Two-photon microscopy: visualization of kidney dynamics.

The introduction of two-photon microscopy, along with the development of new fluorescent probes and innovative computer software, has advanced the study of intracellular and intercellular processes in the tissues of living organisms. Researchers can now determine the distribution, behavior, and interactions of labeled chemical probes and proteins in live kidney tissue in real time without fixation artifacts. Chemical probes, such as fluorescently labeled dextrans, have extended our understanding of dynamic events with subcellular resolution. To accomplish expression of specific proteins in vivo, cDNAs of fluorescently labeled proteins have been cloned into adenovirus vectors and infused by micropuncture to induce proximal tubule cell infection and protein expression. The localization and intensity of the expressed fluorescent proteins can be observed repeatedly at different time points allowing for enhanced quantitative analysis while limiting animal use. Optical sections of images acquired with the two-photon microscope can be 3-D reconstructed and quantified with Metamorph, Voxx, and Amira software programs.

Chronic caffeine consumption exacerbates hypertension in rats with polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder frequently associated with renal failure, hypertension, and other abnormalities. The present study determined whether chronic caffeine intake in an animal model of this disease would affect renal structure and function and blood pressure. Heterozygous male Han:Sprague-Dawley rats with ADPKD and normal littermates were provided with either tap water or solutions of caffeine to drink, starting at 1 month of age. When rats were aged 6 months, glomerular filtration rate (GFR) and mean arterial blood pressure (MAP) were measured under Inactin (Byk Gulden, Konstanz, Germany) anesthesia. Caffeine intake had no effect on GFR or cyst development in rats with PKD. MAP was greater in rats with PKD than normal rats and was increased more by caffeine. The hypertensive effect of chronic caffeine intake could not be ascribed to direct pressor effects of angiotensin II. Based on our finding that caffeine exacerbates hypertension in rats with PKD, it may be prudent for patients with ADPKD to limit coffee consumption to four or fewer cups of caffeinated coffee per day, pending studies of humans.

Citrate therapy for polycystic kidney disease in rats.

BACKGROUND: Few treatments are available to slow the progression to renal failure in autosomal dominant polycystic kidney disease (PKD). In an animal model of PKD, the male heterozygous Han:SPRD rat, intake of a solution of potassium citrate plus citric acid (KCitr) from age one to three months prevented a decline in glomerular filtration rate (GFR). The present study tested whether this beneficial effect is sustained and explored handling of citrate and ammonia in normal and cystic kidneys. METHODS: Rats were provided with tap water or citrate solutions to drink, and clearance and survival studies were performed. RESULTS: The GFRs of rats with PKD that consumed KCitr from one month of age were normal at six months of age, while those of their counterparts on water were about one third of normal. Long-term KCitr treatment extended the average life span of rats with PKD from 10 to 17 months. Compared with normal rats, water-drinking rats with PKD had higher plasma [citrate], renal cortical [citrate], and fractional excretion of citrate, and lower rates of renal citrate consumption, ammonia synthesis, and ammonia excretion. Cortical PNH3 was not elevated in cystic kidneys. Intake of Na3 citrate/citric acid solution or K3 citrate solution, but not ammonium citrate/citric acid solution, prevented a decline in GFR in three-month-old rats with PKD. CONCLUSIONS: Rats with PKD show abnormal renal handling of citrate and ammonia. Citrate salts that have an alkalinizing effect preserve GFR and extend survival.

Effects of potassium citrate/citric acid intake in a mouse model of polycystic kidney disease.

The kidney function in a model of autosomal dominant polycystic kidney disease (PKD), the Han:SPRD rat, is dramatically improved by chronic ingestion of a solution of potassium citrate and citric acid (KCitr). This study investigated whether this treatment would also be beneficial in the pcy/pcy mouse, a model of autosomal recessive PKD. Starting at 1 month of age, male CD-1 pcy/pcy and normal CD-1 mice were provided with a solution of 55 mM K(3) citrate/67 mM citric acid or tap water to drink. The pcy/pcy mice on the KCitr solution failed to grow normally and showed elevated plasma urea levels when compared to water-drinking littermates. Growth of normal CD-1 mice was not affected by KCitr intake. The pcy/pcy mice were then provided with a more dilute solution of KCitr to drink: this resulted in greater kidney wet and dry weights and a higher kidney weight/body weight ratio, but no beneficial effects. We conclude that pcy/pcy mice cannot tolerate a high level of KCitr intake and that a lower level is of no benefit. Whether KCitr therapy would be helpful in patients with PKD is still an open question.

Potassium citrate/citric acid intake improves renal function in rats with polycystic kidney disease.

Polycystic kidney disease (PKD) has been shown to be exacerbated by acidosis or a low potassium intake, and there is evidence that administration of alkali might have a beneficial effect. This study determined whether ingestion of potassium citrate and citric acid would ameliorate PKD. Healthy normal and heterozygous littermate Han:SPRD rats with autosomal dominant PKD were provided with either tap water or 55 mM K3citrate/67 mM citric acid solution (KCitr) to drink starting at the age of 1 mo. Renal clearance measurements and histologic assessments were performed when the rats were 3 mo old. KCitr intake did not affect body weight or urine flow, but completely prevented the decline in GFR found in untreated rats with PKD. In rats that drank tap water, left kidney GFR averaged (in microliter/min per 100 g body wt) 503 +/- 78 (n = 9) in normal animals and 242 +/- 56 (n = 6) in rats with PKD. In rats that drank KCitr, GFR averaged 562 +/- 123 (n = 7) in normal animals and 534 +/- 103 (n = 7) in rats with PKD. Kidneys of rats with PKD were approximately double normal size. KCitr treatment did not affect kidney size, but led to fewer interstitial abnormalities and smaller cysts in cystic kidneys. KCitr ingestion led to a significantly lower (P < 0.001) plasma [K+] in rats with PKD (3.3 +/- 0.2 versus 4.1 +/- 0.2 mEq/L in rats on tap water). Chronic KCitr intake in the young heterozygous Han:SPRD rat with PKD yields a modest improvement of kidney histology and a dramatic improvement in GFR. The mechanism of action of KCitr and the long-term effects of this treatment on renal structure and function in PKD deserve further study.

Organic anion secretion in polycystic kidney disease.

This study examined whether organic anion secretion contributes to fluid accumulation in cysts in polycystic kidney disease. Clearance and micropuncture studies were done on young (7 to 16 wk old), mostly male, heterozygous Han:SPRD cystic rats and healthy control littermate rats. Heterozygous Han:SPRD rats manifest a slowly progressive autosomal dominant polycystic kidney disease that closely resembles the human disorder. Left kidney GFR (polyfructosan clearance), in microl/min per 100 g body wt, averaged 331 +/- 36 (SD) in seven healthy rats and 278 +/- 75 in seven cystic rats. The maximal rate of p-aminohippurate (PAH) secretion, in micromol/min per 100 g body wt, averaged 0.94 +/- 0.24 in healthy rats and 0.83 +/- 0.11 in cystic rats. In these young rats, there were no significant differences in GFR or the maximal rate of PAH secretion despite the presence of cystic disease. Using fluorescence microscopy, it was found that 27 of 29 proximal cysts secreted sulfonefluorescein, an organic anion transported by the PAH system. Transmission electron micrographs of superficial cysts that had secreted sulfonefluorescein demonstrated the presence of both normal-appearing and poorly differentiated proximal tubule cells. Segments of superficial proximal convoluted tubules or cysts, isolated by upstream and downstream wax blocks, failed to accumulate fluid when PAH was infused intravenously. With the stationary microperfusion technique, PAH secretion by both normal and cystic nephrons was demonstrated. It is concluded that most proximal cystic epithelia retain the ability to secrete organic anions. Secretion of organic anions, however, does not appear to contribute in any substantial way to fluid accumulation in cysts in the rat kidney.

Pasteurella spp. in sympatric bighorn and domestic sheep.

Domestic sheep were sighted at different times from 1991 to 1993 on four Nevada (USA) ranges occupied by bighorn sheep. Nasal and pharyngeal swab samples were collected from both sheep species and cultured to determine if any strains of Pasteurella spp. were shared on range conditions after contact of the two species. Pasteurella spp. were isolated from all 38 bighorn sheep and 16 of 17 domestic sheep included in this study. The isolates were characterized on the bases of species, biotype, serotype, biogroup, and restriction enzyme analyses (REA) as well as ribotyping of bacterial DNA. A P. haemolytica biotype 3, biogroup 11 isolate from a domestic sheep had biochemical, REA, and ribotype profiles which were identical to those of isolates from three bighorn sheep on the same range. None of the other isolates were found to be common to the two sheep species. Disease was not detected in any of the bighorn populations. However, bighorn sheep populations were extirpated on two ranges while increasing on the other two, including the range on which P. haemolytica biotype 3, biogroup 11 strain was isolated. Declining sheep numbers were not correlated with the presence of any one strain of Pasteurella spp from the sheep.

Role of obstruction in autosomal dominant polycystic kidney disease in rats.

Kidney micropuncture and microdissection studies were carried out on heterozygous 2- to 4-month-old female and male Han:SPRD rats with autosomal dominant polycystic kidney disease (ADPKD) and on normal controls, to determine whether cysts are obstructed. Pressures in proximal tubules and cysts were determined using a servo null device and were recorded before, during, and after intraluminal infusion of an isotonic equilibrium solution at 15 and 50 n1/min. Initial cyst pressures in nine cystic rats averaged 18.5 +/- 5.9 (SD) mm Hg, N = 49, significantly (P < 0.01) higher than in normal proximal tubules in four control rats, 14.3 +/- 1.6 mm Hg, N = 36. Pressures in non-cystic tubules in cystic rats, 16.8 +/- 4.4 mm Hg, N = 25, were not significantly different from pressures in control kidneys or in cysts. When proximal tubules were microinfused at 15 nl/min in control rats, tubule pressure increased by 3.8 +/- 1.2 mm Hg, N = 24. In cysts, the response was highly variable. Twenty out of 33 microinfused cysts (61%) showed responses similar to normal tubules and were considered to be nonobstructed; 13 (39%) showed large pressure increases upon microinfusion, sometimes to values over 100 mm Hg (obstructed cysts). Left kidney inulin clearance (in microliter/min. 100 g body wt) averaged 335 +/- 65 (N = 4) in control rats and 344 +/- 144 (N = 9) in cystic rats; at this early stage of the disease no decline in GFR was seen. Weights of cystic kidneys were twice those of normal animals. Microdissection and scanning electron microscopy revealed the presence of intraluminal casts and debris and constrictions between cysts that would impede fluid flow. We conclude that obstruction is a frequent, early event in PKD and, when present, promotes cyst enlargement. Since many cysts are not obstructed, we suggest that factors other than fixed obstruction initiate cyst formation.

An in vitro test of the cell stretch-proliferation hypothesis of renal cyst enlargement.

In renal cystic disease, fluid accumulation within cyst lumens might stretch cyst walls and in this way stimulate cell proliferation. To test this idea, the effects of mechanical stretch on Madin-Darby canine kidney cells grown as cysts in a hydrated collagen gel or as monolayers on collagen-coated Flexcell membranes were examined. The percentage of cells synthesizing DNA (labeling index) was determined by measuring bromodeoxyuridine incorporation and counting cell numbers. The distension of single cysts for 1 h by the intraluminal injection of saline failed to produce a significant increase in labeling index. The exposure of cysts for 2.5 h to 1 mM dibutyryl cAMP + 0.1 mM isobutylmethylxanthine led to a 37% increase in luminal surface area (due to stimulated fluid secretion) and a 30% increase in labeling index. The stretch (25%) of Madin-Darby canine kidney monolayers approximately doubled the labeling index between 12 and 24 h after starting the stretch. After 48 h, the cell population density was significantly increased (P < 0.001), from 41.9 +/- 0.2 (SE; N = 12) to 48.2 +/- 0.5 (N = 12) cells/10,000 microns2. The labeling index increased linearly with applied stretch, from 7.2 +/- 0.3% (N = 36) with no stretch to 16.2 +/- 1.0% (N = 6) with 30% stretch. Stretch had to be maintained for 8 h or more to produce an increase in labeling index at 18 h. No evidence was obtained for the release of a diffusible growth factor by stretched monolayers. The increase in labeling index induced by stretch was unaffected by 50 microM gadolinium, a stretch-activated channel blocker, but was abolished by 5 micrograms/mL cytochalasin B, an actin microfilament-disrupting agent. It was concluded that prolonged stretch stimulates renal epithelial cells to synthesize DNA. This supports the idea that increased wall tension in renal cysts may stimulate cell proliferation and, thereby, may contribute to cyst enlargement.

NaCl transport by Madin Darby canine kidney cyst epithelial cells.

The mechanism of NaCl transport across the epithelium of intact MDCK cysts grown in a collagen gel matrix was investigated. Double-barreled microelectrodes were used to measure basolateral membrane PD (Vbl), transepithelial PD (Vt), and intracellular (Cli) and intralumenal (Clcy) Cl- activities in cysts under different conditions. In a control Ringer's solution (RS), Cli (60 +/- 1 mM) and Clcy (107 +/- 2 mM) exceeded the values corresponding to electrochemical equilibrium across the basolateral membrane and epithelium, respectively. Cli was reduced by superfusing the cysts with a low Cl- RS (Cli, 20 +/- 3 mM), a low Na+ RS (Cli, 40 +/- 4 mM), or by adding amiloride to the control RS (Cli, 46 +/- 1 mM). Cli was unaffected by removal of either K+ or HCO3- from the RS or by adding furosemide or SITS to the control RS. Vbl in the control RS was -50 +/- 2 mV and was affected only by removal from the RS of K+ (Vbl, -31 +/- 3 mV) or HCO3- (Vbl, -29 +/- 4 mV) or by the addition of SITS to the control RS (Vbl, -59 +/- 5 mV). Vt in control RS was -2 +/- 0.2 mV (lumen negative), and was increased by reducing bath Na+ (Vt, -37 +/- 2 mV) but not by reducing bath Cl-. These data indicate that Cl- is secreted in a basolateral to apical direction by the cyst epithelium. Basolateral Cl- transport probably occurs mainly by an electroneutral Cl-/HCO3- exchanger. Transepithelial Na+ transport seems to occur via a paracellular route which appears to be cation selective. These experiments also support the existence, in the basolateral membrane, of a Na+/K+ ATPase, a Na+/H+ exchanger, and possibly a Na+/HCO3-/CO3(2-) transporter.

Fluid transport in a cultured cell model of kidney epithelial cyst enlargement.

Madin-Darby canine kidney (MDCK) cells, when seeded into collagen gel, from polarized, spherical, epithelial cysts, which grow by a process involving fluid secretion and cell proliferation. These cysts are a useful model for understanding the dynamics of cyst enlargement in renal cystic disease. The hypothesis that MDCK cyst fluid secretion depends upon chloride secretion was tested, and a cell model for this process is presented here. Lumen and epithelial cell volumes were measured by video microscopy in acute experiments. Fluid absorption (-0.073 +/- 0.007 microliters.h-1.cm-2; N = 8) was observed when cysts were superfused with unsupplemented Dublecco's modified Eagle's medium at 36 to 37 degrees C. Fluid secretion (0.221 +/- 0.0016 microliters.h-1.cm-2; N = 25) was seen when 1 mM dibutyryl cAMP plus 0.1 mM 3-isobutyl-1-methylxanthine were added to the superfusate. cAMP-induced fluid secretion was significantly inhibited by basolateral 1 mM ouabain, 0.1 mM furosemide, or 1 mM amiloride. It was not significantly affected by 1 mM chlorothiazide, 0.01 mM bumetanide, or 0.1 mM acetazolamide in the presence of normal bicarbonate/CO2. In the nominal absence of bicarbonate/CO2 fluid secretion was 18% of control. Vasopressin-induced fluid secretion was significantly inhibited by pretreatment of cysts with 0.1 mM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Cyst cell shrinkage in isosmotic chloride-free Ringer's solution (chloride replaced by gluconate) was inhibited by 0.1 mM basolateral DIDS. The results suggest that chloride-bicarbonate exchange in the basolateral membrane of MDCK cyst epithelial cells plays a critical role in cyst fluid secretion.

Glomerular and proximal tubular morphology after single nephron obstruction.

This study examined the effect of blocking proximal tubule lumens on glomerular and early proximal tubular morphology. Single nephrons in the rat kidney were blocked with wax by micropuncture. After one day, one week, or one month of obstruction, the kidneys were fixed with glutaraldehyde by intravascular perfusion, and nephron structure was examined by light and electron microscopy. Following obstruction, glomerular changes developed more slowly than tubular changes. After one day, the only change noted in some glomeruli was the presence of inflammatory cells. The only tubule change upstream to the block was a focal loss of apical microvilli. This is in contrast to the severe damage previously reported (Evan, Tanner: Kidney Int 30: 818-827, 1986) in downstream proximal tubule segments at this time. After one month of obstruction, glomerular size was decreased and the glomerular filtration membrane was abnormal. Tubular cell size was decreased, apical microvilli were lost, basolateral interdigitations were reduced, and mitochondria were fewer and abnormally oriented. Interstitial fibrosis was present. Changes in nephron structure develop slowly after obstruction, perhaps because continued filtration and reabsorption maintain nephron integrity. Eventually, blocked nephrons atrophy, probably because of reduced blood flow, disuse, and inflammatory responses.

Proximal tubule morphology after single nephron obstruction in the rat kidney.

This study examined the effects on proximal tubule morphology of blocking single nephrons with paraffin wax for one day, one week, or one month in the rat. Proximal tubule lumens were blocked with a short column of wax using micropuncture. Chronically blocked and control (normal) tubules were fixed by either intravascular or intraluminal perfusion of glutaraldehyde solution. Proximal tubule segments downstream to the wax block were examined by light and transmission electron microscopy. Intraluminal Alcian blue dye, serial sectioning, and nephron microdissection techniques were used to identify nephrons. One day after obstruction, all proximal tubule cells downstream to the block were injured. Some recovery was seen. S1 and S2 segments showed more severe damage than S3 segments. Alcian blue, which normally is excluded from cells, entered the cytoplasm of some damaged S1-S2 cells. After one week of obstruction, the tubule appeared to have reconstituted itself, but cells were less differentiated than normal. One month after obstruction, blocked tubules were atrophied. Tubule cells were simplified and were surrounded by a thickened basement membrane. The results suggest that prolonged proximal tubule blockade produces injury and atrophy of the proximal tubule probably due to ischemia and interruption of normal reabsorptive activity.

Glomerular blood flow after single nephron obstruction in the rat kidney.

This study examined the effects of kidney tubule lumen obstruction on glomerular blood flow (GBF) in anesthetized rats. GBF was estimated using microspheres having 9 micron diameter and averaged 239 +/- 10 nl/min in normal nephrons of 41 control rats. Tubule blockade with either paraffin wax or castor oil produced identical results. GBF after 1-2 h of obstruction did not differ from normal. After 1 day, GBF averaged two-thirds of normal, and after 1 wk GBF averaged one-third of normal. The hemodynamic changes produced by obstruction for 1 wk were diminished by chronic administration of high doses of the converting enzyme inhibitor captopril or acute administration of the angiotensin antagonist saralasin. The results suggest that angiotensin contributes to the vasoconstriction produced by prolonged obstruction. Nephrons blocked with castor oil contained oil 1 wk later, had a GBF of 88 +/- 24 nl/min, and were atrophied. We conclude that chronic single nephron obstruction produces progressive vasoconstriction, that this response is in part angiotensin mediated, and that the end result is nephron atrophy.

Tubuloglomerular feedback after nephron or ureteral obstruction.

The effects of 1 day of single nephron, unilateral ureteral (UUO), and bilateral ureteral (BUO) obstruction on tubuloglomerular feedback were studied in anesthetized rats. Stop-flow pressure (SFP) was measured as an index of glomerular capillary pressure before, during, and after loop of Henle microperfusion. Tubuloglomerular feedback (delta SFP) showed an increased sensitivity to low loop perfusion rates and an increased maximal response after 1 day of single nephron obstruction or relief of UUO. Tubuloglomerular feedback was not significantly different from normal after release of BUO. Whole kidney glomerular filtration rate (GFR) was about 10% of normal after release of ureteral obstruction, and single nephron glomerular filtration rate (SNGFR) averaged one-third of normal. Paired measurements of SNGFR from proximal and distal tubules revealed no significant differences in control or in BUO kidneys, but a significant proximal-distal SNGFR difference was observed after UUO. The results suggest that tubuloglomerular feedback does not significantly contribute to the low GFR after release of BUO; after release of UUO, approximately one-fourth of the fall in GFR may be due to activation of the feedback mechanism.

Angiotensin in the hemodynamic response to chronic nephron obstruction.

Microsphere techniques were employed to investigate the role of intrarenal angiotensin generation in producing the arteriolar constriction associated with 24-h tubular obstruction in rats. In each animal, glomerular blood flow (GBF) and nephron vascular resistance were determined for normal and oil-blocked superficial cortical nephrons. In 17 control rats, GBF of normal and blocked nephrons averaged 226 +/- 12 and 130 +/- 9 nl/min, respectively (P less than 0.001). Captopril treatment in five rats (10 mg/kg orally) improved GBF to blocked nephrons to 252 +/- 31 nl/min. Saralasin treatment in six rats (10 micrograms . kg-1 . min-1 i.v.) lessened the difference between GBF of normal and obstructed nephrons. In six rats subjected to a high salt intake and deoxycorticosterone injections, GBF to obstructed nephrons was improved to 181 +/- 21 nl/min. Since both pharmacologic interruption of angiotensin activity and renin suppression were associated with improved GBF of blocked nephrons, these observations support a role for angiotensin as a local factor controlling glomerular hemodynamics of chronically obstructed nephrons.

Nephron obstruction and tubuloglomerular feedback.

The effects of chronic tubule blockade with paraffin on estimated glomerular capillary pressure (GCP) and single nephron glomerular filtration rate (SNGFR) were examined in anesthetized rats using micropuncture techniques. Blockade of individual proximal and distal tubule lumens for 26 hr resulted in identical reductions in GCP (by about 10 mm Hg). Proximal tubules were also blocked for about 6 hr with and without a hole in the tubular wall just upstream to the block; the hole prevented an increase in proximal tubular pressure. GCP was decreased in the block-plus-hole nephrons, suggesting that an increase in pressure in Bowman's space is not the stimulus leading to a fall in GCP. Blockade of tubules for 26 hr reduced SNGFR from 20.4 +/- 1.0 to 12.0 +/- 0.8 nl . min-1 . 100 g of body wt-1. In conclusion, chronic tubular blockade results in reductions in GCP and SNGFR unrelated to changes in tubular pressure. We suggest that this tubuloglomerular feedback response is related to decreased fluid delivery to the distal tubule macula densa.

Effect of albumin microinfusion on proximal tubular pressure.

The present study tested the idea that protein in tubular fluid is responsible for an increase in resistance of Henle's loops in the nephrotic syndrome. Single proximal tubules of anesthetized, normal rats were microinfused with an isotonic equilibrium solution containing 0--250 mg/100 ml bovine serum albumin while proximal tubular pressure (PTP) was continuously recorded. Microinfusion at 16 and 40 nl/min produced significant (p less than 0.01) increments in PTP. There was no significant effect (p greater than 0.50) of albumin. These results suggest that moderate concentrations of albumin in tubular fluid do not cause an increase in resistance to tubular fluid flow.