The AE4 transporter mediates kidney acid-base sensing.

The kidney plays a key role in the correction of systemic acid-base imbalances. Central for this regulation are the intercalated cells in the distal nephron, which secrete acid or base into the urine. How these cells sense acid-base disturbances is a long-standing question. Intercalated cells exclusively express the Na+-dependent Cl-/HCO3- exchanger AE4 (Slc4a9). Here we show that AE4-deficient mice exhibit a major dysregulation of acid-base balance. By combining molecular, imaging, biochemical and integrative approaches, we demonstrate that AE4-deficient mice are unable to sense and appropriately correct metabolic alkalosis and acidosis. Mechanistically, a lack of adaptive base secretion via the Cl-/HCO3- exchanger pendrin (Slc26a4) is the key cellular cause of this derailment. Our findings identify AE4 as an essential part of the renal sensing mechanism for changes in acid-base status.

A new role for the neuronal ubiquitin C-terminal hydrolase-L1 (UCH-L1) in podocyte process formation and podocyte injury in human glomerulopathies.

Glomerular epithelial cell (podocyte) injury is characterized by foot process retraction, slit diaphragm reorganization, and degradation of podocyte-specific proteins. However, the mechanisms underlying podocyte injury are largely unknown. The ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a key modulator of ubiquitin modification in neurons. Like neurons, UCH-L1 expression was associated with an undifferentiated status in cultured human podocytes, whereas differentiation and arborization decreased UCH-L1 and monoUb expression. Inhibition of UCH-L1 induced time and concentration-dependent process formation with alpha-actinin-4 distribution to the cell membrane and processes. An immunohistochemical approach was used to evaluate whether UCH-L1 expression was associated with podocyte injury in 15 different human glomerular diseases. Whereas normal kidneys expressed no UCH-L1 and little ubiquitin, a subset of human glomerulopathies associated with podocyte foot process effacement (membranous nephropathy, SLE class V, FSGS) de novo expressed UCH-L1 in podocyte cell bodies, nuclei, and processes. Interestingly, UCH-L1 expression correlated with podocyte ubiquitin content and internalization of the podocyte-specific proteins nephrin and alpha-actinin-4. In contrast, minimal change glomerulonephritis, a reversible disease, demonstrated minimal UCH-L1 and ubiquitin expression with intact alpha-actinin-4 but internalized nephrin. Glomerular kidney diseases typically not associated with foot process effacement (SLE class IV, ANCA+ necrotizing GN, amyloidosis, IgA nephritis) expressed intermediate to no UCH-L1 and ubiquitin. These studies show a role for UCH-L1 and ubiquitin modification in podocyte differentiation and injury.

Rapid development of severe end-organ damage in C57BL/6 mice by combining DOCA salt and angiotensin II.

The C57BL/6 mouse strain serves as the genetic background of many transgenic and gene knockout models; however, this strain appears to be resistant to hypertension-induced renal injury. We developed a new model of hypertensive end-organ damage in C57BL/6 mice by combining deoxycorticosterone acetate (DOCA) and salt with angiotensin II infusion. The systolic blood pressure (SBP) was significantly elevated in DOCA salt-angiotensin II mice compared to control mice or mice treated individually with DOCA salt or angiotensin II. Hypertensive glomerular damage, increased expression of profibrotic and inflammatory genes, albuminuria, tubular casts, increased plasma cholesterol, cardiac hypertrophy, and fibrosis were found in mice treated with DOCA salt-angiotensin II. The SBP in the angiotensin II-infused group was further increased by increasing the infusion rate; only mild injury was observed in these mice, suggesting that blood pressure was not a causal factor. Removal of DOCA and the angiotensin pump lowered blood pressure to normal; however, albuminuria along with the glomerular and cardiac damage did not completely resolve. Our study describes a new model of hypertensive end-organ damage and repair in C57BL/6 mice.

A pitfall of glomerular sieving: profibrotic and matrix proteins derive from the Bowman's capsule and not the glomerular tuft in rats with renovascular hypertension.

BACKGROUND: The glomeruli in the non-clipped kidney of rats with 2-kidney, 1-clip hypertension are a classical model for studying the mechanisms of glomerular injury. METHODS: In the present study, we compared the glomerular expression of PAI-1 and collagen I alpha1 mRNA from glomeruli isolated by the classic technique of sieving with the recently developed technique of tissue laser microdissection. For quantification of mRNA from both methods, real-time PCR was used. RESULTS: Real-time PCR revealed a 9.0 +/- 1.3- and a 7.1 +/- 0.2-fold induction of PAI-1 and collagen I alpha 1, respectively, in the glomeruli from hypertensive rats isolated by sieving. However, in situ hybridization and microdissection revealed that expression of both mRNAs was mainly from the Bowman's capsule and not from the glomerular tuft (10.7 +/- 1.3- and 7.2 +/- 0.6-fold higher induction in whole glomeruli compared with tuft alone). CONCLUSION: This emphasizes that studies focusing on processes in the mesangium, endothelial cells or podocytes should not rely on glomeruli obtained by sieving. Rather, a technique like the laser microdissection or in situ hybridization should be applied which allows the clear separation of different glomerular and periglomerular compartments.

Antihypertensive therapy upregulates renin and (pro)renin receptor in the clipped kidney of Goldblatt hypertensive rats.

Recently, a (pro)renin receptor has been identified which mediates profibrotic effects independent of angiotensin II. Because antihypertensive therapy induces renal injury in the clipped kidney of two kidney-1-clip hypertensive rats, we examined the regulation of renin and the (pro)renin receptor in this model. Hypertensive Goldblatt rats were treated with increasing doses of the vasopeptidase inhibitor AVE 7688 after which the plasma renin and prorenin as well as the renal renin and (pro)renin receptor expression were measured. The vasopeptidase inhibitor dose-dependently lowered blood pressure, which was associated with a massive increase in plasma prorenin and renin as well as increased renal renin expression. The (pro)renin receptor was upregulated in the clipped kidney of the Goldblatt rat indicating a parallel upregulation of renin and its receptor in vivo. Immunohistochemistry showed a redistribution of renin upstream from the glomerulus in preglomerular vessels and renin staining in tubular cells. Expression of the (pro)renin receptor was increased in the vessels and tubules. This upregulation was associated with thickening of renin-positive vessels and tubulointerstitial damage. We propose that renin and the (pro)renin receptor may play a profibrotic role in the clipped kidney of Goldblatt rats treated for hypertension.

Antihypertensive therapy induces compartment-specific chemokine expression and a Th1 immune response in the clipped kidney of Goldblatt hypertensive rats.

The present study examined the pathogenesis of interstitial inflammation and fibrosis in antihypertensively treated rats with two-kidney, one-clip hypertension. Hypertensive rats were randomized into four groups: no treatment and moderate, intermediate, and intensified lowering of blood pressure with increasing doses of a vasopeptidase inhibitor for 6 wk. The vasopeptidase inhibitor dose dependently lowered blood pressure. The tubulointerstitial damage was accompanied by a diffuse infiltration of mononuclear cells and circumscript mononuclear inflammatory cell cluster formation consisting mainly of T cells and to a lesser degree of macrophages and B cells. Real-time PCR analyses showed a dose-dependent induction of MCP-1 and the Th1-type chemokines IP10 and Mig as well as their receptor CXCR3 and the Th1 cytokine IFN-gamma. In situ hybridization and laser microdissection revealed a strong expression of these Th1-associated transcripts in the clusters and, in the case of MCP-1, also diffusely in the interstitium. The inflammation was accompanied by the appearance of myofibroblasts and synthesis of the fibrogenic factor plasminogen activator inhibitor-1 as well as the collagenase matrix metalloproteinase-2, leading to collagen I upregulation and interstitial scarring. No inflammation or fibrosis was found in normotensive rats treated with the vasopeptidase inhibitor. The renal injury in the clipped kidney is accompanied by compartment-specific chemokine expression and cell cluster formation of Th1 specificity associated with upregulation of fibrogenic proteins and matrix metalloproteinases. These findings suggest that the Th1 chemokines IP10 and Mig as well as their receptor CXCR3 are potential targets for therapeutic interventions in ischemic nephropathy.