Macula Densa Research Articles

Macula Densa SGLT1-NOS1-Tubuloglomerular Feedback Pathway, a New Mechanism for Glomerular Hyperfiltration during Hyperglycemia.

Glomerular hyperfiltration is common in early diabetes and is considered a risk factor for later diabetic nephropathy. We propose that sodium-glucose cotransporter 1 (SGLT1) senses increases in luminal glucose at the macula densa, enhancing generation of neuronal nitric oxide synthase 1 (NOS1)-dependent nitric oxide (NO) in the macula densa and blunting the tubuloglomerular feedback (TGF) response, thereby promoting the rise in GFR. We used microperfusion, micropuncture, and renal clearance of FITC-inulin to examine the effects of tubular glucose on NO generation at the macula densa, TGF, and GFR in wild-type and macula densa-specific NOS1 knockout mice. Acute intravenous injection of glucose induced hyperglycemia and glucosuria with increased GFR in mice. We found that tubular glucose blunts the TGF response in vivo and in vitro and stimulates NO generation at the macula densa. We also showed that SGLT1 is expressed at the macula densa; in the presence of tubular glucose, SGLT1 inhibits TGF and NO generation, but this action is blocked when the SGLT1 inhibitor KGA-2727 is present. In addition, we demonstrated that glucose increases NOS1 expression and NOS1 phosphorylation at Ser1417 in mouse renal cortex and cultured human kidney tissue. In macula densa-specific NOS1 knockout mice, glucose had no effect on NO generation, TGF, and GFR. We identified a novel mechanism of acute hyperglycemia-induced hyperfiltration wherein increases in luminal glucose at the macula densa upregulate the expression and activity of NOS1 via SGLT1, blunting the TGF response and promoting glomerular hyperfiltration.

Tubule-vascular feedback in renal autoregulation.

Afferent arteriole (Af-Art) diameter regulates pressure and flow into the glomerulus, which are the main determinants of the glomerular filtration rate. Thus, Af-Art resistance is crucial for Na+ filtration. Af-Arts play a role as integrative centers, where systemic and local systems interact to determine the final degree of resistance. The tubule of a single nephron contacts an Af-Art of the same nephron at two locations: in the transition of the thick ascending limb to the distal tubule (macula densa) and again in the connecting tubule. These two sites are the anatomic basis of two intrinsic feedback mechanisms: tubule-glomerular feedback and connecting tubule-glomerular feedback. The cross communications between the tubules and Af-Arts integrate tubular Na+ and water processing with the hemodynamic conditions of the kidneys. Tubule-glomerular feedback provides negative feedback that tends to avoid salt loss, and connecting tubule-glomerular feedback provides positive feedback that favors salt excretion by modulating tubule-glomerular feedback (resetting it) and increasing glomerular filtration rate. These feedback mechanisms are also exposed to systemic modulators (hormones and the nervous system); however, they can work in isolated kidneys or nephrons. The exaggerated activation or absence of any of these mechanisms may lead to disequilibrium in salt and water homeostasis, especially in extreme conditions (e.g., high-salt diet/low-salt diet) and may be part of the pathogenesis of some diseases. In this review, we focus on molecular signaling, feedback interactions, and the physiological roles of these two feedback mechanisms.

Effect of nonsteroidal anti-inflammatory drugs in children with Bartter syndrome.

Bartter syndrome (BS) is a salt-wasting tubulopathy with induced expression of cyclooxygenase-2 in the macula densa, leading to increased prostaglandin production and hyperreninemia. Nonsteroidal anti-inflammatory drugs (NSAIDs) are currently used in BS; however, there is limited information on the impact of NSAIDs at treatment initiation or the potential utility of plasma renin level to guide therapy in patients with BS. We included 19 patients with BS treated with NSAIDs between 1994 and 2016. We assessed serum levels of renin, aldosterone, electrolytes, calcium, phosphorus, vitamin D, and intact parathyroid hormone (iPTH) before and after treatment initiation. We also recorded modifications in sodium and potassium supplements and changes in urine calcium. Median age at diagnosis was 0.9months [IQR 0-6.9]. Seven patients had BS types 1 or 2, 12 had BS type 3 and two had no mutation identified. There was a trend towards a decrease in sodium chloride supplementation after initiation of NSAIDs. When defining response to treatment based on the normalization of plasma renin level, responders had a greater reduction in their electrolytes supplementation. NSAIDs treatment was associated with a reduction in urine calcium. Before treatment, half of the patients had elevated iPTH, but iPTH normalized following initiation of NSAIDs in all but one patient. This study confirms that NSAIDs reduce urine wasting of sodium and calcium in patients with BS. Monitoring serum renin levels may be useful to identify the lowest effective dose of NSAIDs that optimizes reduction of urine electrolyte losses.

The potential for improving cardio-renal outcomes by sodium-glucose co-transporter-2 inhibition in people with chronic kidney disease: a rationale for the EMPA-KIDNEY study.

ABSTRACT Diabetes is a common cause of chronic kidney disease (CKD), but in aggregate, non-diabetic diseases account for a higher proportion of cases of CKD than diabetes in many parts of the world. Inhibition of the renin–angiotensin system reduces the risk of kidney disease progression and treatments that lower blood pressure (BP) or low-density lipoprotein cholesterol reduce cardiovascular (CV) risk in this population. Nevertheless, despite such interventions, considerable risks for kidney and CV complications remain. Recently, large placebo-controlled outcome trials have shown that sodium-glucose co-transporter-2 (SGLT-2) inhibitors reduce the risk of CV disease (including CV death and hospitalization for heart failure) in people with type 2 diabetes who are at high risk of atherosclerotic disease, and these effects were largely independent of improvements in hyperglycaemia, BP and body weight. In the kidney, increased sodium delivery to the macula densa mediated by SGLT-2 inhibition has the potential to reduce intraglomerular pressure, which may explain why SGLT-2 inhibitors reduce albuminuria and appear to slow kidney function decline in people with diabetes. Importantly, in the trials completed to date, these benefits appeared to be maintained at lower levels of kidney function, despite attenuation of glycosuric effects, and did not appear to be dependent on ambient hyperglycaemia. There is therefore a rationale for studying the cardio-renal effects of SGLT-2 inhibition in people at risk of CV disease and hyperfiltration (i.e. those with substantially reduced nephron mass and/or albuminuria), irrespective of whether they have diabetes.

Role of connecting tubule glomerular feedback in obesity related renal damage.

Zucker obese rats (ZOR) have higher glomerular capillary pressure (PGC) that can cause renal damage. PGC is controlled by afferent (Af-Art) and efferent arteriole (Ef-Art) resistance. Af-Art resistance is regulated by factors that regulate other arterioles, such as myogenic response. In addition, it is also regulated by 2 intrinsic feedback mechanisms: 1) tubuloglomerular feedback (TGF) that causes Af-Art constriction in response to increased NaCl in the macula densa and 2) connecting tubule glomerular feedback (CTGF) that causes Af-Art dilatation in response to an increase in NaCl transport in the connecting tubule via the epithelial sodium channel. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that increased CTGF contributes to TGF attenuation, which in turn increases PGC in ZOR. We performed a renal micropuncture experiment and measured stop-flow pressure (PSF), which is an indirect measurement of PGC in ZOR. Maximal TGF response at 40 nl/min was attenuated in ZOR (4.47 ± 0.60 mmHg) in comparison to the Zucker lean rats (ZLR; 8.54 ± 0.73 mmHg, P < 0.05), and CTGF was elevated in ZOR (5.34 ± 0.87 mmHg) compared with ZLR (1.12 ± 1.28 mmHg, P < 0.05). CTGF inhibition with epithelial sodium channel blocker normalized the maximum PSF change in ZOR indicating that CTGF plays a significant role in TGF attenuation (ZOR, 10.67 ± 1.07 mmHg vs. ZLR, 9.5 ± 1.53 mmHg). We conclude that enhanced CTGF contributes to TGF attenuation in ZOR and potentially contribute to progressive renal damage.

A3226 NOS1 in kidney macula densa controls the development of hypertension during pregnancy

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Abstract 002: Macula Densa SGLT1-NOS1-TGF Pathway -- A New Mechanism for Glomerular Hyperfiltration in Diabetes

Glomerular hyperfiltration is a common observation in early diabetes and considered as risk factor for diabetic nephropathy. The mechanisms underlying glomerular hyperfiltration have not been fully clarified. In the present study, we tested a novel SGLT1-NOS1 -TGF (sodium-glucose cotransporter 1 - neuronal nitric oxide synthase – tubuloglomerular feedback) pathway with a hypothesis that tubular glucose is sensed by SGLT1 of the macula densa, which enhances macula densa NOS1 activity and blunts TGF response, therefore increasing glomerular filtration rate (GFR). NO generation by the macula densa, measured by DAF-2 DA in isolated perfused juxtaglomerular apparatus (JGA), increased by 78.4±9.1% when tubular glucose (glucose) was increased from 100 mg/dl to 300 mg/dl (n=4, p&lt;0.01 vs 100 mg/dl). A selective SGLT1 inhibitor KGA-2727 (10 -6 M) blocked this glucose-induced NO generation. Human kidney biopsy tissues were cultured in normal glucose (100 mg/dl) and high glucose (300 mg/dl) media for 30 minutes with hanging drop technique. Protein levels of P-NOS1 ser1417 increased by 63.1±9.2% and P-NOS1 ser847 decreased by 47.4±5.9% (n=4, p&lt;0.01 vs normal glucose) with high glucose. TGF response was significantly reduced from 3.8±0.2 μm to 2.4±0.2 μm (n=4, p&lt;0.05 vs 100 mg/dl), measured in isolated and double perfused JGAs when tubular glucose was increased from 100 mg/dl to 300 mg/dl. The glucose-induced TGF inhibition was blocked in the presence of SGLT1 inhibition (n=4). GFR, measured in conscious mice with a single bolus injection of FITC-inulin, increased by 19.1±3.5% in response to an acute intravenous injection of 50 μl 2M glucose in C57BL/6 mice (n=4, p&lt;0.01 vs baseline). Then, we repeated the experiments in macula densa specific NOS1 KO (KO) mice. Both glucose-induced TGF (ΔP sf was from 7.8±1.3 to 7.3±2.1 mmHg) and GFR (from 223±15.7 to 240±6.9 μl/min) (n=5) were blocked in the KO mice. We conclude that increase of tubular glucose concentration activates NOS1 by phosphorylation of NOS1 ser1417 via SGLT1 in the macula densa, which increases GFR by inhibiting TGF response.

Abstract P218: Downregulation of Renal Tumor Necrosis Factor-Alpha Production in Response to Hypotonic Stress Facilitates Adaptive Increases in Renal Cortical Angiotensinogen and Nkcc2b

We previously showed that diverse stimuli activate the thick ascending limb of Henle’s loop (TAL) to synthesize tumor necrosis factor-alpha (TNF), which subsequently acts as an endogenous inhibitor of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). Moreover, renal-specific silencing of TNF unmasks salt-dependent increases in blood pressure via a mechanism involving NKCC2A. The objective of the present study was to determine the effects of TNF on NKCC2 isoform and angiotensinogen (AGT) expression in the renal cortex under conditions of hypotonic stress. Mice given a low salt (0.02% NaCl) diet (LS) for 7 days exhibited a 62±7.4% decrease (p&lt;0.5, n=4) in TNF mRNA expression in the renal cortex, compared with mice ingesting a normal salt diet (NSD), which was associated with a concomitant 4-fold increase in renal cortical AGT mRNA accumulation (p&lt;0.05, n=4). Mice ingesting LS also exhibited about a 63% increase (p&lt;0.05, n=4) in cortical TAL phospho-NKCC2 (pNKCC2) expression and a 3-fold increase in NKCC2B mRNA abundance without a concurrent change in NKCC2A mRNA accumulation. The increases in AGT and NKCC2B mRNA abundance were increased by 6-fold (p&lt;0.05, n=4 ) and 5-fold (p&lt;0.05, n=4), respectively, in mice ingesting LS that also received an intrarenal injection of a lentivirus construct designed to specifically silence TNF in the kidney (U6-TNF-ex4) compared with mice injected with control lentivirus (U6). The effects of a single intrarenal injection of murine recombinant TNF (5ng/g body weight) or saline control for 24 hr on renal AGT and NKCC2 mRNA levels were then determined in mice that ingested LS for 7 days. Administration of TNF inhibited the increase of AGT and NKCC2B mRNA abundance by approximately 42±5.9% and 49±6.5% respectively, p&lt;0.05, n=4 ) in mice exposed to hypotonic stress. Similarly, intrarenal injection of TNF inhibited the increase in pNKCC2 by approximately 54% (p&lt;0.05, n=4 ) in renal cortex from mice given LS for 7 days. Collectively, these findings suggest that downregulation of renal TNF production in response to hypotonic conditions contributes to the regulation of sodium chloride reabsorption via adaptive increases in AGT as well as a selective effect on NKCC2B, which is exclusively expressed by macula densa cells in the renal cortex.

H+-ATPase B1 subunit localizes to thick ascending limb and distal convoluted tubule of rodent and human kidney.

The vacuolar-type H+-ATPase B1 subunit is heavily expressed in the intercalated cells of the collecting system, where it contributes to H+ transport, but has also been described in other segments of the renal tubule. This study aimed to determine the localization of the B1 subunit of the vacuolar-type H+-ATPase in the early distal nephron, encompassing thick ascending limbs (TAL) and distal convoluted tubules (DCT), in human kidney and determine whether the localization differs between rodents and humans. Antibodies directed against the H+-ATPase B1 subunit were used to determine its localization in paraffin-embedded formalin-fixed mouse, rat, and human kidneys by light microscopy and in sections of Lowicryl-embedded rat kidneys by electron microscopy. Abundant H+-ATPase B1 subunit immunoreactivity was observed in the human kidney. As expected, intercalated cells showed the strongest signal, but significant signal was also observed in apical membrane domains of the distal nephron, including TAL, macula densa, and DCT. In mouse and rat, H+-ATPase B1 subunit expression could also be detected in apical membrane domains of these segments. In rat, electron microscopy revealed that the H+-ATPase B1 subunit was located in the apical membrane. Furthermore, the H+-ATPase B1 subunit colocalized with other H+-ATPase subunits in the TAL and DCT. In conclusion, the B1 subunit is expressed in the early distal nephron. The physiological importance of H+-ATPase expression in these segments remains to be delineated in detail. The phenotype of disease-causing mutations in the B1 subunit may also relate to its presence in the TAL and DCT.

Abstract 065: Enhanced Glomerular Capillary Pressure and Tubuloglomerular Feedback (tgf) in Obese Alms1 (alstrom Syndrome 1) Knock Out Rats

The ALMS1 gene has been associated to decreased renal function (lower GFR) and hypertension in genome-wide studies humans. The role of ALMS1 in renal dysfunction is unclear. Our lab has identified the ALMS1 protein as an interacting partner with the Na/K/2Cl cotransporter NKCC2, which mediates NaCl absorption by the thick ascending limb. We also found that rats with ALMS1 deletion exhibited increased NKCC2 activity, hypertension and progressive obesity. NKCC2 also mediates the tubuloglomerular feedback (TGF) mechanism. TGF is initiated by an increase in NaCl transport via NKCC2 in the macula densa, which causes subsequent afferent arteriolar constriction and decreases GFR. We hypothesized that ALMS1 deletion enhances TGF and reduces GFR in rats. To test this we studied homozygous ALMS1 knockout (KO) and littermates wild-type Dahl SS (WT) rats. We measured TGF using in vivo renal micropuncture technique. TGF was calculated as a reduction in stop flow pressure (P SF ) during simultaneous perfusion of late proximal tubule incrementally from 0 to 20 and then 40 nl/min. P SF is an index of glomerular capillary pressure (P GC ) and increased P GC is linked to renal dysfunction. Increasing the perfusion rate from 0 to 20nl/min decreased P SF in ALMS1 KO rats but not in WT rats, indicating enhanced TGF sensitivity (ALMS1: 45.5±2.9 to 41.4±3.0 mmHg, p&lt;0.05 vs. WT: 34.2±1.4 to 31.4±1.9 mmHg, N.S). Baseline P GC was elevated (ALMS1: 46.9±3.3 vs. WT: 34.9±1.5 mmHg, p&lt;0.05) while renal interstitial hydrostatic pressure (RIHP) was reduced in ALMS1 KO (ALMS1: 2.6±0.9 vs. WT: 5.2±0.5 mmHg, p&lt;0.05). We then measured GFR (calculated from inulin clearance) and found it was reduced in ALMS1 KO rats (ALMS1: 0.5±0.1 vs. WT: 1.6±0.3 ml/min/100gBW, p&lt;0.05). Mean arterial pressure was higher in ALMS1 KO rats (ALMS1: 146±2 vs. WT: 113±3 mmHg, p&lt;0.05). We concluded that ALMS1 is involved in the control of glomerular hemodynamics. Deletion of ALMS1 enhanced TGF sensitivity and this may be in part responsible for decreased GFR. It is likely that increased TGF-sensitivity, enhanced glomerular capillary pressure and hypertension combine to reduce GFR in ALMS1 KO rats. These are the first data supporting a role of ALMS1 in glomerular hemodynamics.

FISIOPATOLOGÍA DE LA ENFERMEDAD RENAL DIABÉTICA

La enfermedad renal por diabetes es una complicación mi- crovascular en la cual se producen, a nivel renal, cambios hemodinámicos, metabólicos, estrés oxidativo y aumento de la inflamación. Esto determina un aumento de la matriz ex- tracelular mesangial, engrosamiento de la membrana basal, pérdida de pies podocitarios y disrupción de células tubulares con fibrosis y proteinuria. El aumento de la expresión de los receptores SGLT-1 y SGLT-2, con la consecuente y marcada reducción de la oferta de Na+ a la mácula densa, se interpreta erróneamente como una reducción del volumen plas- mático por el aparato yuxtaglomerular a través del “feedback túbulo-glomerular”, lo que genera vasodilatación de la arteriola aferente y consecuente aumento de la presión intraglomeru- lar. Esto modifica el concepto clásico glomerular y se inter- preta que podría haber una lesión inicial a nivel tubular.

Impact of Diabetes and an SGLT2 Inhibitor on Glomerular Number and Volume in db/db Mice as Estimated by Synchrotron Radiation Micro-CT at SPring-8

To investigate the impact of diabetes and luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor on the numbers and volumes of glomeruli in the whole kidneys, we performed CT imaging of diabetic db/db mice using synchrotron radiation at SPring-8. We found that there was no significant difference in the total glomerular number among mice. Diabetic mice had significantly larger glomerular volume in the mid-cortex compared to lean control db/m mice. Luseogliflozin reduced whole kidney volume, but not the glomerular hypertrophy in db/db mice. Luseogliflozin also decreased mesangial expansion and tubular damage in db/db mice. Luseogliflozin augmented hypoxia of S3 segment of proximal tubules in the juxtamedullary region and macula densa, as well as the number of intracellular vesicles in juxtaglomerular cells, and sustained upregulated renal renin expression in db/db mice. Luseogliflozin ameliorated hyperglycemia, glomerular and tubular injury, but not albuminuria. Db/db mice decreased the expression of renal megalin protein compared with that in db/m mice. Luseogliflozin-treated db/db mice also reduced the expression of megalin, accompanied with decreased reabsorbing urinary albumin as endocytotic ligand of megalin. In conclusion, our results suggest that oxygen metabolism and/or humoral factors more than blood glucose levels might determine the glomerular number and volume in diabetic kidney. Funding: This work was partially supported by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI grant no. 15K09372 to Y.T.), and by a Research Grant from the Japan Diabetes Foundation and Asahikawa Medical University (Research grant for Innovative Research in Life Science). Conflicts of Interests: Y.T. was also supported by several foundational grants, including grants from MSD, K.K., Eli Lilly and Company, Sanofi K.K., Takeda Pharmaceutical Co. and Taisho Pharmaceutical Co. No other potential conflicts of interest relevant to this article were reported. Ethical Approval Statement: These experiments were performed with the approval of the SPring-8 Proposal Review Committee (2012B1772, 2013A1655, 2013B1739).

Effects of SGLT2 Inhibitors on Renin-Aldosterone System for One Month and Six Months in Type 2 Diabetes

Previous study has shown SGLT2 inhibitors affect tubule-glomerular feedback in macula densa associated with decreased glomerular filtration rate. Thereby, plasma renin activity(PRA) may acutely increase after treatment with SGLT2 inhibitors. However chronic activation of PRA-aldosterone system may introduce deterioration of atherosclerosis and fibrosis of heart muscle. The purpose of this study is to examine alterations of PRA and aldosterone concentration (PAC) after treatment with SGLT2 inhibitor in type 2 diabetic patients (T2D). We measured PRA and PAC in T2D (age 68±18 years old, men: female = 12:6) after treatment with SGLT2 inhibitors (tofogliflozin: 7 cases, empagliflozin: 6cases, canagliflozin: 3 cases) for 1 months and 6months (8 cases, age 63±16 years old, men: female = 7:1) from 2014 to 2017. Moreover, we could measured each metabolic parameters such as blood glucose, HbA1c, urinary Na/creatinine and blood pressure before and after treatment with SGLT2 inhibitors (SGLT2i) for 1month and 6months in T2D. We analyzed these results with paired t test using JMP12.2.0. SGLT2i significantly increased PRA for only 1month, but not PAC for 1 month and 6 months as follows; before PRA 1.9±2.4 ng/ml/h, 5.8±9.2 ng/ml/h for 1month (p&amp;lt;0.05) and 2.5±3.1 ng/ml/h for 6months, before PAC 104±65 pg/ml, 105±55 pg/ml for 1 month and 123±85 pg/ml for 6 months, respectively. Moreover, when we examined each metabolic parameter before and after treatment for 1 month, each body weight, BS, and HbA1c was significantly decreased (p&amp;lt;0.05). SGLT2i increases urinary sodium excretion, which affects macula densa in juxta-glomerular apparatus, and finally stimulates secretion of renin for 1month, but not for 6 months. The alteration of aldosterone were not observed after treatment with SGLT2 inhibitors for 1 and 6 months. These results suggest that SGLT2 inhibitors acutely affects PRA but not PAC, and chronic activation of PRA and PAC, which might cause cardiovascular events are not found. Disclosure I. Mori: None. T. Ishizuka: None.

Return of the (pro)renin receptor: a novel role in macula densa signaling.

Return of the (pro)renin receptor: a novel role in macula densa signaling.

The macula densa prorenin receptor is essential in renin release and blood pressure control.

The prorenin receptor (PRR) was originally proposed to be a member of the renin-angiotensin system (RAS); however, recent work questioned their association. The present paper describes a functional link between the PRR and RAS in the renal juxtaglomerular apparatus (JGA), a classic anatomical site of the RAS. PRR expression was found in the sensory cells of the JGA, the macula densa (MD), and immunohistochemistry-localized PRR to the MD basolateral cell membrane in mouse, rat, and human kidneys. MD cell PRR activation led to MAP kinase ERK1/2 signaling and stimulation of PGE2 release, the classic pathway of MD-mediated renin release. Exogenous renin or prorenin added to the in vitro microperfused JGA-induced acute renin release, which was inhibited by removing the MD or by the administration of a PRR decoy peptide. To test the function of MD PRR in vivo, we established a new mouse model with inducible conditional knockout (cKO) of the PRR in MD cells based on neural nitric oxide synthase-driven Cre-lox recombination. Deletion of the MD PRR significantly reduced blood pressure and plasma renin. Challenging the RAS by low-salt diet + captopril treatment caused further significant reductions in blood pressure, renal renin, cyclooxygenase-2, and microsomal PGE synthase expression in cKO vs. wild-type mice. These results suggest that the MD PRR is essential in a novel JGA short-loop feedback mechanism, which is integrated within the classic MD mechanism to control renin synthesis and release and to maintain blood pressure.

Wnt signaling regulates macula densa structure and function

The macula densa (MD), a plaque of 15–20 specialized cells, sits at the vascular pole of the glomerulus and plays a critical role in maintaining renal blood flow and glomerular filtration rate via tubuloglomerular feedback. Changes in renin state of the juxtaglomerular apparatus are known to be associated with alterations in MD cell morphology, although the underlying mechanism and importance of this phenomenon have been elusive. The canonical Wnt‐β catenin signaling pathway is known to regulate several cellular processes including embryonic development, cell proliferation, differentiation and homoeostasis. Glycogen synthase kinase 3 beta (GSK3b), a serine/threonine kinase, is involved in the phosphorylation and subsequent destablization of β catenin; thus, GSK3b plays a critical role in regulating the Wnt‐β catenin pathway. Lithium is a commonly used inhibitor of GSK3b in the kidney. Given the importance of Wnt‐β catenin in maintaining epithelial cell structure, the present studies aimed to explore its role in regulating MD morphology and function. Wild‐type mice were treated with lithium chloride for 2 weeks (40 mmol/kg chow for 7 days, and 60 mmol LiCl/kg chow subsequently), followed by harvesting and histological analysis of the kidneys. Lithium treatment caused a significant increase in the number of proliferating MD cells as quantified by Ki67 and EdU staining. As compared to control mice, MD cell number in the plaque was higher in lithium‐treated groups with several hyperplastic MD plaques observed containing &gt; 40 cells. MD nucleus‐to‐cell volume ratio was 30% higher after lithium treatment. Using a O‐propargyl‐puromycin incorporation‐based protein synthesis imaging assay, a 3‐fold increase in protein synthesis was observed in MD cells after lithium treatment. Identical MD cell morphological alterations were observed in the newly generated nNOS‐Ctnnb1(Ex3) mice, a Wnt gain‐of‐function genetic model, confirming MD cell Wnt specificity of lithium treatment. In summary, Wnt signaling is an important regulator of MD morphology and cell biology, which may be involved in traditional renin and hemodynamic and novel tissue remodeling functions of MD cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SPAK disruption increases the glomerular filtration rate

The thick ascending limb (TAL) of the kidney reabsorbs approximately 25% of filtered NaCl via its luminal Na‐K‐Cl cotransporter (NKCC2). In specialized TAL cells constituting the macula densa (MD), NKCC2 serves to sense the luminal NaCl concentration for paracrine adjustments of the glomerular filtration rate (GFR) to the needs of the body. The catalytically active full‐length (FL) SPAK and OSR1 kinases activate NKCC2 by phosphorylation. In addition to the FL‐SPAK, TAL cells express truncated SPAK variants (SPAK2 and KS‐SPAK) arising from alternative splicing or proteolytic cleavage. SPAK2 and KS‐SPAK exert strong dominant‐negative effects on FL‐SPAK/OSR1‐dependent NKCC2 phosphorylation. This study addresses the role of SPAK variants in regulation of NKCC2 in MD cells with respect to the modulation of GFR.SPAK variants, OSR1, NKCC2 and phospho‐NKCC2 were analyzed in kidneys of wild type (WT) and SPAK‐deficient (SPAK−/−) mice and cultured TAL and MD cells using immunohistochemistry, immunoblotting and quantitative PCR. Key juxtaglomerular components involved in GFR regulation such as cyclooxygenase 2 (COX2), nitric oxide synthase 1 (NOS1) and renin were compared between the two mouse genotypes. Effects of SPAK deficiency on GFR were evaluated by transcutaneous assessment of FITC‐sinistrin clearance in freely moving mice.Immunohistochemical analysis revealed that NKCC2 phosphorylation levels were significantly higher in TAL cells of SPAK−/− kidneys compared to WT, supporting the presence of the dominant‐negative SPAK2 and KS‐SPAK isoforms in WT TAL cells. In contrast, NKCC2 phosphorylation was not significantly altered in SPAK‐deficient MD cells, suggesting minor effects of truncated SPAK variants on NKCC2 and a redundant role of FL‐SPAK in this cell type. In line with this, immunoblotting of lysates from cultured TAL and MD cells showed predominant abundance of SPAK2 and KS‐SPAK in TAL cells, whereas MD cells mainly expressed FL‐SPAK. Both TAL and MD cells showed substantial OSR1 levels, which may explain the FL‐SPAK redundancy. Juxtaglomerular COX2, NOS1 and renin levels as well as the FITC‐sinistrin clearance were substantially higher in SPAK−/− which might reflect increased NKCC2‐mediated salt reabsorption in preceding TAL and preserved NKCC2‐mediated sensing of luminal NaCl in MD cells.Our results describe differential expression of SPAK variants in TAL and MD cells and demonstrate that SPAK disruption stimulates GFR likely due increased NKCC2 activity in TAL prior to MD resulting in reduced NaCl concentration at the MD site.Support or Funding InformationFunding: DFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, R01DK098141 to JAM; DFG Grant MU2924/2‐2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

NNOS in Embryonic Kidney Contributes to Glomerular Maturation

The regulatory role of neuronal nitric oxide synthase (nNOS) in renal and glomerular hemodynamics is well established. In addition, nNOS is involved in cellular migration in other organs, such as the brain. The highest level of nNOS expression occurs on day 6 after birth (P6), when the mouse kidney is still developing. The purpose of this study was to explore a novel, non‐traditional role of nNOS in renal development and glomerular maturation. To determine the timepoints and localization of nNOS expression in embryonic kidney, fluorescent reporter nNos+/−‐Cre+/+/mTmG+/+ mice were induced with tamoxifen at different timepoints during pregnancy. The specific inhibitor of nNOS, 7‐Nitroindazole (7‐NI) was administered daily by ip. injections (20 mg/kg) from E16.5 to P7 in pregnant Pod+/+‐Cre+/+/Confetti+/+ mice. Fetal kidneys were harvested after birth at P7 and further processed for histological analysis. During embryonic kidney development, nNOS expression is detectable in the medullary region as early as E16.5. At E19.5, nNOS is expressed in macula densa cells at the juxtaglomerular apparatus and in a few cells of the medullary collecting duct. At P3, nNOS was localized in the same cell types as in earlier stages. Pharmacological inhibition of nNOS for 10 days by daily 7‐NI treatment did not alter the kidney/body weight ratio compared to vehicle treatment, albeit the 7‐NI treated animal group had growth retardation (n=4 each). The diameter of glomeruli was 20% smaller, and the cumulative podocyte number in glomeruli was decreased by 40% in 7‐NI treated animals (n=4). Albeit 7‐NI treatment resulted in the reduction of body weight likely due to nonrenal effects, the embryonic presence of nNOS in macula densa cells and the negative impact of nNOS inhibition on glomerular diameter and podocyte number suggest a novel, non‐traditional role for nNOS in embryonic kidney development and glomerular maturation.Support or Funding InformationResearch support NIH NIDDK R01 DK64324This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

COX-2-derived PGE2 triggers hyperplastic renin expression and hyperreninemia in aldosterone synthase-deficient mice

Pharmacological inhibition or genetic loss of function defects of the renin angiotensin aldosterone system (RAAS) causes compensatory renin cell hyperplasia and hyperreninemia. The triggers for the compensatory stimulation of renin synthesis and secretion in this situation may be multimodal. Since cyclooxygenase-2 (COX-2) expression in the macula densa is frequently increased in states of a defective RAAS, we have investigated a potential role of COX-2 and its derived prostaglandins for renin expression and secretion in aldosterone synthase-deficient mice (AS−/−) as a model for a genetic defect of the RAAS. In comparison with wild-type mice (WT), AS−/− mice had 9-fold and 30-fold increases of renin mRNA and of plasma renin concentrations (PRC), respectively. Renin immunoreactivity in the kidney cortex of AS−/− mice was 10-fold higher than in WT. Macula densa COX-2 expression was 5-fold increased in AS−/− kidneys relative to WT kidneys. Treatment of AS−/− mice with the COX-2 inhibitor SC-236 for 1 week lowered both renal renin mRNA and PRC by 70%. Hyperplastic renin cells in AS−/− kidneys were found to express the prostaglandin E2 receptors EP2 and EP4. Global deletion of EP2 receptors did not alter renin mRNA nor PRC values in AS−/− mice. Renin cell-specific inducible deletion of the EP4 receptor lowered renin mRNA and PRC by 25% in AS−/− mice. Renin cell-specific inducible deletion of the EP4 receptor in combination with global deletion of the EP2 receptor lowered renin mRNA and PRC by 70–75% in AS−/− mice. Lineage tracing of renin-expressing cells revealed that deletion of EP2 and EP4 leads to a preferential downregulation of perivascular renin expression. Our findings suggest that increased macula densa COX-2 activity in AS−/− mice triggers perivascular renin expression and secretion via prostaglandin E2.

Lack of contribution of nitric oxide synthase to cholinergic vasodilation in murine renal afferent arterioles.

We have previously reported significant increases in neuronal nitric oxide synthase (NOS) immunostaining in renal arterioles of angiotensin type 1A receptor (AT1A) knockout mice, and in arterioles and macula densa cells of AT1A/AT1B knockout mice. The contribution of nitric oxide derived from endothelial and macula densa cells in the maintenance of afferent arteriolar tone and acetylcholine-induced vasodilation was functionally determined in kidneys of wild-type, AT1A, and AT1A/AT1B knockout mice. Acetylcholine-induced changes in arteriolar diameters of in vitro blood-perfused juxtamedullary nephrons were measured during control conditions, in the presence of the nonspecific NOS inhibitor, Nω-nitro-l-arginine methyl ester (NLA), or the highly selective neuronal NOS inhibitor, N5-(1-imino-3-butenyl)-l-ornithine (VNIO). Acetylcholine (0.1 mM) produced a significant vasoconstriction in afferent arterioles of AT1A/AT1B mice (-10.9 ± 5.1%) and no changes in afferent arteriolar diameters of AT1A knockout mice. NLA (0.01-1 mM) or VNIO (0.01-1 μM) induced significant dose-dependent vasoconstrictions (-19.8 ± 4.0% 1 mM NLA; -7.8 ± 3.5% 1 μM VNIO) in afferent arterioles of kidneys of wild-type mice. VNIO had no effect on afferent arteriole diameters of AT1A knockout or AT1A/AT1B knockout mice, suggesting nonfunctional neuronal nitric oxide synthase. These data indicate that acetylcholine produces a significant renal afferent arteriole vasodilation independently of nitric oxide synthases in wild-type mice. AT1A receptors are essential for the manifestation of renal afferent arteriole responses to neuronal nitric oxide synthase-mediated nitric oxide release.