High glucose induces trafficking of prorenin receptor and stimulates profibrotic factors in the collecting duct.

Growing evidence indicates that prorenin receptor (PRR) is upregulated in collecting duct (CD) of diabetic kidney. Prorenin is secreted by the principal CD cells, and is the natural ligand of the PRR. PRR activation stimulates fibrotic factors, including fibronectin, collagen, and transforming growth factor-ß (TGF-ß) contributing to tubular fibrosis. However, whether high glucose (HG) contributes to this effect is unknown. We tested the hypothesis that HG increases the abundance of PRR at the plasma membrane of the CD cells, thus contributing to the stimulation of downstream fibrotic factors, including TGF-ß, collagen I, and fibronectin. We used streptozotocin (STZ) male Sprague-Dawley rats to induce hyperglycemia for 7 days. At the end of the study, STZ-induced rats showed increased prorenin, renin, and angiotensin (Ang) II in the renal inner medulla and urine, along with augmented downstream fibrotic factors TGF-ß, collagen I, and fibronectin. STZ rats showed upregulation of PRR in the renal medulla and preferential distribution of PRR on the apical aspect of the CD cells. Cultured CD M-1 cells treated with HG (25 mM for 1 h) showed increased PRR in plasma membrane fractions compared to cells treated with normal glucose (5 mM). Increased apical PRR was accompanied by upregulation of TGF-ß, collagen I, and fibronectin, while PRR knockdown prevented these effects. Fluorescence resonance energy transfer experiments in M-1 cells demonstrated augmented prorenin activity during HG conditions. The data indicate HG stimulates profibrotic factors by inducing PRR translocation to the plasma membrane in CD cells, which in perspective, might be a novel mechanism underlying the development of tubulointerstitial fibrosis in diabetes mellitus.

Expression pattern of apoptosis-inducing factor in the kidneys of streptozotocin-induced diabetic rats.

BACKGROUND: It is believed that tubulo-interstitial fibrosis and atrophy in diabetic patients are directly associated with the progression of chronic kidney disease, CKD. AIF is one of the crucial factors responsible for mitochondrial apoptosis, however, it can also promote cell survival independently from its role in apoptosis, and therefore can be potentially used as a tool in prevention of the onset of CKD in diabetic patients. Our aim was to investigate the significance of AIF expression in the development of CKD by observing the expression of AIF in 2 weeks' and 2 months' kidneys of diabetic rats compared to their controls. METHODS: Male Sprague-Dawley rats were treated with 55 mg/kg streptozotocin (model of type 1 diabetes mellitus; DM group) or citrate buffer (control). After 2 weeks and 2 months kidney samples were collected and analysed in different renal areas. RESULTS: Characteristic morphologic changes were found between the 2 months' control and 2 months' diabetic groups. Those changes, including fibrosis and possible replacement of podocytes with connective tissue were mainly present in the glomeruli. AIF expression was seen in the both cortex, and in the collecting ducts of the medulla. Strong intensity of AIF expression was seen in proximal and distal convoluted tubules in both diabetic groups. In the control groups the glomeruli showed no AIF staining but moderate staining was seen in both diabetic groups. Overall, the percentage of AIF positive cells in the glomeruli was the lowest. The greatest rise in cell positivity was displayed from the 2 weeks' control group to 2 weeks' diabetes group (38 %) in glomeruli. The cell positivity of the 2 weeks' diabetic group is significantly reduced to 18 % in the 2 months' diabetic group in glomeruli. A similar pattern was seen in the proximal tubular cells (92 % positivity 2 weeks diabetic groups; 89 % positivity 2 months diabetic groups), as well as in the distal tubules. The highest percentage of AIF positive cells was seen in the collecting ducts, more than 80 % in all groups. CONCLUSIONS: Our study provides insight into AIF expression pattern during short term diabetes model, confirming possible dual role of AIF, not only in apoptosis but also in cell function and homeostasis, and proving AIF as potential therapeutic target and marker of advancement of CKD.

Gentamicin-Induced Acute Kidney Injury in an Animal Model Involves Programmed Necrosis of the Collecting Duct.

BACKGROUND: Gentamicin is a potent aminoglycoside antibiotic that targets gram-negative bacteria, but nephrotoxicity limits its clinical application. The cause of gentamicin-induced AKI has been attributed mainly to apoptosis of the proximal tubule cells. However, blocking apoptosis only partially attenuates gentamicin-induced AKI in animals. METHODS: Mice treated with gentamicin for 7 days developed AKI, and programmed cell death pathways were examined using pharmacologic inhibitors and in RIPK3-deficient mice. Effects in porcine and murine kidney cell lines were also examined. RESULTS: Gentamicin caused a low level of apoptosis in the proximal tubules and significant ultrastructural alterations consistent with necroptosis, occurring predominantly in the collecting ducts (CDs), including cell and organelle swelling and rupture of the cell membrane. Upregulation of the key necroptotic signaling molecules, mixed lineage kinase domain-like pseudokinase (MLKL) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3), was detected in gentamicin-treated mice and in cultured renal tubule cells. In addition, gentamicin induced apical accumulation of total and phosphorylated MLKL (pMLKL) in CDs in mouse kidney. Inhibiting a necroptotic protein, RIPK1, with necrostatin-1 (Nec-1), attenuated gentamicin-induced necrosis and upregulation of MLKL and RIPK3 in mice and cultured cells. Nec-1 also alleviated kidney inflammation and fibrosis, and significantly improved gentamicin-induced renal dysfunction in mice. Furthermore, deletion of RIPK3 in the Ripk3-/- mice significantly attenuated gentamicin-induced AKI. CONCLUSIONS: A previously unrecognized role of programmed necrosis in collecting ducts in gentamicin-induced kidney injury presents a potential new therapeutic strategy to alleviate gentamicin-induced AKI through inhibiting necroptosis.

Resveratrol diminishes bisphenol A-induced oxidative stress through TRPM2 channel in the mouse kidney cortical collecting duct cells.

Bisphenol A (BisPH-A) is a latent danger that threatens our health, which we frequently exposure in our modern life (e.g. the widespread use of drinking water in plastic pet bottles). But the BisPH-A induced transient receptor potential melastatin 2 (TRPM2)-mediated oxidative stress and apoptosis in these cells has not been studied yet. Calcium (Ca2+) plays an important role in a versatile intracellular signal transduction that works over a wide range to regulate oxidative stress processes. TRPM2 is activated by oxidative stress and it has emerged as an important Ca2+ signaling mechanism in a variety of cells, contributing many cellular functions including cell death. Resveratrol (RESV), which belongs to the polyphenol group, acts as an antioxidant, eliminating cellular oxidative stress and increasing the body's resistance to diseases. The current study aimed to elucidate the effect of antioxidant resveratrol on TRPM2-mediated oxidative stress induced by BisPH-A exposure in the mouse kidney cortical collecting duct cells (mpkCCDcl4). The cells were divided into four groups as control, resveratrol (50 µM for 24 h), BisPH-A (100 µM for 24 h) and BisPH-A + RESV. Intracellular free Ca2+ concentrations and TRPM2 channel currents were high in BisPH-A treated cells, but decreased with resveratrol treatment. In addition, BisPH-A induced mitochondrial membrane depolarization, reactive oxygen species (ROS), caspase 3, caspase 9 and apoptosis values were decreased by the resveratrol treatment. In conclusion, resveratrol protected cells from BisPH-A induced oxidative damage. In this study, we showed that TRPM2 channel mediates this protective effect of resveratrol.

Chloroquine attenuates lithium-induced NDI and proliferation of renal collecting duct cells.

Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na+-K+-2Cl- cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and ß-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.

Potassium depletion induces cellular conversion in the outer medullary collecting duct altering Notch signaling pathway.

Potassium depletion affects AQP2 expression and the cellular composition of the kidney collecting duct. This, in turn, contributes to the development of a secondary form of nephrogenic diabetes insipidus and hypokalemic nephropathy. Here we show that after 14 days of potassium depletion, the cellular fraction of A-type intercalated cells increases while the fraction of principal cells decreases along the outer medullary collecting duct in rats. The intercalated cells acquired a novel distribution pattern forming rows of cells attached to each other. These morphological changes occur progressively and reverse after 7 days of recovery on normal rat chow diet. The cellular remodeling mainly occurred in the inner stripe of outer medulla similar to the previously seen effect of lithium on the collecting duct cellular profile. The cellular remodeling is associated with the appearance of cells double labelled with both specific markers of principal and type-A intercalated cells. The appearance of this cell type was associated with the downregulation of the Notch signaling via the Hes1 pathways. These results show that the epithelium of the collecting duct has a high degree of plasticity and that Notch signaling likely plays a key role during hypokalemia.

Short-chain fatty acid mitigates adenine-induced chronic kidney disease via FFA2 and FFA3 pathways.

Short-chain fatty acids (SCFAs), including acetate, butyrate, and propionate, are produced when colonic bacteria in the human gastrointestinal tract ferment undigested fibers. Free fatty acid receptor 2 (FFA2) and FFA3 are G-protein-coupled receptors recently identified as SCFA receptors that may modulate inflammation. We previously showed through in vitro experiments that SCFAs activate FFA2 and FFA3, thereby mitigating inflammation in human renal cortical epithelial cells. This study used a murine model of adenine-induced renal failure to investigate whether or not SCFAs can prevent the progression of renal damage. We also examined whether or not these FFA2 and FFA3 proteins have some roles in this protective mechanism in vivo. Immunohistochemical analyses of mouse kidneys showed that FFA2 and FFA3 proteins were expressed mainly in the distal renal tubules and collecting tubules. First, we observed that the administration of propionate mitigated the renal dysfunction and pathological deterioration caused by adenine. Consistent with this, the expression of inflammatory cytokines and fibrosis-related genes was reduced. Furthermore, the mitigation of adenine-induced renal damage by the administration of propionate was significantly attenuated in FFA2-/- and FFA3-/- mice. Therefore, the administration of propionate significantly protects against adenine-induced renal failure, at least in part, via the FFA2 and FFA3 pathways. Our data suggest that FFA2 and FFA3 are potential new therapeutic targets for preventing or delaying the progression of chronic kidney disease.

Epac1 null mice have nephrogenic diabetes insipidus with deficient corticopapillary osmotic gradient and weaker collecting duct tight junctions.

AIM: The cAMP-mediator Epac1 (RapGef3) has high renal expression. Preliminary observations revealed increased diuresis in Epac1-/- mice. We hypothesized that Epac1 could restrict diuresis by promoting transcellular collecting duct (CD) water and urea transport or by stabilizing CD paracellular junctions to reduce osmolyte loss from the renal papillary interstitium. METHODS: In Epac1-/- and Wt C57BL/6J mice, renal papillae, dissected from snap-frozen kidneys, were assayed for the content of key osmolytes. Cell junctions were analysed by transmission electron microscopy. Urea transport integrity was evaluated by urea loading with 40% protein diet, endogenous vasopressin production was manipulated by intragastric water loading and moderate dehydration and vasopressin type 2 receptors were stimulated selectively by i.p.-injected desmopressin (dDAVP). Glomerular filtration rate (GFR) was estimated as [14 C]inulin clearance. The glomerular filtration barrier was evaluated by urinary albumin excretion and microvascular leakage by the renal content of time-spaced intravenously injected 125 I- and 131 I-labelled albumin. RESULTS: Epac1-/- mice had increased diuresis and increased free water clearance under antidiuretic conditions. They had shorter and less dense CD tight junction (TJs) and attenuated corticomedullary osmotic gradient. Epac1-/- mice had no increased protein diet-induced urea-dependent osmotic diuresis, and expressed Wt levels of aquaporin-2 (AQP-2) and urea transporter A1/3 (UT-A1/3). Epac1-/- mice had no urinary albumin leakage and unaltered renal microvascular albumin extravasation. Their GFR was moderately increased, unless when treated with furosemide. CONCLUSION: Our results conform to the hypothesis that Epac1-dependent mechanisms protect against diabetes insipidus by maintaining renal papillary osmolarity and the integrity of CD TJs.

Molecular characterization of an aquaporin-2 mutation causing a severe form of nephrogenic diabetes insipidus.

The water channel aquaporin 2 (AQP2) is responsible for water reabsorption by kidney collecting duct cells. A substitution of amino acid leucine 137 to proline in AQP2 (AQP2-L137P) causes Nephrogenic Diabetes Insipidus (NDI). This study aimed to determine the cell biological consequences of this mutation on AQP2 function. Studies were performed in HEK293 and MDCK type I cells, transfected with wildtype (WT) AQP2 or an AQP2-L137P mutant. AQP2-L137P was predominantly detected as a high-mannose form of AQP2, whereas AQP2-WT was observed in both non-glycosylated and complex glycosylated forms. In contrast to AQP2-WT, the AQP2-L137P mutant did not accumulate on the apical plasma membrane following stimulation with forskolin. Ubiquitylation of AQP2-L137P was different from AQP2-WT, with predominance of non-distinct protein bands at various molecular weights. The AQP2-L137P mutant displayed reduced half-life compared to AQP2-WT. Treatment of cells with chloroquine increased abundance of AQP2-WT, but not AQP2-L137P. In contrast, treatment with MG132 increased abundance of AQP2-L137P but not AQP2-WT. Xenopus oocytes injected with AQP2-WT had increased osmotic water permeability when compared to AQP2-L137P, which correlated with lack of the mutant form in the plasma membrane. From the localization of the mutation and nature of the substitution it is likely that AQP2-L137P causes protein misfolding, which may be responsible for the observed functional defects. The data suggest that the L137P mutation results in altered AQP2 protein maturation, increased AQP2 degradation via the proteasomal pathway and limited plasma membrane expression. These combined mechanisms are likely responsible for the phenotype observed in this class of NDI patients.

Concomitant multiple anomalies of renal vessels and collecting system.

Although anomalies of renal vessels and collecting system are relatively frequent, their concomitant occurrence is a rare event. During dissection of a 75-year-old male formalin-embalmed cadaver, we found multiple variations in the renal vessels and renal collecting system. Both kidneys were normal in size and anteriorly malrotated, with duplex collecting system and duplex ureter. One ureter drained the upper part of the kidney and the second ureter drained the lower part of the kidney. Superior and inferior collecting systems were separated by renal parenchyma. The right kidney had two renal arteries, the first renal artery (main renal artery) originating from the abdominal aorta, passing behind the inferior vena cava (IVC) and entering the kidney through the superior and inferior renal hilum. The second artery was the inferior polar artery. In addition, the right kidney had two renal veins as well. Three renal tributaries emerged from the upper and lower portion of the right renal hilum, and they joined to form the main renal vein which drained into the IVC. The lower renal vein was the inferior polar vein. The left kidney had four renal arteries (two hilar arteries and two polar arteries). The main left renal vein emerged from both superior and inferior left renal hilum, passed in front of the abdominal aorta and drained into the IVC. The left kidney also had the inferior polar vein which was divided behind the aorta (retro aortic vein) into two venous trunks. These venous trunks drained separately into posteromedial aspect of the IVC. Finally, the right testicular vein was formed by two tributaries and drained into the IVC, whereas the two left testicular veins drained separately into the left main renal vein.

Integrin-Linked Kinase Deficiency in Collecting Duct Principal Cell Promotes Necroptosis of Principal Cell and Contributes to Kidney Inflammation and Fibrosis.

BACKGROUND: Necroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied. METHODS: We performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown. RESULTS: Ilk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-ß signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals' injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice. CONCLUSIONS: The study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

Cell-specific image-guided transcriptomics identifies complex injuries caused by ischemic acute kidney injury in mice.

The kidney's inherent complexity has made identifying cell-specific pathways challenging, particularly when temporally associating them with the dynamic pathophysiology of acute kidney injury (AKI). Here, we combine renal cell-specific luciferase reporter mice using a chemoselective luciferin to guide the acquisition of cell-specific transcriptional changes in C57BL/6 background mice. Hydrogen peroxide generation, a common mechanism of tissue damage, was tracked using a peroxy-caged-luciferin to identify optimum time points for immunoprecipitation of labeled ribosomes for RNA-sequencing. Together, these tools revealed a profound impact of AKI on mitochondrial pathways in the collecting duct. In fact, targeting the mitochondria with an antioxidant, ameliorated not only hydrogen peroxide generation, but also significantly reduced oxidative stress and the expression of the AKI biomarker, LCN2. This integrative approach of coupling physiological imaging with transcriptomics and drug testing revealed how the collecting duct responds to AKI and opens new venues for cell-specific predictive monitoring and treatment.

Differences in acidosis-stimulated renal ammonia metabolism in the male and female kidney.

Renal ammonia excretion is a critical component of acid-base homeostasis, and changes in ammonia excretion are the predominant component of increased net acid excretion in response to metabolic acidosis. We recently reported substantial sex-dependent differences in basal ammonia metabolism that correlate with sex-dependent differences in renal structure and expression of key proteins involved in ammonia metabolism. The purpose of the present study was to investigate the effect of sex on the renal ammonia response to an exogenous acid load. We studied 4-mo-old C57BL/6 mice. Ammonia excretion, which was less in male mice under basal conditions, increased in response to acid loading to a greater extent in male mice, such that maximal ammonia excretion did not differ between the sexes. Fundamental structural sex differences in the nonacid-loaded kidney persisted after acid loading, with less cortical proximal tubule volume density in the female kidney than in the male kidney, whereas collecting duct volume density was greater in the female kidney. To further investigate sex-dependent differences in the response to acid loading, we examined the expression of proteins involved in ammonia metabolism. The change in expression of phosphoenolpyruvate carboxykinase and Rh family B glycoprotein with acid loading was greater in male mice than in female mice, whereas Na+-K+-2Cl- cotransporter and inner stripe of the outer medulla intercalated cell Rh family C glycoprotein expression were significantly greater in female mice than in male mice. There was no significant sex difference in glutamine synthetase, Na+/H+ exchanger isoform 3, or electrogenic Na+-bicarbonate cotransporter 1 variant A protein expression in response to acid loading. We conclude that substantial sex-dependent differences in the renal ammonia response to acid loading enable a similar maximum ammonia excretion response.

Claudin-7 Modulates Cl- and Na+ Homeostasis and WNK4 Expression in Renal Collecting Duct Cells.

Claudin-7 knockout (CLDN7-/-) mice display renal salt wasting and dehydration phenotypes. To address the role of CLDN7 in kidneys, we established collecting duct (CD) cell lines from CLDN7+/+ and CLDN7-/- mouse kidneys. We found that deletion of CLDN7 increased the transepithelial resistance (TER) and decreased the paracellular permeability for Cl- and Na+ in CLDN7-/- CD cells. Inhibition of transcellular Cl- and Na+ channels has no significant effect on TER or dilution potentials. Current-voltage curves were linear in both CLDN7+/+ and CLDN7-/- CD cells, indicating that the ion flux was through the paracellular pathway. The impairment of Cl- and Na+ permeability phenotype can be rescued by CLDN7 re-expression. We also found that WNK4 (its mutations lead to hypertension) expression, but not WNK1, was significantly increased in CLDN7-/- CD cell lines as well as in primary CLDN7-/- CD cells, suggesting that the expression of WNK4 was modulated by CLDN7. In addition, deletion of CLDN7 upregulated the expression level of the apical epithelial sodium channel (ENaC), indicating a potential cross-talk between paracellular and transcellular transport systems. This study demonstrates that CLDN7 plays an important role in salt balance in renal CD cells and modulating WNK4 and ENaC expression levels that are vital in controlling salt-sensitive hypertension.

Loss of primary cilia increases polycystin-2 and TRPV4 and the appearance of a nonselective cation channel in the mouse cortical collecting duct.

Flow-related bending of cilia results in Ca2+ influx through a polycystin-1 (Pkd1) and polycystin-2 (Pkd2) complex, both of which are members of the transient receptor potential (TRP) family (TRPP1 and TRPP2, respectively). Deletion of this complex as well as cilia result in polycystic kidney disease. The Ca2+ influx pathway has been previously characterized in immortalized collecting duct cells without cilia and found to be a 23-pS channel that was a multimere of TRPP2 and TRPV4. The purpose of the present study was to determine if this TRPP2 and TRPV4 multimere exists in vivo. Apical channel activity was measured using the patch-clamp technique from isolated split-open cortical collecting ducts from adult conditional knockout mice with (Ift88flox/flox) or without (Ift88-/-) cilia. Single tubules were isolated for measurements of mRNA for Pkd1, Pkd2, Trpv4, and epithelial Na+ channel subunits. The predominant channel activity from Ift88flox/flox mice was from epithelial Na+ channel [5-pS Na+-selective channels with long mean open times (475.7 ± 83.26 ms) and open probability > 0.2]. With the loss of cilia, the predominant conductance was a 23-pS nonselective cation channel (reversal potential near 0) with a short mean open time (72 ± 17 ms), open probability < 0.08, and a characteristic flickery opening. Loss of cilia increased mRNA levels for Pkd2 and Trpv4 from single isolated cortical collecting ducts. In conclusion, 23-pS channels exist in vivo, and activity of this channel is elevated with loss of cilia, consistent with previous finding of an elevated-unregulated Ca2+-permeable pathway at the apical membrane of collecting duct cells that lack cilia.

Roscovitine blocks collecting duct cyst growth in Cep164-deficient kidneys.

Nephronophthisis is an autosomal recessive kidney disease with high genetic heterogeneity. Understanding the functions of the individual genes contributing to this disease is critical for delineating the pathomechanisms of this disorder. Here, we investigated kidney function of a novel gene associated with nephronophthisis, CEP164, coding a centriolar distal appendage protein, using a Cep164 knockout mouse model. Collecting duct-specific deletion of Cep164 abolished primary cilia from the collecting duct epithelium and led to rapid postnatal cyst growth in the kidneys. Cell cycle and biochemical studies revealed that tubular hyperproliferation is the primary mechanism that drives cystogenesis in the kidneys of these mice. Administration of roscovitine, a cell cycle inhibitor, blocked cyst growth in the cortical collecting ducts and preserved kidney parenchyma in Cep164 knockout mice. Thus, our findings provide evidence that therapeutic modulation of cell cycle activity can be an effective approach to prevent cyst progression in the kidney.

Modulation of renin angiotensin system components by high glucose levels in the culture of collecting duct cells.

Diabetes mellitus and its complications have become a major health concern in Western countries. Increased activity of the intrarenal renin-angiotensin system (RAS) contributes to diabetic nephropathy (DN). We previously reported that in mesangial cells, the high glucose concentration (HG) leads to upregulation of angiotensin-converting enzyme (ACE) messenger RNA, suggesting that ACE was modulated by angiotensin II (Ang II) release. However, this relation in the collecting duct has not yet been studied. We, therefore, aimed to evaluate RAS modulation in inner medullary collecting duct cells (IMCD) exposed to HG. The IMCD were divided into normal glucose (5 mM D-glucose, NG), high glucose (30 mM, HG), and mannitol (30 mM, M) groups. The cells were cultured 48 hr in their respective media. The intracellular and extracellular ACE activity was measured using hippuryl-His-Leu as substrate via a fluorimetric assay and expression was analyzed using western blot analysis. ACE activity, intracellular (27%) and extracellular (22%), was significantly lower in the HG group than in NG and M. ACE2 activity and Ang 1-7 levels were higher in the intracellular compartment. Our data suggest that the HG cannot modify ACE synthesis in IMCD cells but can modulate its activity. The decrease in ACE activity may result in decreased levels of Ang II to protect the IMCD against proliferative and inflammatory deleterious effects of this peptide. Conversely, the increase of ACE2 generating high levels of Ang 1-7, a vasodilator peptide, suggesting that this peptide can induce glucose uptake and protect cells against oxidative stress, which can elicit insulin resistance.

Aquaporin-3 deficiency slows cyst enlargement in experimental mouse models of autosomal dominant polycystic kidney disease.

Human autosomal dominant polycystic kidney disease (ADPKD) is characterized by bilateral renal cysts that lead to a decline in kidney function. Previous studies reported aquaporin (AQP)-3 expression in cysts derived from collecting ducts in ADPKD. To study the role of AQP3 in cyst development, we generated 2 polycystic kidney disease (PKD) mouse models: kidney-specific Pkd1 knockout mice and inducible Pkd1 knockout mice, each without and with AQP3 deletion. In both models, kidney sizes and cyst indexes were significantly reduced in AQP3-null PKD mice compared with AQP3-expressing PKD mice, with the difference seen mainly in collecting duct cysts. AQP3-deficient kidneys showed significantly reduced ATP content, increased phosphorylated (p)-AMPK, and decreased p-ERK and p-mammalian target of rapamycin (mTOR). In a matrix-grown Madin-Darby canine kidney cyst model, AQP3 expression promoted cyst enlargement and was associated with increased expression of hypoxia-inducible factor 1-α and glucose transporter 1 and increased glucose uptake. Our data suggest that the slowed renal cyst enlargement in AQP3 deficiency involves impaired energy metabolism in the kidney through AMPK and mTOR signaling and impaired cellular glucose uptake. These findings implicate AQP3 as a novel determinant of renal cyst enlargement and hence a potential drug target in ADPKD.-Wang, W., Geng, X., Lei, L., Jia, Y., Li, Y., Zhou, H., Verkman, A. S., Yang, B. Aquaporin-3 deficiency slows cyst enlargement in experimental mouse models of autosomal dominant polycystic kidney disease.

Insulin receptor signaling regulates renal collecting duct and intercalated cell antibacterial defenses.

People with diabetes mellitus have increased infection risk. With diabetes, urinary tract infection (UTI) is more common and has worse outcomes. Here, we investigate how diabetes and insulin resistance impact the kidney's innate defenses and urine sterility. We report that type 2 diabetic mice have increased UTI risk. Moreover, insulin-resistant prediabetic mice have increased UTI susceptibility, independent of hyperglycemia or glucosuria. To identify how insulin resistance affects renal antimicrobial defenses, we genetically deleted the insulin receptor in the kidney's collecting tubules and intercalated cells. Intercalated cells, located within collecting tubules, contribute to epithelial defenses by acidifying the urine and secreting antimicrobial peptides (AMPs) into the urinary stream. Collecting duct and intercalated cell-specific insulin receptor deletion did not impact urine acidification, suppressed downstream insulin-mediated targets and AMP expression, and increased UTI susceptibility. Specifically, insulin receptor-mediated signaling regulates AMPs, including lipocalin 2 and ribonuclease 4, via phosphatidylinositol-3-kinase signaling. These data suggest that insulin signaling plays a critical role in renal antibacterial defenses.

Klotho gene-modified BMSCs showed elevated antifibrotic effects by inhibiting the Wnt/ß-catenin pathway in kidneys after acute injury.

Klotho is a protein primarily expressed in renal tubular epithelial cells. Studies have suggested that Klotho is an antiaging protein that reduces renal fibrosis after acute kidney injury (AKI) and inhibits stem cell senescence. Bone marrow mesenchymal stem cells (BMSCs) have consistent proliferation ability and multidirectional differentiation ability and have been used to treat tissue injury. Thus, we hypothesized that Klotho expressed in BMSCs could increase the renal protective effects of BMSCs. To verify the hypothesis, we isolated BMSCs from C57BL/6 mice, transfected them with Klotho-GFP-adenovirus and investigated the change in BMSC proliferation. We then transplanted Klotho-GFP-BMSCs into mice with AKI and investigated the therapeutic effect compared with that of sham-treated mice and GFP-BMSC-transplanted mice. Kidney fibrosis after ischemia/reperfusion injury (IRI) was relieved by BMSC transplantation, and the antifibrotic effect of BMSCs was significantly enhanced by overexpressing the Klotho gene. Mechanistic studies showed that Klotho increased pluripotency gene expression in BMSCs. Klotho produced by Klotho-GFP-BMSCs inhibited the Wnt/ß-catenin pathway in renal tubular epithelial cells (TECs). Klotho-GFP-BMSCs showed increased proliferative ability and more potent immuno-regulation ability than did GFP-BMSCs. Our findings suggested that Klotho gene-modified BMSCs may be a better choice for cell therapy after AKI.