Conversion of extracellular ATP into adenosine: a master switch in renal health and disease.

ATP and its ultimate degradation product adenosine are potent extracellular signalling molecules that elicit a variety of pathophysiological functions in the kidney through the activation of P2 and P1 purinergic receptors, respectively. Extracellular purines can modulate immune responses, balancing inflammatory processes and immunosuppression; indeed, alterations in extracellular nucleotide and adenosine signalling determine outcomes of inflammation and healing processes. The functional activities of ectonucleotidases such as CD39 and CD73, which hydrolyse pro-inflammatory ATP to generate immunosuppressive adenosine, are therefore pivotal in acute inflammation. Protracted inflammation may result in aberrant adenosinergic signalling, which serves to sustain inflammasome activation and worsen fibrotic reactions. Alterations in the expression of ectonucleotidases on various immune cells, such as regulatory T cells and macrophages, as well as components of the renal vasculature, control purinergic receptor-mediated effects on target tissues within the kidney. The role of CD39 as a rheostat that can have an impact on purinergic signalling in both acute and chronic inflammation is increasingly supported by the literature, as detailed in this Review. Better understanding of these purinergic processes and development of novel drugs targeting these pathways could lead to effective therapies for the management of acute and chronic kidney disease.

Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: methodological pitfalls and their solution.

Pimonidazole adduct immunohistochemistry is one of the few available methods for assessing renal tissue hypoxia at the cellular level. It appears to be prone to artifactual false positive staining under some circumstances. Here, we assessed the nature of this false positive staining and, having determined how to avoid it, reexamined the nature of cellular hypoxia in rat models of kidney disease. When a mouse-derived anti-pimonidazole primary antibody was used, two types of staining were observed. First, there was diffuse staining of the cytoplasm of tubular epithelial cells, which was largely absent when the primary antibody was omitted from the incubation protocol or in tissues known not to contain pimonidazole adducts. Second, there was staining of the apical membranes of tubular epithelial cells, debris within the lumen of renal tubules, including tubular casts, and the interstitium; this latter staining was present even when the primary antibody was omitted from the incubation protocol. Such false positive staining was particularly prominent in acutely injured kidneys. It could not be avoided by preincubation of sections with a mouse IgG blocking reagent. Furthermore, preadsorption of the secondary antibody against rat Ig abolished all staining; however, when a rabbit-derived polyclonal anti-pimonidazole primary antibody was used, the false positive staining was largely avoided. Using this method, we confirmed the presence of hypoxia, localized mainly to the tubular epithelium, in the acute phase of severe renal ischemia-reperfusion injury, adenine-induced chronic kidney disease, and polycystic kidney disease. We conclude that this new method provides improved detection of renal cellular hypoxia.

Epitope-specificities of HLA antibodies: the effect of epitope structure on Luminex technique-dependent antibody reactivity.

The search of HLA antibodies is currently more accessible by solid-phase techniques (Luminex) in the immunized patients leading to an expansion of the antibody patterns. The aim of this study was to investigate low median fluorescence intensity value in unexpected reactivity patterns. Here, we performed HLAMatchmaker analyses to evaluate the potential functional epitopes that can elicit HLA-specific alloantibody responses in a pregnancy-sensitized woman with an epitope defined by the 82LR. Surprisingly, in according to the registry of HLA epitopes, we found that 82LR epitope covered all allelic specificities of our unexpected antibody patterns, shared between Bw4-positive HLA-B antigen and HLA-A23, -A24, -A25 and -A32. This finding is consistent with the verification of HLA ABC epitope recorded in the website-based HLA Epitope Registry and addresses the importance of determining HLA antibody epitope-specificities on Luminex technique-dependent antibody reactivity.

HLA incompatible combined liver-kidney transplantation: dynamics of antibody modulation revealed by a novel approach to HLA antibody characterisation.

This case report confirms the utility of simultaneous liver transplantation in allowing successful kidney transplantation in the face of preformed, high levels of DSA, which would under normal circumstances be associated with hyperacute rejection and kidney graft failure. Antibody characterisation in terms of epitope specificity is more accurate and informative than antibodies described as "antigen-specific" and we suggest a method for identifying and tracking these antibodies; i.e. follow the epitope reaction not the antigen reactions. We consider that this will give a better insight into the behaviour and pathogenicity of HLA-specific sera. In the case presented here this approach has revealed some novel features of the post transplant antibody response in a sensitised recipient. These illustrate three phenomena which challenge current dogmas; an early resynthesis of DSA does not necessarily cause AMR, high levels of DSA can spontaneously modulate, and measurement of antibodies in terms of antigen specificity can give misleading results.

Coordinate regulation of 11 beta-HSD and Ke 6 genes in cpk mouse: implications for steroid metabolic defect in PKD.

Polycystic kidney disease (PKD) is characterized by multiple renal cysts, which ultimately result in renal failure. We have reported the identification of a new gene, Ke 6, within the major histocompatibility complex, which is downregulated in two different mouse models of heritable PKD (N. Aziz, M. Maxwell, B. St.-Jacques, and B.M. Brenner. Mol. Cell. Biol. 13: 1847-1853, 1993). The Ke 6 gene gives rise to two transcripts, Ke 6a and Ke 6b. Ke 6a protein has significant homology to several mammalian and bacterial steroid dehydrogenases. The homology of Ke 6a protein to specific functional domains of the human and rat 11 beta-hydroxysteroid dehydrogenase enzyme (11 beta-HSD), which inactivates glucocorticoids, is substantial. We report here that the Ke 6 gene and the 11 beta-HSD gene are regulated in the same aberrant pattern in the cpk mouse. The strong evidence for a critical role of steroids in cystogenesis leads us to propose that a possible elevation of intrahepatic and intrarenal steroid levels occurs in the cpk mouse as a result of repression of steroid metabolic enzymes, which ultimately leads to development of cysts.

Specific changes in the basement membrane of the proximal tubules in the murine polycystic kidney detected by the novel anti-basement membrane monoclonal antibody D28.

In order to analyze renal abnormalities in mice with polycystic kidney disease (PKD), we produced a series of monoclonal antibodies reactive with the murine kidney by hybridizing P3U1 myeloma cells with spleen cells from DBA/2 mice immunized with the kidney of adult-type PKD mice, DBA/2FG-pcy. One clone, D28, reacted specifically with the basement membrane of the proximal tubules of DBA/2 mice and DBA/2FG-pcy mice. It did not react with other parts of the murine kidney nor with other tissues such as the skin, ovary, fallopian tube, testis, lung and small intestine. While other components such as collagen IV, laminin and the core protein of proteoglycan could be found, the D28 epitope could not be found in the basement membrane of renal cysts formed in adult-type (DBA/2FG-pcy) and infant-type (C57BL/6J-cpk) PKD mice. The D28 epitope did not, however, disappear from the basement membrane of proximal tubules in other types of renal abnormalities. These results suggest that the formation of renal cysts in the proximal tubules is associated with an alteration to the proximal tubule-specific structure of the basement membrane. The D28 monoclonal antibody should prove a useful tool with which to analyze basement membrane-associated abnormalities in genetic PKD.

Characterization of the anti-A antibody binding in an ABO-incompatible living donor renal transplantation.

A 57-year-old female, blood group B, with polycystic kidney disease, received an ABO-identical, HLA-A,B,DR 5-mismatched renal allograft in 1986. Due to graft artery thrombosis and vascular rejection, she lost the kidney 6 months after transplantation and developed HLA antibodies with a panel reactivity of 99%. Despite 5 years on a European waiting list for highly immunized patients, she was not offered a second kidney. An attempt to remove her HLA antibodies by plasmapheresis combined with cyclophosphamide therapy did not succeed. Her 53-year-old HLA-identical, but ABO-incompatible sister (blood group A1), was then accepted as a donor. After immunoadsorption on Biosynsorb-A columns, transplantation was performed. The post-transplant course was uneventful without any signs of rejection. Studies on the anti-A antibody binding characteristics before and after immunoadsorption and after transplantation, showed that IgM and IgG antibodies recognizing the A trisaccharide epitope based on the type 1, 2, and 4 core saccharide chains, were effectively removed by Biosynsorb-A adsorption, but the column failed to remove anti-A antibodies recognizing the A type 3 antigen. These antibodies probably requires part of the core saccharide chain for binding. The presence of these antibodies did not seem to influence the outcome of the ABO-incompatible transplantation.

Panlobar nephroblastomatosis with cystic dysplasia: an unusual case with diffuse renal involvement studied by immunohistochemistry.

A unilateral cystic renal process discovered prenatally was removed in a neonate. Dysplastic cysts were associated with diffuse (both intra- and perilobar) nephroblastomatosis. We describe a comprehensive immunohistological study confirming the transition observed on simple histology between the different structures: nephrogenic rests (CD9+, CD24+/-, CD56+/-), glomeruloid bodies (CD10++, CD35++), ducts lined by columnar epithelium (CD9+, CD24+, CD56++), cysts lined by cuboidal or thin epithelium (some cells CD10+, CD26+, others EMA+, CD24+). Although no typical S-shaped bodies are seen, small cysts and ducts with a columnar epithelium are considered similar. The dysplastic primitive ducts are KL1++, vimentin+/-, CD9+, CD24+. With a view to assessing dysplastic preneoplastic potential, the value of CD56 and Ki67 as activation antigens with possible prognostic significance is discussed.

Basement membrane antigens in renal polycystic disease.

Status of basement membrane antigens in renal polycystic disease was investigated. Antibodies directed against various components of basement membrane, including anti-heparan sulfate proteoglycan, Type IV collagen, laminin, and fibronectin, were employed. Their reactivities with basement membranes of normal and cystic segments of the renal tubules were ascertained by indirect immunofluorescence. The tissues were obtained either from kidneys of patients with adult (autosomal dominant) polycystic disease or from rats with renal cystic change induced by administration of 2-amino-4,5-diphenylthiazole HCl. The human and rat tissues that had undergone cystic change exhibited essentially similar results. A loss of reactivity to anti-heparan sulfate proteoglycan antibodies was observed. The reactivity toward anti-Type IV collagen and laminin either remained unchanged or was focally increased. The reactivity toward fibronectin, normally absent, increased dramatically in the peritubular regions and interstitium. The results indicate that there is an imbalance in various antigenic components associated with renal tubular cystic disease in rat and man, which may have a fundamental role in the pathogenesis of this disorder.

Glomerulosclerosis and renal cysts in mice transgenic for the early region of SV40.

Considerable evidence indicates that genetic determinants play a major role in the pathogenesis of a variety of human and experimentally-induced renal diseases. There are, however, no firm data to indicate which genes or types of genes can induce or promote renal disease. The recently acquired ability to make specific alterations in the genetic background of an animal affords a unique opportunity to assess the effect(s) of a given gene on the structure and function of an organ of interest. Such modifications have been carried out in the creation of transgenic mice. We examined mice transgenic for the transforming gene encoding large T-antigen which is present in the early region of simian virus 40 (SV40). Renal lesions were present in most animals. While there was some heterogeneity in the type and severity of the renal lesions observed, a majority of the older mice displayed glomerulosclerosis and/or proliferative tubular lesions which in some were associated with multiple, large tubular cysts. The appearance of these lesions in mice transgenic for a transforming gene suggests that renal expression of a gene which controls cell proliferation may be associated with the development of glomerulosclerosis and renal cysts. These findings indicate a possible role for other transforming genes, or oncogenes, in the pathogenesis of glomerulosclerosis and cystic renal disease in humans and other animal models.

Increased incidence of sensitization among patients with polycystic kidney disease following pretransplant blood transfusions.

Following pretreatment with at least 5 blood transfusions to 58 nontransfused uremic patients, 23 (40%) formed lymphocytotoxic antibodies against B cells and 9 (15%) against T cells as well. Significantly more (p less than 0.01) patients with polycystic kidney disease (6/16) formed T cell antibodies compared to patients with other diseases. The presence of antibodies delayed kidney transplantation, since significantly more (p less than 0.01) patients without antibodies (28 out of 35) received kidney grafts than patients with antibodies (9 out of 23). 5 patients received kidney grafts despite the occurrence of antibodies against donor B cells, but 3 of the patients lost their grafts within 1 month. In vitro lymphocyte subpopulations were studied in 4 patients before and after each of the planned blood transfusions. No persistent changes in lymphocyte responses to phytohemagglutinin and in mixed lymphocyte culture were seen. T cell subpopulations identified by monoclonal antibodies were unchanged, but the proportion of macrophages/monocytes (OKMl-positive cells) increased from 22 +/- 6 to 46 +/- 10% (p less than 0.05).

Increased vulnerability of the donor organ in related kidney transplants for certain diseases.

The one-year kidney transplant survival rates from parental donors into recipients with pyelonephritis (PN) was 79% as compared with the low rate of 62% for polycystic disease (PC) and diabetes mellitus (DM). Even more striking was the 42% one-year graft survival in systemic lupus erythematosus (SLE) patients receiving parental donor grafts. HLA-identical sibling donor transplants into patients with DM had a low survival rate of 75% as compared with 90% in PN patients. These results were analyzed for interactions of donor type and disease by comparing the relative survival rates among types of donors within each recipient disease. After taking into account higher overall risks attributable to medical complications inherent in the different disease categories, related donor grafts into patients with PC, SLE, and DM have lower graft survival rates than would be expected from differences in cadaver donor rates by disease. In practical terms, for related donor transplants into patients with SLE, DM, and PC, it may be necessary to consider the vulnerability of the donor organ as another factor.

Absence of linkage between adult polycystic kidney disease and the major histocompatibility system.

Four multiplex families affected with adult polycystic kidney disease were investigated for segregation of the disease with haplotypes bearing HLA-A and B antigens and Bf allotypes. In no case did the disease travel with specific haplotypes within families showing an absence of linkage between the disease and the major histocompatability system. In addition, the disease was not associated with any HLA-A or B antigen or Bf allotype either within or among the families studied.

Anti-HLA immunisation in 130 haemodialysed patients.

Anti-HLA immunisation has been studied in 130 patients treated by haemodialysis between 1969 and 1978. Until 1977 blood transfusions were restricted (number of transfusions per patient averaged 3.42 units). However 50% of the patients were found to be immunised. The frequency of immunisation was higher in women patients (63%) than in men (39.7%). This difference shows a good correlation with the frequency of pregnancies (r = 0.849). The percentage of immunisation increases in parallel with the number of months of dialysis (r = 0.95). The capacity to eliminate HBS Ag seems to be related to the capacity for anti-HLA immunisation: 64% of the patients transiently positive for HBS antigen develop anti-HLA antibodies and only 26.7% when antigenaemia is persistent. Since 1977, 24 patients have been transfused with two units of whole blood once a week for three weeks, every six months. Antibodies appeared in only seven of them who had been transfused some years ago. The GLA system and transplantation play a small part. The age and the type of nephropathy seem to have no effect. A few patients developed antibodies for no apparent reason. Possibly bacterial or viral infections, or venous allografts were responsible.