Micropuncturing the nephron

Abstract

To achieve the role of the kidney in maintaining body homeostasis, the renal vasculature, the glomeruli, and the various segments of the nephron and the collecting duct system have to fulfill very diverse and specific functions. These functions are dependent on a complex renal architecture and are regulated by systemic hemodynamics, hormones, and nerves. As a consequence, to better understand the physiology of the kidney, methods are necessary that allow insights on the function of these diverse structures in the physiological context of the intact kidney. The renal micropuncture technique allows direct access to study superficial nephrons in vivo. In this review, the application of micropuncture techniques on the single nephron level is outlined as an approach to better understand aspects of glomerular filtration, tubular transport, and tubulo-glomerular communication. Studies from the author's lab, including experiments in gene-targeted mice, are briefly presented to illustrate some of the approaches and show how they can further advance our understanding of the molecular mechanisms involved in the regulation of kidney function.

Figure 1. Preparation of the kidney for micropuncture experiments

(A) Following a flank incision, the perirenal fat and adrenal gland are carefully separated from the kidney. Without applying tension or torsion, the kidney is carefully overturned and placed in a Lucite cup such that the dorsal kidney surface faces up (B). In rats and mice, the kidney capsule should not be removed as it provides protection, and stabilizes inserted micropuncture pipettes. (C) In the rat, the ureter is catheterized for collection of urine; this is less useful in mice because of a tendency of catheters to obstruct. To immobilize the kidney, the Lucite cup is lined with moleskin (D) and cotton soaked in saline is carefully placed around the kidney. 2-3% agar is dripped onto the cotton to prevent leakage of the superfused mineral oil (37°C) and establish an oil layer over the kidney surface of 1-2 mm (to keep the kidney surface warm and protected). Adapted from (5).

Figure 2. Identification of structures on the kidney surface accessible by micropuncture

The nephron of a Munich-Wistar-Fromter rat is mapped by injecting small amounts of blue dye into Bowman’s space and following it moving along the tubular system (1 to 4).

Figure 3. Reabsorption profiles of glucose and K⁺ along the proximal tubule in mice using the fractional reabsorption of fluid as a reference for the puncturing sites

The fractional reabsorption of fluid, glucose and K⁺ was determined in individual proximal collections. Glucose is rapidly and nearly quantitatively reabsorbed within the very early proximal tubule (low fractional reabsorption of fluid). In contrast, fractional reabsorption of K⁺ is negative in the early proximal tubule indicating net K⁺ fluxes into the lumen. Proximal tubular K⁺ secretion serves to counteract the depolarizing effects of electrogenic Na⁺ reabsorption (e.g. Na⁺-glucose cotransport); the K⁺ secretion is controlled by the regulatory β-subunit KCNE1, and mediated by an unknown K⁺ channel in the early proximal tubule and by KCNQ1 in the late proximal tubule (65; 66). Adapted from (66).

Figure 4. Reabsorption profiles of Ca²⁺ along the nephron and along the distal segments using luminal K⁺ concentration as a reference for puncturing sites

A) Ca²⁺ reabsorption was unaffected in TRPV5 -/- mice up to the last surface loop of the proximal tubule (LPT); in contrast, mean Ca²⁺ delivery to puncturing sites within the distal segments (DS) accessible to micropuncture as well as urine (U) was significantly enhanced in TRPV5 -/- mice. * p<0.001 vs. TRPV5 +/+. B) The aim then was to try to directly assess Ca²⁺ reabsorption along the distal segments. Luminal K⁺ concentration was used as a reference for the puncturing site with low and high concentrations indicating early and late aspects of the DS, respectively. Whereas the Ca²⁺ delivery profile indicated net reabsorption along the distal segments in TRPV5 +/+ mice, fractional Ca²⁺ delivery actually increased in TRPV5 -/- mice, demonstrating a defect in Ca²⁺ reabsorption along the distal segments. Finally, comparing the fractional Ca²⁺ delivery in the late distal segment with the values in urine indicated significant compensation in the collecting duct of TRPV5 -/- (consistent with expression of TRPV6 in that segment). Adapted from (25).

Figure 5. The transtubular osmotic gradient in late proximal tubular fluid (Δ osmolality)

Δ osmolality, i.e., the difference in osmolality between plasma and late proximal tubular fluid, is about -12 mmHg in AQP1 wild-type (+/+) mice (i.e. a little more hypotonic in proximal tubular fluid) consistent with the principle that water follows the reabsorption of electrolytes. Δ osmolality of AQP1 knockout (-/-) mice was significantly increased to about -40 mmHg. This demonstrates the functional importance of the water channel AQP1 for near-isosmolar reabsorption in the proximal tubule, and argues against a significant paracellular water reabsorption pathway and thus against a mechanism of solvent-drag in the proximal tubule. * p<0.001 vs. AQP1 +/+. Adapted from (73).

Figure 6. Tubuloglomerular feedback (TGF) assessed by retrograde perfusion of the macula densa

The TGF response was assessed as the change in SNGFR (by paired proximal collections) during retrograde perfusion of the macula densa segment from the early distal tubule with artificial tubular perfusate containing either 10 or 50 mM NaCl to induce minimum and maximum stimulation of TGF. The fall in SNGFR observed in control experiments was completely absent when the local adenosine A₁ receptor (A₁R) activation was “clamped” by pharmacological inhibition of adenosine generation and adding back constant amounts of an A₁R agonist. These data indicate that local adenosine concentrations must fluctuate for normal TGF to occur, indicating that adenosine is a mediator of TGF. Adapted from (54).