Profile of chronic kidney disease related-mineral bone disorders in newly diagnosed advanced predialysis diabetic kidney disease patients: A hospital based cross-sectional study.

AIM: Chronic kidney disease related-mineral bone disorder (CKD-MBD) has been poorly studied in pre-dialysis Indian CKD population. There are limited data on the pattern of these disturbances in diabetic CKD patients. Therefore, a study was conducted to find out the profile of mineral bone disorders in T2DM patients with pre-dialysis CKD. METHODS: In this cross-sectional design, diabetic patients with newly-diagnosed stage 4 and 5 CKD were evaluated. Serum levels of calcium, phosphorus, intact parathyroid hormone (iPTH), 25 hydroxy vitamin D and total alkaline phosphatase (ALP) were measured in all patients. Bone mineral density (BMD) was measured using dual-energy X-ray absorptiometry (DXA). RESULTS: A total of 72 eligible patients participated (44 males, 28 females; age 54.2±11.7). Patients with CKD Stage 5 had a lower level of corrected serum calcium and significantly higher level of inorganic phosphorus, total ALP and iPTH as compared to stage 4 patients. Overall, 38.5% were hypocalcemic, 31.43% were hyperphosphatemic. 24.2% of CKD subjects were vitamin D deficient (<10ng/ml) and 41.4% having vitamin D insufficiency (10-20ng/ml). In stage 4, hyperparathyroidism (iPTH>110pg/ml) was detected in nearly 43% of patients. In stage 5, only 32% patients was found to have hyperparathyroidism (iPTH>300pg/ml). There was a good correlation between iPTH and total ALP (r=0.5, p=0.0001) in this cohort. 25 (OH) vitamin D was inversely correlated with ALP (r=-0.39, P=0.001) and showed negative correlation with urine ACR (r=-0.37, P=0.002). As a group, the osteoporotic CKD subjects exhibited higher iPTH (220.1±153.8 vs. 119±108pg/ml, p<0.05) as compared to those who were osteopenic or had normal bone density. There was significant correlation between BMD and iPTH (adjusted r=-0.436; P=0.001). In the multivariate regression model, we found intact PTH to predict BMD even after adjustment of all the confounders. CONCLUSION: The current study showed that adynamic bone disease is prevalent even in pre-dialysis CKD population. High bone turnover disease may not be the most prevalent type in diabetic CKD. However, it could contribute to the development of osteoporosis in CKD subjects. Serum total ALP can serve as a biochemical marker to identify pattern of bone turnover where intact PTH is not available.

Membranous nephropathy as a rare renal manifestation of IgG4-related disease.

IgG4-related disease, a newly described immune-mediated disorder with tissue infiltration of IgG4-positive plasma cells, has been reported in nearly every organ. In the kidney, it manifests as IgG4-related tubulointerstitial nephritis (TIN) but may also present as membranous nephropathy. We report a patient with IgG4 renal disease who had membranous nephropathy as well as TIN.

Depression and anxiety as potential correlates of post-transplantation renal function and quality of life.

The objective of this study was to determine anxiety and depression and its relationship with quality of life (QOL) in renal transplant (RT) recipients. A total of 105 consecutive patients were assessed cross-sectionally at least 3 months after RT. Hospital Anxiety and Depression Scale was applied to assess anxiety and depression. QOL was assessed through the abbreviated version of World Health Organization QOL scale. Patients' awareness of illness and treatment was assessed through Structured Interview for Renal Transplantation. Nine (8.57%) patients had syndromal anxiety and 9 (8.57%) had syndromal depression. Both these groups had significantly lower scores in almost all domains of QOL compared with their non-anxious and non-depressed counterparts. There were a higher number of hospitalizations and episodes of complication or rejection in post-RT patients with anxiety as compared to those without (P = 0.001). Syndromal depression and anxiety are associated with poor QOL and syndromal anxiety is associated with significantly higher number of hospitalizations, rejections and complications in post-RT patients.

Regulation of PECAM-1 in endothelial cells during cell growth and migration.

Endothelial cells (EC) that form the inner lining of blood vessels remain quiescent in the normal adult vasculature except during angiogenesis and reendothelialization, which result in EC proliferation and migration. EC placed in culture at subconfluent density also undergo cell multiplication and movement. This report demonstrates that whereas in confluent EC in a compact monolayer, the EC-EC adhesion molecule platelet-endothelial cell adhesion molecule-1 (PECAM-1) is strongly expressed at cell borders, little or no PECAM-1 immunostaining is detected in sparse or migrating cultured EC. Consistent with this observation, steady-state PECAM-1 mRNA expression was much lower in subconfluent EC than in confluent EC. The absence of PECAM-1 expression in sparse EC appeared not to be linked to ability to proliferate, since PECAM-1 expression remained low even in the presence of nitric oxide (NO) or mitomycin C, agents that inhibit EC growth. However, another growth-inhibitory agent, TGF-beta 1, did not alter PECAM-1 staining. Based on these observations, it is hypothesized that cell-associated mechanical forces underlying cell tensegrity regulate PECAM-1 expression.

Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

Growth regulation of cultured endothelial cells by inflammatory cytokines: mitogenic, anti-proliferative and cytotoxic effects.

The inflammatory cytokines tumor necrosis factor-alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) have angiogenic properties but generally inhibit cultured endothelial cell (EC) proliferation. Investigations into the growth-regulatory effects of these two agents on a variety of cultured EC types showed that they exert mitogenic, anti-proliferative or cytotoxic effects depending upon cell type and cytokine combinations. The anti-proliferative effect was distinct from cytotoxicity. Nitric oxide (NO) release from EC, examined as a potential mechanism underlying some of these effects, did not appear to mediate the anti-proliferative effects of these cytokines. However, NO also seemed to have a bimodal effect on EC proliferation depending upon whether the NO was endogenous or exogenous. These data underscore the diversity in cytokine and NO effects on cultured EC which, if reproducible in vivo, may be partly responsible for the variable and sometimes contradictory results obtained with regards to the role of inflammatory cytokines and NO on angiogenesis.

Inhibition of endothelial cell proliferation and bFGF-induced phenotypic modulation by nitric oxide.

S-nitroso-N-acetyl-D,L-acetylpenicillamine (SNAP), a chemical donor of NO, inhibited serum- and basic fibroblast growth factor (bFGF)-stimulated cultured endothelial cell (EC) proliferation in a dose-dependent manner. The inhibitory effect of NO was reversible after washoff of SNAP-containing media. Measurement of nitrate and nitrite in the media of SNAP-treated EC indicated that decomposition of SNAP into NO reached a stable level at or before 24 h; proliferation of EC was significantly inhibited for another 48 h and recovered thereafter if no additional SNAP was added. The level of NO produced by inhibitory concentrations of SNAP was comparable to NO levels produced by the induction of inducible nitric oxide synthase (iNOS) in smooth muscle cells or retinal pigmented epithelial cells. The growth-inhibitory effect of NO was unlikely to be due to cytotoxicity since 1) cells never completely lost their proliferative capacity even after 10 days of exposure to repeated additions of SNAP, 2) the inhibitory effect was reversible upon removal of NO and with the passage of time, and 3) NO did not reduce the number of cells that were growth-arrested with TGF-beta 1. In addition to its mitogenic effect, bFGF induced pronounced phenotypic changes, including suppression of contact inhibition, altered cell morphology, and scattering of the cells, in BPAEC cultures, whereas cells treated simultaneously with bFGF and NO did not exhibit these changes. These observations suggest that NO contributes to the regulation of angiogenesis and reendothelialization, processes that require EC proliferation, migration, and differentiation.

Comparison of normal and tumorigenic endothelial cells: differences in thrombospondin production and responses to transforming growth factor-beta.

Cultured endothelial cells constitutively synthesize significant levels of thrombospondin, an extracellular matrix-associated protein with reported anti-anti-angiogenic properties. However, two murine endothelial cell lines, bEND.3 and Py-4-1, which have been immortalized with polyoma T oncogenes and which generate vascular malformations in vivo, produce little or no thrombospondin though bEND.3 (but not Py-4-1) growth is inhibited by the addition of exogenous thrombospondin. In addition, Py-4-1 cells are not growth-inhibited by transforming growth factor-beta, a potent endothelial inhibitor. These results indicate that these two cell lines may be useful tools in understanding the role and mechanism of action of thrombospondin and transforming growth factor-beta in endothelial cell biology. A role for thrombospondin in vascular development is further suggested by the observation of significant differences in the levels of thrombospondin mRNA and protein between capillary and aortic endothelial cells. Transforming growth factor-beta-1 treatment of normal endothelial cells increases steady-state levels of thrombospondin mRNA and protein and results in extensive deposition of thrombospondin into the extracellular matrix. In contrast, transforming growth factor-beta-1 has little effect on thrombospondin levels in the tumorigenic endothelial cell lines. In view of our earlier finding that contact between endothelial cells and mural cells generates activated transforming growth factor-beta-1, and the fact that thrombospondin is present in a fibrillar network around vascular structures in vitro, we speculate that modulation of thrombospondin production and distribution by transforming growth factor-beta may be a physiological process to enjoin stabilization of vessels and cessation of vessel growth.

Endothelial cell regulation by transforming growth factor-beta.

Pronounced changes including growth inhibition, increased matrix deposition and suppression of cell-associated proteolytic activity, take place in endothelial cells (EC) upon the application of TGF-beta. Interrelationships between these effects have shed some light on the mechanism of action of TGF-beta and on its role in regulating EC function vis-a-vis angiogenesis. For instance, preliminary evidence has indicated that increased levels of certain matrix components may be partly responsible for the antiproliferative action of TGF-beta. In addition, TGF-beta and bFGF have opposing effects on cellular proteolytic balance which may contribute to the antagonistic effect that TGF-beta has on bFGF-induced EC growth and possibly to the anti-angiogenic effect exerted by TGF-beta under certain circumstances. Of particular interest in this regard is the fact that physical contact between EC and vascular mural cells in EC:mural cell cocultures has been found to generate active TGF-beta, thus further implicating TGF-beta in the maintenance of the quiescent, differentiated aggregation of EC as found in vascular structures in vivo. While more information is needed to define what, if any role TGF-beta plays in endothelial differentiation, it is to be noted that many of the cellular and biochemical processes affected by TGF-beta are linked to differentiation. It is therefore possible that the growth inhibition of EC by TGF-beta primes them for differentiation and/or is critical for the maintenance of a differentiated state.

A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.

Three tightly linked genes encoding human type I keratins: conservation of sequence in the 5'-untranslated leader and 5'-upstream regions of coexpressed keratin genes.

We have isolated and subcloned three separate segments of human DNA which share strong sequence homology with a previously sequenced gene encoding a type I keratin, K14 (50 kilodaltons). Restriction endonuclease mapping has demonstrated that these three genes are tightly linked chromosomally, whereas the K14 gene appears to be separate. As judged by positive hybridization-translation and Northern blot analyses, the central linked gene encodes a keratin, K17, which is expressed in abundance with K14 and two other type I keratins in cultured human epidermal cells. None of these other epidermal keratin mRNAs appears to be generated from the K17 gene through differential splicing of its transcript. The sequence of the K17 gene reveals striking homologies not only with the coding portions and intron positions of the K14 gene, but also with its 5'-noncoding and 5'-upstream sequences. These similarities may provide an important clue in elucidating the molecular mechanisms underlying the coexpression of the two genes.

Enteropathogenicity of Klebsiella pneumoniae strains isolated from stools of diarrhoeal patients and other clinical specimens: an experimental study.

In recent years strains of Klebsiella pneumoniae have been implicated as intestinal pathogen. Sixty three strains isolated from faeces of children with or without diarrhoea and other specimens of extraintestinal infections were tested for their enteropathogenicity in biological models. Thirty nine of them isolated from different sources caused accumulation of fluid in rabbit ileal loops comparable to that caused by toxigenic V. cholerae 569B. Ten strains, however, required 1-3 serial passages in rabbit gut before giving a positive loop reaction. Culture filtrates of these 39 strains also caused fluid accumulation comparable to that caused by live cells indicating elaboration of enterotoxic substance(s) in the medium. The enterotoxic factor was found to be heat-stable. However, the pattern of time course of fluid accumulation in ileal loops indicated elaboration of an additional heat labile factor. Most of the strains produced cytoxic factor as well. The mode of action of the toxin/s in secretion of fluid in experimental model possibly involve cAMP and prostaglandin. However, further studies are necessary to elucidate the exact mode of action of the enterotoxins. None of the strains tested was found to be enteroinvasive.