High baseline ROMK activity in mouse late distal convoluted and early connecting tubule (DCT2/CNT) probably contributes to aldosterone-independent K+ secretion

The renal outer medullary K+ channel (ROMK) is co-localized with the epithelial Na+ channel (ENaC) in late distal convoluted tubule (DCT2), connecting tubule (CNT) and cortical collecting duct (CCD). ENaC-mediated Na+ absorption generates the electrical driving force for ROMK-mediated tubular K+ secretion which is critically important for maintaining renal K+ homeostasis. ENaC activity is aldosterone-dependent in late CNT and early CCD (CNT/CCD) but aldosterone-independent in DCT2 and early CNT (DCT2/CNT). This suggests that under baseline conditions with low plasma aldosterone ROMK-mediated K+ secretion mainly occurs in DCT2/CNT. Therefore, we hypothesized that baseline ROMK activity is higher in DCT2/CNT than in CNT/CCD. To test this hypothesis, patch-clamp experiments were performed in DCT2/CNT and CNT/CCD microdissected from mice maintained on standard diet. In single-channel recordings from outside-out patches we detected typical ROMK channel activity in both DCT2/CNT and CNT/CCD and confirmed that ROMK is the predominant K+ channel in the apical membrane. Amiloride-sensitive (ΔIami) and tertiapin-sensitive (ΔITPNQ) whole-cell currents were determined to assess ENaC and ROMK activity, respectively. As expected, baseline ΔIami was high in DCT2/CNT (~370 pA) but low in CNT/CCD (~60 pA). Importantly, ΔITPNQ was significantly higher in DCT2/CNT than in CNT/CCD (~810 pA versus ~350 pA). We conclude that high ROMK activity in DCT2/CNT is critical for aldosterone-independent renal K+ secretion under baseline conditions. A low potassium diet significantly reduced ENaC but not ROMK activity in DCT2/CNT. This suggests that modifying ENaC activity in DCT2/CNT plays a key regulatory role in adjusting renal K+ excretion to dietary K+ intake.

ENaC and ROMK channels in the connecting tubule regulate renal K+ secretion

We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K+ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe). In more distal parts of the CNT (aldosterone-sensitive portion [CNTas]), these currents were minimal. This functional difference correlated with alterations in the intracellular location of ENaC, which was at or near the apical membrane in CNTe and more cytoplasmic in the CNTas. ROMK activity, measured as TPNQ-sensitive currents, was substantial in both segments. A mathematical model of the rat nephron suggested that K+ secretion by the CNTe predicted from these currents provides much of the urinary K+ required for K balance on this diet. In animals fed a K-deficient diet (0.1% K), both ENaC and ROMK currents in the CNTe decreased by ∼50%, predicting a 50% decline in K+ secretion. Enhanced reabsorption by a separate mechanism is required to avoid excessive urinary K+ losses. In animals fed a diet supplemented with 3% K, ENaC currents increased modestly in the CNTe but strongly in the CNTas, while ROMK currents tripled in both segments. The enhanced secretion of K+ by the CNTe and the recruitment of secretion by the CNTas account for the additional transport required for K balance. Therefore, adaptation to increased K+ intake involves the extension of robust K+ secretion to more distal parts of the nephron.

Abstract P025: Hyperinsulinemia Causes Renal Hyperperfusion And Increases Glomerular Basement Membrane Thickness Independent Of Renal Perfusion Pressure And Glycemic Status: Potential Role Of Connecting Tubule Glomerular Feedback?

Obesity is often associated with hyperinsulinemia (HI) and renal damage. However, the role of HI in Obesity related Renal Damage (ORD) is unclear. Renal hyperperfusion/hyperfiltration plays a major role in ORD. Normally, the kidneys autoregulate blood flow by two feedback mechanisms: 1.) Tubuloglomerular feedback (TGF), a vasoconstrictor mechanism and 2.) Connecting Tubule Glomerular Feedback (CTGF), a vasodilator mechanism. Previously, we found that Zucker obese rats had higher CTGF (TGF was unchanged) and were hyperinsulinemic before the development of ORD. Epithelial sodium channel (ENaC) initiates CTGF and Insulin is a known ENaC activator. Hypothesis: HI increases renal cortical blood flow (CBF) by increasing CTGF and causes renal damage. To isolate the effect of HI from the blood glucose (BG) level, HI-euglycemic clamp was created in normal anesthetized Sprague Dawley (SD) rats by simultaneous intravenous (IV) infusion of 10% glucose and insulin. Average baseline BG in non-fasted anesthetized SD rats was 199.0±31.6 mmol/L. Insulin infusion increased CBF significantly by 12.2 ± 1.3% (n=3, p<0.05) from the baseline even before the BG level starts decreasing. Insulin was further infused to attain normoglycemic condition (96.0±2.3 mmol/L) and this was associated with additional increase in CBF by 19.2 ± 4.5% (p<0.05) from the baseline. Subsequent ENaC inhibition by benzamil (BZ) (400 μg/kg, IV) completely reversed the insulin-induced increase in CBF. Neither Insulin nor BZ treatment altered the renal perfusion pressure (RPP) suggesting insulin-induced increase in CBF was independent of RPP. In a separate group of SD rats, renal-HI (1.8 IU/kg/day) was created in only one of the two kidneys for 6 weeks using renal subcapsular catheter to measure glomerular basement membrane (GBM) thickness (a marker of renal damage). GBM was significantly thickened in insulin-infused kidney compared to vehicle-infused kidney (199.4±17 vs. 145.5±3.6nm, n=3, p<0.05). Conclusion: Acute HI increased CBF, that was completely reversed by ENaC inhibition implying a possible role of enhanced CTGF. Chronic renal-HI caused GBM thickening. Perspective: HI observed in obesity or type-2 diabetes may cause renal hyperperfusion by increasing CTGF and contribute to the ORD.

Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2

BackgroundThe progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1, which encodes endothelin 1 in the connecting tubule/collecting duct.MethodsTo determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct–specific Dot1l conditional knockout mice (Dot1lAC), Dot1l and Edn1 double-knockout mice (DEAC), and Edn1 connecting tubule/collecting duct–specific conditional knockout mice (Edn1AC), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription.ResultsIn each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter.ConclusionsOur study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.

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.

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.

Connecting tubule glomerular feedback mediates tubuloglomerular feedback resetting after unilateral nephrectomy

Unilaterally nephrectomized rats (UNx) have higher glomerular capillary pressure (PGC) that can cause significant glomerular injury in the remnant kidney. PGC is controlled by the ratio of afferent (Af-Art) and efferent arteriole resistance. Af-Art resistance in turn is regulated by two 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 (ENaC). Resetting of TGF post-UNx can allow systemic pressure to be transmitted to the glomerulus and cause renal damage, but the mechanism behind this resetting is unclear. Since CTGF is an Af-Art dilatory mechanism, we hypothesized that CTGF is increased after UNx and contributes to TGF resetting. To test this hypothesis, we performed UNx in Sprague-Dawley (8) rats. Twenty-four hours after surgery, we performed micropuncture of individual nephrons and measured stop-flow pressure (PSF). PSF is an indirect measurement of PGC. Maximal TGF response at 40 nl/min was 8.9 ± 1.24 mmHg in sham-UNx rats and 1.39 ± 1.02 mmHg in UNx rats, indicating TGF resetting after UNx. When CTGF was inhibited with the ENaC blocker benzamil (1 μM/l), the TGF response was 12.29 ± 2.01 mmHg in UNx rats and 13.03 ± 1.25 mmHg in sham-UNx rats, indicating restoration of the TGF responses in UNx. We conclude that enhanced CTGF contributes to TGF resetting after UNx.