Renal allograft rejection: immunohistochemistry of inflammatory cellular subsets and vascular lesions

We have examined sixty-seven surgically removed allograft kidneys to identify the different leukocyte subsets of interstitial infiltration and the early vascular lesions which characterize renal allograft rejection. Histochemical and immunohistochemical results (mouse monoclonal antibodies anti: Leu 1, Leu 3a-3b, Leu 7, Leu 2a, OK Ia-Dr, OKB2, Leu M1, Leu M3; rabbit heteroclonal antibodies anti -: IgA, IgG, IgM, C3, fibrinogen, lysozyme; lectins-ABC: RCA, WGA, UEA) and routine histochemical staining have shown an increase of T-helper and T-activated lymphocyte subsets in acute rejection. Neutrophilic leucocytes were present in hyperacute rejection; macrophages were also noted. In chronic rejection, several lymphocyte subsets, in different ratios, were identified. Monocyte/macrophage leukocytes were the most abundant cell population. IgA deposits were noted on tubular epithelia in hyperacute and chronic rejections. IgM deposits were observed in vascular walls in chronic rejection. C3 and fibrinogen deposits were seen in glomerular capillaries and in arterial walls, although in different ratios, in all cases of renal allograft rejections. We have generally seen weak reactions to IgG deposits. Histochemical analysis of lectin receptors has given different results according to the type of rejection considered. In hyperacute rejection, receptors for WGA were found both on glomerular endothelial cells and on the tubular brush border. In the latter, receptors for RCA were also found. In acute rejection, receptors for UEA and WGA were found in a lower number of cases of acute vascular rejection. In acute cellular rejection, receptors for RCA, UEA and WGA were recognized in tubular epithelia. In acute vascular rejection, as well as in chronic rejection, only receptors for WGA were present on tubular epithelia and on capillary loop endothelium. The use of anti-human lysozyme antibodies has yielded the following results: in acute and hyperacute rejection, when renal failure occurred, we saw a high ratio of lysozyme, either coarsely granular or diffuse in the proximal tubular epithelia. Lysozyme was found in myelocyte/macrophage cells within capillary loops and arterial walls, when acute necrotizing vasculitis was present. In acute rejection, proximal tubular cells were lysozyme-negative or lysozyme-positive only segmentally, especially when obliterative vasculitis by fibrointimal proliferation was present and renal function progressively failed. In most of the chronic rejections, tubular cells were lysozyme-negative.     

Acute and chronic effects of chemically induced unilateral renal disease in rats.

Chemically induced unilateral renal disease was associated with a high incidence of proteinuria, diuresis, a morphological spectrum ranging from perinephritis to acute tubular or cortical necrosis, and unilateral or bilateral glomerular fibrinogen deposition during the first 2 wk after induction. Later, a decrease in proteinuria and return to normal urine output was not infrequently followed by recurrent proteinuria, hypergammaglobulinemia, morphological alterations, and deposition of IgG and beta1C on the glomerular basement membranes and mesangium of the contralateral kidney and the treated kidney. Intercapillary deposition of fibrinogen in association with IgG and beta1C was occasionally observed in one or both kidneys. The morphologic, immunohistologic, serologic, and chemical findings suggest that this model may be useful for further defining the course and prognosis of unilateral renal disease produced by vascular insufficiency.     

The morphology of allograft rejection in Styela plicata (Urochordata: Ascidiacea)

Summary This study identifies three discrete processes responsible for the rejection of tunic tissue transplanted between individuals of the solitary ascidian Styela plicata. The first stage of rejection is characterized by the destruction of blood vascular components within incompatible allografts. In the second phase, dense boundaries of extracellular material are deposited between grafts and the surrounding host tunic, effectively amputating the transplanted tissues. Finally, detached transplants undergo a gradual necrosis which results in the total degeneration of extracellular graft matrices. Of these three phases, the initial cellular depletion of allografts is responsible for the immunological specificity that is characteristic of histocompatibility in S. plicata. The subsequent amputation and necrosis of extracellular graft matrices are taken to be non-specific consequences of the initial cellular reactivity.     

Characterisation of cellular infiltration and adhesion molecule expression in CBA/CaH-kdkd mice with tubulointerstitial renal disease

 CBA/CaH-kdkd mice develop a spontaneous and chronic tubulointerstitial renal disease which is characterised by mononuclear cell infiltration, tubular collapse and cystic dilatation of tubules. The pathogenic mechanisms of renal injury have not been fully elucidated in this model. We have analysed the nature of infiltrating cells and the expression of MHC class II antigens, cytokines and adhesion molecules in CBA/CaH-kdkd kidneys at various disease stages. Using immunohistochemical techniques we found that kdkd kidneys are characterised by abundant macrophage and dendritic cell infiltration with fewer T cells with CD4+ and CD8+ phenotypes. Interestingly, MHC class II antigens were not induced on renal tubules. The proinflammatory cytokine, TNF-α, was markedly enhanced in kdkd kidney (up to fourfold), whereas the T cell-specific cytokine, IFN-γ, increased less (less than twofold). ICAM-1 and VCAM-1 were markedly overexpressed by injured proximal tubules. ICAM-2 and PECAM-1 were constitutively expressed on glomerular capillaries and vascular endothelium in normal kidneys and did not change in CBA/CaH-kdkd mice. In conclusion, tubulointerstitial nephritis in CBA/CaH-kdkd mice is characterised by prominent macrophage infiltration and abundant expression of ICAM-1 and VCAM-1 on injured renal tubules. The lack of MHC class II antigens on injured tubules suggests that the kd gene defect could generate a secondary renal inflammatory response which is characterised by prominent macrophage infiltration and a relative scarcity of T cells. Accepted: 10 June 1997     

Oxalosis associated with an Aspergillus niger fungus ball. Report of a case

During a seventeen day period an A. niger fungus ball evolved within a healed tuberculous cavity of a patient. Symptoms were a cough with a chocolate brown expectoration and dyspnea. The patient died and necropsy was performed. Crystals of calcium oxalate were deposited in the cavity lining and in the adjacent tissue of the lung. Fibrosis, mononuclear infiltration and intraalveolar purulent exudate were seen in these tissues. Some small vessels presented recent thrombosis and deposition of calcium oxalate. The bronchus connected with the cavity presented a disrupted epithelial layer, edema, polymorphonuclear infiltration and birefringent crystals. Scattered areas of tubular atrophy, glomerular sclerosis and lymphoid infiltration were seen in the cortex of the kidney. Oxalate crystals were also seen within the renal tubuli.Bolsistas do CNPq (Brazil).     

Immunogenicity of allograft components: II. Relative immunogenicity of rat kidney parenchymal versus “passenger” cells

Well-perfused adult DA kidneys were enzymatically dispersed under conditions which do not affect the expression of cell surface major histocompatibility antigens. The kidney cell suspensions were separated via sedimentation at unit gravity into three fractions: I, rapidly sedimenting (>6.5 mm/hr) enriched for kidney tubular and glomerular cells and depleted of passenger leukocytes (76 and 8%, respectively); II, intermediate (5.1–6.0 mm/hr) mixed population equivalent to the unseparated kidney cell suspension (52% tubular and glomerular cells, 20% endothelial cells, and 28% passenger leukocytes); and III, slow sedimenting (<5.0 mm/hr) enriched for passenger leukocytes (63%). The three isolated fractions were analyzed for their ability to accelerate allograft rejection in the “primed heart rejection assay.” The cells in fraction I were unable to reduce heart allograft survival, while the cells from fraction III reduced it significantly. Cells from fraction II were intermediary effective. The results are in agreement with the hypothesis that the urine-producing apparatus of rat kidney is relatively nonimmunogenic, while the main stimulus for graft rejection is provided by the “passenger” cell component.     

Protective effect of Tribulus terrestris linn on liver and kidney in cadmium intoxicated rats

Administration of cadmium (Cd) significantly increased the peroxidation markers such as malondialdehyde and protein carbonyls along with significant decrease in antioxidant markers such as super oxide dismutase and reduced glutathione in liver and kidney tissues. Cadmium also caused a significant alteration in hepatic and renal functional markers in serum viz. total protein, albumin, alanine transaminase, blood urea nitrogen and creatinine. Prominent pathological changes observed in liver were severe vascular and sinusoidal congestion with diffuse degenerative changes and mononuclear infiltration into peripheral areas, while the kidney showed vascular and glomerular congestion, cloudy swelling of tubular epithelium. Coadministration of ethonolic extract of T. terrestris or vitamin E along with Cd significantly reversed the Cd induced changes along with significant reduction in Cd load.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency. Morphological, enzyme histochemical and immunohistochemical studies

An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were accompanied by hematuria and tubular necroses including and collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.     

In vivo effects of monoclonal antibody to ICAM-1 (CD54) in nonhuman primates with renal allografts

These studies test whether allograft rejection can be blocked by interference with leukocyte adhesion, using a murine IgG2a mAb (R6.5) reactive with monkey ICAM-1 (CD54). In 16 Cynomolgus renal allograft recipients, R6.5 was administered prophylactically as the sole immunosuppressive agent for 12 days (0.01 to 2 mg/kg/day). Survival in 14 recipients with technically successful grafts was significantly prolonged (24.2 +/- 2.4 vs 9.2 +/- 0.6 days for controls; p less than 0.001). Intercellular adhesion molecule-1 (CD54) (ICAM-1) was expressed on vascular endothelium in the kidney and other organs in the monkey in a pattern similar to that in humans. During cellular rejection in controls, ICAM-1 expression increased on endothelial cells, infiltrating mononuclear leukocytes and tubular cells. Biopsies during R6.5 administration showed decreased T cell infiltration (CD2, CD8, CD4) compared with controls and decreased arterial endothelial inflammation. No changes occurred in circulating T cells, aside from variable coating with mIgG. In six of eight other recipients R6.5 administration (0.5 to 2 mg/kg/day for 10 days) reversed preexisting rejection that resulted from taper of Cyclosporine to subtherapeutic levels. Responding grafts showed decreased edema and hemorrhage but no consistent change in the infiltrate. At 1 h after the first dose, mouse IgG deposited primarily on the graft vascular endothelium without any change in the inflammatory infiltrate. Mouse IgG also deposited on the endothelium of normal organs without eliciting an inflammatory response and was cleared from the endothelium within 4 days. Inasmuch as the principal site of binding was the vascular endothelium, we hypothesize that the antibody blocks adhesion to graft ICAM-1 molecules on the vessels. Anti-ICAM-1 also binds to recipient cells and may interfere with Ag presentation and/or T cell interactions. Whatever the mechanism(s), these studies indicate that an anti-ICAM-1 antibody inhibits T cell mediated injury in vivo, and that ICAM-1 is a critical molecule in the pathogenesis of allograft rejection.     

The role of cyclosporin in prolonging survival in vascularized bone allografts

It is known that experimental vascularized bone allografts are subject to host rejection. To be useful clinically, this rejection response would need to be controlled. Cyclosporin is a potent immunosuppressant whose precise role in vascularized bone allograft transplantation has not been established. Using a proven reliable vascularized knee allograft model in inbred rats, cyclosporin was used postoperatively both continuously and short term (14 days after transplant) at 10 mg/kg per day as recipient treatment. Across a strong histocompatibility barrier, continuous cyclosporin was required for long-term graft survival. Short-term therapy delayed rejection for 4 to 6 weeks. However, across a weak histocompatibility barrier, short-term therapy was as effective as continuous therapy in achieving long-term graft survival. The implication is that a limited course of cyclosporin may be clinically successful in sustaining vascularized bone allograft survival, provided the genetic disparity between graft and host has been minimized by genetic matching techniques.     

A light microscopic study of the mononuclear cells infiltrating skin homografts in the garter snake, Thamnophis sirtalis (Reptilia: Colubridae)

The rejection of skin homografts in the snake, Thamnophis sirtalis is preceded by an infiltration of mononuclear cells into the graft bed. The initial arrangement of infiltrating cells in perivascular halos suggests that these cells emigrate from the blood stream of the host. A cytological study showed that the vast majority of the cells can be classified as small and mediumsized lymphocytes, monocytes and macrophages. Early stages of infiltration were associated with large proportions of lymphocytes while later stages were characterized by a predominance of macrophages. It was concluded that the mononuclear cells associated with graft rejection include large proportions of lymphocytes and macrophages and not just one kind of lymphoid cell.     

IFN-gamma alters the pathology of graft rejection: protection from early necrosis

We studied the effect of host IFN-gamma on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-gamma genes) IFN-gamma genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-gamma (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-gamma to GKO hosts reduced but did not fully prevent the effects of IFN-gamma deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-gamma has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-gamma produced in large quantities in the allograft.     

The Sequence of Events in the Development of Bilateral Renal Cortical Necrosis Accompanying the Generalized Shwartzman Reaction

The generalized Shwartzman reaction, or Shwartzman-like conditions, were induced in a variety of experimental mammalian species by systemic injections of disintegrated cells of Gram negative bacteria, live Salmonella cholerae-suis or Liquoid. A comparative study of the renal lesions showed that the initial step in the development of bilateral cortical necrosis is stagnation and disintegration of red cells in glomerular capillaries. The glomerular “microthrombi” consist mainly of erythrocytic debris, which frequently has staining properties akin to those of fibrin; even wide-spread glomerular “thrombosis” is not accompanied by obvious destruction of renal parenchyma. A second step is necrotic mural lesions in afferent arteries, with ensuing thrombosis. These vascular lesions lead to the formation of individual infarcts which fuse to form total bilateral cortical necrosis in fulminant cases of the generalized Shwartzman reaction.     

Kidney atrophy vs hypertrophy in diabetes: which cells are involved?

One of the first structural changes in diabetic nephropathy (DN) is the renal enlargement. These changes resulted in renal hypertrophy in both glomerular and tubular cells. Shrink in the kidney size, which described as kidney atrophy resulted from the loss of nephrons or abnormal nephron function and lead to loss of the kidney function. On the other hand, increase in kidney size, which described as hypertrophy resulted from increase in proximal tubular epithelial and glomerular cells size. However overtime, tubular atrophy and tubulointerstitial fibrosis occurs as subsequent changes in tubular cell hypertrophy, which is associated with the infiltration of fibroblast cells into the tubulointerstitial space. The rate of deterioration of kidney function shows a strong correlation with the degree of tubulointerstitial fibrosis. A consequence of long-standing diabetes/hyperglycemia may lead to major changes in renal structure that occur but not specific only to nephropathy. Identifying type of cells that involves in renal atrophy and hypertrophy may help to find a therapeutic target to treat diabetic nephropathy. In summary, the early changes in diabetic kidney are mainly includes the increase in tubular basement membrane thickening which lead to renal hypertrophy. On the other hand, only renal tubule is subjected to apoptosis, which is one of the characteristic morphologic changes in diabetic kidney to form tubular atrophy at the late stage of diabetes.     

Predilection of Segmental Glomerulosclerosis Lesions for the Glomerulotubular Junction Area in Type 1 Diabetic Patients: A Novel Mapping Method

The location of segmental glomerular lesions in relation to the vascular or tubular pole may have diagnostic or prognostic significance. We have developed a model-based method to estimate the distance from a glomerular lesion to a given landmark (vascular or tubular pole) or the glomerular center and applied this to biopsies from 5 microalbuminuric, 5 normoalbuminuric and 7 proteinuric type 1 diabetic patients and 5 normal controls. The distance from each glomerular adhesion to the glomerulotubular junction was measured and divided by the glomerular radius, allowing comparability among different glomeruli, assuming a spherical shape for Bowman''s capsule, an assumption which was validated. The frequency of adhesions in 6 glomerular zones with equal height (zone I adjacent to the glomerulotubular junction and zones II–VI progressively farther away) was determined: 59% of adhesions were in zone I, 15% in zone II, 16% in zone III, 7% in zone IV and 3% in zone VI (adjacent to the hilus). In glomeruli with only one adhesion, 82% of these were in zone I. This new method accurately localizes segmental lesions within glomeruli and revealed a marked predilection in type 1 diabetic patients for segmental sclerosis to develop at the glomerulotubular junction.     

Study of Cynomolgus monkey (Macaca fascicularis) MhcDRB (Mafa-DRB) polymorphism in two populations

Cynomolgus monkey is one of the macaque species currently used as an animal model for experimental surgery and medicine, in particular, to experiment new drugs or therapy protocols designed for the prevention of allograft rejection. In this field, it is of utmost importance to select histoincompatible recipient–donor pairs. One way to ensure incompatibility between donor and recipient is to check their major histocompatibility complex (MHC) genotypes at the loci playing a determinant role in histocompatibility. We report in this paper on the cynomolgus monkey DRB polymorphism evidenced by sequencing of amplified exon 2 separated either by denaturing gradient gel electrophoresis (DGGE), or by cloning. By the study of 253 unrelated animals from two populations (Mauritius and The Philippines), we characterized 50 exon 2 sequences among which 28 were identical to sequences already reported in Macaca fascicularis or other macaque species (Macaca mulatta, Macaca nemestrina). By cloning and sequencing DRB cDNA, we revealed two additional DRB alleles. Out of the 20 haplotypes that we defined here, only two were found in both populations. The functional impact of DR incompatibility was studied in vitro by mixed lymphocyte culture.     

Enhancement of maternal and fetal nephrotoxicity of salicylate by zinc deficiency

Summary An oral dose of 700 mg/kg salicylic acid was given to normal and Zn-deficient rats at day 16 of gestation. Maternal and fetal kidneys were studied at day 19 of gestation. Zn-deficiency did not cause any lesions detectable by semi-thin section light microscopy, electron microscopy, enzyme histochemistry and immunohistochemistry. Salicylate may lead only to small morphological, enzymatic and cytoskeletal defects in the maternal and fetal kidney. However, enzyme activities decreased in plasma membranes, mitochondria, lysosomes, endoplasmic reticulum and peroxisomes in all segments of the tubular apparatus when salicylate was given to Zn-deficient rats. Cytoskeletal proteins such as keratin in the glomerular cells and epithelial lining of the collecting ducts and vimentin in vascular endothelial cells of the maternal kidney were also affected. In addition, the epithelial cells of the collecting ducts, which were comparatively less damaged, accumulated high amounts of fat. In severe cases, the enzymatic and cytoskeletal lesions were aecompanied by hematuria and tubular necroses including the collecting ducts in the renal papilla. In less severe cases reduced activities of brush border hydrolases were the only sign of disturbed renal function in maternal rats indicating that membrane alteration and loss of membrane-bound enzymes are the primary defects. In the fetal kidneys, mitotic activity of the cells of the nephron anlagen and collecting ducts was reduced and enzymatic and morphological differentiation were disturbed. As a consequence less mature nephrons and collecting ducts occurred.Dedicated to Professor Zdenek Lojda on the occasion of his 60th birthdaySupported by the German Research Foundation (Sfb 174)     

Spontaneous lesions in beagle dogs used in toxicity studies

Spontaneous histopathologic lesions were examined in 276 young beagle dogs (8 to 15 months old) used in toxicity studies. Mononuclear cell infiltration in the liver was observed with the highest incidence (more than 95%). Calcium deposition in the kidney, mononuclear cell infiltration in the salivary glands, pigmentation in the spleen, and cortical atrophy in the thymus were seen with relatively high incidences (more than 30%). The incidence of mononuclear cell infiltration in the gallbladder, vacuolization of the tubular epithelium in the kidney, and vacuolization of the zona glomerulosa cell in the adrenal gland showed differences between the sexes. No abnormalities were seen in the spinal cord, sternum, and femur. Although most lesions observed are considered to be age-related, several were considered to be due to congenital anomalies observed in some organs.     

Mouse Models for Graft Arteriosclerosis

Graft arteriosclerois (GA), also called allograft vasculopathy, is a pathologic lesion that develops over months to years in transplanted organs characterized by diffuse, circumferential stenosis of the entire graft vascular tree. The most critical component of GA pathogenesis is the proliferation of smooth muscle-like cells within the intima. When a human coronary artery segment is interposed into the infra-renal aortae of immunodeficient mice, the intimas could be expand in response to adoptively transferred human T cells allogeneic to the artery donor or exogenous human IFN-γ in the absence of human T cells. Interposition of a mouse aorta from one strain into another mouse strain recipient is limited as a model for chronic rejection in humans because the acute cell-mediated rejection response in this mouse model completely eliminates all donor-derived vascular cells from the graft within two-three weeks. We have recently developed two new mouse models to circumvent these problems. The first model involves interposition of a vessel segment from a male mouse into a female recipient of the same inbred strain (C57BL/6J). Graft rejection in this case is directed only against minor histocompatibility antigens encoded by the Y chromosome (present in the male but not the female) and the rejection response that ensues is sufficiently indolent to preserve donor-derived smooth muscle cells for several weeks. The second model involves interposing an artery segment from a wild type C57BL/6J mouse donor into a host mouse of the same strain and gender that lacks the receptor for IFN-γ followed by administration of mouse IFN-γ (delivered via infection of the mouse liver with an adenoviral vector. There is no rejection in this case as both donor and recipient mice are of the same strain and gender but donor smooth muscle cells proliferate in response to the cytokine while host-derived cells, lacking receptor for this cytokine, are unresponsive. By backcrossing additional genetic changes into the vessel donor, both models can be used to assess the effect of specific genes on GA progression. Here, we describe detailed protocols for our mouse GA models.     

Kidney transplantation between congenic versus standard inbred strains of rats: I The significance ofH-1 and non-H-1 gene differences

Concepts of “antigenic strength” in organ transplantation have been evaluated in relation to orthotopic kidney allografts inH-1 congenic strains of rats. Untreated recipients reject fully allogeneic kidneys possessing singleH-1 differences as acutely as kidneys displaying multiple histocompatibility differences. Heterozygozity for H-1 specificities as well as for H-1 plus non-H-1 specificities (semiallogeneic kidneys) favors long term survival (autoenhancement), especially when the specific immune response genes of the recipient lead to reduced reactivity. In active enhancement, the transplantedH-1 congenic kidneys, devoid of additional weak antigens, retain prolonged functional integrity. Weak non-H-1 antigens substantially influence the successful establishment of specific enhancement in an adverse way, either as additive immunogens or as target sites for the effector arm of the rejection response.