Expression of silencer of death domains and death-receptor-3 in normal human kidney and in rejecting renal transplants

We have previously reported the pattern of cellular expression of tumor necrosis factor receptors (TNFR) in human kidney and their altered expression in transplant rejection. We have extended our studies to examine the expression of Silencer of Death Domains (SODD), a protein that binds to the cytoplasmic portion of TNFR1 to inhibit signaling in the absence of ligand. In normal human kidney SODD is expressed in glomerular endothelial cells where it colocalizes with TNFR1. During acute rejection both SODD and TNFR1 are lost from glomeruli, but we found strong expression of SODD on the luminal surface of tubular epithelial cells. This occurs in the absence of detectable TNFR1 expression, suggesting that SODD could interact with other proteins at these sites. Several other members of the TNF superfamily, including Fas and death receptors (DR)-3, -4, and -5, also contain intracellular death domains, but SODD only interacts with the death domain of DR3. We therefore studied the expression of DR3 in human kidney, and report that this death receptor is up-regulated in renal tubular epithelial cells and endothelial cells of some interlobular arteries, in parallel with SODD, during acute transplant rejection. In less severe rejection episodes, DR3 and SODD were more focally induced, generally at sites of mononuclear cell infiltrates. In ischemic allografts, eg, with acute tubular necrosis but no cellular rejection, DR3 was induced on tubular epithelial cells and on glomerular endothelial cells. These data confirm that TNF receptor family members are expressed in a regulated manner during renal transplant rejection, and identify DR3 as a potential inducible mediator of tubular inflammation and injury.     

Direct acute tubular damage contributes to Shigatoxin‐mediated kidney failure

The pathogenesis and therapy of Shigatoxin 2 (Stx2)‐mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2‐producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2‐receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild‐type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule‐specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2‐associated kidney failure (n = 11, aged 22–44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement‐inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31–17.60) mg/dl, lactate dehydrogenase 1944 (753–2792) U/l, platelets 33 (19–124)/nl and haemoglobin 6.2 (5.2–7.8) g/dl; median (range)], all patients were discharged after 33 (range 19–43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84–2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66–1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2‐mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Renal ACE2 expression in human kidney disease

Angiotensin-converting enzyme 2 (ACE2) is a recently discovered homologue of angiotensin-converting enzyme (ACE) that is thought to counterbalance ACE. ACE2 cleaves angiotensin I and angiotensin II into the inactive angiotensin 1-9, and the vasodilator and anti-proliferative angiotensin 1-7, respectively. ACE2 is known to be present in human kidney, but no data on renal disease are available to date. Renal biopsies from 58 patients with diverse primary and secondary renal diseases were studied (hypertensive nephropathy n = 5, IgA glomerulopathy n = 8, minimal change nephropathy n = 7, diabetic nephropathy n = 8, focal glomerulosclerosis n = 5, vasculitis n = 7, and membranous glomerulopathy n = 18) in addition to 17 renal transplants and 18 samples from normal renal tissue. Immunohistochemical staining for ACE2 was scored semi-quantitatively. In control kidneys, ACE2 was present in tubular and glomerular epithelium and in vascular smooth muscle cells and the endothelium of interlobular arteries. In all primary and secondary renal diseases, and renal transplants, neo-expression of ACE2 was found in glomerular and peritubular capillary endothelium. There were no differences between the various renal disorders, or between acute and chronic rejection and control transplants. ACE inhibitor treatment did not alter ACE2 expression. In primary and secondary renal disease, and in transplanted kidneys, neo-expression of ACE2 occurs in glomerular and peritubular capillary endothelium. Further studies should elucidate the possible protective mechanisms involved in the de novo expression of ACE2 in renal disease.     

上海地区肾综合征出血热尸检肾组织病毒检测

目的：研究上海地区肾综合征出血热（ＨＦＲＳ）尸检病例肾脏组织中病毒ＲＮＡ及抗原的分布及定位，方法：用核酸原位分子杂交方法和细胞免疫组化方法，检测了１７例ＨＦＲＳ尸检肾组织中病毒，结果：１７例肾组织中１５例可检测到病毒ＲＮＡ及抗原，病毒ＲＮＡ主要位于肾组织血管壁，毛细血管内皮细胞脉冲有小球毛细血管内皮细胞，病毒抗原主要分布于肾间质管内皮细胞和血管壁中，部分位于肾曲管上皮细胞，以病毒包涵体样颗粒出现。     

Hypoxia induces renal tubular epithelial cell apoptosis in chronic renal disease.

Renal tubular atrophy characterizes chronic progressive renal disease, but the molecular mechanisms of renal tubular cell (RTC) deletion are unclear. Because glomerular sclerosis leads to impaired peritubular blood flow, we tested the hypothesis that chronic hypoxia contributes to RTC apoptosis. Tubule hypoxia in mice with progressive renal disease (Os/+) was assessed by injecting EF5, a nitroimidazole compound that preferentially binds to cells undergoing anaerobic metabolism. Hypoxic tubules, as determined by direct immunofluorescence with anti-EF5 antibodies, were identified in kidneys from Os/+ mice, but not in age-matched controls (+/+) at 12 weeks, coincident with the onset of glomerular pathology. Hypoxia can cause apoptosis, but apoptotic RTCs were rare and equivalent in number in 12 week Os/+ and +/+ kidneys. However, by 16 weeks apoptotic RTCs were significantly more frequent in Os/+ versus +/+ mice, demonstrating that tubule hypoxia preceded RTC apoptosis. Importantly, apoptotic RTCs co-localized to hypoxic, but not normoxic tubules, indicating that tubular atrophy may result from hypoxic stimulation of RTC apoptosis. We have previously demonstrated enhanced, diffuse expression of the Fas apoptosis receptor in Os/+ tubules, providing a potential intermediary between hypoxia and apoptosis. To determine whether hypoxia stimulates Fas-dependent apoptosis, RTCs were cultured within a hypoxia chamber or in the presence of the cyanide analog, sodium azide. Both in vitro hypoxic conditions stimulated RTC plasma membrane Fas expression, and caused RTC apoptosis upon ligation with agonistic Fas antibodies. The data suggest that in the context of progressive renal disease, chronic hypoxia stimulates Fas-dependent RTC apoptosis, which represents the first definitive link between hypoxia and tubular atrophy. We believe that hypoxic induction of RTC apoptosis provides a unifying mechanism for the pathogenesis of tubular atrophy, and this paradigm identifies novel targets for chronic renal failure therapy.     

Changes at the glomerulo-tubular junction in renal transplants

We studied by microscopy 377 biopsies, nephrectomies, and necropsy kidneys from 123 human renal transplants. We discovered two common abnormalities of the renal corpuscle, both affecting the glomerulo-tubular junction. Adhesion of the tip of the glomerular tuft to the origin of the tubule, as reported in various non-transplant glomerulopathies, was seen in 197 specimens (52 per cent). This change was common in material showing acute or chronic vascular rejection and glomerulopathy, and was almost universal in transplants that had been in place for over 1 year. Another change at the glomerulo-tubular junction, not previously highlighted, consisted of an infiltrate of lymphocytes or neutrophil polymorphs into the epithelium at the tubular origin. This change was seen in 145 specimens (38 per cent) and was associated with cellular rejection and ascending infection. These changes are of importance because they show two responses of the kidney to injury that involve the glomerulo-tubular junction and thus suggest that this part of the kidney has some specific properties that have been largely neglected up to now.     

Morphological evidence that A-CAM is a major intercellular adhesion molecule in human kidney

We have used immunocytochemistry to identify the major primary adhesion molecule of the cadherin class in human kidney. In frozen sections of kidney, A-CAM was detected using the monoclonal antibody GC4 on the surface of renal tubular epithelial cells. Renal tubular epithelium did not express L-CAM. No cadherin reactivity was found on the glomerular epithelial cells. Cultured renal tubular epithelium was studied by immunofluorescence and immunogold methods. A-CAM was found at the contact points of adjacent epithelial cells, the phenotype of which was confirmed by the demonstration of cytokeratins using the antibody CAM 5.2. The A-CAM molecule in human kidney had an Mr of 130 kD in Western blotting experiments. These results lead us to conclude that A-CAM is the major cadherin of adult human renal epithelium.     

Polycystic kidney disease in SBM transgenic mice: role of c-myc in disease induction and progression.

SBM mouse is a unique transgenic model of polycystic kidney disease (PKD) produced by dysregulation of c-myc in the kidneys. Our previous demonstration that c-myc is overexpressed in human autosomal polycystic kidney disease (ADPKD) prompted us to investigate the pathogenetic role of c-myc in the induction and progression of the cystogenic phenotype in our mouse model. In young SBM kidneys, c-myc was two- to threefold increased with persistent expression levels into adulthood, an age when c-myc is normally undetectable. In situ hybridization analysis of the c-myc transgene demonstrated intense signal specifically overlying glomerular and tubular epithelium of developing cysts in fetal and young kidneys. Increased expression of c-myc correlated with the initiation and progression of the PKD phenotype as evidenced by early tubular and glomerular cysts at E16.5. Cyst number and size increased with age, with co-development of glomerular and tubular epithelial hyperplasia. Consistently, the mean renal proliferative index was increased approximately 5- to 20-fold in noncystic and cystic tubules of newborn SBM animals compared with littermate controls. Similarly, in fetal and newborn kidneys the tubular apoptotic indices were increased approximately three- to ninefold over controls. Both proliferation and apoptotic rates in cystic tubules approached levels in developing tubules from the normal nephrogenic zone. We conclude that the pathogenesis of PKD hinges on a critical imbalance in c-myc regulation of the opposing processes of cell proliferation and apoptosis, recapitulating the cellular phenomena in developing fetal kidney.     

Pathologic diagnosis of renal transplantation

Eighty-seven renal allografts were examined at our department between 1980 and 1989. In these specimens, 24 cases involved so-called "1 hour biopsy", including a case of donor carrying IgA nephropathy. Nine cases showed glomerulonephritis, of which three cases are suspected as de novo membranous nephropathy. Acute rejection, glomerular, tubular and vascular type were seen in 11 cases, and chronic rejection in 20 cases. Four cases showed tubular injury which may be associated with cyclosporine nephrotoxicity. At diagnosis of acute cellular rejection, immunohistological analysis was performed to characterize the composition of the micronuclear cells. In all cases we examined, T lymphocyte is dominant, and CD8+ (Leu 2) cells and CD4+ (Leu 3) cells were seen in various percentages. The Leu M1(+) macrophage was also seen in glomerular capillary lumen or peritubular space. Whether the expression of HLA-DR antigens is altered in renal transplants was examined. Expression of DR antigens increased considerably on renal tubular cells and glomerular capillary endothelial, and may be a markers of acute rejection.     

Increased expression of apoptosis-associated proteins in puromycin aminonucleoside nephrosis

Increased apoptosis has been reported in acute puromycin aminonucleoside nephrosis (PAN). The aim of this study was to investigate if increased apoptosis is related to increased expression of apoptosis-associated proteins (AAP) in this model of nephrosis. Sprague-Dawley rats were made nephrotic by intraperitoneal injection of one dose of puromycin aminonucleoside. Renal tissues were obtained at 1, 2 and 7 weeks after injection and apoptosis was investigated by TUNEL and by electron microscopy. Fas, Fas ligand, p53, Bax and Bcl-2 expressions were analyzed by the respective monoclonal and polyclonal antibodies, using indirect immunofluorescence. In the glomerulus of nephrotic animals, increased apoptosis was accompanied with increased expression of p53, Fas and Bax. In the interstitium, high expression of apoptosis, Fas, Fas-L and Bax were observed and in tubules increased apoptosis was accompanied with increased expression of p53, Fas and Fas-L. Bcl-2 was increased in interstitium and tubules during PAN. The incidence of apoptosis during PAN was correlated with the expression of AAP in glomerulus (p53), interstitium (Fas, Fas-L and Bax) and tubules (Fas, Fas-L, p53 and Bcl-2). There was correlation between Fas and Fas-L expression in interstitium and tubules. About 4% of glomerular and 25% of tubular p53 positive cells were apoptotic cells. The data suggest that increased local expression of AAP could contribute to renal apoptosis in the glomerular, interstitial and tubular compartments during this experimental model of nephrosis.     

Fractalkine expression in human renal inflammation.

Immune and inflammatory human renal disease is associated with heavy mononuclear cell infiltration. The trafficking of these cells to extravascular sites is directed by local production of chemokines. Fractalkine is the first described cell-surface anchored chemokine and has potent mononuclear cell-directed adhesion and chemotactic properties. The purpose of this study was to analyse the expression and distribution of fractalkine in human renal inflammation. In situ hybridization and immunohistochemistry were used to study renal biopsies from 15 patients with predominant glomerular inflammation (vasculitic glomerulonephritis) and 15 with predominant tubular and interstitial inflammation (acute renal allograft rejection). Controls comprised non-inflammatory glomerulonephritis and normal tissue. Fractalkine mRNA was predominantly expressed in the major compartment, glomerular or tubulointerstitial, affected by disease and with the strongest expression localized to vascular sites local to inflammation. In acute renal allograft rejection, there was increased expression of fractalkine mRNA by tubular epithelial cells. There was no expression of fractalkine by infiltrating leukocytes and there was only sparse expression in control tissue. Fractalkine mRNA expression correlated with infiltrating leukocyte subsets. Immunohistochemistry confirmed this pattern of expression, with serial section co-localization showing fractalkine expression in areas with macrophage (CD68+) and T cell (CD3+) infiltrates. These expression patterns show that fractalkine is a strong candidate for directing mononuclear cell infiltration in human renal inflammation.     

特异性小干扰RNA沉默CHOP对肾小管上皮细胞凋亡的影响

目的:研究C/EBP同源蛋白(CHOP)对肾小管上皮HK2细胞凋亡的影响。方法:采用qPCR法检测急性肾损伤患者及健康对照者血清中CHOP的mRNA水平。体外培养肾小管上皮HK2细胞,随机分为对照组、阴性组和si-CHOP组,si-CHOP组和阴性组分别转染CHOP小干扰RNA(siRNA)和阴性对照siRNA,通过转化生长因子β1(TGF-β1)诱导细胞损伤。MTT法检测细胞的活力,流式细胞术检测细胞的凋亡率, Western blot检测细胞中细胞核抗原Ki-67、增殖细胞核抗原(PCNA)、caspase-3和cleaved caspase-3的蛋白水平。结果:与健康对照者相比,急性肾损伤患者血清中CHOP的表达量显著增加(P0.05);转染CHOP siRNA显著降低肾小管上皮细胞HK2中CHOP的水平,与对照组相比差异具有统计学意义(P0.05)。敲减CHOP的表达显著增加肾小管上皮HK2细胞的活力(P0.05),降低其凋亡率(P0.05),增加Ki-67和PCNA的表达量(P0.05),下调cleaved caspase-3的蛋白水平(P0.05)。结论:在急性肾损伤患者血清中CHOP的水平增加。敲减CHOP表达通过调节增殖和凋亡相关蛋白的表达抑制肾小管上皮HK2细胞的凋亡。     

Acute humoral xenograft rejection: destruction of the microvascular capillary endothelium in pig-to-nonhuman primate renal grafts

The major cause of xenograft loss beyond hyperacute rejection is a form of injury, traditionally termed delayed xenograft rejection (DXR), whose pathogenesis is unknown. Here we analyze the immunologic and morphologic features of DXR that develops in pig kidney xenografts transplanted into nonhuman primates. Kidneys from miniature swine were transplanted into cynomolgus monkeys (n = 14) or baboons (n = 11) that received regimens aimed to induce mixed chimerism and tolerance. No kidney was rejected hyperacutely. Morphologic and immunohistochemical studies were performed on serial biopsies, and an effort was made to quantify the pathologic features seen. The early phase of DXR (Days 0-12) was characterized by focal deposition of IgM, IgG, C3, and scanty neutrophil and macrophage infiltrates. The first abnormality recognized was glomerular and peritubular capillary endothelial cell death as defined by in situ DNA nick-end labeling (TUNEL). Damaged endothelial cells underwent apoptosis and, later, frank necrosis. The progressive phase developed around Day 6 and was characterized by progressive deposition of IgM, IgG, C3, and prominent infiltration of cytotoxic T cells and macrophages, with a small number of NK cells. Thrombotic microangiopathy developed in the glomeruli and peritubular capillaries with TUNEL+ endothelial cells, platelet aggregation, and destruction of the capillary network. Only rare damaged arterial endothelial cells and tubular epithelial cells were observed, with rare endothelialitis and tubulitis. In the advanced phase of DXR, interstitial hemorrhage and infarction occurred. During the development of DXR, the number of TUNEL+ cells increased, and this correlated with progressive deposition of antibody. The degree of platelet aggregation correlated with the number of TUNEL+ damaged endothelial cells. We conclude that peritubular and glomerular capillary endothelia are the primary targets of renal DXR rather than tubular epithelial cells or arterial endothelium and that the earliest detectable change is endothelial cell death. DXR was characterized by progressive destruction of the microvasculature (glomeruli and peritubular capillaries) and formation of fibrin-platelet thrombi. Both cytotoxic cells and antibodies potentially mediate the endothelial damage in DXR; however, in this model, DXR is largely humorally mediated and is better termed "acute humoral xenograft rejection."     

Antibody-Mediated Rejection of Human Renal Allografts: An Electron Microscopic Study of Peritubular Capillaries

The role of humoral rejection in acute and chronic rejection of human renal allografts other than in hyperacute rejection has not been well established, and its importance may be underestimated. Recently, a specific histological pattern of antibody-mediated rejection of renal allografts has been recognized. The antigens targeted by this mode of rejection are not well defined but are likely located on the endothelium of small vessels (arterioles and glomerular and peritubular capillaries). In both cellular and humoral rejection, the microvasculature of transplanted organs appears to be a main target of injury. This study describes the ultrastructural changes of peritubular capillaries, over a period of up to 8 months, in 14 biopsy specimens obtained from 5 renal allograft recipients diagnosed with “pure” antibody-mediated rejection. In peritubular capillaries, there is progression of injury from necrosis of endothelial cells with lifting and denudation of basement membrane to complete disappearance of capillaries. Acutely, acute tubular necrosis is a constant finding. At 2 to 3 months posttransplantation, the remaining capillaries are dilated, misshapen, and distorted, and are surrounded by a reduplicated and thickened basement membrane. These changes are associated with increased interstitial fibrosis and tubular atrophy, comparable to a sort of renal “asphyxial” death. The author concludes that in “pure” antibody-mediated rejection, the endothelium of peritubular capillaries is a main target of injury. The potential role of antibody-mediated rejection in acute and chronic rejection of renal allografts needs to be explored further.     

血浆置换与免疫球蛋白高敏受者脱敏治疗肾移植

背景：高敏受者肾移植前应用静脉注射免疫球蛋白尚无统一方案,而国内应用较少。 目的：探讨采用血浆置换联合静脉注射免疫球蛋白干预的方法,对肾移植高敏受者进行脱敏治疗的可行性及效果分析。 方法：28例肾移植患者进行人类白细胞抗原交叉配型,并进行脱敏及血浆置换联合静脉注射免疫球蛋白,观察排斥反应发生率和移植肾存活时间及功能。 结果与结论：28例脱敏患者均未发生超急性排斥反应,9例(32%)发生急性排斥反应,其中5例(18%)为急性体液性排斥,所有排斥反应均逆转。平均随访(50±24)个月,移植后1和2年平均血肌酐分别为(112.18±17.20)和(129.78±36.52) μmol/L。移植肾12和48个月年存活率分别为95.0%和78.0%。提示采用血浆置换联合静脉注射免疫球蛋白能有效地对高敏受者进行脱敏治疗,移植后急性体液性排斥发生率高是该方案的主要问题,随访表明近期效果可以,远期效果有待观察。     

血糖波动对糖尿病大鼠肾小球内皮细胞和肾小管上皮细胞凋亡的影响

目的:观察血糖波动和持续高血糖对糖尿病大鼠肾小球血管内皮细胞和肾小管上皮细胞凋亡和Bax、Bcl-2表达的影响。方法:SD大鼠24只,均分为正常对照组、糖尿病持续高血糖组、糖尿病血糖波动组。采用链脲佐菌素（STZ）60 mg/kg腹腔注射诱发糖尿病，血糖波动组每天定时腹腔注射超短效胰岛素类似物诺和锐，并错时给予葡萄糖，造成1 d中血糖浓度大幅度波动模型。制模4周后，免疫组化法检测肾组织Bcl-2和Bax蛋白表达，原位缺口末端标记法（TUNEL）检测肾小球血管内皮细胞和肾小管上皮细胞凋亡。结果:糖尿病血糖波动组的肾脏凋亡细胞明显多于、肾小球Bcl-2蛋白表达少于、肾小管Bax表达明显多于糖尿病持续高血糖组。结论:糖尿病大鼠血糖明显波动可加速肾小管上皮细胞凋亡。     

Peripolar cells and other granulated epithelial cells in renal biopsies.

The peripolar cell is a recently described glomerular epithelial cell which is situated within Bowman's capsule at the vascular pole. It contains cytoplasmic granules which contain plasma proteins, although it may also have a secretory function. The relationship between peripolar cells, other granulated glomerular epithelial cells and tubular epithelial cells is unclear. We have studied 242 biopsies from 19 types of renal disease for peripolar cells, other granulated epithelial cells and granulated tubular epithelial cells. Peripolar cells were most numerous in mesangioproliferative glomerulonephritis, IgA nephropathy, focal segmental glomerulosclerosis, membranous glomerulonephritis and lupus nephropathy. Other granulated glomerular epithelial cells were most prominent in diffuse lupus nephropathy, focal glomerulonephritis, acute vascular transplant rejection, crescentic glomerulonephritis and mesangioproliferative glomerulonephritis. Granulation of the tubular epithelium was most prominent in minimal change nephrotic syndrome and amyloidosis. It is likely that the granules in tubular epithelial cells represent lysosomes containing plasma proteins which have been absorbed from the tubular fluid. However, granulation of glomerular cells may represent a more specific response to glomerular damage. In addition, peripolar cells are prominent in only certain diseases, suggesting a specialized function.