. Furthermore, on the 1st day a relevant increase of enzyme activity (at least +50% over the basal value) was observed in the same number of patients (67%) for AAP and GGT. The concordance between GGT and AAP variations was high and statistically significant. Finally, different variables (osmolarity, dose of CM, and baseline renal function of the patients) had a similar effect on urinary excretion of AAP and GGT. The repeatability of duplicated determinations of GGT resulted better than that of AAP. In conclusion, the good concordance of the results of GGT with those of AAP justifies the use of GGT as a marker for tubular effects due to CM. Furthermore, the measurement of GGT has a better repeatability than that of AAP.

Tubular Toxicity Is the Main Renal Effect of Contrast Media

The aim of this study is to evaluate the effects of contrast media on both tubular and glomerular function. Different parameters of tubular and glomerular function were determined before and at 1, 3, and 5 days after the intravascular administration of contrast media in 100 adult renal patients (plasma creatinine 0.6-10.8 mg/dL, mean: 1.3). Urinary activities of five tubular enzymes (alanine aminopeptidase, gamma-glutamyltransferase, alkaline phosphatase, lactate dehydrogenase, N-acetyl-beta-D-glucosaminidase) increased significantly on the first day after the administration of contrast media, indicating a tubular damage. Glomerular filtration rate and the conventional tests of glomerular function (plasma creatinine, creatinine clearance, and urinary proteins) presented only slight variations after the administration of contrast media. In conclusion, contrast media principally affected the renal tubule (as demonstrated by enzymuria), while their effects on glomerular function were very mild.

Effects of Devil's Claw (Harpagophytum procumbens) on the multidrug transporter ABCB1/P-glycoprotein.

UNLABELLED Devils Claw (Harpagophytum procumbens) a plant native to Southern Africa, has historically been used in traditional medicine to treat a wide range of diseases and currently is widely employed as anti-inflammatory and pain-relieving natural remedy in Europe and other parts of the world. AIM OF THE STUDY Little is known about possible herb-drug interactions arising from effects of Devils Claw on the major drug metabolizing enzymes or transporters. This study evaluated in vitro the effects of Devils Claw on the multidrug transporter ABCB1/P-glycoprotein. MATERIALS AND METHODS The effects of three commercially available Devils Claw preparations and that of pure harpagoside were studied in the human kidney (HK-2) proximal tubule cell line, constitutively expressing ABCB1/P-glycoprotein (P-gp). Pgp activity and expression were tested by the calcein-AM test and by Western blotting, respectively. RESULTS Commercial preparations inhibited P-gp activity, even if to a different extent, while pure harpagoside was almost ineffective. In cells cultured for three days in the presence of Devils Claw preparations or pure harpagoside, a dose-dependent P-gp upregulation was found. CONCLUSIONS Our results demonstrate for the first time that Devils Claw may interact with the multidrug transporter ABCB1/P-gp, the effect not appearing strictly related to the harpagoside relative content. Modulation of both P-gp activity and P-gp expression by Devils Claw raise the possibility of herb-drug interactions, to be further explored in depth.