TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis.

Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.

Longitudinal changes in MRI markers in a reversible unilateral ureteral obstruction mouse model: preliminary experience.

PURPOSE: To evaluate longitudinal changes in renal oxygenation and diffusion measurements in a model of reversible unilateral ureteral obstruction (rUUO) which has been shown to induce chronic renal functional deficits in a strain dependent way. C57BL/6 mice show higher degree of functional deficit compared with BALB/c mice. Because hypoxia and development of fibrosis are associated with chronic kidney diseases and are responsible for progression, we hypothesized that MRI measurements would be able to monitor the longitudinal changes in this model and will show strain dependent differences in response. Here blood oxygenation level dependent (BOLD) and diffusion MRI measurements were performed at three time points over a 30 day period in mice with rUUO. MATERIALS AND METHODS: The studies were performed on a 4.7T scanner with the mice anesthetized with isoflurane before UUO, 2 and 28 days postrelease of 6 days of obstruction. RESULTS: We found at the early time point (∼2 days after releasing the obstruction), the relative oxygenation in C57Bl/6 mice were lower compared with BALB/c. Diffusion measurements were lower at this time point and reached statistical significance in BALB/c CONCLUSION: These methods may prove valuable in better understanding the natural progression of kidney diseases and in evaluating novel interventions to limit progression.

Cell cycle arrest in a model of colistin nephrotoxicity.

Colistin (polymixin E) is an antibiotic prescribed with resurging frequency for multidrug resistant gram negative bacterial infections. It is associated with nephrotoxicity in humans in up to 55% of cases. Little is known regarding genes involved in colistin nephrotoxicity. A murine model of colistin-mediated kidney injury was developed. C57/BL6 mice were administered saline or colistin at a dose of 16 mg/kg/day in 2 divided intraperitoneal doses and killed after either 3 or 15 days of colistin. After 15 days, mice exposed to colistin had elevated blood urea nitrogen (BUN), creatinine, and pathologic evidence of acute tubular necrosis and apoptosis. After 3 days, mice had neither BUN elevation nor substantial pathologic injury; however, urinary neutrophil gelatinase-associated lipocalin was elevated (P = 0.017). An Illumina gene expression array was performed on kidney RNA harvested 72 h after first colistin dose to identify differentially expressed genes early in drug treatment. Array data revealed 21 differentially expressed genes (false discovery rate < 0.1) between control and colistin-exposed mice, including LGALS3 and CCNB1. The gene signature was significantly enriched for genes involved in cell cycle proliferation. RT-PCR, immunoblot, and immunostaining validated the relevance of key genes and proteins. This murine model offers insights into the potential mechanism of colistin-mediated nephrotoxicity. Further studies will determine whether the identified genes play a causative or protective role in colistin-induced nephrotoxicity.

Endotoxemia alters tight junction gene and protein expression in the kidney.

Intact tight junctional (TJ) proteins are required for tubular ion transport and waste excretion. Disruption of TJs may contribute to a decreased glomerular filtration rate in acute kidney injury (AKI) via tubular backleak. The effect of LPS-mediated AKI on murine TJs has not been studied extensively. We hypothesized LPS endotoxin administration to mice would disrupt tubular TJ proteins including zonula occludens-1 (ZO-1), occludin, and claudins. ZO-1 and occludin immunofluorescence 24 h post-LPS revealed a marked change in localization from the usual circumferential fencework pattern to one with substantial fragmentation. Renal ZO-1 expression was significantly reduced 24 h after LPS (decrease of 56.1 ± 7.4%, P < 0.001), with subsequent recovery. ZO-1 mRNA expression was increased 24 h post-LPS (4.34 ± 0.87-fold, P = 0.0019), suggesting disruption of ZO-1 protein is not mediated by transcriptional regulation, but rather by degradation or changes in translation. Similarly, claudin-4 protein expression was decreased despite elevated mRNA. LPS administration resulted in dephosphorylation of occludin and fragmented tubular redistribution. Protein expression of claudin-1, and -3 was increased after LPS. ZO-1, occludin, and claudin-1, -3, and -4 gene expression were increased 48 h after LPS, suggesting a renal response to strengthen TJs following injury. Interestingly, reduced mRNA expression was found only for claudin-8. This study provides further support that LPS-induced AKI is associated with structural injury and is not merely due to hemodynamic changes.

A new model of the distal convoluted tubule.

The Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney is a key determinant of Na(+) balance. Disturbances in NCC function are characterized by disordered volume and blood pressure regulation. However, many details concerning the mechanisms of NCC regulation remain controversial or undefined. This is partially due to the lack of a mammalian cell model of the DCT that is amenable to functional assessment of NCC activity. Previously reported investigations of NCC regulation in mammalian cells have either not attempted measurements of NCC function or have required perturbation of the critical without a lysine kinase (WNK)/STE20/SPS-1-related proline/alanine-rich kinase regulatory pathway before functional assessment. Here, we present a new mammalian model of the DCT, the mouse DCT15 (mDCT15) cell line. These cells display native NCC function as measured by thiazide-sensitive, Cl(-)-dependent (22)Na(+) uptake and allow for the separate assessment of NCC surface expression and activity. Knockdown by short interfering RNA confirmed that this function was dependent on NCC protein. Similar to the mammalian DCT, these cells express many of the known regulators of NCC and display significant baseline activity and dimerization of NCC. As described in previous models, NCC activity is inhibited by appropriate concentrations of thiazides, and phorbol esters strongly suppress function. Importantly, they display release of WNK4 inhibition of NCC by small hairpin RNA knockdown. We feel that this new model represents a critical tool for the study of NCC physiology. The work that can be accomplished in such a system represents a significant step forward toward unraveling the complex regulation of NCC.

Abnormal immune complex processing and spontaneous glomerulonephritis in complement factor H-deficient mice with human complement receptor 1 on erythrocytes.

Complement receptor 1 (CR1) on human erythrocytes (Es) and complement factor H (CFH) on rodent platelets perform immune adherence, which is a function that allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1(hu)Tg/CFH(-/-) mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing Ag, IgG and C3 bound to Es in CR1(hu)Tg/CFH(-/-) mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.

Chronic kidney disease induced in mice by reversible unilateral ureteral obstruction is dependent on genetic background.

Chronic kidney disease (CKD) begins with renal injury; the progression thereafter depends upon a number of factors, including genetic background. Unilateral ureteral obstruction (UUO) is a well-described model of renal fibrosis and as such is considered a model of CKD. We used an improved reversible unilateral ureteral obstruction (rUUO) model in mice to study the strain dependence of development of CKD after obstruction-mediated injury. C57BL/6 mice developed CKD after reversal of three or more days of ureteral obstruction as assessed by blood urea nitrogen (BUN) measurements (>40 mg/dl). In contrast, BALB/c mice were resistant to CKD with up to 10 days ureteral obstruction. During rUUO, C57BL/6 mice exhibited pronounced inflammatory and intrinsic proliferative cellular responses, disruption of renal architecture, and ultimately fibrosis. By comparison, BALB/c mice had more controlled and measured extrinsic and intrinsic responses to injury with a return to normal within several weeks after release of ureteral obstruction. Our findings provide a model that allows investigation of the genetic basis of events during recovery from injury that contribute to the development of CKD.

Renal FcRn reclaims albumin but facilitates elimination of IgG.

The widely distributed neonatal Fc receptor (FcRn) contributes to maintaining serum levels of albumin and IgG in adults. In the kidney, FcRn is expressed on the podocytes and the brush border of the proximal tubular epithelium. Here, we evaluated the role of renal FcRn in albumin and IgG metabolism. Compared with wild-type controls, FcRn(-/-) mice had a lower t((1/2)) for albumin (28.7 versus 39.9 h) and IgG (29.5 versus 66.1 h). Renal loss of albumin could account for the former, suggested by the progressive development of hypoalbuminemia in wild-type mice transplanted with FcRn-deficient kidneys. Furthermore, serum albumin levels returned to normal in FcRn(-/-) recipients of wild-type kidneys after removing the native FcRn-deficient kidneys. In contrast, renal loss could not account for the enhanced elimination of IgG in FcRn(-/-) mice. These mice had minimal urinary excretion of native and labeled IgG, which increased to wild-type levels in FcRn(-/-) recipients of a single FcRn-sufficient kidney (t((1/2)) of IgG was 21.7 h). Taken together, these data suggest that renal FcRn reclaims albumin, thereby maintaining the serum concentration of albumin, but facilitates the loss of IgG from plasma protein pools.

The extended multidomain solution structures of the complement protein Crry and its chimeric conjugate Crry-Ig by scattering, analytical ultracentrifugation and constrained modelling: implications for function and therapy.

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crry contains only short complement/consensus repeat (SCR) domains. X-ray and neutron scattering was performed on recombinant rat Crry containing the first five SCR domains (rCrry) and mouse Crry with five SCR domains conjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to determine their solution structures at medium resolution. The radius of gyration R(G) of rCrry was determined to be 4.9-5.0 nm, and the R(G) of the cross-section was 1.2-1.5 nm as determined by X-ray and neutron scattering. The R(G) of mCrry-Ig was 6.6-6.7 nm, and the R(G) of the cross-section were 2.3-2.4 nm and 1.3 nm. The maximum dimension of rCrry was 18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated that rCrry and mCrry-Ig have molecular mass values of 45,000 Da and 140,000 Da, respectively, in agreement with their sequences, and sedimentation equilibrium data supported these determinations. Time-derivative velocity experiments gave sedimentation coefficients of 2.4S for rCrry and 5.4S for mCrry-Ig. A medium-resolution model of rCrry was determined using homology models that were constructed for the first five SCR domains of Crry from known crystal and NMR structures, and linked by randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structures that best accounted for the scattering and ultracentrifugation data. These were shorter than the most extended rCrry models as the result of minor bends in the inter-SCR orientations. The mCrry-Ig solution data were modelled starting from a fixed structure for rCrry and the crystal structure of mouse IgG1, and was based on conformational searches of the hinge peptide joining the mCrry and Fc fragments. The best-fit models showed that the two mCrry antennae in mCrry-Ig were extended from the Fc fragment. No preferred orientation of the antennae was identified, and this indicated that the accessibility of the antennae for the molecular targets C4b and C3b was not affected by the covalent link to Fc. A structural comparison between Crry and complement receptor type 1 indicated that the domain arrangement of Crry SCR 1-3 is as extended as that of the CR1 SCR 15-17 NMR structure.

TNF causes changes in glomerular endothelial permeability and morphology through a Rho and myosin light chain kinase-dependent mechanism.

A key function of the endothelium is to serve as a regulated barrier between tissue compartments. We have previously shown that tumor necrosis factor (TNF) plays a crucial role in lipopolysaccharide (LPS)-induced acute kidney injury, in part by causing injury to the renal endothelium through its receptor TNFR1. Here, we report that TNF increased permeability to albumin in primary culture mouse renal endothelial cells, as well as human glomerular endothelial cells. This process occurred in association with changes in the actin cytoskeleton and was associated with gaps between previously confluent cells in culture and decreases in the tight junction protein occludin. This process was dependent on myosin light chain activation, as seen by its prevention with Rho-associated kinase and myosin light chain kinase (MLCK) inhibitors. Surprisingly, permeability was not blocked by inhibition of apoptosis with caspase inhibitors. Additionally, we found that the renal glycocalyx, which plays an important role in barrier function, was also degraded by TNF in a Rho and MLCK dependent fashion. TNF treatment caused a decrease in the size of endothelial fenestrae, dependent on Rho and MLCK, although the relevance of this to changes in permeability is uncertain. In summary, TNF-induced barrier dysfunction in renal endothelial cells is crucially dependent upon the Rho/MLCK signaling pathway.

Distinct and separable roles of the complement system in factor H-deficient bone marrow chimeric mice with immune complex disease.

Plasma complement factor H (Cfh) is a potent complement regulator, whereas Cfh on the surface of rodent platelets is responsible for immune complex processing. For dissection between the two, bone marrow chimeras between Cfh-deficient (Cfh(-/-)) and wild-type C57BL/6 mice were created. Platelet Cfh protein was tracked with the Cfh status of the bone marrow donor, indicating that platelet Cfh is of intrinsic origin. In an active model of immune complex disease, Cfh(-/-) mice that were reconstituted with wild-type bone marrow had levels of platelet-associated immune complexes comparable to those of wild-type mice and were protected against the excessive glomerular deposition of immune complexes seen in Cfh(-/-) mice, yet these mice still developed glomerular inflammation. In contrast, wild-type mice with Cfh(-/-) bone marrow had reduced platelet-associated immune complexes and extensive glomerular deposition of complement-activating immune complexes, but they did not develop glomerular pathology. The large quantities of glomerular C3 in wild-type mice with Cfh(-/-) bone marrow were in the form of iC3b and C3dg, whereas active C3b remained in Cfh(-/-) recipients of wild-type bone marrow. These data show that plasma Cfh limits complement activation in the circulation and other accessible sites such as the glomerulus, whereas platelet Cfh is responsible for immune complex processing.

Rat glomerular epithelial cells produce and bear factor H on their surface that is up-regulated under complement attack.

BACKGROUND: Factor H is a potent complement inhibitory molecule that is primarily produced by the liver and appears in plasma as a soluble protein. Yet there is evidence that other cells, including those in the kidney, can produce factor H, and that it can be cell-associated as well as present as a plasma protein. Here we studied factor H in rat glomerular epithelial cells (GEC). METHODS: A polyclonal antibody to factor H was used to identify factor H protein. A polymerase chain reaction (PCR)-based strategy was utilized to clone the full-length cDNA of GEC factor H. The relative quantity of factor H mRNA was measured by quantitative reverse transcription (RT)-PCR in cultured GEC exposed to complement activation and in the passive Heymann nephritis (PHN) model of membranous nephropathy. RESULTS: By immunofluorescence microscopy, factor H protein was present on the plasma membranes of cultured GEC. Based upon Western blot studies, this appeared to be the full-length 150 kD factor H protein. Factor H cDNA cloned from GEC was identical to the newly deposited sequence for rat liver factor H cDNA. In cultured GEC in which complement was activated, factor H mRNA increased over time. Similarly, in the PHN model in which complement was activated on GEC in vivo, factor H mRNA and protein also increased over time. CONCLUSION: Cultured GEC and glomeruli express factor H mRNA and protein. As modeled both in vitro and in vivo in the rat, factor H is up-regulated in membranous nephropathy. This is likely to be a direct response of GEC to complement attack and may represent a protective response of this cell.

Isolation and characterization of a novel rat factor H-related protein that is up-regulated in glomeruli under complement attack.

The factor H family in humans is composed of seven distinct proteins, including factor H-related proteins (FHR) 1-5. All members contain tandemly arranged short consensus repeats (SCR) typical of the regulators of complement activation gene family. FHR-5 is unusual for this group of proteins, as it was initially identified as a component of immune deposits in glomerular diseases. During our cloning of the cDNA for rat factor H from glomerular epithelial cells (GEC), we identified an alternative 2729-bp cDNA transcript. The translated sequence encoded a protein containing 11 SCRs, most similar to SCRs 7-15 and 19-20 in native rat factor H, which is the same basic structure of human FHR-5. As such, this rat protein was termed FHR. Recombinant rat FHR produced in a eukaryotic expression system had a molecular mass of 78 kDa. In functional studies, recombinant FHR bound C3b and inhibited the complement alternative pathway in a dose-dependent fashion. Given the prominent expression of FHR-5 in human membranous nephropathy, a disease in which complement activation occurs in the vicinity of GEC, the expression of FHR in a rat model of this disease was evaluated. In both in vitro and in vivo models of complement activation on the GEC, FHR mRNA was up-regulated by a factor of 3-6-fold compared with controls in which complement could not be activated. Thus, we have identified a novel factor H family member in rats. This FHR protein is analogous to human FHR-5, both in structure and in potential involvement in glomerular immune complex diseases.

A complement-dependent model of thrombotic thrombocytopenic purpura induced by antibodies reactive with endothelial cells.

Thrombotic thrombocytopenic purpura (TTP) is an immunologically mediated disease characterized by thrombocytopenia, hemolytic anemia, and pathologic changes in various organs, including the kidney, which are secondary to widespread thromboses. Central to TTP is platelet activation, which may occur from a variety of mechanisms, including endothelial cell activation or injury. In this study, injection of K6/1, a monoclonal antibody with widespread reactivity toward endothelia, led to dose-dependent thrombocytopenia in rats. This was magnified if animals were preimmunized with mouse IgG, thereby resulting in an accelerated autologous phase of injury. In this setting, significant anemia also resulted. Rats injected with K6/1 developed renal injury, consisting of tubular damage and glomerular thrombi. Thrombocytopenia and renal morphological abnormalities were eliminated if animals were complement depleted with cobra venom factor prior to K6/1 injection and worsened when the activity of the ubiquitous complement regulator Crry was inhibited with function-neutralizing antibodies. Therefore, we have developed a complement-dependent model of TTP in rats by injecting monoclonal antibodies reactive with endothelial cells. Antibody-directed complement activation leads to stimulation of platelets, through direct interactions with complement fragments and/or indirectly through endothelial cell activation or injury, with the subsequent development of TTP.

Administration of a soluble recombinant complement C3 inhibitor protects against renal disease in MRL/lpr mice.

Complement receptor 1-related gene/protein y (Crry) in rodents is a potent membrane complement regulator that inhibits complement C3 activation by both classical and alternative pathways. To clarify the role of complement in lupus nephritis, MRL/lpr mice were given Crry as a recombinant protein (Crry-Ig) from 12 to 24 wk of age. Control groups were given saline or normal mouse IgG. Sera and urine were collected biweekly. Only 1 of 20 (5%) Crry-Ig-treated mice developed renal failure (BUN > 50 mg/dl) compared with 18 of 38 (47.4%) mice in control groups (P = 0.001). BUN levels at 24 wk were reduced from 68.8 +/- 9.7 mg/dl in control groups to 38.5 +/- 3.9 mg/dl in the Crry-Ig-treated group (P < 0.01). Urinary albumin excretion at 24 wk was also significantly reduced from 5.3 +/- 1.4 mg/mg creatinine in the control groups to 0.5 +/- 0.2 mg/mg creatinine in the Crry-Ig-treated group (P < 0.05). Of the histologic data at 24 wk, there was a significant reduction in scores for glomerulosclerosis and C3d, IgG, IgG3, and IgA staining intensity in glomeruli in complement-inhibited animals. Crry-Ig-treated animals were also protected from vasculitic lesions. Although there was no effect on relevant autoimmune manifestations such as anti-double stranded DNA titers or cryoglobulin IgG3 levels, circulating immune complex levels were markedly higher in complement-inhibited animals. Thus, inhibition of complement activation with Crry-Ig significantly reduces renal disease in MRL/lpr lupus mice. The data support the strategy of using recombinant complement C3 inhibitors to treat human lupus nephritis.

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

BACKGROUND: Decay-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. METHODS: Function-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. RESULTS: DAF 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. CONCLUSION: In 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.