Fumio Niimura

Murine double nullizygotes of the angiotensin type 1A and 1B receptor genes duplicate severe abnormal phenotypes of angiotensinogen nullizygotes.

Rodents are the unique species carrying duplicated angiotensin (Ang) type 1 (AT1) receptor genes, Agtr1a and Agtr1b. After separately generating Agtr1a and Agtr1b null mutant mice by gene targeting, we produced double mutant mice homozygous for both Agtr1a and Agtr1b null mutation (Agtr1a-/-; Agtr1b-/-) by mating the single gene mutants. Agtr1a-/-, Agtr1b-/- mice are characterized by normal in utero survival but decreased ex utero survival rate. After birth they are characterized by low body weight gain, marked hypotension, and abnormal kidney morphology including delayed maturity in glomerular growth, hypoplastic papilla, and renal arterial hypertrophy. These abnormal phenotypes are quantitatively similar to those found in mutant mice homozygous for the angiotensinogen gene (Agt-/-), indicating that major biological functions of endogenous Ang elucidated by the abnormal phenotypes of Agt-/- are mediated by the AT1 receptors. Infusion of Ang II, AT1 blockers, or an AT2 blocker was without effect on blood pressure in Agtr1a-/-; Agtr1b-/- mice, indicating that AT2 receptor does not exert acute depressor effects in these mice lacking AT1 receptors. Also, unlike Agt-/- mice, some Agtr1a-/-; Agtr1b-/- mice have a large ventricular septum defect, suggesting that another receptor such as AT2 is functionally activated in Agtr1a-/-, Agtr1b-/- mice.

Autophagy Protects the Proximal Tubule from Degeneration and Acute Ischemic Injury

Autophagy is a bulk protein degradation system that likely plays an important role in normal proximal tubule function and recovery from acute ischemic kidney injury. Using conditional Atg5 gene deletion to eliminate autophagy in the proximal tubule, we determined whether autophagy prevents accumulation of damaged proteins and organelles with aging and ischemic renal injury. Autophagy-deficient cells accumulated deformed mitochondria and cytoplasmic inclusions, leading to cellular hypertrophy and eventual degeneration not observed in wildtype controls. In autophagy-deficient mice, I/R injury increased proximal tubule cell apoptosis with accumulation of p62 and ubiquitin positive cytoplasmic inclusions. Compared with control animals, autophagy-deficient mice exhibited significantly greater elevations in serum urea nitrogen and creatinine. These data suggest that autophagy maintains proximal tubule cell homeostasis and protects against ischemic injury. Enhancing autophagy may provide a novel therapeutic approach to minimize acute kidney injury and slow CKD progression.

Angiotensin-independent mechanism for aldosterone synthesis during chronic extracellular fluid volume depletion.

Wild-type (Agt+/+) and homozygous angiotensinogen deletion mutant (Agt-/-) littermates were placed on normal (NS) or low Na diet (LS) for 2 weeks. Plasma aldosterone levels (P(aldo)) were comparable during NS, and similarly elevated during LS in Agt+/+ and Agt-/-. Moreover, in both, the elevation in P(aldo) was accompanied by marked increase in adrenal zona glomerulosa cells and adrenal P450aldo mRNA. Agt-/- mice were distinguished from Agt+/+ mice by their higher plasma K level, by approximately 1.5 and approximately 3.8 mEq/liter during NS and LS, respectively. Within the Agt-/- group, P(aldo) was directly proportional to plasma K. The importance of K for the hyperaldosteronism during dietary Na restriction was verified by the observation that superimposition of K restriction led to hypotension in Agt+/+ and uniform death in Agt-/- mice along with a reduction in P(aldo) by 75 and 90%, respectively. Thus, suppression of potassium, but not angiotensin, led to a marked attenuation of hyperaldosteronism during dietary Na restriction. Therefore, (a) a powerful angiotensin-independent mechanism exists for the hyperaldosteronism during LS; (b) high K is a central component of this mechanism; (c) contrary to current belief, the tonic effect of high K on aldosterone synthesis and release does not require an intact renin-angiotensin system; and (d) normally, intermediary feedback signals for hyperaldosteronism, i.e., both hypotension and high K, are effectively masked by aldosterone actions.

Liver Angiotensinogen Is the Primary Source of Renal Angiotensin II

Angiotensin II content in the kidney is much higher than in the plasma, and it increases more in kidney diseases through an uncertain mechanism. Because the kidney abundantly expresses angiotensinogen mRNA, transcriptional dysregulation of angiotensinogen within the kidney is one potential cause of increased renal angiotensin II in the setting of disease. Here, we observed that kidney-specific angiotensinogen knockout mice had levels of renal angiotensinogen protein and angiotensin II that were similar to those levels of control mice. In contrast, liver-specific knockout of angiotensinogen nearly abolished plasma and renal angiotensinogen protein and renal tissue angiotensin II. Immunohistochemical analysis in mosaic proximal tubules of megalin knockout mice revealed that angiotensinogen protein was incorporated selectively in megalin-intact cells of the proximal tubule, indicating that the proximal tubule reabsorbs filtered angiotensinogen through megalin. Disruption of the filtration barrier in a transgenic mouse model of podocyte-selective injury increased renal angiotensin II content and markedly increased both tubular and urinary angiotensinogen protein without an increase in renal renin activity, supporting the dependency of renal angiotensin II generation on filtered angiotensinogen. Taken together, these data suggest that liver-derived angiotensinogen is the primary source of renal angiotensinogen protein and angiotensin II. Furthermore, an abnormal increase in the permeability of the glomerular capillary wall to angiotensinogen, which characterizes proteinuric kidney diseases, enhances the synthesis of renal angiotensin II.

Autophagy guards against cisplatin-induced acute kidney injury.

Autophagy is a highly conserved bulk protein degradation pathway involved in cellular homeostasis. Although emerging evidence indicates involvement of autophagy in various conditions, efforts to clarify the role of autophagy in renal tubules are beginning to be elucidated. In the present study, we examined the hypothesis that autophagy guards against acute kidney injury (AKI) by modulating several deteriorative pathways that lead to tubular cell death using a cisplatin-induced model of AKI. Cisplatin treatment of GFP-LC3 (green fluorescent protein-microtubule-associated protein 1 light chain 3) transgenic mice induced autophagy in kidney proximal tubules in a time-dependent manner. Proximal tubule-specific autophagy-deficient mice exhibited more severe cisplatin-induced AKI than did control mice, as assessed via kidney function and morphologic findings. In addition, cisplatin induced more severe DNA damage and p53 activation, concomitant with an increase in apoptotic cell number, and a massive accumulation of protein aggregates in autophagy-deficient proximal tubules. Cisplatin treatment significantly increased reactive oxygen species-producing damaged mitochondria in immortalized autophagy-deficient proximal tubular cells when compared with autophagy-retrieved control cells. In conclusion, autophagy guards kidney proximal tubules against AKI, possibly by alleviating DNA damage and reactive oxygen species production and by eliminating toxic protein aggregates. Enhancing autophagy may provide a novel therapeutic option to minimize AKI.

Permanent Genetic Tagging of Podocytes: Fate of Injured Podocytes in a Mouse Model of Glomerular Sclerosis

Injured podocytes lose differentiation markers. Therefore, the true identity of severely injured podocytes remains unverified. A transgenic mouse model equipped with a podocyte-selective injury induction system was established. After induction of podocyte injury, mice rapidly developed glomerulosclerosis, with downregulation of podocyte marker proteins. Proliferating epithelial cells accumulated within Bowmans space, as seen in collapsing glomerulosclerosis. In this study, the fate of injured podocytes was pursued. Utilizing Cre-loxP recombination, the podocyte lineage was genetically labeled with lacZ in an irreversible manner. After podocyte injury, the number of lacZ-labeled cells, which were often negative for synaptopodin, progressively declined, correlating with glomerular damage. Parietal epithelial cells, but not lacZ-labeled podocytes, avidly proliferated. The cells proliferating within Bowmans capsule and, occasionally, on the outer surface of the glomerular basement membrane were lacZ-negative. Thus, when podocytes are severely injured, proliferating parietal epithelial cells migrate onto the visceral site, thereby mimicking proliferating podocytes.

Autoimmunity to the Sodium-Level Sensor in the Brain Causes Essential Hypernatremia

Na(x) is the sodium-level sensor of body fluids in the brain involved in sodium homeostasis. Na(x)-knockout mice do not stop ingesting salt even when dehydrated. Here we report a case with clinical features of essential hypernatremia without demonstrable hypothalamic structural lesions, who was diagnosed as a paraneoplastic neurologic disorder. The patient had autoantibodies directed against Na(x), along with a ganglioneuroma composed of Schwann-like cells robustly expressing Na(x). The removal of the tumor did not reduce the autoantibody levels or relieve the symptoms. Intravenous injection of the immunoglobulin fraction of the patients serum into mice induced abnormalities in water/salt intake and diuresis, which led to hypernatremia. In the brains of these mice, cell death was observed along with focal deposits of complement C3 and inflammatory infiltrates in circumventricular organs where Na(x) is specifically expressed. Our findings thus provide new insights into the pathogenesis of hypernatremia relevant to the sodium-level-sensing mechanism in humans.

Podocyte injury enhances filtration of liver-derived angiotensinogen and renal angiotensin II generation

Intrarenal angiotensin II is increased in kidney diseases independently of plasma angiotensin II and is thought to promote progressive deterioration of renal architecture. Here we investigated the mechanism of enhanced renal angiotensin II generation in kidney glomerular diseases. For this, kidney- or liver-specific angiotensinogen gene (Agt) knockout was superimposed on the mouse model of inducible podocyte injury (NEP25). Seven days after induction of podocyte injury, renal angiotensin II was increased ninefold in NEP25 mice with intact Agt, accompanied by increases in urinary albumin and angiotensinogen excretion, renal angiotensinogen protein, and its mRNA. Kidney Agt knockout attenuated renal Agt mRNA but not renal angiotensin II, renal, or urinary angiotensinogen protein. In contrast, liver Agt knockout markedly reduced renal angiotensin II to 18.7% of that of control NEP25 mice, renal and urinary angiotensinogen protein, but not renal Agt mRNA. Renal angiotensin II had no relationship with renal Agt mRNA, or with renal renin mRNA, which was elevated in liver Agt knockouts. Kidney and liver dual Agt knockout mice showed phenotypes comparable to those of liver Agt knockout mice. Thus, increased renal angiotensin II generation upon severe podocyte injury is attributed to increased filtered angiotensinogen of liver origin resulting from loss of macromolecular barrier function of the glomerular capillary wall that occurs upon severe podocyte injury.

Autophagy protects kidney proximal tubule epithelial cells from mitochondrial metabolic stress

Chronic metabolic stress is related to diseases, whereas autophagy supplies nutrients by recycling the degradative products. Cyclosporin A (CsA), a frequently used immunosuppressant, induces metabolic stress via effects on mitochondrial respiration, and thereby, its chronic usage is often limited. Here we show that autophagy plays a protective role against CsA-induced metabolic stress in kidney proximal tubule epithelial cells. Autophagy deficiency leads to decreased mitochondrial membrane potential, which coincides with metabolic abnormalities as characterized by decreased levels of amino acids, increased tricarboxylic acid (TCA) ratio (the levels of intermediates of the latter part of the TCA cycle, over levels of intermediates in the earlier part), and decreased products of oxidative phosphorylation (ATP). In addition to the altered profile of amino acids, CsA decreased the hyperpolarization of mitochondria with the disturbance of mitochondrial energy metabolism in autophagy-competent cells, i.e., increased TCA ratio and worsening of the NAD+/NADH ratio, coupled with decreased energy status, which suggests that adaptation to CsA employs autophagy to supply electron donors from amino acids via intermediates of the latter part of the TCA cycle. The TCA ratio of autophagy-deficient cells was further worsened with decreased levels of amino acids in response to CsA, and, as a result, the deficiency of autophagy failed to adapt to the CsA-induced metabolic stress. Deterioration of the TCA ratio further worsened energy status. The CsA-induced metabolic stress also activated regulatory genes of metabolism and apoptotic signals, whose expressions were accelerated in autophagy-deficient cells. These data provide new perspectives on autophagy in conditions of chronic metabolic stress in disease.

Angiotensin Receptor Blocker Protection Against Podocyte-Induced Sclerosis Is Podocyte Angiotensin II Type 1 Receptor-Independent

In the present study, we tested the hypothesis that the renoprotective effect of an angiotensin receptor blocker depends on the angiotensin II type 1 (AT1) receptor on podocytes. For this purpose, we generated podocyte-specific knockout mice for the AT1 gene (Agtr1a) and crossed with NEP25, in which selective podocyte injury can be induced by immunotoxin, anti-Tac(Fv)-PE38. Four weeks after the addition of anti-Tac(Fv)-PE38, urinary albumin:creatinine ratio was not attenuated in Agtr1a knockout/NEP25 mice (n=18) compared with that in control NEP25 mice (n=13; 8.08±2.41 in knockout versus 4.84±0.73 in control). Both strains of mice showed similar degrees of sclerosis (0.66±0.17 versus 0.82±0.27 on a 0 to 4 scale) and downregulation of nephrin (5.78±0.45 versus 5.65±0.58 on a 0 to 8 scale). In contrast, AT1 antagonist or an angiotensin I–converting enzyme inhibitor, but not hydralazine, remarkably attenuated proteinuria and sclerosis in NEP25 mice. Moreover, continuous angiotensin II infusion induced microalbuminuria similarly in both Agtr1a knockout and wild-type mice. Thus, angiotensin inhibition can protect podocytes and prevent the development of glomerulosclerosis independent of podocyte AT1. Possible mechanisms include inhibitory effects on AT1 of other cells or through mechanisms independent of AT1. Our study further demonstrates that measures that directly affect only nonpodocyte cells can have beneficial effects even when sclerosis is triggered by podocyte-specific injury.