Faulty Podocyte Hypoxia Sensing—A Novel Pathway for Rapidly Progressive Glomerulonephritis
Authors: M. Ding, S. Cui, C. Li, S. Jothy, V. Haase, B. Steer, P. Marsden, J. Pippin, S. Shankland, M. Rastaldi, C. Cohen, M. Kretzler, S. Quaggin
Journal: Journal of the American Society of Nephrology (2006)

Rapidly progressive glomerulonephritis (RPGN) is an important nephrologic emergency. Based on underlying immunohistology, RPGN is usually categorized into 3 groups: immune complex–associated RPGN, antibasal membrane antibody–associated RPGN (Goodpasture’s disease), and so-called pauciimmune RPGN with the two partially overlapping entities of Wegener’s granulomatosis and microscopic polyangiitis. In the latter variant, antineutrophil cytoplasmic antibodies (ANCA) (1,2) directed against either proteinase 3 (PR3) or myeloperoxidase (MPO) are found in the great majority of cases. At least in the case of MPO, the elegant experiments of Xiao et al. (3) documented that these antibodies are pathogenetic. A certain proportion of patients with RPGN, 20% according to a study in Wessex (4), markedly less in our experience, fail to present with ANCA. It is for the latter type of ANCA-negative RPGN that the experiments of Ding et al. (5) are of particular clinical relevance, but the implications of the identified mechanisms go considerably beyond this narrow issue and provide more general insights into a novel mechanism of acutely progressive glomerular injury. The conventional concept postulates that in RPGN antineutrophil antibodies interact with polymorphonuclear cells in the intracapillary space and secondarily induce crescent formation (inside→out). In contrast, in the model established by Ding et al. (5) the podocyte has been identified as the primary culprit, suggesting in this model the alternative sequence outside→in. The authors used CRE-lox technology to selectively deactivate the gene coding for the von Hippel-Lindau (vHL) gene product in mouse podocytes. The vHL protein plays a crucial role in oxygen sensing (6): under normoxic conditions proline molecules of the oxygen sensing protein HIF1α are oxydised so that hypoxia-inducible factor 1α (HIF1α) is recognized, captured by vHL, and delivered to the proteasome for proteolytic breakdown (7). In brief, vHL acts as a negative regulator. Under hypoxic conditions the prolines of HIF1α are no longer oxydised, HIF1α accumulates, dimerizes with HIF1β, and increases transcription of downstream genes, thus triggering responses such as increased glucose uptake, glycolysis, angiogenesis, vascular remodeling, and many other processes (8). Loss of vHL function, as produced in the experiment of Ding et al. (5), will imitate the effects of hypoxia and result in the constitutive expression of known HiF target genes. Despite the loss of the vHL gene in the podocytes, the mice were healthy up to the age of 4 wk. At the age of 3 wk, the mice developed proteinuria and dilated capillary loops were seen by histologic analysis. At 4 wk, however, the explosive onset of hematuria, proteinuria, and renal insufficiency was noted. Histologic examination now showed crescentic glomerulonephritis with prominent fibrin deposition and fibrinoid necrosis. ANCA-specific antibodies were not seen. It is currently widely assumed that crescents are derived from proliferation of parietal epithelium with an admixture of infiltrating macrophages, but in a series of studies Ding et al. identified podocytes as the main component of the crescents (5). In this experiment it appears that podocytes lost their terminally differentiated postmitotic phenotype and reentered the cell cycle as shown by pulsing with bromodeoxyuridine. The most important question then was to identify the downstream genes activated in this model of a hypoxia analog in the genetically manipulated podocytes. As expected, examination of isolated glomeruli identified increased HiF1α and HiF2α protein levels. Gene expression profiling confirmed that downstream target genes of the vHL-HiF pathway were upregulated, among others the chemokine receptor CXCR4 and its only ligand, stromal-derived factor (SDF1/CXCligand12). At this point some background information is helpful. The chemokine receptor CXCR4 entered the limelight because of its relation to HIV. HIV enters target cells via the viral envelope glycoprotein (Env) in a coordinated interaction with the CD4 receptor and a chemokine coreceptor (CCR5 or CXCR4) (9,10). This knowledge has led to promising efforts to develop CXCR4 inhibitors (11). To understand its role in renal disease, however, it is relevant to draw attention to its role in the crosstalk between tumor cells and their microenvironment (12). Mesenchymal or marrow-derived stromal cells, which constitute a large proportion of nonneoplastic cells in the tumor microenvironment, secrete the respective ligand SDF-1, which interacts with CXCR4 and attracts tumor cells. The importance of this system is illustrated by the fact that both SDF-1 and CXCR4 knockouts are embryonic lethal (13). SDF-1 is thought to play an important role in aspects such as metastatic tumor spread, tumor cell survival, tumor angiogenesis, and others. In analogy to the above connection to HIV, great hope is currently placed into the potential benefit from CXCR4 antagonists for the treatment of malignant tumors. SDF-1 expression in tumors is triggered by hypoxia (14) and promotes tumor cell proliferation (15) and stem cell trafficking (16), of interest with respect to the findings in the kidney as described below. The role of the SDF-1/CXCR4 system goes far beyond oncology: SDF-1 has been identified, for instance, as an agent promoting negative inotropy in the heart (17), and the role of this system in RPGN may not come as a complete surprise after it was documented in kidney organogenesis (18). By immunostaining and real-time PCR it was shown that SDF-1 was upregulated in the podocytes of mice with conditional vHL knockout. To prove the functional importance of this system, neutralizing anibodies to the receptor of SDF-1 (i.e., CXCR4) were administered before the onset of overt renal disease. This delayed, but failed to prevent, the onset of RPGN. Ding et al. (5) went one step further. To assess whether the SDF-1/CXCR4 system fully accounts for the phenotype, or is only one aggravating factor, they generated transgenic mice expressing CXCR4 selectively in their podocytes. This maneuver caused proliferation of podocytes as well as some glomerular disease, and at 6 mo of age caused hematuria and proteinuria, but failed to reproduce the full-blown scenario of RPGN, suggesting that other systems are also involved in the pathogenesis of vHL knockout–associated RPGN. Finally, to provide evidence for the functional relevance in human disease, the authors examined glomeruli of patients with RPGN using real-time PCR and immunostaining. They found upregulation of HiF target genes including CXCR4, increased immunostaining for CXCR4, and, as sign of podocyte dedifferentiation, reduced staining for synaptopodin. Presumably because the well-perfused glomerulus appeared to be an unlikely target for hypoxic damage, the HiF/vHL system had not been on the agenda of renal investigators, in contrast to the tubulointerstitium, where the great importance of hypoxia had long been postulated (19) and recently confirmed by elegant experiments (20). Of course, the conditional knockout of vHL is not hypoxia per se. It is of interest that RPGN does not appear to be more frequent in vHL disease and the coincidence of renal cell carcinoma, a state of known (albeit local intratumoral) mutation of the vHL gene, may be no more than coincidence of frequent conditions (21,22). The merit of this paper by Ding et al. is to have drawn attention to a potentially very important system in the pathogenesis of glomerular disease, which so far had not been very high on the agenda of nephrologists.Eberhard Ritz: Feature Editor