Difference in estimated GFR with two different formulas in elderly individuals.

The aim of this study was to characterize the differences between the prediction of GFR with Cockcroft-Gault formula (CG=(140-age)/(72 x PCr (mg/ml), for females multiplied by 0.85) and the new formula based on the multicenter trial of the Modification of Diet in Renal Diseases (MDRD=186 x P (Cr) (-1.154) x age(-0.203); 0.742 if patient is female) in elderly subjects. The study involved 100 individuals aged 65-111 years (mean age 88.3+/-14.7; 79 females and 21 males). In all subjects GFR was estimated according to both formulas mentioned above and expressed in ml/min/1.73 m2. Thereafter we calculated the difference between MDRD and CG (MDRD-CG) and analyzed its determinants in every subject. Mean GFR, obtained with MDRD was 76.0+/-24.0, whereas according to CG 67.9+/-18.6 (p < 0.0001). However, the mean MDRD-CG was up to 30.0+/-26.6 which means that MDRD results were much higher in comparison with CG. Using the multiple linear regression analysis we showed that MDRD-CG strongly depend on age (p < 0.0001), BMI (p < 0.0001) and serum creatinine concentration (p<0.0001). However, the gender has not effect on MDRD-CG value. The values of MDRD-CG strongly and positively correlated with age (r=0.7027, p < 0.0001) and negatively both with body mass index (r=-0.7171, p < 0.0001) and serum creatinine (r=-0.5590, p < 0.0001). In summary, our results show that the difference between MDRD and CG strongly depends on age, BMI and Scr. Investigators should be aware of these differences and take it into account in elderly.

In situ upregulation of IL-10 reflects the activity of human glomerulonephritides.

A reciprocal regulation between the expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10, demonstrated in monocytes and mesangial cells, provides a rationale for new therapeutic approaches in glomerulonephritis (GN). Administration of IL-10 to mice with antibody-mediated GN attenuated the severity of glomerular lesions. Recently, however, it has been shown that the genetically determined predominance of Th1 or Th2 cytokines results in different glomerular responses to the same planted antigen, but in an equally severe impairment of renal function. We looked for the expression of IL-10 and TNF-alpha in 111 renal biopsy specimens with proliferative and nonproliferative forms of GN and in 10 control kidneys, by means of immunocytochemistry, in situ hybridization, or reverse-transcriptase polymerase chain reaction (RT-PCR). Six patients had acute endocapillary GN (AGN), 10 patients had pauci-immune GN due to microscopic polyangiitis (MP), 48 patients had immunoglobulin-A (IgA)-GN, 18 patients had idiopathic membranous GN (IMGN), 12 patients had minimal change disease (MCD), and 13 patients had focal segmental glomerulosclerosis (FSGS) and four other forms of GN. Antibodies against monocytes (CD14) and macrophages (CD68) were applied to attribute the expression of TNF-alpha and IL-10 to resident renal or infiltrating cells. We show that mRNAs for TNF-alpha and IL-10 are detected by RT-PCR and in situ hybridization in the normal kidney. A constitutive expression of TNF-alpha protein is observed in mesangial cells, smooth muscle cells in renal arteries, and in the interstitium. A trace immunoreactivity for IL-10 is restricted to arterial smooth muscle cells, distal tubular epithelial cells, and some interstitial cells. Upregulation of both cytokines is found in glomerular diseases. The expression of TNF-alpha increases in mesangial areas in MCD, IMGN stages I/II, and IgA-GN with minor glomerular abnormalities, that is, under conditions with a generally well-preserved glomerular structure. Conversely, marked glomerular proliferation in IgA-GN and, particularly, acute vascular lesions in MP, are accompanied by a significant upregulation of IL-10 (at the mRNA and protein level). Patients with nephrotic-range proteinuria show a significant increase in tubulointerstitial expression of IL-10, whereas the immunoreactivity for TNF-alpha reflects the extent of interstitial fibrosis. Thus, our results confirm previous suggestions that proinflammatory and antiinflammatory cytokines are produced in situ by resident renal cells and contribute to the natural course of human GN.

Protein and mRNA expression of CD56/N-CAM on follicular epithelial cells of the human thyroid.

In order to confirm CD56/N-CAM antigen prevalence in the human thyroid and to compare its expression on thyrocytes and NK cells, an expression of CD56/N-CAM antigen was searched for on isolated thyroid follicular cells and NK cells by flow cytometry. In addition, mRNA for CD56 was searched for in RNA isolated from human thyroid samples and few other organs using dot blot hybridization assay to prove the existence of mechanisms for active synthesis of the protein in question. The isolated cells from the follicular epithelium of 22 various pathological thyroid tissue specimens were examined for the expression of CD56/N-CAM in terms of the percentage of positive cells and the mean fluorescence intensity (MFI). Blood lymphocytes were tested in parallel. The total RNA isolated from thyroid and control tissue specimens was subjected to dot-blot hybridization assay using CD56/N-CAM cDNA probe. All thyroid specimens expressed CD56/N-CAM, but the obtained values differed depending on the tissue examined and the CD56 antibody used. There were no significant differences between the non-malignant thyroid cells of various histology, while cells in the carcinoma group had a much lower MFI, especially the median value. CD56 expression on NK cells from the donors' blood had a homogeneous distribution but the mean and median values of FI were almost three times lower than those on the thyroid cells. Dot blot hybridization came out positive with the RNA isolated from the thyroid specimens and also from the RNA isolated from the tonsil and lymph nodes, but came out negative with the RNA isolated from human and rat kidneys. These results strongly suggest that thyroid follicular epithelial cells express both protein and mRNA of CD56/N-CAM, thus being able to synthesis the relevant antigen. The protein expression seems to be affected by the malignant transformation of the thyroid cells. NK cells have apparently lower CD56/NCAM expression than thyroid cells.

Idiopathic myelofibrosis with extramedullary hematopoiesis in the kidneys.

Extramedullary hematopoiesis is a common finding in idiopathic myelofibrosis and is usually found in liver and spleen. We report on a patient with biopsy-proven myeloid metaplasia and fibrosis of the renal parenchyma as a rare cause of chronic renal failure. The renal biopsy specimen showed numerous infiltrates of hematopoietic cells expressing growth factors like M-CSF, GM-CSF, IL-1beta and PDGF while TGF-beta was not elevated. These findings suggest that hematopoietic growth factors play a key role in the pathogenesis of this condition causing proliferating fibrosis and enlargement of the kidneys.

[Urinary excretion of alpha-1-microglobulin and complement components in patients with chronic glomerulonephritis]. / Wydalanie alpha-1-mikroglobuliny i skladowych dopelniacza z moczem chorych na prezewlekle klebkowe zapalenie nerek.

The urinary excretion of alpha-1-micro-albumin (alpha 1m) and complement components (CCs) was evaluated in the urine of 49 patients suffering from chronic glomerulonephritis (GN). Nephrotic syndrome (NS) was shown in 18 cases and increased serum creatinine level (Pcr: 115.0-159.1 mumol/l) in 9 patients. The most frequent CCs presence and the highest values of alpha 1m excretion were found in patients with membranous GN. In the early phase of the disease the alpha 1m urinary excretion was higher in subjects with NS than in those not showing the feature of it, independently of the morphological basis of the disease. Also CCs were detected mainly in the nephrotic patients. As the glomerular filtration improved a significant decrease in the urinary alpha 1m excretion was observed. The application of steroid immunosuppressive therapy resulted in the decrease of alpha 1m as well as CCs excretion. The results seem to point out that the increased alpha 1m and CCs excretion may be secondary to the activity of glomerular alternations as well as to the disturbances in glomerular blood flow.

The effect on renal structure and function of late-life-introduced caloric restriction (CR) in rats.

Previous studies have shown that life-long caloric restriction in rats protects the kidneys from age-dependent injury. In this study, we analyzed whether late-life-introduced caloric restriction has a similar effect. The study lasted 12 months. Three groups of animals were analyzed: rats fed "ad libitum" (AD, n = 9), rats on 60% caloric restriction (CR, n = 9), and rats fed "ad libitum" for the first six months of their life then switched to 60% caloric restriction thereafter (LCR, n = 9). At the end of the study kidney function was assessed and kidney samples were analyzed histologically. Serum creatinine and urine albumin were higher in AD than in both CR and LCR (P < 0.001). Creatinine clearance (Cl(cr)) corrected for body weight was lowest in AD and comparable in CR and LCR. Similarly Cl(cr) corrected for kidney weight was lower in AD than in both CR and LCR (P < 0.05). Severe albuminuria was observed only in AD. In CR and LCR the amount of albumin excreted was comparable (AD vs. CR, P < 0.0001; AD vs. LCR, P < 0.001). In morphometric analysis, the mean size of the glomeruli was higher in AD than in both CR and LCR (P < 0.01). Similar results were found for the mesangial area (AD vs. CR, P < 0.001; AD vs. LCR, P < 0.01) and for mesangial cell counts (AD vs. CR, P < 0.001; AD vs. LCR, P < 0.05). No difference was found between CR and LCR in morphometry. In conclusion, our study indicates that late-life introduction of caloric restriction reverses most of the structural and functional changes observed in the kidneys of "ad libitum"-fed rats.

[The effect of tonsillectomy on the level of circulating immune complexes and urine changes in patients with glomerulonephritis]. / Wplyw tonsylektomii na poziom krazacych kompleksów immunologicznych i zmiany w moczu u chorych na klebuszkowe zapalenie nerek.

The influence of tonsillectomy on circulating immune complexes (C.I.C.) level, proteinuria and erythrocyturia was studied in 42 patients with chronic tonsillitis (Ch.T.) and urine abnormalities. The level of C.I.C. was examined by two methods: the 3.5% polyethyleneglycol (PEG) precipitation method and the 125I-C1q binding method. After tonsillectomy, bacteriological analysis of removes facial tonsilla was performed in 7 patients and morphological analysis in 11. Renal biopsy was done in 28 patients. The control group was consisted of 18 patients with Ch.T. without urine abnormalities. The presence of C.I.C. was established in 48% of patients with urine abnormalities using PEG method and in 33% with 125I-C1q binding method. Mean values of C.I.C. in patients with proteinuria or erythrocyturia were statistically higher than in the control group. After tonsillectomy, transitory increase of C.I.C. level was observed in 60% of patients, accompanied by augmentation in urine changes, especially proteinuria. During one year of observation, significant decrease in C.I.C. levels detected by PEG method, as well as in proteinuria and in erythrocyturia was found. In 10 patients urine abnormalities disappeared. No differences between both groups of patients were found in the results of bacteriological and morphological studies of removed tonsilla. However, the normalisation of urine changes was noticed in patients without hypertension and in whom renal disease did not exceed two years. Renal histology revealed mesangocapillary proliferative Gn in 14, mesangial proliferative Gn in 11, and focal/segmental glomerulosclerosis in 3 patients. In one patient with mesangial proliferative Gn complete retreat of urine changes was observed. We suggest that the presence of Ch.T. influences on the C.I.C. detectability in patients with chronic glomerulonephritis. The tonsillectomy can lead do the decrease of C.I.C. levels, as well as to the decrease of proteinuria and/or erythrocyturia. Serum C.I.C. examination seems to be helpful in qualifying patients with Ch.T. for tonsillectomy, in immunological monitoring after the operation and in later prognosis in case of chronic glomerulonephritis.

Expression of two alternative transcription forms of platelet-derived growth factor-A chain in the normal human kidney and in glomerulonephritis.

PURPOSE: Up to now, a role of platelet-derived growth factor (PDGF)-AA in glomerulonephritis (GN) remains unclear. PDGF-A chain may be produced in two forms, as a result of the alternative splicing. MATERIAL AND METHODS: We examined the expression of this growth factor in the renal tissue of 57 patients with GN and seven normal kidneys (NK). The gene expression of PDGF-A was examined by reverse transcriptase-polymerase chain reaction. Sets of primers allowing distinction between the two forms of transcripts were used. Specificity of the PCR products was confirmed by restriction enzyme analysis and sequencing. The expression of PDGF-AA/AB was also evaluated by immunohistochemistry. RESULTS: Compared to NK, the expression of PDGF-A gene was higher in the renal tissue with GN. This expression was higher in non-proliferative GN (NPGN) than in proliferative forms of GN (PGN) (1.24 +/- 0.34 vs. 0.86 +/- 0.14). In NK, both forms of transcripts (N = 4) or only the short one (N = 3) were found. In 45.5% of patients with NPGN, only the short form could be detected. In contrast, in 68.6% of patients with PGN both or only the longer form of transcripts were found. In NK, a faint staining for PDGF-AA/AB was observed within glomerular capillaries, whereas a statistically significant increase in this protein expression was particularly stated in NPGN. These results suggest that the production of the longer PDGF-A chain variant is associated with glomerular cells' proliferation. However, the higher expression of PDGF-AA/AB protein in NPGN could indicate an essential role of this growth factor in the maintaining the glomerular architecture.

Can von Willebrand factor, platelet-endothelial cell adhesion molecule-1 and thrombomodulin be used as alternative markers of endothelial cell injury in human glomerulonephritis?

PURPOSE: There is growing evidence that endothelial cells (EC) are active participants of an inflammatory process in glomeruli. MATERIAL AND METHODS: We compared the glomerular expression of three EC-coupled molecules, i.e. platelet-endothelial cell adhesion molecule-1 (PECAM-1 or CD31), von Willebrand factor (vWF) and thrombomodulin (TM) in 60 patients with glomerulonephritis (GN) and five normal kidneys (NK). The alkaline phosphatase anti-alkaline phosphatase method was used to examine the expression of these proteins in the biopsy specimens. RESULTS: In NK, the expression of CD31 and vWF comprised the whole glomerular network. In contrast, the expression of TM was much lower and localized mainly to EC at the vascular pole and adjacent areas. In GN, the glomerular staining for CD31 and vWF was significantly reduced. A fall in the expression of both these EC antigens was more pronounced in proliferative forms of GN (PGN) than in non-proliferative GN (NPGN) (CD31: NPGN vs. PGN, p < 0.02; vWF: NPGN vs. PGN, p < 0.05). In addition, a linear relationship between the expression of CD31 and vWF was found in GN (r = 0.8, p < 0.001). Conversely to CD31 and vWF, a marked increase in glomerular reactivity for TM was observed in all the patients with GN (GN: 2.12 +/- 0.32, NK: 0.95 +/- 0.05, p < 0.02). However, the highest expression of TM was found in membranoproliferative GN and lupus GN. CONCLUSIONS: Our results suggest that CD31 and vWF may be used as markers of glomerular EC loss during GN, whereas TM staining seems to reflect EC activation in response to circulating and/or released in situ procoagulant factors.

Kidney function estimated with different formulas in centenarians.

PURPOSE: There are growing doubts about the accuracy of Cockcroft-Gault formula (CG) used for the estimation of creatinine clearance, especially in elderly. Recently, the authors of the multicenter trial of the Modification of Diet in Renal Diseases (MDRD) have proposed a new equation. Moreover, Baracskay et al. (B), proposed the special formula for the estimation of kidney function (KF) in elderly. The aim of our study was to compare the results of KF calculated with these three formulas in centenarians. MATERIAL AND METHODS: The study involved 50 centenarian subjects aged 100-111 years (41 females and 9 males) who participated in Polish Centenarians Program. In all of them KF was estimated with the CG, B and MDRD formulas. RESULTS: In the whole population examined, the mean KF according to CG was lower in comparison to both others (p < 0.001 vs both B and MDRD). Also, in females CG results were the lowest (p < 0.001 vs both B and MDRD). In contrast, KF calculated according to CG and B did not differ in males. The results of the MDRD formula significantly exceeded the two others also in males (p < 0.001 vs CG and B). No impact of gender on the obtained results could be found when CG and MDRD were used. However, according to B, the mean values for females were higher (p < 0.01). CONCLUSIONS: KF calculated with the CG, B and MDRD formulas significantly differed in the centenarians examined. Thus, further studies, which include a reference standard, are necessary to answer the question which of these mathematical formulas is the most reliable for the calculation of KF in the elderly.

Expression of cytokines and growth factors in human glomerulonephritides.

Numerous experimental studies point to the potential role of cytokines and growth factors in the pathogenesis of renal disease. However, from the various autocrine and paracrine mediators identified in vitro and in animal models, so far only a few have been demonstrated in selected human glomerulopathies. We examined two types of glomerulonephritis (GN): extracapillary GN with anti-neutrophil cytoplasmic autoantibodies (ANCA), an example of an acute form of GN, and mesangial IgA GN, usually a chronic form of GN, with immunocytochemistry, in situ hybridization and the polymerase chain reaction. Normal renal tissue from tumour nephrectomies served as a control. In ANCA-positive GN with active renal lesions (crescents, glomerular and vascular necrosis), infiltrating mononuclear cells in glomeruli and in the interstitium expressed interleukin (IL)-1 beta, tumour necrosis factor (TNF)-alpha, IL-2, interferon (IFN)-gamma, platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. Cytokine expression was also observed in activated resident cells, including endothelial cells, capsular epithelial cells, smooth muscle cells of vessel walls, fibroblasts and some tubular epithelial cells. In addition, we noted an increase in the cytokine and growth factor receptors TNF-R, IL-1R type II, IL-2R, IFN-gamma R and PDGF beta-R. In contrast, in mesangial IgA-GN, IL-1 beta, TNF-alpha, IFN-gamma and IL-2 were usually absent in glomeruli. Mesangial expansion in this disorder was accompanied by an increased expression of PDGF, PDGF beta-R, TGF-beta and IL-6 in mesangial areas. In both conditions a good correlation was observed between cytokine expression at the mRNA (in situ hybridization) and protein level (immunocytochemistry).(ABSTRACT TRUNCATED AT 250 WORDS)

PDGF and TGF-beta contribute to the natural course of human IgA glomerulonephritis.

PDGF and TGF-beta are known mediators of mesangial cell proliferation and matrix expansion. The presence of these regulatory factors was examined in 30 renal biopsies from patients with IgA glomerulonephritis (IgA-GN) at the mRNA and protein level. Normal renal tissue served as control. The mRNA expression of PDGF A/B chains, PDGF-beta R and TGF-beta 1 was evaluated by means of RT/PCR with subsequent Southern blot hybridization and/or non-radioactive in situ hybridization. In addition, PDGF-AB/BB, PDGF-beta R, TGF-beta isoforms (beta 1, beta 1 + 2, beta 2 + 3), the small TGF-beta 1 latency associated peptide (TGF-beta 1 LAP) and the extracellular matrix proteins tenascin and decorin were analyzed by immunocytochemistry. The expression of growth factors was correlated with light microscopic and clinical features. Compared to normal control kidneys, an increased expression of PDGF-BB/PDGF-beta R mRNAs and the corresponding proteins was observed in all biopsies with IgA-GN. Up-regulation was related to the degree of glomerular proliferation and the extent of fibrosing interstitial lesions. In contrast, there was a discordance between TGF-beta 1 mRNA and protein expression (evaluated by immunocytochemistry). In all biopsies, irrespective of the stage of the disease, abundant TGF-beta 1 transcripts were detected, whereas TGF-beta 1 immunoreactivity was expressed to a lesser degree and disclosed a more variable staining pattern. In patients with significant proliferative glomerular lesions and minor tubulointerstitial alterations, TGF-beta 1 positivity was confined to areas of glomerular proliferation, whereas in cases with more severe histology including sclerosing lesions TGF-beta 1 immunoreactivity was less prominent. The distribution and the intensity of TGF-beta 1 LAP staining commonly exceeded the positivity noted for TGF-beta 1, indicating only limited TGF-beta 1 activation. A decreased reactivity for tenascin accompanied the morphological features of glomerular sclerosis. The staining patterns and the fact that only very few inflammatory cells, particularly CD68 positive monocytes/macrophages, were detected in glomeruli confirm that predominantly resident glomerular cells (mesangial and endothelial cells) are the major source of up-regulated growth factor production in IgA-GN. Since the expression of PDGF-AB/BB paralleled the severity of proliferative glomerular changes, PDGF seems to represent a potential indicator of activity in this condition. It is suggested that an imbalance between PDGF and TGF-beta (by restricted translation and/or activation) production contribute to the progressive nature of IgA-GN.

In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease.

A dysregulated secretion of contra-inflammatory cytokines such as interleukin-10 (IL-10) could play a role in the pathogenesis of inflammatory bowel disease (IBD). We have investigated the expression of IL-10 in gut tissues from patients with Crohn's disease (CD), ulcerative colitis (UC) and controls by mRNA in situ hybridization and immunohistochemistry. Intestinal epithelial cells were found to express IL-10 mRNA and IL-10 protein in all of the tissues investigated without any major differences in the expression patterns. However, compared with noninflamed gut, significantly increased numbers of mononuclear cells (MNCs) producing IL-10 were present in inflamed gut, both in CD and UC. This cytokine was expressed most prominently by inflammatory infiltrates enriched in macrophages, although T cells seem to contribute to its production as well. Elevated IL-10 expression in IBD was mainly detected in the submucosa, whereas IL-10 production by lamina propria cells remained comparably low. In contrast, the expression of IL-1beta mRNA was preferentially increased in the lamina propria. Our data argue against a general deficiency in IL-10 production in IBD. The results suggest rather that the local production of IL-10 by mucosal MNCs in IBD is insufficient to down-regulate pro-inflammatory cytokines such as IL-1beta in the lamina propria compartment.

Podocytes are the major source of IL-1 alpha and IL-1 beta in human glomerulonephritides.

To address the question of in situ production of IL-1 alpha and IL-1 beta in proliferative and non-proliferative forms of human glomerulonephritis (GN), we performed immunocytochemical and in situ hybridization studies on renal biopsies from patients with mesangial IgA-GN (N = 38), idiopathic membranous GN (MGN; N = 12), minimal change disease (MCD; N = 9), focal segmental glomerulosclerosis (FSGS; N = 5) and acute endocapillary GN (AGN; N = 3). Normal kidneys (N = 10) served as controls. Concomitantly, the expression of IL-1 receptor type I (IL-1 RI), IL-1 receptor type II (IL-1 RII) and of IL-1 receptor antagonist (IL-1 RA) was analyzed. Antibodies against antigens expressed on podocytes (PP-44), endothelial cells (CD31) and monocytes/macrophages (CD11b, CD14, CD68) were applied to attribute the expression of IL-1/IL-1 related peptides to intrinsic glomerular and/or blood-derived infiltrating cells. Our results demonstrate that IL-1 RII is constitutively expressed on endothelial cells, and its expression can be induced in proximal tubular cells and in the interstitium. In diseased glomeruli podocytes are capable of producing IL-1 alpha/beta. In MGN and MCD/FSGS, the expression of both IL-1 forms is particularly noted in early stages of the disease and is not only accompanied by a marked reactivity for IL-1 RI, but also for IL-1 RA. In segmental sclerosing lesions in FSGS and in IgA-GN with marked glomerular proliferation and/or sclerosis, a reduced expression of the PP-44 antigen and a diminished ability of podocytes to produce IL-1/IL-1 related peptides are noted. These results suggest that intrinsic glomerular production of IL-1 may be of relevance for the protection of glomeruli from continuing injury.