Expression of type I collagen mRNA in glomeruli of rats with passive Heymann nephritis

Expression of type I collagen mRNA in glomeruli of rats with passive Heymann nephritis. In passive Heymann nephritis (PHN) glomeruli exhibit marked basement membrane expansion around subepithelial immune deposits but they fail to show any change in mRNA levels of type IV collagen, laminin or fibronectin by Northern and slot-blot analysis, or in the amount or distribution of type IV collagen or laminin by immunohistology for up to 12 weeks after disease onset. On the other hand, in situ hybridization (ISH) revealed the appearance of positive cells exhibiting mRNA for the α1 chain of rat type I collagen two to three weeks after the onset of PHN in all glomeruli of all rats. Positive cells persisted for at least eight weeks. In many glomeruli, the location of the clusters of silver grains suggested that they were in visceral epithelial cells. In controls injected with normal sheep IgG, and in early PHN (<11 days after sheep anti-Fx1A), glomeruli were negative but cells in the renal capsule and adventitia of vessels showed strong ISH and served as positive controls. RNAse pre-treatment and the “sense” probe gave appropriately negative results. RNA from PHN glomeruli contained an α1 type I collagen transcript of the same size as that from rat fibroblasts. These results show that the evolution of glomerular basement membrane expansion in rat membranous nephropathy coincides with the induction of a matrix gene that is not normally expressed in glomerular cells. Further, they suggest that the intercalation of ectopically-expressed matrix molecules may contribute to the production of a disorganized basement membrane

Transgenic Expression of Human LAMA5 Suppresses Murine Lama5 mRNA and Laminin α5 Protein Deposition

Laminin α5 is required for kidney glomerular basement membrane (GBM) assembly, and mice with targeted deletions of the Lama5 gene fail to form glomeruli. As a tool to begin to understand factors regulating the expression of the LAMA5 gene, we generated transgenic mice carrying the human LAMA5 locus in a bacterial artificial chromosome. These mice deposited human laminin α5 protein into basement membranes in heart, liver, spleen and kidney. Here, we characterized two lines of transgenics; Line 13 expressed ∼6 times more LAMA5 than Line 25. Mice from both lines were healthy, and kidney function and morphology were normal. Examination of developing glomeruli from fetal LAMA5 transgenics showed that the human transgene was expressed at the correct stage of glomerular development, and deposited into the nascent GBM simultaneously with mouse laminin α5. Expression of human LAMA5 did not affect the timing of the mouse laminin α1–α5 isoform switch, or that for mouse laminin β1–β2. Immunoelectron microscopy showed that human laminin α5 originated in both glomerular endothelial cells and podocytes, known to be origins for mouse laminin α5 normally. Notably, in neonatal transgenics expressing the highest levels of human LAMA5, there was a striking reduction of mouse laminin α5 protein in kidney basement membranes compared to wildtype, and significantly lower levels of mouse Lama5 mRNA. This suggests the presence in kidney of a laminin expression monitor, which may be important for regulating the overall production of basement membrane protein

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes

Decay-accelerating factor expression in the rat kidney is restricted to the apical surface of podocytes.BackgroundDecay-accelerating factor (DAF) has inhibitory activity toward complement C3 and C5 convertases. DAF is present in human glomeruli and on cultured human glomerular visceral epithelial cells (GEC). We studied the distribution and function of rat DAF.MethodsFunction-neutralizing antibodies (Abs) were raised against DAF. The distribution of DAF in vivo was determined by immunoelectron microscopy. Functional studies were performed in cultured GEC and following IV injection of anti-DAF Abs into rats.ResultsDAF was present exclusively on the apical surfaces of GEC, and was not present on the basal surfaces of GEC, nor other glomerular or kidney cells. DAF was functionally active on cultured GEC, and served to limit complement activation in concert with CD59, an inhibitor of C5b-9 formation. Upon injection into normal rats, anti-DAF F(ab′)2 Abs bound to GEC in vivo, yet there was no evidence for complement activation and animals did not develop abnormal albuminuria. Anti-megalin complement-activating IgG Abs were “planted” on GEC, which activated complement as evidenced by the presence of C3d on GEC. Attempts to inhibit DAF function with anti-DAF Abs did not affect the quantity of complement activation by these anti-megalin Abs, nor did it lead to development of abnormal albuminuria. In contrast, in the puromycin aminonucleoside model of GEC injury and proteinuria, anti-DAF Abs slowed the recovery from renal failure that occurs in this model.ConclusionIn cultured rat GEC, DAF is an effective complement regulator. In vivo, DAF is present on GEC apical surfaces. Yet, it appears that DAF is not essential to prevent complement activation from occurring under normal circumstances and in those cases in which complement-activating Abs are present on the basal surfaces of GEC in vivo. However, in proteinuric conditions, DAF appears to be protective to GEC

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Epitope Mapping of the Laminin Molecule in Murine Skin Basement Membrane Zone: Demonstration of Spatial Differences in Ultrastructural Localization

Results of studies performed to date with polyclonal anti-laminin antibodies have been conflicting as to the ultrastructural localization of this glycoprotein in skin basement membrane zone (BMZ). Whereas initial reports suggested its presence solely within the lamina lucida (LL), others have suggested that laminin is instead an exclusive component of the lamina densa (LD). In an attempt to more critically address this issue, we have examined both intact and partially separated (via 1M NaCl) murine skin BMZ by indirect immunoelectron microscopy via a two-step immunoperoxidase technique on unfixed cryopreserved tissue, utilizing nine well-characterized monoclonal antibodies with binding specificity for laminin. Localization of the sites of the epitopes recognized by these antibodies on isolated laminin molecules was previously determined by rotary shadowing and by biochemical analyses on enzymatic fragments of laminin. Whereas at least faint immunoreactants were detected in both regions with eight of nine antibodies, predominant staining was noted within the LL with three of eight and within (and even sparsely below) the LD in three of eight. One antibody bound solely to the LL; another bound equally within both regions. Although some overlap was noted, it appears that the epitope on the distal portion of the long arm of the laminin molecule resides primarily within the skin LD, whereas epitopes on more central portions of the short arms are present within the LL or within both LL and LD. The findings of stratification of laminin epitopes within skin BMZ supports a similar recent observation in mouse kidney and suggests that portions of the laminin molecule span both LD and LL, and that there may be a non-random spatial orientation for the laminin molecule within murine skin BMZ