Podocytes produce homeostatic chemokine stromal cell-derived factor-1/CXCL12, which contributes to glomerulosclerosis, podocyte loss and albuminuria in a mouse model of type 2 diabetes.

AIMS/HYPOTHESIS: Chemokine (C-X-C motif) ligand 12 (CXCL12) (also known as stromal cell-derived factor-1 [SDF-1]-alpha) is a homeostatic chemokine with multiple roles in cell homing, tumour metastasis, angiogenesis and tissue regeneration after acute injuries. However, its role in chronic diseases remains poorly defined, e.g. in chronic glomerular diseases like diabetic glomerulosclerosis. We hypothesised that CXCL12 may have a functional role during the evolution of diabetic glomerulosclerosis, either by assisting glomerular repair or by supporting the maladaptive tissue remodelling in response to hyperglycaemia and glomerular hyperfiltration. METHODS: To define the functional role of CXCL12 in the progression of glomerular disease, we used the CXCL12-specific inhibitor NOX-A12, an L: -enantiomeric RNA oligonucleotide (Spiegelmer). A mouse model of type 2 diabetes (db/db mice) was used. Male db/db mice, uni-nephrectomised at 6 weeks of age, received subcutaneous injections with a PEGylated form of NOX-A12, non-functional control Spiegelmer or vehicle on alternate days from 4 to 6 months of age. RESULTS: Immunostaining localised renal CXCL12 production to glomerular podocytes in db/db mice with early or advanced diabetic nephropathy. CXCL12 inhibition significantly reduced the degree of glomerulosclerosis, increased the number of podocytes, prevented the onset of albuminuria and maintained the peritubular vasculature without affecting blood glucose levels, body weight or glomerular macrophage infiltration. CONCLUSIONS/INTERPRETATION: We conclude that podocytes produce CXCL12, which contributes to proteinuria and glomerulosclerosis in our mouse model of type 2 diabetes. This novel pathomechanism provides the first evidence that CXCL12 could be a therapeutic target in (diabetic) glomerulosclerosis.

Characterization of a protocatechuate catabolic gene cluster from Rhodococcus opacus 1CP: evidence for a merged enzyme with 4-carboxymuconolactone-decarboxylating and 3-oxoadipate enol-lactone-hydrolyzing activity.

The catechol and protocatechuate branches of the 3-oxoadipate pathway, which are important for the bacterial degradation of aromatic compounds, converge at the common intermediate 3-oxoadipate enol-lactone. A 3-oxoadipate enol-lactone-hydrolyzing enzyme, purified from benzoate-grown cells of Rhodococcus opacus (erythropolis) 1CP, was found to have a larger molecular mass under denaturing conditions than the corresponding enzymes previously purified from gamma-proteobacteria. Sequencing of the N terminus and of tryptic peptides allowed cloning of the gene coding for the 3-oxoadipate enol-lactone hydrolase by using PCR with degenerate primers. Sequencing showed that the gene belongs to a protocatechuate catabolic gene cluster. Most interestingly, the hydrolase gene, usually termed pcaD, was fused to a second gene, usually termed pcaC, which encodes the enzyme catalyzing the preceding reaction, i.e., 4-carboxymuconolactone decarboxylase. The two enzymatic activities could not be separated chromatographically. At least six genes of protocatechuate catabolism appear to be transcribed in the same direction and in the following order: pcaH and pcaG, coding for the subunits of protocatechuate 3,4-dioxygenase, as shown by N-terminal sequencing of the subunits of the purified protein; a gene termed pcaB due to the homology of its gene product to 3-carboxy-cis,cis-muconate cycloisomerases; pcaL, the fused gene coding for PcaD and PcaC activities; pcaR, presumably coding for a regulator of the IclR-family; and a gene designated pcaF because its product resembles 3-oxoadipyl coenzyme A (3-oxoadipyl-CoA) thiolases. The presumed pcaI, coding for a subunit of succinyl-CoA:3-oxoadipate CoA-transferase, was found to be transcribed divergently from pcaH.

Evolutionary relationship between chlorocatechol catabolic enzymes from Rhodococcus opacus 1CP and their counterparts in proteobacteria: sequence divergence and functional convergence.

Biochemical investigations of the muconate and chloromuconate cycloisomerases from the chlorophenol-utilizing strain Rhodococcus opacus (erythropolis) 1CP had previously indicated that the chlorocatechol catabolic pathway of this strain may have developed independently from the corresponding pathways of proteobacteria. To test this hypothesis, we cloned the chlorocatechol catabolic gene cluster of strain 1CP by using PCR with primers derived from sequences of N termini and peptides of purified chlorocatechol 1,2-dioxygenase and chloromuconate cycloisomerase. Sequencing of the clones revealed that they comprise different parts of the same gene cluster in which five open reading frames have been identified. The clcB gene for chloromuconate cycloisomerase is transcribed divergently from a gene which codes for a LysR-type regulatory protein, the presumed ClcR. Downstream of clcR but separated from it by 222 bp, we detected the clcA and clcD genes, which could unambiguously be assigned to chlorocatechol 1,2-dioxygenase and dienelactone hydrolase. A gene coding for a maleylacetate reductase could not be detected. Instead, the product encoded by the fifth open reading frame turned out to be homologous to transposition-related proteins of IS1031 and Tn4811. Sequence comparisons of ClcA and ClcB to other 1,2-dioxygenases and cycloisomerases, respectively, clearly showed that the chlorocatechol catabolic enzymes of R. opacus 1CP represent different branches in the dendrograms than their proteobacterial counterparts. Thus, while the sequences diverged, the functional adaptation to efficient chlorocatechol metabolization occurred independently in proteobacteria and gram-positive bacteria, that is, by functionally convergent evolution.

The putative regulator of catechol catabolism in Rhodococcus opacus 1CP--an IclR-type, not a LysR-type transcriptional regulator.

The catechol catabolic genes catABC from Rhodococcus opacus 1CP have previously been characterized by sequence analysis of the insert cloned on plasmid pRER1. Now, a 5.1-kb DNA fragment which overlaps with the insert of pRER1 was cloned, yielding pRER2, and subjected to sequencing. Besides three other open reading frames, a gene was detected ca 200 bp upstream of the catechol 1,2-dioxygenase gene catA, which is obviously transcribed divergently from catABC. The protein which can be deduced from this gene, CatR, resembles members of the PobR subfamily of IclR-type regulatory proteins. This finding was unexpected, as all catechol and chlorocatechol gene clusters known thus far from proteobacteria are under control of LysR-type regulators. It was not possible to inactivate catR by homologous recombination. However, heterologously expressed CatR in vitro bound specifically to the intergenic region between catR and catA thereby providing a first indication for a possible involvement of CatR in the regulation of catechol catabolism.

Characterization of catechol catabolic genes from Rhodococcus erythropolis 1CP.

The biochemical characterization of the muconate and the chloromuconate cycloisomerases of the chlorophenol-utilizing Rhodococcus erythropolis strain 1CP previously indicated that efficient chloromuconate conversion among the gram-positive bacteria might have evolved independently of that among gram-negative bacteria. Based on sequences of the N terminus and of tryptic peptides of the muconate cycloisomerase, a fragment of the corresponding gene has now been amplified and used as a probe for the cloning of catechol catabolic genes from R. erythropolis. The clone thus obtained expressed catechol 1,2-dioxygenase, muconate cycloisomerase, and muconolactone isomerase activities. Sequencing of the insert on the recombinant plasmid pRER1 revealed that the genes are transcribed in the order catA catB catC. Open reading frames downstream of catC may have a function in carbohydrate metabolism. The predicted protein sequence of the catechol 1,2-dioxygenase was identical to the one from Arthrobacter sp. strain mA3 in 59% of the positions. The chlorocatechol 1,2-dioxygenases and the chloromuconate cycloisomerases of gram-negative bacteria appear to be more closely related to the catechol 1,2-dioxygenases and muconate cycloisomerases of the gram-positive strains than to the corresponding enzymes of gram-negative bacteria.