Genes Expressed by Both Mesangial Cells and Bone Marrow–Derived Cells Underlie Genetic Susceptibility to Crescentic Glomerulonephritis in the Rat
Jennifer Smith*,
Ping-Chin Lai,
Jacques Behmoaras,
Candice Roufosse,
Gurjeet Bhangal*,
John P McDaid*,
Timothy Aitman,
Frederick WK Tam*,
Charles D. Pusey* and
H. Terence Cook
* Section of Renal Medicine, Department of Histopathology and Physiological Genomics and Medicine Group, MRC Clinical Sciences Centre, Imperial College London, London, United Kingdom; and Kidney Institute, Department of Nephrology, School of Medicine, Chang Gung University, Chang Gung Memorial Hospital, Taipei, Taiwan
Address correspondence to: Prof. H. Terence Cook, Department of Histopathology, Hammersmith Campus, Imperial College London, Du Cane Road, London, W12 0NN, UK. Phone: +44-20-8383-2009; Fax: +44-20-8383-814l; E-mail: t.cook{at}imperial.ac.uk
Received for publication July 13, 2006.
Accepted for publication March 29, 2007.
The Wistar-Kyoto (WKY) rat shows marked susceptibility to crescenticglomerulonephritis. In the model of nephrotoxic nephritis (NTN)that is induced by a small dose of nephrotoxic globulin, WKYrats developed crescents in 80 ± 2% of glomeruli at day10, whereas no crescents were seen in Lewis rats. This was associatedwith marked increase in monocyte chemoattractant protein-1 synthesisin WKY glomeruli. It was posited whether susceptibility dependedon circulating cells or intrinsic renal cells. Bone marrow (BM)isografts from WKY to WKY or Lewis to Lewis did not affect susceptibilityto NTN. When BM was transferred from WKY to Lewis rats, crescentsdeveloped in 35 ± 9% of glomeruli 10 d after inductionof NTN, indicating that susceptibility could be transferredby BM cells. However, crescents were also seen in WKY rats thatwere given Lewis marrow. For assessment of the contributionof intrinsic renal cells, kidneys from WKY or Lewis rats weretransplanted into F1 animals. In NTN, the ratio of crescentsin the transplanted kidney to the native kidney was significantlyhigher for WKY-to-F1 than for Lewis-to-F1 transplants, demonstratingthat the kidney itself also influences susceptibility. Mesangialcell responses were then examined in the two strains. Mesangialcells that were derived from WKY rats synthesized significantlymore monocyte chemoattractant protein-1 basally and after stimulationwith heat-aggregated rabbit IgG or TNF-. These results showthat susceptibility to NTN in the WKY rat depends on both circulatingand intrinsic renal cells and that there are genetic differencesbetween the strains in mesangial responses to inflammatory stimuli.
The Wistar-Kyoto (WKY) rat shows a marked susceptibility tocrescentic glomerulonephritis. We and others (1–3) haveshown that, in this strain, administration of a very small doseof nephrotoxic serum (NTS), that would be subnephritogenic inother strains, leads to severe crescentic glomerulonephritis.WKY rats are also more susceptible than other strains to crescenticglomerulonephritis after immunization with glomerular basementmembrane antigen (4–6). A detailed study of the time courseof nephrotoxic nephritis (NTN) in WKY rats (1) showed developmentof glomerular fibrinoid necrosis at day 4, crescent formationin the majority of glomeruli by day 11, and progression to severescarring with renal failure by 6 wk. Crescent formation is dependenton circulating macrophages (7) and CD8+ cells (2) but independentof complement (8). The ease with which crescents can be inducedin this strain and the reproducibility of the NTN model hasled to the use of WKY rats in a number of studies to test theeffect of therapeutic interventions on crescent formation. Strategiesthat have been shown to ameliorate disease in this model includeadministration of IL-4 (9), blockade of TNF- (10), neutralizationof the fractalkine receptor (11), and administration of a leukotrieneB4 receptor antagonist (12).
We have been particularly interested in the reason for thismarked susceptibility of the WKY strain to crescent formation.We recently published a detailed account of the genetics ofthis susceptibility using as a nonsusceptible strain for comparisonthe Lewis rat, which shares the same MHC haplotype (RT1–1)(13). When given a single dose of 0.1 ml of rabbit NTS, WKYrats developed crescents in 80 ± 2%) of glomeruli byday 10, but no crescents were seen in Lewis rats. We found thatF1 rats, generated by crossing WKY and Lewis rats, showed intermediatesusceptibility and that susceptibility in F2 rats spanned therange of the parental strains. A genome-wide linkage analysison the F2 population identified two major quantitative traitloci (QTL) on chromosomes 13 and 16, designated crescentic glomerulonephritis1 (Crgn1) and Crgn2, both of which were linked to crescent formationand proteinuria. Several additional linkages to crescent formationand proteinuria were detected on other chromosomes and weredesignated Crgn3 through Crgn7.
The locus on chromosome 13 included the gene that codes forthe subunit of the activatory Fc receptor Fcgr3. We showedthat most laboratory strains of rat express two forms of Fcgr3but that, in the WKY rat, a newly identified paralogue, Fcgr3-relatedsequence (Fcgr3-rs), was absent. Loss of Fcgr3-rs leads to enhancedantibody-dependent cytotoxicity in macrophages from WKY rats,a phenomenon that was originally described by Rennke et al.(3).
These data indicate that one major determinant of susceptibilityto crescentic glomerulonephritis in the rat is an enhanced Fc-receptor–drivenmacrophage response to intraglomerular Ig. However, the roleof genes at the other QTL that we have identified remains unknown,and neither is it clear whether susceptibility to crescentsis entirely determined by circulating cells or whether intrinsicrenal factors also make a contribution. In an attempt to elucidatethis, we carried out bone marrow (BM) and kidney transplantsbetween the susceptible WKY and resistant Lewis strains. Ourresults show that susceptibility depends on both circulatingcells and the kidney itself. We also demonstrate that mesangialcells from WKY rats show enhanced proinflammatory responsesthat may partly explain the contribution of the kidney to susceptibilityto crescent formation.
Animals
Wistar-Kyoto (WKY/NCrl) and Lewis (LEW/Crl) rats were purchasedfrom Charles River (Margate, UK). F1 rats were generated byintercrossing the two strains. All procedures were performedin accordance with the United Kingdom Animals (Scientific Procedures)Act.
BM Transplantation
Femurs were removed from donor rats. Marrow was flushed outusing RPMI with 10% FBS (Sigma-Aldrich, Poole, UK), 100 U/mlpenicillin, and 100 g/ml streptomycin (Invitrogen, Paisley,UK). Cells were then washed, resuspended in fresh medium at5 x 107 cells/ml, and kept on ice. Recipient rats were irradiatedwith 12 Gy at 0.57 Gy/min, using gamma rays from a cesium sourceirradiator (IBL 637; CIS Bio International, Saclay, France).They were administered an intravenous injection of 0.2 ml ofBM cell preparation within 2 h of irradiation. Rats were lefta minimum of 8 wk to recover and to reconstitute their BM. Ratswere weighed every 2 wk and at the end of the experiment.
Renal Transplantation
Orthotopic transplantation of the left kidney was performedas described previously (14) with removal of the recipient'sown left kidney at the time. After transplantation, rats wereallowed to recover for 6 to 8 d before induction of NTN.
NTN
NTS was prepared in rabbits by standard methods. NTN was inducedin male rats by intravenous injection of 0.1 ml of NTS. Ninedays later, urine was collected by placing rats into metaboliccages for 24 h with free access to food and water. Proteinuriawas determined by the sulfosalicylic acid method (15). On day10 after induction of NTN, rats were killed under isofluraneanesthesia and blood was collected from the abdominal aorta.Samples of kidney, skin, liver, colon, and lung were fixed in10% formal saline, processed, and embedded in paraffin wax.In some cases, glomeruli were isolated by sieving as describedpreviously (16), and 2000 glomeruli were plated in six-wellplates (Nunc, Roskilde, Denmark) in DMEM (Life Technologies,Paisley, UK). After 48 h of incubation, supernatants and celllayers (glomeruli+cells) were collected for sandwich ELISA andquantitative reverse transcription–PCR (qRT-PCR), respectively.
Assessment of BM Chimerism
Genomic DNA was extracted from whole blood using the PuregeneDNA Purification kit (Gentra Systems, Minneapolis, MN). A totalof 100 ng of DNA from the BM transplant recipient was amplifiedby PCR using fluorescence-labeled primers for a polymorphicmarker (D13Rat77) that distinguishes the donor allele from therecipient allele. A standard curve was prepared by mixing varyingpercentages of WKY and Lewis genomic DNA. After separation ofPCR products in an automated DNA sequencer, ABI 3700 or 3730XL(Applied Biosystems, Warrington, UK), allelic-peak height ratioswere calculated for each sample using GeneMapper Software 3.7(ABS, Warrington, UK), and the percentage of donor or recipientDNA of the sample was determined from the standard curve.
Histology and Immunohistochemistry
Crescent formation was assessed by counting the number of crescentsin 100 consecutive glomeruli in periodic acid-Schiff–stainedsections. Macrophages were identified by immunoperoxidase stainingwith mAb ED-1 (Serotec, Oxford, UK) and CD8+ cells were stainedwith mAb MRC OX8 (Serotec) as described previously (9). Thetotal number of immunostained cells in 25 consecutive glomeruliwas counted and converted to a mean count per glomerular crosssection (gcs).
BM-Derived Macrophage Culture
Femurs from adult WKY and Lewis rats were isolated and flushedwith Hanks buffer (Life Technologies). Total BM-derived cellswere plated and cultured for 7 d in DMEM (Life Technologies)that contained 25 mM HEPES (Sigma), 25% L929 conditioned medium,25% decomplemented FBS (F-539; M.B. Meldrum, Bourne End, UK),penicillin (100 U/ml; Invitrogen), streptomycin (100 µg/ml;Invitrogen, Paisley, UK), and l-glutamine (2 mM; Invitrogen).These cells were characterized as macrophages by ED-1 staining.Macrophages were made quiescent in serum-free medium for 24h and then stimulated with TNF- (2 ng/ml). Control macrophageswere unstimulated. Monocyte chemoattractant protein 1 (MCP-1)protein and mRNA levels were measured by sandwich ELISA andqRT-PCR, respectively.
Mesangial Cell Culture
Glomeruli from Lewis and WKY rats were isolated by sieving.Purified glomeruli were digested with collagenase type 1 (Sigma;750 U/ml) for 20 min. Partially digested glomeruli were culturedin 25-cm2 tissue culture flasks at 600 glomeruli/ml in RPMI1640 medium (Invitrogen) that contained 20% decomplemented FBS(F-539), penicillin (100 U/ml; Invitrogen), streptomycin (100µg/ml; Invitrogen), and l-glutamine (2 mM, Invitrogen)and was supplemented with insulin-transferrin-selenite (Sigma).The cultures were maintained at 37°C with 5% CO2 for 6 d,allowing glomerular mesangial cells to grow out. Medium waschanged every 2 to 3 d thereafter. By days 21 to 28, when thecell outgrowth reached confluence, cells were subcultured. Thesecells were characterized by both FACS staining and immunofluorescencestaining using cells that were cultured on coverslips. Theywere positive for Thy-1.1 antigen, myosin, and desmin and negativefor pancytokeratin, OX-1, ED-1, and OX-23.
Comparison of Mesangial Cell Responses between Strains
For making the culture conditions comparable, mesangial cellsfrom different strains of rats were passaged into 48-well cultureplates at the same time. Passage 10 cells were used for eachexperiment. Confluent mesangial cells were stimulated with eitherheat-aggregated rabbit IgG (Sigma) or TNF- (R&D, Abingdon,UK) in serum-free medium. Heat-aggregated IgG was prepared asdescribed previously, with modifications (17). After stimulationfor 24 h, mesangial cell supernatants were harvested and centrifugedto remove cellular contaminants. These supernatants were eitherexamined immediately with sandwich ELISA or stored at –20°C.In each experiment, two wells of cells from each group weretrypsinized and counted in a hemocytometer. The viability ofthe cells was checked by 0.2% trypan blue stain (Invitrogen).MCP-1 was measured in the supernatant by sandwich ELISA as describedpreviously (10).
RNA Extraction and qRT-PCR
Total RNA was isolated from the whole kidney, isolated glomeruli,or macrophages using the Trizol-method. Total RNA concentrationwas determined by using Nanodrop spectrophotometer (LabtechInt., Ringmer, UK). MCP-1 and glyceraldehyde-3-phosphate dehydrogenasegenes forward and reverse primers were as follows: MCP-1 forwardprimer 5'ATGCAGTTAATGCCCCACTC-3', MCP-1 reverse primer 5'TTCCTTATTGGGGTCAGCAC-3'
Real-time RT-PCR was performed on a ABI 7500 Sequence DetectionSystem (Applied Biosystems, Warrington, UK) using SYBR Green(Stratagene, Cambridge, UK). A total of 200 ng of total RNAwas used for qRT-PCR, and all of the samples were amplifiedin triplicate. After the initial reverse transcription (30 minat 50°C and 10 min at 95°C), the samples were cycled40 times at 95°C for 30 s and 60°C for 45 s. Resultswere than exported to 7500 Fast system SDS software (ABS), andCt values were determined for MCP-1 and glyceraldehyde-3-phosphatedehydrogenase. The relative expression levels were then determinedby using the 2–Ct method.
Statistical Analyses
Results are expressed as means ± SEM. Comparisons betweengroups were analyzed by Mann-Whitney U test.
MCP-1 Synthesis by Glomeruli from Native Kidneys with NTN
We previously showed that, after a small dose of NTS, WKY ratsdevelop severe crescentic glomerulonephritis, whereas no crescentsare seen in Lewis rats. It is known that administration of antibodiesto MCP-1 reduces crescent formation in WKY NTN (18). We thereforestudied glomerular MCP-1 synthesis and mRNA levels in WKY andLewis rats 5 d after induction of NTN. As shown in Figure 1,glomeruli from WKY rats showed markedly higher levels of secretedMCP-1 protein and mRNA compared with Lewis rats.
Figure 1. Monocyte chemoattractant protein 1 (MCP-1) production from ex vivo cultured glomeruli after nephrotoxic nephritis (NTN) induction. Four rats from each strain were killed at day 5, and 2000 glomeruli were cultured in DMEM. Supernatants were harvested at 48 h. MCP-1 production was measured in supernatants by sandwich ELISA (A) and in glomeruli by quantitative reverse transcription–PCR (qRT-PCR; B).
BM Transplantation
BM transplants were carried out from WKY to Lewis and from Lewisto WKY. As controls, we carried out isologous transplants ineach strain. In WKY-to-Lewis transplants, the reconstitution,as measured as a percentage of donor DNA in peripheral bloodcells, was 95 ± 3% (n = 6), and in Lewis-to-WKY transplants,it was 90 ± 1% (n = 6). To monitor for graft-versus-hostdisease, we assessed body weight and histology of skin, liver,colon, and lung (19,20). We found no evidence of graft-versus-hostdisease on histologic examination. Percentage weight gain frombefore transplantation to the end of the experiment was similarin WKY rats that were given WKY marrow (16 ± 3%) or Lewismarrow (17 ± 2%). In Lewis rats, percentage weight gainwas higher in those that were given WKY marrow (47 ±4%) than in those with an isologous transplant (34 ±4%), although this did not reach statistical significance (P= 0.064).
Figure 2 shows the results of NTN induction in the rats thatreceived a transplant at 10 d, and Figure 3 shows representativehistology. We found that Lewis rats that received Lewis BM showedno crescents (Figures 2 and 3A), whereas WKY rats that weregiven WKY BM showed marked crescent formation that was comparableto that seen in unmanipulated WKY rats (Figures 2 and 3B). Therefore,the BM transplantation procedure itself did not affect susceptibilityto nephritis. In resistant Lewis rats that were given WKY BM,there was clear and significant glomerulonephritis with proteinuria,macrophage infiltration, and crescent formation (Figures 2 and3C). The number of glomerular CD8+ cells was also significantlyhigher in WKY-to-Lewis than in Lewis-to-Lewis transplants (5.5± 0.4 versus 0.7 ± 0.3 cells/gcs; P = 0.001).However, the numbers of crescents that were seen in these ratswere still considerably less than in WKY rats. In WKY rats thatwere given Lewis BM, there was a significant reduction in crescentformation compared with WKY rats after isologous transplantation.These results show that susceptibility to crescent formationcan be transferred by BM-derived cells but suggest that, aswell as the effect of BM-derived factors, there are other factors,possibly intrinsic to the kidney, that may also affect developmentof crescents.
Figure 2. NTN after bone marrow (BM) transplantation. Rats received a transplant of BM after 12 Gy of irradiation. After 8 wk, rats were given NTS and killed 10 d later. Glomerular crescents (A), proteinuria (B), and glomerular macrophages (C) were analyzed. Lewis-to-Lewis control transplants did not develop crescents. In WKY rats that were given WKY BM, crescent formation was similar to that in unmanipulated WKY rats. There was crescentic glomerulonephritis in Lewis rats that were given WKY BM. In WKY rats that were given Lewis BM, the severity of disease was reduced.
Figure 3. Renal histology in NTN after BM or kidney transplantation. No crescents were seen in glomeruli in a Lewis rat that was given a Lewis BM transplant (A). In a WKY rat that was given a WKY BM transplant, there was crescent formation similar to that seen in unmanipulated WKY rats (B). Crescents were seen in Lewis rat kidneys after transplantation of WKY BM (C) or when transplanted into an F1 (WKY x Lewis) rat (D). All periodic acid-Schiff stain. Magnification, x200.
Kidney Transplantation
To investigate the possibility that the kidney itself contributesto susceptibility to glomerulonephritis, we examined NTN inkidney transplants. We first performed control isografts fromLewis to Lewis (n = 2) and showed that these rats did not developcrescents after induction of NTN, whereas crescentic glomerulonephritisdid develop in WKY-to-WKY grafts (n = 4). We then transplantedkidneys from WKY to Lewis and vice versa but found, despitethat these strains share the same MHC haplotype, that therewas acute cellular rejection, presumably associated with mismatchesat minor histocompatibility antigens. We previously showed (13)that F1 rats that are derived from crossing WKY with Lewis havean intermediate susceptibility to NTN, so we decided to studytransplants from the parental strains into F1 rats, which donot undergo rejection. NTS was given 6 to 8 d after transplantation,and rats were killed 10 d later. Crescents were found in resistantLewis kidneys that were transplanted into F1 rats (31 ±10% crescents; n = 5; Figure 3D). There is some variabilityin the susceptibility of F1 rats to crescentic nephritis; however,if there were no effect of the kidney itself on susceptibility,then we would expect the ratio of the percentage of crescentsin the transplanted kidney to the native kidney to be the samewhatever the strain of the donor kidney. In fact, as shown inFigure 4, there is a significantly higher ratio in transplantsof WKY kidneys, demonstrating that kidneys from this straindevelop more crescents in F1 rats than Lewis kidneys. Therewere also significantly more macrophages in glomeruli of WKYkidneys that were transplanted into F1 rats than in Lewis kidneysthat were transplanted into F1 rats (33 ± 2 versus 25± 3 cells/gcs; P = 0.015). We extracted RNA from transplantand native kidneys and measured MCP-1 mRNA by real-time qRT-PCR.As shown in Figure 5, the relative expression of MCP-1 in transplantedcompared with native kidneys was significantly higher when thedonor was WKY.
Figure 4. Ratio of crescents in transplanted to native kidney. Kidneys were transplanted from Lewis or WKY rats into Lewis x WKY F1 rats. NTN was induced, and rats were killed 10 d later. Each symbol represents the ratio of percentage crescents in the transplanted kidney to that in the native kidney. The ratio is significantly higher for WKY transplants.
Figure 5. MCP-1 expression as assessed by qRT-PCR after induction of NTN in rats with kidney transplants. Lewis kidneys or WKY kidneys were transplanted into WKY x Lewis F1 rats. NTN was induced, and rats were killed 10 d later. MCP-1 mRNA levels normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels are shown as relative to F1 kidneys (expression level = 1) in LEWF1 (A) and in WKYF1 transplants (B); MCP-1 mRNA levels are also presented as transplanted to native relative expression in LEWF1 and WKYF1 rats (C).
Mesangial Cell Studies
We reasoned that if susceptibility to crescent formation dependedin part on the kidney, then this might be due to a differencein mesangial cell responses to inflammatory stimuli. Mesangialcells synthesize MCP-1, and administration of antibodies toMCP-1 reduces crescent formation in WKY NTN (18). In addition,as we have shown here that there is considerably enhanced glomerularsynthesis of MCP-1 in NTN in WKY compared with Lewis rats. Wetherefore studied mesangial cell synthesis of MCP-1 in WKY andLewis rats. Figure 6 shows MCP-1 production by mesangial cellsthat were stimulated with heat-aggregated IgG. Significantlymore MCP-1 is secreted by WKY compared with Lewis mesangialcells, but, interesting, unstimulated WKY mesangial cells alsoproduced more MCP-1 than Lewis mesangial cells. Stimulationwith TNF- also led to significantly more MCP-1 secretion byWKY mesangial cells (Figure 7). These experiments were carriedout by using replicate wells of cell lines that were derivedfrom single WKY or Lewis rats. We were concerned that this mightnot represent a true strain difference but merely incidentalselection during the process of deriving the cell lines. Wetherefore set up mesangial cell lines from several rats, andFigure 8 shows a comparison of MCP-1 production by mesangialcell lines that were derived from four Lewis rats and five WKYrats. There was significantly higher MCP-1 synthesis by WKYmesangial cells with or without stimulation by rabbit IgG. Therefore,experiments from multiple mesangial cell lines that were derivedfrom several Lewis and WKY rats confirmed that WKY rat mesangialcells produced more MCP-1 than those from Lewis rats. We alsocompared the synthesis of MCP-1 by BM-derived macrophages fromWKY and Lewis rats and found that macrophages from WKY ratscontained more MCP-1 mRNA and secreted more MCP-1, but, in contrastto mesangial cells, there was no effect of TNF- (Figure 9).
Figure 6. MCP-1 production from WKY and Lewis mesangial cells after stimulation with heat-aggregated IgG (AIgG). Mesangial cells were made quiescent by culturing in serum-free medium for 24 h and then stimulated with 200 µg/ml AIgG for another 24 h. Control cells were unstimulated. MCP-1 was measured in cell supernatants by sandwich ELISA.
Figure 7. MCP-1 production from WKY and Lewis mesangial cells after stimulation with TNF- (2 ng/ml). Mesangial cells were made quiescent by culturing in serum-free medium for 24 h and then stimulated with TNF- (2 ng/ml) for another 24 h. Control cells were unstimulated. MCP-1 was measured in cell supernatants by sandwich ELISA.
Figure 8. Comparison of MCP-1 production from Lewis and WKY mesangial cells when stimulated with 200 µg/ml AIgG. Mesangial cells that were derived from four Lewis and five WKY rats were used, and each point represents the mesangial cell response (six pooled replicates) from one rat. Control cells were unstimulated. MCP-1 was measured in cell supernatants by sandwich ELISA.
Figure 9. MCP-1 production from WKY and Lewis BM-derived macrophages. Macrophages were made quiescent by culturing in serum-free medium for 24 h and then stimulated with TNF- (2 ng/ml). Control macrophages were unstimulated. MCP-1 protein and mRNA levels were measured by sandwich ELISA and qRT-PCR, respectively. One-way ANOVA followed by Bonferroni test was applied, and all of the samples were compared with Lewis rat.
The unique susceptibility of the WKY rat to crescentic glomerulonephritishas made it a very useful model system for studying therapeuticinterventions, but little attention has been paid to the reasonsfor why this strain is so susceptible. We believe that understandingthis is likely to lead to important insights into the mechanismsof crescent formation. We previously reported the results ofa genome-wide linkage study that showed that crescent formationwas strongly linked to QTL on chromosomes 13 and 16 (13) withless strong linkage to five other QTL. We further showed thatFcgr3, a candidate gene in the chromosome 13 QTL, has multiplecopies in the rat and that a novel form of Fcgr3, which we havenamed Fcgr3-rs, is absent from the WKY genome. Loss of Fcgr3-rsis associated with enhanced macrophage antibody–dependentcytotoxicity and Fc-mediated phagocytosis. We therefore hypothesizedthat susceptibility to NTN depended, at least in part, on increasedreactivity of circulating macrophages to antibody bound in glomeruli.However, our genetic analysis showed that there must be othergenes contributing to the phenotype, so we thought that it wasimportant to discover whether susceptibility was entirely dependenton the functions of circulating cells.
We initially used BM transplant experiments to test the roleof circulating cells. These experiments showed unequivocallythat the susceptibility to crescents could be transferred toresistant Lewis rats by WKY BM. This is consistent with a majorrole for Fc receptor–mediated responses in susceptibilitybut, of course, does not exclude the possibility that theremay be other functional differences in WKY and Lewis macrophagesor other leukocytes that contribute to susceptibility. However,even with 95% reconstitution by donor BM, we saw crescents inonly a mean of 35% of glomeruli of Lewis rats compared with89% in control WKY rats that received isologous BM. Similarly,although transplantation of Lewis BM into WKY rats led to significantlyfewer crescents (51%), it did not abolish the susceptibilityto crescent formation. This suggested that there was an importantcontribution to severity of crescentic glomerulonephritis thatwas not dependent on BM-derived cells. This intrinsic contributioncould be either from the kidney itself or possibly from an extrarenal,BM-independent effect, for example from the endocrine system.To test directly whether there was an intrinsic renal influence,we therefore studied kidney transplants.
The kidney transplant experiments were made more complex bythe fact that there was rejection when we transplanted kidneysbetween the strains. We therefore made use of the fact, whichwe had shown previously, that F1 rats have an intermediate susceptibilityto glomerulonephritis compared with the parental strains. Itis possible to transplant from parental strains into F1 animalswithout rejection because the recipient animals share all ofthe antigens of the donor. It may be that the process of transplantationitself could have an effect on the severity of crescentic glomerulonephritisin the transplanted kidney, and, in addition, there is somevariability between the susceptibility of the F1 animals. Therefore,to take account of this, we compared the severity of glomerulonephritisin the transplanted kidney with that in the native kidney. Theresults with kidney transplantation confirmed the findings withBM transplantation, in that Lewis kidneys developed crescenticglomerulonephritis when placed in an appropriate (WKY) environment.However, the severity of glomerulonephritis in transplantedLewis kidneys relative to native kidneys in F1 recipients wassignificantly less than that of transplanted WKY kidneys, indicatingthat the kidney itself makes a contribution to susceptibilityto glomerulonephritis. It is noteworthy that whereas four ofsix WKY transplanted kidneys showed similar disease severityto the recipient F1 kidneys, the Lewis transplanted kidneysshowed clearly less disease, suggesting the presence of protectivefactors in Lewis kidney. We found a similar effect on MCP-1mRNA levels and glomerular macrophage numbers, which were higherin WKY kidneys than in Lewis kidneys that were transplantedinto F1 animals. This contrasts with experiments in the Thy-1model of mesangial proliferative glomerulonephritis in ratsusing kidney transplants, which showed that influx of macrophageswas entirely dependent on circulating cells (21). Conversely,in the same Thy-1 model, experiments that used two substrainsof Lewis rat with different susceptibility to glomerulosclerosisshowed that this susceptibility is governed by genes that areexpressed by the kidney (22). As far as we are aware, our studyis the first to show a naturally occurring genetic susceptibilityto glomerular inflammation that is dependent on the kidney.
There is considerable evidence from studies in mice that intrinsicglomerular cells contribute to the severity of glomerular inflammation.For example, in a series of elegant experiments, the group ofTipping and Holdsworth studied mice in which BM is transplantedbetween wild-type mice and mice that are genetically engineeredto lack selected cytokines, allowing the construction of chimeraswith cytokine deficiency in BM-derived cells or intrinsic renalcells (23). They showed that intrinsic renal cell productionof TNF- (24), IFN- (25), and IL-12 (26) and intrinsic renalcell expression of CD40 (27) contribute to induction of crescenticglomerulonephritis in a mouse model of NTN. It is not possibleto be certain from their experiments, but a likely intrarenalsource of these cytokines is the glomerular mesangial cell.We previously showed that rat mesangial cells that are stimulatedwith IL-1 produce macrophage chemotactic factors, includingMCP-1, and that culture supernatants from stimulated mesangialcells are able to suppress macrophage activation as assessedby nitric oxide synthesis (28). All of these studies indicatean important cross-talk between mesangial cells and inflammatorycells in the mediation of glomerulonephritis. It is thereforenot entirely unexpected that in the WKY rat, where several differentgenes are involved in susceptibility, one or more of these couldcontrol mesangial cell function.
To define further the possible role of the mesangial cell inthe susceptibility of the WKY rat to glomerulonephritis, weexamined the response of cultured mesangial cells to variousstimuli. We first studied responses to aggregated rabbit IgGbecause rabbit IgG is used as the nephrotoxic globulin in ourmodel. In response to aggregated IgG, we found that WKY mesangialcells made considerably more MCP-1 than did Lewis mesangialcells. One possible explanation for this result is that it isdue to the absence of Fcgr3-rs, as we have shown for WKY macrophages.However, our previous studies in mice showed no role for intrinsicrenal cell expression of Fc receptors in mouse NTN (29). Wetherefore looked at responses to other stimuli and showed thatthere was also enhanced synthesis of MCP-1 by WKY mesangialcells in response to TNF- (Figure 6) and LPS (data not shown).It is interesting that we also showed enhanced MCP-1 synthesisby WKY macrophages, although this was not further increasedby TNF-. The cellular mechanism that determines this enhancedresponse remains to be elucidated. We are currently breedingrats that are congenic for the QTL that we have described onchromosomes 13 and 16, and these should allow us to determinewhich of these loci are responsible for the phenotypes shownby macrophages and mesangial cells in the WKY rat.
Our results indicate that the unique susceptibility of the WKYrat to crescentic glomerulonephritis is due to several genes.One of these is Fcgr3, which is responsible for the enhancedresponses of WKY macrophages to immune complexes, although othergenes may also be involved in the macrophage phenotype. Transferof BM-derived cells is sufficient to transfer susceptibilityto crescentic glomerulonephritis to a nonsusceptible strain,but the full-blown crescentic nephritis that is seen in theWKY rat is also dependent on genes that control intrinsic renalcell responses and in particular the response of mesangial cellsto proinflammatory stimuli. This is the first demonstrationof naturally occurring genetic differences in mesangial cellphenotype and raises the intriguing possibility that similargenetic variation may occur in human mesangial cell responsesand could influence development of glomerulonephritis.
Tam FWK, Smith J, Morel D, Karkar AM, Thompson EM, Cook HT, Pusey CD: Development of scarring and renal failure in a rat model of crescentic glomerulonephritis.
Nephrol Dial Transplant 14
: 1658
–1666, 1999[Abstract/Free Full Text]
Kawasaki K, Yaoita E, Yamamoto T, Kihara I: Depletion of CD8 positive cells in nephrotoxic serum nephritis of WKY rats.
Kidney Int 41
: 1517
–1526, 1992[Medline]
Granados R, Mendrick DL, Rennke HG: Antibody-induced crescent formation in WKY rats: Potential role of antibody-dependent cell cytotoxicity (ADCC) in vivo [Abstract].
Kidney Int 37
: 414A
, 1990
Sado Y, Naito I, Akita M, Okigaki T: Stain specific responses of inbred rats on the severity of experimental autoimmune glomerulonephritis.
J Clin Lab Immunol 19
: 193
–199, 1986[Medline]
Bolton WK, May WJ, Sturgill BC: Proliferative autoimmune glomerulonephritis in rats: A model for autoimmune glomerulonephritis in humans.
Kidney Int 44
: 294
–306, 1993[Medline]
Reynolds J, Mavromatidis K, Cashman SJ, Evans DJ, Pusey CD: Experimental autoimmune glomerulonephritis (EAG) induced by homologous and heterologous glomerular basement membrane in two substrains of Wistar-Kyoto rat.
Nephrol Dial Transplant 13
: 44
–52, 1998[Abstract/Free Full Text]
Isome M, Fujinaka H, Adhikary LP, Kovalenko P, El Shemi AG, Yoshida Y, Yaoita E, Takeishi T, Takeya M, Naito M, Suzuki H, Yamamoto T: Important role for macrophages in induction of crescentic anti-GBM glomerulonephritis in WKY rats.
Nephrol Dial Transplant 19
: 2997
–3004, 2004[Abstract/Free Full Text]
Shike T, Isome M, Mizuno M, Suzuki J, Matsuo S, Yamamoto T, Suzuki H: Implication of the complement system in the induction of anti-glomerular basement membrane glomerulonephritis in WKY rats.
Pathol Int 53
: 757
–761, 2003[CrossRef][Medline]
Cook HT, Singh SJ, Wembridge DE, Smith J, Tam FWK, Pusey CD: Interleukin 4 ameliorates crescentic glomerulonephritis in Wistar Kyoto rats.
Kidney Int 55
: 1319
–1326, 1999[CrossRef][Medline]
Khan SB, Cook HT, Bhangal G, Smith J, Tam FW, Pusey CD: Antibody blockade of TNF-alpha reduces inflammation and scarring in experimental crescentic glomerulonephritis.
Kidney Int 67
: 1812
–1820, 2005[CrossRef][Medline]
Feng L, Chen S, Garcia GE, Xia Y, Siani MA, Botti P, Wilson CB, Harrison JK, Bacon KB: Prevention of crescentic glomerulonephritis by immunoneutralization of the fractalkine receptor CX3CR1 rapid communication.
Kidney Int 56
: 612
–620, 1999[CrossRef][Medline]
Suzuki S, Kuroda T, Kazama JI, Imai N, Kimura H, Arakawa M, Gejyo F: The leukotriene B4 receptor antagonist ONO-4057 inhibits nephrotoxic serum nephritis in WKY rats.
J Am Soc Nephrol 10
: 264
–270, 1999[Abstract/Free Full Text]
Aitman TJ, Dong R, Vyse TJ, Norsworthy PJ, Johnson MD, Smith J, Mangion J, Roberton-Lowe C, Marshall AJ, Petretto E, Hodges MD, Bhangal G, Patel SG, Sheehan-Rooney K, Duda M, Cook PR, Evans DJ, Domin J, Flint J, Boyle JJ, Pusey CD, Cook HT: Copy number polymorphism in Fcgr3 predisposes to glomerulonephritis in rats and humans.
Nature 439
: 851
–855, 2006[CrossRef][Medline]
Lameijer JM, Smith J, Mowbray JF: Effect of kidney perfusion on renal allograft survival in azathioprine treated rats.
Br J Exp Pathol 53
: 130
–137, 1972[Medline]
Baker FJ, Silverton RE, Luckcock ED:
An Introduction to Medical Laboratory Technology, London, Butterworth, 1966
Cook HT, Cattell V, Smith J, Salmon JA, Moncada S: Effect of a thromboxane synthetase inhibitor on eicosanoid synthesis and glomerular injury during acute unilateral glomerulonephritis in the rat.
Clin Nephrol 26
: 195
–202, 1986[Medline]
Hora K, Satriano JA, Santiago A, Mori T, Stanley ER, Shan Z, Schlondorff D: Receptors for IgG complexes activate synthesis of monocyte chemoattractant peptide 1 and colony-stimulating factor 1.
Proc Natl Acad Sci U S A 89
: 1745
–1749, 1992[Abstract/Free Full Text]
Wada T, Yokoyama H, Furuichi K, Kobayashi KI, Harada K, Naruto M, Su SB, Akiyama M, Mukaida N, Matsushima K: Intervention of crescentic glomerulonephritis by antibodies to monocyte chemotactic and activating factor (MCAF/MCP-1).
FASEB J 10
: 1418
–1425, 1996[Abstract]
Blazar BR, Taylor PA, Snover DC, Bluestone JA, Vallera DA: Nonmitogenic anti-CD3F(ab')2 fragments inhibit lethal murine graft-versus-host disease induced across the major histocompatibility barrier.
J Immunol 150
: 265
–277, 1993[Abstract]
Fontaine P, Perrault C: Diagnosis of graft-versus-host disease across minor histocompatibility barriers.
Transplantation 49
: 1177
–1179, 1990[Medline]
de Heer E, Prodjosudjadi W, Davidoff A, van der Wal A, Bruijn JA, Paul LC: Control of monocyte influx in glomerulonephritis in transplanted kidneys in the rat.
Lab Invest 78
: 1327
–1337, 1998[Medline]
Aben JA, Hoogervorst DA, Paul LC, Borrias MC, Noble NA, Border WA, Bruijn JA, de Heer E: Genes expressed by the kidney, but not by bone marrow-derived cells, underlie the genetic predisposition to progressive glomerulosclerosis after mesangial injury.
J Am Soc Nephrol 14
: 2264
–2270, 2003[Abstract/Free Full Text]
Tipping PG, Timoshanko J: Contributions of intrinsic renal cells to crescentic glomerulonephritis.
Nephron Exp Nephrol 101
: e173
–e178, 2005[CrossRef][Medline]
Timoshanko JR, Sedgwick JD, Holdsworth SR, Tipping PG: Intrinsic renal cells are the major source of tumor necrosis factor contributing to renal injury in murine crescentic glomerulonephritis.
J Am Soc Nephrol 14
: 1785
–1793, 2003[Abstract/Free Full Text]
Timoshanko JR, Holdsworth SR, Kitching AR, Tipping PG: IFN-gamma production by intrinsic renal cells and bone marrow-derived cells is required for full expression of crescentic glomerulonephritis in mice.
J Immunol 168
: 4135
–4141, 2002[Abstract/Free Full Text]
Timoshanko JR, Kitching AR, Holdsworth SR, Tipping PG: Interleukin-12 from intrinsic cells is an effector of renal injury in crescentic glomerulonephritis.
J Am Soc Nephrol 12
: 464
–471, 2001[Abstract/Free Full Text]
Largen PJ, Tam FWK, Rees AJ, Cattell V: Rat mesangial cells have a selective role in macrophage recruitment and activation.
Exp Nephrol 3
: 34
–39, 1995[Medline]
Tarzi RM, Davies KA, Robson MG, Fossati-Jimack L, Saito T, Walport MJ, Cook HT: Nephrotoxic nephritis is mediated by Fcgamma receptors on circulating leukocytes and not intrinsic renal cells.
Kidney Int 62
: 2087
–2096, 2003[CrossRef]
This article has been cited by other articles:
P. G. Tipping Crescentic nephritis--is it in your genes?
Nephrol. Dial. Transplant.,
October 1, 2008;
23(10):
3065 - 3066.
[Full Text][PDF]
Top
Abstract
Introduction
. These results show that susceptibility to NTN in the WKY rat depends on both circulating and intrinsic renal cells and that there are genetic differences between the strains in mesangial responses to inflammatory stimuli. 
(25), and IL-12 (26) and intrinsic renal cell expression of CD40 (27) contribute to induction of crescentic glomerulonephritis in a mouse model of NTN. It is not possible to be certain from their experiments, but a likely intrarenal source of these cytokines is the glomerular mesangial cell. We previously showed that rat mesangial cells that are stimulated with IL-1
produce macrophage chemotactic factors, including MCP-1, and that culture supernatants from stimulated mesangial cells are able to suppress macrophage activation as assessed by nitric oxide synthesis (28). All of these studies indicate an important cross-talk between mesangial cells and inflammatory cells in the mediation of glomerulonephritis. It is therefore not entirely unexpected that in the WKY rat, where several different genes are involved in susceptibility, one or more of these could control mesangial cell function. 
F1 (A) and in WKY
