Cytokines in human renal interstitial fibrosis. I. Interleukin-1 is a paracrine growth factor for cultured fibrosis-derived kidney fibroblasts.

We studied the role of interleukin-1 beta (IL-1 beta) and basic fibroblast growth factor (bFGF) in the proliferative response of transformed human renal interstitial fibroblast cell lines established from either a kidney with glomerulonephritis and interstitial fibrosis or a normal kidney in comparison to primary human foreskin fibroblasts. Growth of fibrosis-derived renal fibroblasts was inhibited in the presence of IL-1 receptor antagonist (IL-1Ra) by 35% (P < 0.005), suggesting that these cells produce IL-1 and possess IL-1 receptors as part of paracrine growth. In contrast, spontaneous proliferation of fibroblasts derived from a normal kidney or normal skin were not inhibited by IL-1Ra. In fibrosis-derived but not in normal renal cells, fibronectin synthesis was increased 2.2-fold (P < 0.01) in the presence of IL-1Ra. Addition of exogenous IL-1 beta or bFGF stimulated proliferation of skin fibroblasts. In contrast, growth of fibrosis-derived renal fibroblasts was stimulated by IL-1 beta and unchanged by bFGF. Growth of normal kidney fibroblasts was unaffected by bFGF and inhibited by IL-1 beta. We conclude that compared to normal fibroblasts, fibrosis-derived renal fibroblasts have a different cytokine-response profile, are IL-1-dependent, produce IL-1 as a paracrine growth factor and do not proliferate to bFGF, a classical fibroblast growth factor.

Cytokines in human renal interstitial fibrosis. II. Intrinsic interleukin (IL)-1 synthesis and IL-1-dependent production of IL-6 and IL-8 by cultured kidney fibroblasts.

We compared cytokine production from transformed human fibroblast cell lines derived from either a kidney with interstitial fibrosis or a normal kidney to that from primary human foreskin fibroblasts. Fibrosis-derived as well as normal renal fibroblasts, but not skin fibroblasts, spontaneously produced the chemokine, IL-8, and the growth promoting cytokine, IL-6. Spontaneous IL-8 and IL-6 synthesis by renal fibroblasts was dependent on the intrinsic release of IL-1, since blocking IL-1 receptors with IL-1 receptor antagonist (IL-1Ra) partially inhibited the constitutive production of these cytokines. Both kidney cell lines had detectable mRNA and protein for IL-1 alpha and IL-1 beta. Renal and skin fibroblasts stimulated by picomolar concentrations of exogenous IL-1 or TNF-alpha produced large amounts of IL-6 and IL-8, whereas nanomolar concentrations of basic fibroblast growth factor did not. Fibrosis-derived cells expressed less high affinity IL-1 receptors (600 receptors/cell; KD = 0.6 pM) compared to normal renal fibroblasts (1000 receptors/cell). However, fibrosis-derived renal fibroblasts produce three- to fourfold more IL-8 and IL-6 in response to picomolar concentrations of IL-1 beta compared to cells derived from a normal kidney. As this enhanced production is not due to increased numbers of IL-1 receptors, we speculate that post-receptor responsiveness to either endogenous or exogenous IL-1 is greater in fibrosis-derived renal fibroblasts than in cells from normal kidneys.

Reduction of background problems in nonradioactive northern and Southern blot analyses enables higher sensitivity than 32P-based hybridizations.

An improved chemiluminescence-based RNA/DNA detection procedure offering a widely applicable alternative to the conventional 32P labeling employed in molecular biology is described. Even highly sensitive applications such as Northern blot analysis of low-copy RNAs are shown to be feasible now without radioactive labeling. Improved quality of nonradioactive detection was obtained by the use of digoxigenin-labeled nucleotides in combination with dioxetane substrates which are decomposed by the hydrolysis of alkaline phosphatase. Previously existing problems involving unacceptably high background signals in nonradioactive labeling procedures were eliminated by the application of a modified RNA/DNA transfer, hybridization, and detection protocol. The data presented here delineate a system consistently superior to radioactivity and should considerably increase the usefulness of nonradioactively labeled probes detected by chemiluminescence.

Human renal tubular cells as a cytokine source: PDGF-B, GM-CSF and IL-6 mRNA expression in vitro.

The pathogenesis of renal interstitial fibrosis is not fully understood at present. As renal interstitial fibrosis occurs mainly in association with interstitial cell infiltrates, several cytokines, e.g. PDGF, TGF-beta, IL-6, are thought to play a major role in the induction and progression of renal interstitial fibrosis. In order to prove whether renal tubular epithelial cells produce cytokines which are known to modulate renal interstitial fibroblasts, RNA was isolated from human renal tubular epithelial cells and from human renal fibroblasts. The expression of specific cytokines and growth factors was analysed by Northern blot analysis using cDNA probes for IL-1 alpha, IL-1 beta, IL-6, INF-gamma, TNF-alpha, a-FGF, b-FGF, GM-CSF, PDGF-B and oligonucleotides for IL-2 and TGF-beta 1. In renal tubular epithelial cells from normal and diseased kidneys, mRNA transcripts for PDGF-B, IL-6 and GM-CSF could be detected. Quantitative analysis of the mRNA transcripts of the cytokines expressed revealed consistently higher amounts of GM-CSF and PDGF-B mRNA in tubular epithelial cells from diseased in comparison to normal kidneys. Furthermore, higher amounts of GM-CSF and PDGF-B mRNA transcripts were detected in tubular epithelial cells derived from kidneys with interstitial fibrosis as compared to tubular epithelial cells from diseased but nonfibrotic kidneys. Renal fibroblasts from normal and fibrotic kidneys showed a weak signal for IL-6, but PDGF-B and GM-CSF were not expressed in these cells. By means of colony assays, using human bone marrow cells in the presence or absence of neutralizing antibodies, the release of biologically active colony-stimulating factors GM-CSF and M-CSF by renal tubular epithelial cells from normal and diseased kidneys could be demonstrated. The present results probably indicate interactions between renal tubular epithelial cells and renal fibroblasts in the progression of renal diseases.

Human cytomegalovirus in rejected kidney grafts; detection by polymerase chain reaction.

Human cytomegalovirus (CMV) infections are frequently associated with graft rejection in the immunosuppressed patients following organ transplantation. Thirty-four tissue samples from rejected kidneys and 18 samples from normal adult kidneys obtained from autopsies were investigated for the presence of CMV-DNA by the polymerase chain reaction (PCR) and by immunohistochemistry. DNA extracted from renal tissues after proteinase K digestion was specifically amplified in 32 cycles using primers which flank a 147 bp DNA fragment of the immediate early CMV gene and analysed by slot-blot hybridization with digoxigenin-labelled detection oligonucleotides. CMV-DNA was detected by PCR in a range from 0.1 fg up to 100 fg in 14 (41%) rejected kidney transplants. Comparative immunohistological analysis revealed presence of CMV in only three biopsies of these rejected kidneys. Furthermore, CMV-DNA was also found in four of 18 (22%) normal donor kidneys. These results reveal that CMV is often present in rejected kidneys and that the infection can be transferred from the donor to the recipient, since the normal adult kidney appears to be a frequent site of latency for CMV. No differences in local immunological changes, characterized by interstitial mononuclear leukocyte infiltration as well as by aberrant expression of HLA-class II antigens and of ICAM1 on proximal tubular epithelial cells, could be detected by further immunohistological analysis between grafted kidneys at late stage of rejection with and without CMV infection.

Human cytomegalovirus in immunoglobulin A nephropathy: detection by polymerase chain reaction.

Human cytomegalovirus (HCMV) has been suspected to participate in the pathogenesis of IgA nephropathy (IgAN). However, with regard to the presence of HCMV in the renal tissue of IgAN, conflicting results have been reported using a variety of different techniques. Renal biopsies of 29 patients with IgAN, of 7 with focal segmental glomerulosclerosis (FSGS) and of 11 normal kidneys were analyzed for the presence of HCMV-DNA using the polymerase chain reaction. HCMV-DNA was detected by hybridization with digoxigenin-labelled probes in 14 of 19 analyzed frozen renal biopsies from patients with IgAN. However, only 1 of 17 renal biopsies of IgAN embedded in paraffin was positive for HCMV-DNA. Furthermore, HCMV-DNA was detected in 4 of 18 frozen normal kidneys but in none of the tissues from patients with FSGS. The present results provide further evidence of an association between the presence of HCMV in renal tissue and IgAN.

Urinary detected renal antigens in the early diagnosis of kidney graft rejection.

The determination of renal antigens in the urine with an immunoassay, based on monoclonal antibodies (moabs), is a noninvasive test system for the analysis and monitoring of renal injury. New moabs allowing an immunohistologic dissection of the human nephron were generated by a direct intrasplenic immunization of mice with pathologic urine samples. A sandwich enzyme immunoassay was developed to quantitate renal cell membrane antigens in the urine samples. A sandwich enzyme immunoassay was developed to quantitate renal cell membrane antigens in the urine. While antigen excretion in healthy individuals is low, preliminary data of a clinical investigation suggest the usefulness of these assay systems in diagnosis of tubular injury in human kidney transplant recipients. The immunoassay can provide very early hints of renal graft rejection prior to the appearance of clinical symptoms or the detection by routine clinical laboratory investigations.

Differential regulation of HLA class I genes by interferon.

Allele-specific differences in the regulation of HLA class I genes by type I interferon (IFN) were observed after transfection of eight HLA-B, -A, or -C genes into mouse L cells. HLA-B7 and -Bw64 gene expression was significantly more inducible by type I IFN than the genes coding for HLA-B27, HLA-B51, HLA-B38, HLA-B39, HLA-Cw3, and HLA-A2 antigens. Modification of the 5' end of HLA-B7 and HLA-B27 genes revealed the presence of enhancer sequences responding to IFN treatment in the 5' untranslated region of HLA-B7, but not of HLA-B27 and suggested further, independently acting enhancer elements downstream of the transcription initiation site. Comparison of 5' enhancer region sequences in correlation with type I IFN inducibility of the different HLA class I alleles indicated that the exchange of only two nucleotides in the interferon response sequence (IRS) or enhancer A region of HLA-B7 or -Bw64 could account for nonregulated promoters in all other HLA-A, -B, or -C alleles analyzed. Thus, type I IFN stimulation of HLA class I genes in mouse L cells appears to predominantly operate in most alleles by a mechanism targeted to enhancer sequences downstream of the gene's transcription initiation site.

Genetic and serological heterogeneity of the supertypic HLA-B locus specificities Bw4 and Bw6.

Gene cloning and sequencing of the HLA-B locus split antigens B38 (B16.1) and B39 (B16.2) allowed localization of their subtypic as well as their public specificities HLA-Bw4 or -Bw6 to the alpha-helical region of the alpha 1 domain flanked by the amino acid positions 74-83. Comparison of their amino acid sequences with those of other HLA-B-locus alleles established HLA-Bw6 to be distinguished by Ser at residue 77 and Asn at residue 80. In contrast, HLA-Bw4 is characterized by at least seven different patterns of amino acid exchanges at positions 77 and 80-83. Reactivity patterns of Bw4- or Bw6-specific monoclonal antibodies reveal two alloantigenic epitopes contributing to the HLA-Bw4 or -Bw6 specificity residing next to the region of highest diversity of the alpha 1 domain.