A mathematical model of the rat kidney IV: whole-kidney response to hyperkalemia

The renal response to acute hyperkalemia is mediated by increased K secretion within connecting tubule (CNT), flux that is modulated by tubular effects (e.g. aldosterone) in conjunction with increased luminal flow. There is ample evidence that peritubular K blunts Na reabsorption in proximal tubule, thick ascending Henle limb, and distal convoluted tubule (DCT). While any such reduction may augment CNT delivery, the relative contribution of each is uncertain. The kidney model of this lab was recently advanced with representation of cortical labyrinth and medullary ray. Model tubules capture the impact of hyperkalemia to blunt Na reabsorption within each upstream segment. However, this forces the question of the extent to which increased Na delivery is transmitted past macula densa and its tubuloglomerular feedback (TGF) signal. Beyond increasing macula densa Na delivery, peritubular K is predicted to raise cytosolic Cl and depolarize macula densa cells, which may also activate TGF. Thus, although upstream reduction in Na transport may be larger, it appears that the DCT effect is critical to increasing CNT delivery. Beyond the flow effect, hyperkalemia reduces ammoniagenesis and reduced ammoniagenesis enhances K excretion. What this model provides is a possible mechanism. When cortical NH4 is taken up via peritubular Na,K(NH4)-ATPase, it acidifies principal cells. Consequently, reduced ammoniagenesis increases principal cell pH, thereby increasing conductance of both ENaC and ROMK, enhancing K excretion. In this model, aldosterone's effect on principal cells, diminished DCT Na reabsorption, and reduced ammoniagenesis, all provide relatively equal and additive contributions to renal K excretion.

Knockout of Macula Densa Neuronal Nitric Oxide Synthase Increases Blood Pressure in db/db Mice

Hypertension is a common comorbid condition in patients with diabetes. The pathogenesis of hypertension in diabetes has not been fully clarified. Primary tubular hyperreabsorption may contribute, which may be counteracted by glomerular hyperfiltration in the early diabetic kidney. In this study, we hypothesize that in early diabetes, the macula densa neuronal nitric oxide synthase (NOS1)-derived nitric oxide (NO) production is enhanced, which blunts tubuloglomerular feedback (TGF) response, promotes glomerular hyperfiltration, and maintains normal blood pressure; conversely, insufficient NO generation by the macula densa induces hypertension by lowering glomerular filtration rate and thus inhibiting natriuresis. To test this hypothesis, we examined the changes of macula densa NOS1 expression and phosphorylation as well as NO production, TGF response, glomerular filtration rate, sodium excretion, and blood pressure in a murine model of leptin receptor-deficient (db/db) diabetes with or without macula densa-specific NOS1 deletion. We found that db/db mice presented reduced fractional renal sodium excretion and only a small increase in blood pressure, associated with upregulated expression and activity of macula densa NOS1, inhibited TGF response, and glomerular hyperfiltration. Genetic knockout of macula densa NOS1 restored the TGF response and attenuated glomerular hyperfiltration in db/db mice but also further reduced fractional renal sodium excretion and substantially increased blood pressure. In conclusion, the present study demonstrates that in the early stage of leptin receptor-deficient diabetes, the upregulation of macula densa NOS1 inhibits TGF and increases glomerular filtration rate, which counteracts renal sodium retention and limits the rise in blood pressure.

Macula Densa NOS1β Modulates Renal Hemodynamics and Blood Pressure During Pregnancy: Role in Gestational Hypertension

Background: Regulation of renal hemodynamics and blood pressure (BP) via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the β-splice variant of nitric oxide synthase 1 (NOS1β) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1β in the macula densa, thus blunting TGF, allowing glomerular filtration rate (GFR) to increase and BP to decrease. Methods: We employed sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radio-telemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1β/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. Results: Macula densa NOS1β was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1β, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1β expression increased in normotensive pregnancies but decreased in spontaneous gestational hypertensive pregnancies. Conclusions: Macula densa NOS1β plays a critical role in control of renal hemodynamics and BP during pregnancy.

A new view of macula densa cell microanatomy

Although macula densa (MD) cells are chief regulatory cells in the nephron with unique microanatomical features, they have been difficult to study in full detail due to their inaccessibility and limitations in earlier microscopy techniques. The present study used a new mouse model with a comprehensive imaging approach to visualize so far unexplored microanatomical features of MD cells, their regulation and functional relevance. MD-GFP mice with conditional and partial induction of green fluorescent protein (GFP) expression, which specifically and intensely illuminated only single MD cells were used with fluorescence microscopy of fixed tissue and live MD cells in vitro and in vivo with complementary electron microscopy (EM) of rat, rabbit, and human kidney. An elaborate network of major and minor cell processes here named maculapodia were found at the cell base, projecting towards other MD cells and the glomerular vascular pole. The extent of maculapodia showed up-regulation by low dietary salt intake and female gender. Time-lapse imaging of maculapodia revealed highly dynamic features including rapid outgrowth and an extensive vesicular transport system. EM of rat, rabbit, and human kidneys, and three-dimensional (3D) volume reconstruction in optically cleared whole-mount MD-GFP mouse kidneys further confirmed the presence and projections of maculapodia into the extraglomerular mesangium and the afferent and efferent arterioles. The newly identified dynamic and secretory features of MD cells suggest the presence of novel functional and molecular pathways of cell-to-cell communication in the juxtaglomerular apparatus (JGA) between MD cells and between MD and other target cells.

A new mechanism for the sex differences in angiotensin II-induced hypertension: the role of macula densa NOS1β-mediated tubuloglomerular feedback

Females are protected against the development of angiotensin II (ANG II)-induced hypertension compared with males, but the mechanisms have not been completely elucidated. In the present study, we hypothesized that the effect of ANG II on the macula densa nitric oxide (NO) synthase 1β (NOS1β)-mediated tubuloglomerular feedback (TGF) mechanism is different between males and females, thereby contributing to the sexual dimorphism of ANG II-induced hypertension. We used microperfusion, micropuncture, clearance of FITC-inulin, and radio telemetry to examine the sex differences in the changes of macula densa NOS1β expression and activity, TGF response, natriuresis, and blood pressure (BP) after a 2-wk ANG II infusion in wild-type and macula densa-specific NOS1 knockout mice. In wild-type mice, ANG II induced higher expression of macula densa NOS1β, greater NO generation by the macula densa, and a lower TGF response in vitro and in vivo in females than in males; the increases of glomerular filtration rate, urine flow rate, and Na+ excretion in response to an acute volume expansion were significantly greater and the BP responses to ANG II were significantly less in females than in males. In contrast, these sex differences in the effects of ANG II on TGF, natriuretic response, and BP were largely diminished in knockout mice. In addition, tissue culture of human kidney biopsies (renal cortex) with ANG II resulted in a greater increase in NOS1β expression in females than in males. In conclusion, macula densa NOS1β-mediated TGF is a novel and important mechanism for the sex differences in ANG II-induced hypertension.

Targeted single-cell RNA-seq identifies minority cell types of kidney distal nephron that regulate blood pressure and calcium balance

ABSTRACTA major objective in modern biology is generation of comprehensive atlases of various organs identifying all cell types and their expressed genes. In kidney, extensive data exists for proximal tubule and collecting duct cells, but not for non-abundant intermediate epithelial cell types. Here, we coupled a FACS-enrichment protocol with single-cell RNA-seq analysis to profile the transcriptomes of 9099 cells from the nephron region adjacent to the macula densa. Clusters containing Slc12a3+/Pvalb+ and Slc12a3+/Pvalb- cells were identified as DCT1 and DCT2 cells. The DCT1 cells appear to be heterogeneously associated with variable expression of Slc8a1, Calb1, and Ckb among other mRNAs. No DCT2-specific transcripts were found. The analysis also identified two distinct cell types in the Slc12a1+ portion of Henle’s loop as well as Nos1+/Avpr1a+ macula densa cells. Thus, we identify unexpected cell diversity in the intermediate region of the nephron and create a web-based data resource for these cells.