Oxidative modification of low-density lipoproteins and the outcome of renal allografts at 1 1/2 years.

BACKGROUND: Previous studies reported a significant association between hyperlipidemia of the recipient and chronic allograft nephropathy (CAN). However, the nature and the pathogenic mechanism of circulating lipid abnormalities in CAN remain unclear. METHODS: In a prospective study of 50 consecutive adult recipients of a cadaveric renal allograft, we investigated the impact of lipid abnormalities on the outcome of the graft at 1 1/2 years. Besides morphometric analysis of implantation and protocol biopsies, clinical and biochemical variables were studied at three-month intervals. Plasma concentrations of oxidized low-density lipoprotein (OxLDL) were determined by means of enzyme-linked immunosorbent assay. Immunohistochemical staining for OxLDL and macrophages was performed on paired renal biopsies. Study end points were the fractional interstitial volume and the 24-hour creatinine clearance at 11/2 years. RESULTS: High-density lipoprotein (HDL) cholesterol of the recipient < or =47 mg/dL was a risk factor for the functional (RR = 1.56; 95% CI, 0.978 to 2.497) and the morphological (RR = 2.75; 95% CI, 1.075 to 7.037) outcome of the graft, mainly in patients without acute rejection (RR = 2.03; 95% CI, 1.13 to 3.65, and RR = 4.67; 95% CI, 1.172 to 18.582, respectively). Interstitial accumulation of OxLDL was inversely associated with HDL cholesterol (R = -0.476, P = 0.019), and was associated with a higher density of tubulointerstitial macrophages (R = 0.656, P = 0.001) and a higher fractional interstitial volume at 11/2 years (P = 0.049). CONCLUSION: Decreased HDL cholesterol levels of the recipient adversely affect the outcome of renal allografts through the accumulation of OxLDL in the renal interstitium of the graft. Interstitial accumulation of OxLDL was associated with the presence of macrophages and the development of interstitial fibrosis.

Flow cytometric immunodissection of the human distal tubule and cortical collecting duct system.

BACKGROUND: In recent years, considerable efforts were drawn to isolate human distal tubule (DT) and collecting duct (CD) cells with more or less success. Here, we present a procedure for isolating human DT cells [thick ascending limb (TAL)/distal convoluted tubule (DCT)] and CD system cells (connecting tubule/initial CD) as separate populations within the same kidney specimen, applying monoclonal antibodies in fluorescence-activated cell sorting (FACS) and culturing them. METHODS: We tested antibodies directed against the DT/CD system antigens, epithelial membrane antigen (EMA) and L1-cell adhesion molecule (L1-CAM). Segmental and subsegmental expressions were first assessed by using morphologic and histotopographic criteria, and by comparing sections with adjacent sections stained for expression of well-defined distal subsegment-specific markers. Immunoreactive cells were further characterized by dual immunostaining using cell type-specific markers. As a second step, cells obtained by collagenase digestion of normal renal cortical tissue were flow sorted following labeling with aforementioned antibodies and cultured. RESULTS: EMA expression was found on all cells present in the DT and in the CD system. Its expression was most abundant in TAL and from thereon decreased gradually along the course of the DT and CD system. Flow sorting of all EMA-expressing cells resulted in identification/isolation of DT and CD system cells as a heterogeneous mixture. Flow sorting of only the most strongly EMA-positive cells allowed purification of DT cells only, mainly TAL cells as shown by Tamm-Horsfall protein expression on> 80% of sorted cells. L1-CAM was expressed in only the CD system, and sorting of all L1-CAM-positive cells allowed> 95% purification of CD system cells (connecting tubule/cortical CD). Primary cultures of DT and CD system cells rapidly developed into confluent monolayers, and retained antigenic and functional properties inherent to their segments of origin. CONCLUSION: Our study presents a procedure for isolating and culturing pure populations of human DT cells and CD system cells as separate populations, using antibodies to the best available markers in FACS.

TIMP-1 gene expression and PAI-1 antigen after unilateral ureteral obstruction in the adult male rat.

BACKGROUND: Sustained obstruction of urinary flow invariably leads to inflammation, loss of functional renal structures and progressive deposition of extracellular matrix proteins, culminating in renal fibrosis. Although increased renal tissue inhibitor of matrix metalloproteinase (TIMP-1) expression is one of the early events following experimental hydronephrosis, little is known about its cellular source. Both the recruited macrophage and the resident/recruited (myo)fibroblast have been postulated to be candidate TIMP-1 transcribing cells. Currently, data concerning plasminogen activator inhibitor type 1 (PAI-1) expression in the ligated kidney are unavailable. Our study concentrated on the localization of TIMP-1 expressing cells and PAI-1 immunoreactive cells in the obstructed rat kidney. METHODS: Rats were sacrificed 1, 5, 10, 15, 20 and 26 days after unilateral ureteral obstruction (UUO) or sham-surgery (SOR). Leukocyte (OX-1+), macrophage (ED1+) and neutrophil infiltration were analyzed using specific antibodies or nuclear morphology. alpha-Smooth muscle actin (alpha-SMA) immunostaining was measured morphometrically. Mitotic figures and nuclei with an apoptotic morphology were quantified in hematoxylin-eosin (H&E)-stained sections. TIMP-1 mRNA transcribing cells were localized with in situ hybridization (ISH) and identified by subsequent immunostainings for alpha-SMA and macrophages. PAI-1 antigenicity was evaluated immunohistochemically in SOR, contralateral unobstructed kidneys (CUK), and UUO kidneys. RESULTS: The number of leukocytes and macrophages in the ligated rat kidney increased progressively in time, starting from day 5 post-surgery when compared with CUKs. Neutrophil accumulation in UUO kidneys became apparent from day 5 and large intraluminal leukocyte clusters (neutrophils and macrophages) were found in the lumen of distended tubules, especially at later stages post-obstruction, when collected urine and tissue samples proved to be sterile upon culture. From day 5 on, the number of apoptotic cells started to predominate the number of mitotic cells in the obstructed kidneys. Interstitial alpha-SMA immunoreactivity in the ligated kidney expanded from day 5 on and was most pronounced in the inner stripe of the outer medulla. As early as 24 hours post-ligation, TIMP-1 mRNA transcribing interstitial cells were detected with ISH, while tubular TIMP-1 expression was sparse. Since at that point in time, no interstitial alpha-SMA expressing cells and only few ED1+ macrophages were present, the bulk of the TIMP-1 mRNA transcription occurred in other interstitial cells. Throughout the study period numerous interstitial TIMP-1 expressing cells were detectable in obstructed kidneys and from day 5 after ligation on, we could identify alpha-SMA+ and to a lesser degree ED1+ macrophages as TIMP-1 transcribing cells. In addition, dilated tubules containing intraluminal leukocyte casts were surrounded by a corona of intact neutrophils in H&E-stained sections and ISH showed that similar tubules were encircled by TIMP-1 mRNA expressing cells. PAI-1 immunoreactivity appeared to diminish in the early phase following urinary outlet obstruction, but emerged in damaged tubules from day 5 to 10 on. In later stages post-ligation, PAI-1+ cells and PAI-1 immunoreactive material were found embedded in the extracellular matrix. CONCLUSIONS: Our results confirm that TIMP-1 is active in the early phase of the fibrotic process and we demonstrated that initially TIMP-1 mRNA is transcribed by very few ED1+ macrophages but mainly by other, presently unidentified, interstitial cells. During later stages of post-ligation, both TIMP-1 (transcribed among others by alpha-SMA+ myofibroblasts, ED1+ macrophages, and possibly neutrophils) and PAI-1 are involved in the progression of tubulointerstitial scarring.

Inhibition of the matrix metalloproteinase system in a rat model of chronic cyclosporine nephropathy.

BACKGROUND: Chronic cyclosporine A (CsA)-induced nephropathy is histologically characterized by tubular lesions, the interstitial recruitment of inflammatory cells, arteriolopathy and focal interstitial fibrosis. Recent studies show that the intrarenal inhibition of matrix degradation and recruitment of monocytes/ macrophages into the kidney plays a critical role in the development of renal interstitial fibrosis. METHODS: We examined the expression of components of the matrix metalloproteinase (MMP) system and plasminogen activator inhibitor type-1 (PAI-1) in kidneys from rats injected daily s.c. during three weeks with CsA (10, 15 or 20 mg CsA/kg body wt) or vehicle solution. RESULTS: In all CsA-treated rats, serum creatinine levels were significantly elevated compared to control levels. The extent of CsA-induced atrophy was not influenced by the dosage during a three-week CsA treatment. The administration of CsA did not significantly increase total cortical interstitial collagen deposition, whereas alpha-smooth muscle actin expression was significantly increased in all CsA-treated rats. Analysis of the different subpopulations of inflammatory cells recruited into the chronically injured kidney revealed a marked influx of macrophages into fibrotic cortical foci of CsA-treated rats. The number of cortical macrophages was highest in the group receiving the highest CsA dose. PAI-1 antigen, present in proximal tubular lysosomes in kidneys from all experimental groups, stained very intensely in atrophic tubules in CsA-treated rats. Both stromelysin and interstitial collagenase mRNA were expressed in the kidneys of control rats, but their message transcription remained unaltered after CsA treatment. In contrast, the expression of tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1) was significantly increased after CsA treatment. TIMP-1 mRNA was undetectable in renal sections from sodium-depleted vehicle-treated animals using the in situ hybridization (ISH) technique. ISH of selected renal sections of CsA-treated rats identified the cells responsible for the increased TIMP-1 message transcription after CsA administration, mainly as interstitial cells and also as visceral and parietal epithelial cells. CONCLUSIONS: These results suggest that the locally increased expression of TIMP-1 rather than a decrease of matrix metalloprotease expression, contributes to the development of CsA-induced focal interstitial fibrosis in the rat.

Immunodissection of the human proximal nephron: flow sorting of S1S2S3, S1S2 and S3 proximal tubular cells.

We report on the use of several proximal tubular cell (PTC) surface markets and corresponding antibodies in fluorescence-activated cell sorting (FACS), and their ability to identify and flow sort cells of defined proximal tubular origin (S1S2S3) or of defined proximal subsegmental origin (S1S2 only/S3 only). We tested monoclonal/polyclonal antibodies directed against five different surface peptidases [leucine aminopeptidase (LAP), neutral endopeptidase 24.11 (NEP), dipeptidyl peptidase IV (DPPIV), aminopeptidase A (APA) and gamma-glutamyl transferase (gamma-GT)], the S3 segment-specific marker intestinal type alkaline phosphatase (iAP) and an S1S2 marker (TN20-antigen), originally proposed as a surface marker for interstitial fibroblasts. Segmental (proximal tubular vs. distal tubular) and proximal subsegmental (S1S2 vs. S3) expression of all five surface peptidases and TN20 antigen were first assessed by comparing immunohistochemical staining on normal human kidney tissue with staining for well-known segment-specific differentiation markers (intestinal type alkaline phosphatase, Tamm-Horsfall protein) on adjacent sections. All five peptidases were found to be expressed to a certain degree in all subsegments (S1 S2 and S3) of the proximal nephron, whereas expression was never seen in the more distal parts of the nephron. Flow cytometry was performed on cells obtained following gradient purification of collagenase-digested human renal tissue. Labeling cells for expression of LAP, NEP or DPPIV resulted in high yields of specifically labeled PTC (S1S2S3 origin). Labeling with anti-LAP resulted in the clearest distinction between positive and negative cell subpopulations, and therefore LAP was considered the best PTC marker for use in FACS. iAP histochemical staining on sorted cells showed that flow sorting with monoclonal antibody (moAb) 250 (anti-intestinal type alkaline phosphatase) allowed sorting of S3 cells with > 90% purity. Likewise, moAb TN20 enabled us to obtain a highly purified S1S2 population as confirmed by the absence of iAP on sorted cells.

Cholesterol feeding accentuates the cyclosporine-induced elevation of renal plasminogen activator inhibitor type 1.

Long-term cyclosporine (CsA) therapy is accompanied by the occurrence of hypercholesterolemia and renal interstitial fibrosis. The present study investigates the effect of dietary cholesterol on CsA-induced lipid disturbances in the rat and on CsA nephrotoxicity. Since plasminogen activator inhibitor type 1 (PAI-1) is a major inhibitor of matrix degradation and elevated plasma PAI-1 levels are reported to be associated with increased low-density lipoprotein (LDL) cholesterol, PAI-1 was examined in the kidneys of rats fed a sodium-deficient diet, with or without cholesterol. After nine weeks, both diet groups were subdivided into a CsA-treated group and a vehicle-treated group. Although cholesterol feeding significantly aggravated CsA-induced renal function impairment, CsA-induced histological lesions were comparable in both diet groups. Cholesterol feeding significantly decreased high-density lipoprotein (HDL) cholesterol irrespective of the treatment, while CsA treatment significantly elevated serum triglycerides irrespective of the diet. Cholesterol feeding alone did not increase the number of infiltrating cells in the renal interstitium. In contrast, in both diet groups CsA treatment caused a significant influx of macrophages, while combined treatment with CsA and cholesterol additionally elevated the number of T-helper cells in the cortex. In all rats, PAI-1 immunostaining was found mainly in intracellular vesicles (lysosomes) in proximal tubules, which stained most intensely in fibrotic areas of kidneys from CsA-treated rats. Cholesterol feeding enhanced the CsA-induced elevation of renal PAI-1 immunostaining to a significant level. These results show that, although serum creatinine, PAI-1 staining and T cell influx were significantly increased in the cholesterol-fed CsA-treated group compared to the other groups, renal CsA-induced histological lesions were not influenced by cholesterol feeding after short-term (3 weeks) CsA administration. To what extent the more pronounced proximal tubular PAI-1 (inhibitor of matrix degradation) immunostaining in fibrotic areas in the cortex of cholesterol-fed CsA-treated rats contributes to the progression of CsA-induced renal fibrosis remains to be determined.

Radiolocalisation and imaging of stably HPLAP-transfected MO4 tumours with monoclonal antibodies and fragments.

Immunotargeting of PLAP-expressing tumours was studied for two radioiodinated, highly specific anti-PLAP monoclonal antibodies, 7E8 and 17E3, differing 10-fold in affinity, as well as for 7E8 F(ab')2 fragments. An anti-CEA monoclonal antibody or anti-CD3 F(ab')2 fragments were used as controls. Specific and non-specific targeting was examined in nude mice simultaneously grafted with PLAP-positive tumours derived from MO4 1-4 cells, and CEA-positive tumours, derived from 5583-S cells. Results indicated that (1) MO4 1-4 tumours, with a stable expression of PLAP on the plasma membrane, represent a useful new in vivo model for immunodirected tumour targeting; (2) differences in antibody affinity for PLAP in vitro are not reflected in antibody avidity for tumour cells in vivo; and (3) excellent selective and specific localisation of the PLAP-positive tumours is achieved when 7E8 F(ab')2 fragments are used. The high tumour/blood ratios (10.7 +/- 3.9 at 46 h after injection) were due to a much faster blood clearance of 7E8 F(ab')2 fragments. At this time point, the mean tumour/non-tumour tissue ratio was as high as 34.5, and the mean specific localisation index was 29.0. As expected, the F(ab')2 fragments provided high tumour imaging efficiency on gamma camera recording. These data imply important potentials of the PLAP/anti-PLAP system for immunolocalisation and therapy in patients, but also emphasise that in vitro criteria alone are not reflected in in vivo tumour localisation capacities of antibodies.