Chantal Mandet

KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron

Familial hyperkalemic hypertension (FHHt) is a Mendelian form of arterial hypertension that is partially explained by mutations in WNK1 and WNK4 that lead to increased activity of the Na+-Cl− cotransporter (NCC) in the distal nephron. Using combined linkage analysis and whole-exome sequencing in two families, we identified KLHL3 as a third gene responsible for FHHt. Direct sequencing of 43 other affected individuals revealed 11 additional missense mutations that were associated with heterogeneous phenotypes and diverse modes of inheritance. Polymorphisms at KLHL3 were not associated with blood pressure. The KLHL3 protein belongs to the BTB-BACK-kelch family of actin-binding proteins that recruit substrates for Cullin3-based ubiquitin ligase complexes. KLHL3 is coexpressed with NCC and downregulates NCC expression at the cell surface. Our study establishes a role for KLHL3 as a new member of the complex signaling pathway regulating ion homeostasis in the distal nephron and indirectly blood pressure.

High Sirolimus Levels May Induce Focal Segmental Glomerulosclerosis De Novo

Sirolimus has been associated with high-range proteinuria when used in replacement of calcineurin inhibitors in renal transplant recipients with chronic allograft nephropathy (CAN). Primary FSGS was demonstrated previously in some such patients, but the coexistence of CAN lesions made the interpretation uneasy. However, nephrotic syndrome and FSGS were observed recently in three patients who received sirolimus de novo, without medical history of primary FSGS or CAN. Markers of podocyte differentiation were studied in kidney biopsies of the three patients who received sirolimus de novo and of five patients who switched to sirolimus. All patients developed FSGS lesions of classic type (not otherwise specified), but only switched patients exhibited advanced sclerotic lesions. Immunohistochemistry showed that some podocytes in FSGS lesions had absent or diminished expression of the podocyte-specific epitopes synaptopodin and p57, reflecting dedifferentiation, and had acquired expression of cytokeratin and PAX2, reflecting a immature fetal phenotype. Such a pattern of epitope expression provides evidence for podocyte dysregulation. Moreover, a decrease in vascular endothelial growth factor expression was observed in some glomeruli. In conclusion, sirolimus induces FSGS that is responsible for proteinuria in some transplant patients.

Sequential immunological targeting of chronic experimental arterial allograft.

Arterial wall is the main site involved in the chronic rejection process. The rat aortic allograft model was used here to characterize and describe the sequential evolution of the different targets and effectors of arterial wall immunological injury and response during arterial allograft rejection. Rat abdominal aortae were isografted or allografted from Brown-Norway to Lewis rats. Endothelial and smooth muscle cell injury and humoral and cellular immunological effectors were characterized from 0 to 60 days after transplantation using a battery of specific antibodies. The intimal proliferative response was also characterized over this time. Isografted Brown-Norway aorta adventitia had very few cellular components, which suggests that donor adventitia would be poorly antigenic in allografts. In contrast, allograft adventitia was the site of a major inflammatory cell invasion in which the expression of an adhesion molecule by colonizing capillary endothelial cells could play a main role. This adventitial infiltration continued as long as medial smooth muscle persisted. The luminal endothelial cells disappeared early, probably associated with macrophage margination. In contrast, medial smooth muscle cell disappearance occurred later and was specifically targeted

Immunohistochemical study of the human glomerular C3b receptor in normal kidney and in seventy-five cases of renal diseases: loss of C3b receptor antigen in focal hyalinosis and in proliferative nephritis of systemic lupus erythematosus.

The presence and distribution of C3b receptors in normal human kidneys and in biopsies from 75 patients with renal disease were investigated by immunohistochemical techniques using monospecific rabbit antibody to the 205,000-mol wt glycoprotein that is the C3b receptor of human peripheral blood cells. Anti-C3b receptor bound exclusively to podocytes in normal renal cortex, and was homogeneously distributed on the plasma membrane of these cells. Biosynthesis of the receptor by the podocyte was suggested by the presence of antigenic activity in the Golgi apparatus. Although occupancy of receptor sites following the interaction of kidney sections with aggregated IgG preincubated with normal serum inhibited binding to glomeruli of C3b coated cells, the C3b receptor remained accessible to anti-C3b receptor antibody. No staining of podocytes was found in extra-capillary proliferating cells in rapidly progressive glomerulonephritis (GN). Segmental loss of staining was found in focal hyalinosis, nodular diabetic glomerulosclerosis, and amyloidosis while no detectable C3b receptor antigen was found in severe proliferative nephritis of systemic lupus erythematosus (SLE). Normal staining of podocytes was found in other nephropathies with endocapillary proliferation such as acute GN and mesangial GN and in renal diseases associated with immune deposits containing C3 such as mesangial proliferative and membranous SLE nephritis, idiopathic membranous GN, membranoproliferative GN types I and II, mesangial GN with IgA or C3 deposition and Henoch Schönleins purpura. Loss of C3b receptor antigen in the diffuse proliferative nephritis of SLE distinguishes it both from nonproliferative lupus nephritis and other immunologically mediated proliferative GN.

Parietal Podocytes in Normal Human Glomeruli

Although parietal podocytes along the Bowmans capsule have been described by electron microscopy in the normal human kidney, their molecular composition remains unknown. Ten human normal kidneys that were removed for cancer were assessed for the presence and the extent of parietal podocytes along the Bowmans capsule. The expression of podocyte-specific proteins (podocalyxin, glomerular epithelial protein-1, podocin, nephrin, synaptopodin, and alpha-actinin-4), podocyte synthesized proteins (vascular endothelial growth factor and novH), transcription factors (WT1 and PAX2), cyclin-dependent kinase inhibitor p57, and intermediate filaments (cytokeratins and vimentin) was tested. In addition, six normal fetal kidneys were studied to track the ontogeny of parietal podocytes. The podocyte protein labeling detected parietal podocytes in all of the kidneys, was found in 76.6% on average of Bowmans capsule sections, and was prominent at the vascular pole. WT1 and p57 were expressed in some parietal cells, whereas PAX2 was present in all or most of them, so some parietal cells coexpressed WT1 and PAX2. Furthermore, parietal podocytes coexpressed WT1 and podocyte proteins. Cytokeratin-positive cells covered a variable part of the capsule and did not express podocyte proteins. Tuft-capsular podocyte bridges were present in 15.5 +/- 3.7% of the glomerular sections. Parietal podocytes often covered the juxtaglomerular arterioles and were present within the extraglomerular mesangium. Parietal podocytes were present in fetal kidneys. Parietal podocytes that express the same epitopes as visceral podocytes do exist along Bowmans capsule in the normal adult kidney. They are a constitutive cell type of the Bowmans capsule. Therefore, their role in physiology and pathology should be investigated.

Chronic Blockade of NO Synthase Activity Induces a Proinflammatory Phenotype in the Arterial Wall Prevention by Angiotensin II Antagonism

Chronic blockade of NO production induces hypertension and early occlusive and fibrotic end-stage organ damage owing to vascular lesions in the brain, kidney, and heart. In this study, we evaluated the inflammatory phenotypic changes induced in the arterial wall by chronic N(G)-nitro-L-arginine methyl ester (L-NAME) administration and the effect of an angiotensin II receptor (AT1) antagonist, irbesartan, on these changes. For this purpose, 2 groups of rats received L-NAME in the drinking water (50 mg x kg(-1) x d(-1)) for 2 months. One group received no other treatment and the other was treated with irbesartan (10 mg x kg(-1) x d(-1)). A third group (controls) received neither L-NAME nor irbesartan. After 8 weeks, plasma, aortas, and left ventricles were sampled from all 3 groups. Expression of inducible NO synthase (iNOS) was evaluated at both the mRNA (quantitative reverse transcription-polymerase chain reaction) and the protein (Western blot and immunohistochemistry) level in the aorta. Expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was evaluated by reverse transcription-polymerase chain reaction, Western immunoblotting, and immunohistochemistry; inflammatory cell infiltration by immunohistochemistry; and fibrosis by Sirius red staining. Chronic L-NAME administration induced the expression of iNOS in the aorta, which was localized in smooth muscle cells as shown by immunohistochemistry and NADPH diaphorase activity. ICAM-1 and VCAM-1 expression was also increased in aortas of L-NAME-treated rats. These phenotypic changes of the vascular wall were associated with inflammatory cell infiltration and fibrosis in the heart. All of these pathological phenomena were prevented by the angiotensin II antagonist irbesartan. The proinflammatory phenotypic changes of the vascular wall induced by blockade of NOS activity could be involved in the interaction between endothelial dysfunction and the development of arteriosclerosis.

PTH-independent regulation of blood calcium concentration by thecalcium-sensing receptor

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney

Four Na+/H+exchangers (NHE1 to NHE4) have been detected in the kidney. Renal NHE2 expression sites have not been fully established. We have raised rabbit antisera against an oligopeptide related to the amino acids 652 to 661 of rat NHE2. Western blot analysis of plasma membrane fractions isolated from rat renal cortex showed that affinity-purified anti-NHE2 antibody detected an 85-kDa protein in apical but not in basolateral membranes. The labeling of this 85-kDa protein was specifically blocked by preincubation of the antibody with its monomeric peptide, indicating specific recognition. Indirect immunolabeling was performed on sections of paraformaldehyde-fixed rat kidney embedded in paraffin. Strong staining was seen in the apical membrane of cortical thick ascending limbs, distal convoluted tubules, and connecting tubules. Much weaker apical staining was found in medullary thick ascending limbs of Henle. In the inner medulla, some thin limbs were intensively labeled by the anti-NHE2 antibody. No staining could be detected in any segments of the proximal tubule and collecting duct.

Inflammatory cells and myocardial fibrosis: spatial and temporal distribution in renovascular hypertensive rats

OBJECTIVEnThe fibroblasts producing collagen are co-localized with inflammatory cells in myocardial fibrosis areas of spontaneously hypertensive rats, suggesting that collagen overproduction in this model may be modulated by inflammatory cells. The present study extends these observations to the Goldblatt model of hypertension in which the renin-angiotensin system is activated.nnnMETHODSnInflammatory cells were identified with monoclonal antibodies directed against macrophages (ED1+), T helper (CD4+) and cytotoxic lymphocytes (CD8+), and MHC class II-expressing cells (Ia+). The alkaline phosphatase-anti-alkaline phosphatase (APAAP) immuno-staining technique was used. A new computer-assisted morphometric method was utilized to quantify the inflammatory infiltrate in each cardiac compartment with polarized-light microscopy. Cells responsible for the collagen synthesis were identified by in situ hybridization. The collagen content was estimated by morphometry on left ventricle sections stained with Sirius red, and by biochemical quantification of the hydroxyproline concentration.nnnRESULTSnComputer-assisted morphometry under polarized light was well suited to quantify inflammatory cells labeled by the APAAP technique. Inflammatory cells were co-localized with collagen-synthesizing fibroblasts. The main inflammatory cells were CD4+ lymphocytes > Ia+ > ED1+ > CD8+ cells. These cell densities were increased in hypertensive rats in all cardiac areas compared to control rats except for IA+ cells which were concentrated in microscars. Macrophage density was correlated with plasma renin activity. The inflammatory cell density which best correlated with fibrosis was macrophage density, and which best correlated with systolic blood pressure was macrophage and T helper lymphocyte densities.nnnCONCLUSIONSnOne can speculate that the correlation between macrophage density and blood pressure as well as with plasma renin activity may indicate that angiotensins and/or elevation of blood pressure could participate in the initial signalling which may mobilize inflammatory cells. These inflammatory cells could promote fibrosis by releasing mediators such as growth factors or cytokines which act upon fibroblasts.

Vascular Smooth Muscle Cell Endovascular Therapy Stabilizes Already Developed Aneurysms in a Model of Aortic Injury Elicited by Inflammation and Proteolysis

Objective:To investigate the efficiency of endovascular smooth muscle cell (VSMC) seeding in promoting healing and stability in already-developed aneurysms obtained by matrix metalloproteases (MMPs)-driven injury. Summary Background Data:VSMCs are instrumental in arterial healing after injury and are in decreased number in arterial aneurysms. This cellular deficiency may account for poor healing capabilities and ongoing expansion of aneurysms. Methods:Aneurysmal aortic xenografts in rats displaying extracellular matrix injury by inflammation and proteolysis were seeded endoluminally with syngeneic VSMCs, with controls receiving culture medium only. Diameter, structure, and the destruction/reconstruction balance were assessed. Results:Eight weeks after endovascular infusion, aneurysmal diameter had increased further, from 3.0 ± 0.3 mm to 10.9 ± 6.5 mm (P = 0.009), and medial elastin content had decreased from 36.5 ± 8.5 to 5.2 ± 5.5 surface-percent (S%; P = 0.009) in controls, whereas these parameters remained stable in the seeded group (3.0 ± 0.3 to 2.7 ± 0.2 mm, P = 0.08; 36.5 ± 8.4 to 31.6 ± 9.7 S%, P = 0.22). VSMC seeding was followed by a decrease in mononuclear infiltration. MMP-1, -3, -7, -9, and -12 mRNA contents were sharply decreased in the diseased wall in response to seeding. Tissue inhibitor of metalloproteinase-1, -2, and -3 mRNAs in the intima were increased in a 2 to 10 magnitude in comparison with controls. Gelatin zymography showed the disappearance of MMP-9 activity and reverse zymography a strong increase in tissue inhibitor of metalloproteinase-3 activity in the seeded group. VSMC-seeded aneurysms were rich in collagen and lined with an endothelium instead of a thrombus in controls. Conclusions:VSMCs endovascular seeding restores the healing capabilities of proteolytically injured extracellular matrix in aneurysmal aortas, and stops expansion.