We recently showed in a tetracycline-controlled transgenic mouse model that overexpression of transforming growth factor (TGF)-␤1 in renal tubules induces widespread peritubular fibrosis and focal degeneration of nephrons. In the present study we have analyzed the mechanisms underlying these phenomena.

Activation of the intrarenal renin-angiotensin system (RAS) can elicit hypertension independently from the systemic RAS. However, the precise mechanisms by which intrarenal Ang II increases blood pressure have never been identified. To this end, we studied the responses of mice specifically lacking kidney angiotensin-converting enzyme (ACE) to experimental hypertension. Here, we show that the absence of kidney ACE substantially blunts the hypertension induced by Ang II infusion (a model of high serum Ang II) or by nitric oxide synthesis inhibition (a model of low serum Ang II). Moreover, the renal responses to high serum Ang II observed in wild-type mice, including intrarenal Ang II accumulation, sodium and water retention, and activation of ion transporters in the loop of Henle (NKCC2) and distal nephron (NCC, ENaC, and pendrin) as well as the transporter activating kinases SPAK and OSR1, were effectively prevented in mice that lack kidney ACE. These findings demonstrate that ACE metabolism plays a fundamental role in the responses of the kidney to hypertensive stimuli. In particular, renal ACE activity is required to increase local Ang II, to stimulate sodium transport in loop of Henle and the distal nephron, and to induce hypertension. IntroductionHypertension affects more than 1.5 billion people worldwide and is a key contributor to stroke and cardiovascular and kidney disease. The importance of the renin-angiotensin system (RAS) in the origins of this disorder is underscored by the blood pressure-lowering effects of angiotensin-converting enzyme (ACE) inhibitors and Ang II receptor blockers. However, plasma renin activity, the clinical index used to determine systemic RAS status, is distributed over a wide range in hypertensive subjects (1, 2). This observation prompts the suggestion that alterations in tissue-specific RAS, not detected by plasma renin activity, may underlie hypertension. The kidneys play a central role in long-term blood pressure control through their regulation of sodium and fluid balance. Because renal salt retention is strongly influenced by Ang II and there is a complete RAS along the nephron, it has been suggested that increased local Ang II formation may induce hypertension. Indeed, using gene-targeted mice, we and others have shown that increased intrarenal Ang II formation results in hypertension (3-6). As a whole, these observations suggest that renal Ang II synthesis has important consequences for nephron function and the development of hypertension. However, precisely how the intrarenal generation of Ang II elevates blood pressure is not known. Therefore, we tested the hypothesis that, in conditions in which the intrarenal RAS becomes activated, local Ang II synthesis enhances sodium and water reabsorption along the nephron. In addition, we postulated that inhibiting intrarenal Ang II formation effectively protects against hypertension.

The heteromeric inwardly rectifying Kir4.1/Kir5.1 K + channel underlies the basolateral K + conductance in the distal nephron and is extremely sensitive to inhibition by intracellular pH. The functional importance of Kir4.1/Kir5.1 in renal ion transport has recently been highlighted by mutations in the human Kir4.1 gene ( KCNJ10 ) that result in seizures, sensorineural deafness, ataxia, mental retardation, and electrolyte imbalance (SeSAME)/epilepsy, ataxia, sensorineural deafness, and renal tubulopathy (EAST) syndrome, a complex disorder that includes salt wasting and hypokalemic alkalosis. Here, we investigated the role of the Kir5.1 subunit in mice with a targeted disruption of the Kir5.1 gene ( Kcnj16 ). The Kir5.1 −/− mice displayed hypokalemic, hyperchloremic metabolic acidosis with hypercalciuria. The short-term responses to hydrochlorothiazide, an inhibitor of ion transport in the distal convoluted tubule (DCT), were also exaggerated, indicating excessive renal Na + absorption in this segment. Furthermore, chronic treatment with hydrochlorothiazide normalized urinary excretion of Na + and Ca 2+ , and abolished acidosis in Kir5.1 −/− mice. Finally, in contrast to WT mice, electrophysiological recording of K + channels in the DCT basolateral membrane of Kir5.1 −/− mice revealed that, even though Kir5.1 is absent, there is an increased K + conductance caused by the decreased pH sensitivity of the remaining homomeric Kir4.1 channels. In conclusion, disruption of Kcnj16 induces a severe renal phenotype that, apart from hypokalemia, is the opposite of the phenotype seen in SeSAME/EAST syndrome. These results highlight the important role that Kir5.1 plays as a pH-sensitive regulator of salt transport in the DCT, and the implication of these results for the correct genetic diagnosis of renal tubulopathies is discussed.

We investigated the proliferative capacity of renal proximal tubular cells in healthy rats. Previously, we observed that tubular cells originate from differentiated cells. We now found 1) by application of bromo-deoxyuridine (BrdU) for 14 days and costaining for BrdU, and the G 1-phase marker cyclin D1 that the bulk of cells in the S3 segment of juvenile rats were involved in proliferation; 2) that although the proliferation rate was about 10-fold higher in juvenile rats compared with adult rats, roughly 40% of S3 cells were in G 1 in both groups; 3) that after a strong mitotic stimulus (lead acetate), proliferation was similar in juveniles and adults; 4) that there was a high incidence of cyclin D1-positive cells also in the healthy human kidney; and 5) by labeling dividing cells with BrdU for 2 days before the application of lead acetate and subsequent costaining for BrdU and cell cycle markers, that, although a strong mitotic stimulus does not abolish the period of quiescence following division, it shortens it markedly. Thus the capacity of the proximal tubule to rapidly recruit cells into division relies on a large reserve pool of cells in G 1 and on the shortening of the obligatory period of quiescence that follows division. kidney; cyclin D1; bromodeoxyuridine; stem cells; regeneration HOW THE KIDNEY maintains the homeostasis of tubular epithelial cell number has been a fundamental question for years. Since differentiated cells in most tissues, including renal tubules, have only a limited life span, it is crucial for these cells to be replaced to maintain organ function. Also a variation in tissue mass as seen during physiological adaptation encompasses the production of specialized cells. Furthermore, after injury, the generation of new cells is a prerequisite to restore the normal function of the injured organ.The source of newly formed renal epithelial cells is debated. Some studies show that novel cells are derived from divisions of differentiated cells (1,10,27), and others claim that a subpopulation in the renal tubular epithelial cells function as progenitor cells (5,9,16,19). In addition, hematopoietic stem cells (12,15,21,22) and mesenchymal stem cells (18) have been shown to be able to repopulate the renal tubular system; however, under physiological conditions tubular cells originating from extrarenal stem cells are not detectable in the kidney, and even after severe organ injury these cells constitute only a small population (7).Our previous studies revealed that tubular cell proliferation in healthy rats rely on the division of differentiated cells (25). Furthermore, we were unable to detect any slow-cycling stem cells or rapidly cycling transit amplifying (TA) cells to support a "classic" stem cell system (26).In the present study we address the question whether the capacity to divide is restricted to a small subpopulation of the differentiated cells in S3 or whether it is broadly distributed among the cells in that segment. To that aim we investigated the cycling behavior of tubular cells in...

Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 gene family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause Bartter syndrome, a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

ATPase H-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na-K-2Cl cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

Tubulo-interstitial Wbrosis is a constant feature of chronic renal failure and it is suspected to contribute importantly to the deterioration of renal function. In the Wbrotic kidney there exists, besides normal Wbroblasts, a large population of myoWbroblasts, which are supposedly responsible for the increased production of intercellular matrix. It has been proposed that myoWbroblasts in chronic renal failure originate from the transformation of tubular cells via epithelial-mesenchymal transition (EMT) or from inWltration by bone marrow-derived precursors. Little attention has been paid to the possibility of a transformation of resident Wbroblasts into myoWbroblasts in renal Wbrosis. Therefore we examined the fate of resident Wbroblasts in the initial phase of renal Wbrosis in the classical model of unilateral ureter obstruction (UUO) in the rat. Rats were perfusion-Wxed on days 1, 2, 3 and 4 after ligature of the right ureter. Starting from 1 day of UUO an increasing expression of alpha-smooth muscle actin ( SMA) in resident Wbroblasts was revealed by immunoXuorescence and conWrmed by the observation of bundles of microWlaments and webs of intermediate Wlaments in the electron microscope. Inversely, there was a decreased expression of 5Ј-nucleotidase (5ЈNT), a marker of renal cortical Wbroblasts. The RER became more voluminous, suggesting an increased synthesis of matrix. Intercellular junctions, a characteristic feature of myoWbroblasts, became more frequent. The mitotic activity in Wbroblasts was strongly increased. Renal tubules underwent severe regressive changes but the cells retained their epithelial characteristics and there was no sign of EMT. In conclusion, after ureter ligature, resident peritubular Wbroblasts proliferated and they showed progressive alterations, suggesting a transformation in myoWbroblasts. Thus the resident Wbroblasts likely play a central role in Wbrosis in that model.

An inverse relationship exists between urinary tissue kallikrein (TK) excretion and blood pressure in humans and rodents. In the kidney TK is synthesized in large amounts in the connecting tubule and is mainly released into the urinary fluid where its function remains unknown. In the present study mice with no functional gene coding for TK (TK ؊/؊ ) were used to test whether the enzyme regulates apically expressed sodium transporters. Semiquantitative immunoblotting of the renal cortex revealed an absence of the 70-kDa form of ␥-ENaC in TK ؊/؊ mice. Urinary Na ؉ excretion after amiloride injection was blunted in TK ؊/؊ mice, consistent with reduced renal ENaC activity. Amiloride-sensitive transepithelial potential difference in the colon, where TK is also expressed, was decreased in TK ؊/؊ mice, whereas amiloridesensitive alveolar fluid clearance in the lung, where TK is not expressed, was unchanged. In mice lacking the B2 receptor for kinins, the abundance of the 70-kDa form of ␥-ENaC was increased, indicating that its absence in TK ؊/؊ mice is not kinin-mediated. Incubation of membrane proteins from renal cortex of TK ؊/؊ mice with TK resulted in the appearance of the 70-kDa band of the ␥-ENaC, indicating that TK was able to promote ␥-ENaC cleavage in vitro. Finally, in mouse cortical collecting ducts isolated and microperfused in vitro, the addition of TK in the luminal fluid increased significantly intracellular Na ؉ concentration, consistent with an activation of the luminal entry of the cation. The results demonstrate that TK, like several other proteases, can activate ENaC in the kidney and the colon. Tissue kallikrein5 (TK) 6 is a serine protease that generates kinins locally in many organs, including the kidney, colon, and arteries. In the kidney, TK that is synthesized in large amounts by connecting tubule cells (1) is mainly secreted into the urinary fluid and to a lesser extent to the peritubular interstitium. In the renal interstitium it cleaves locally produced kininogen to yield bradykinin that in turn can activate type-2 (B2) bradykinin receptors. Bradykinin-dependent activation of B2 receptor increases sodium excretion by inhibiting sodium reabsorption in the collecting duct (2). Therefore, the renal kallikrein-kinin system is expected to play a role in renal NaCl balance and blood pressure regulation. Patients with essential hypertension have lower kallikrein levels in their urine (3, 4), and mutant mice lacking B2 receptor also exhibit salt-sensitive hypertension (5). However, inactivation of the TK gene in the mouse does not alter blood pressure (6) even though the decrease in renal and urinary kallikrein activity in TK-deficient mice reproduces the phenotype that has been repeatedly associated with hypertension in human and rat studies (7-9). This finding suggests that low urinary kallikrein excretion observed in hypertensive patients is not a primary cause of high blood pressure (HBP) but rather a consequence of hypertension or of HBPassociated renal defects. An alternative explanation is t...